Thuc Pham Chuc Nang

I HC QUC GIA TP.
H CH MINH TRNG I HC BCH KHOA KHOA K THUT HA HC B MN CNG NGH SINH HC
BK
TP HCM
N CHUYN NGNH
CNG NGH SINH HC NG DNG TRONG SN XUT V CH BIN THC PHM CHC NNG
GVHD : TS. Nguyn Thy Hng SVTH : V Th Ngc An - MSSV: 60600031 Nguyn Kiu Oanh - MSSV: 60601729 Nguyn Th Minh Tm - MSSV: 60602122
TP. H Ch Minh, thng 06/2010
LI CM N
Trong sut 4 nm hc ti trng i hc Bch Khoa, ngnh Cng ngh sinh hc, chng em c trang b mt hnh trang vo i qu bu v mt kin thc chuyn ngnh m chng em yu thch. n chuyn ngnh ny l thnh qu lm vic nghim tc ca tng c nhn trong nhm thc hin sut mt hc k, bc u nh hng cho Lun vn tt nghip, vi ti : Cng ngh sinh hc ng dng trong sn xut v ch bin thc phm chc nng. hon thnh tt n, ngoi cng sc lm vic ca mi c nhn trong nhm khng th khng k n cng lao to ln ca cc thy c lun theo st chng em. Chng em xin gi li cm n chn thnh nht n Qu thy c B mn Cng ngh sinh hc, c bit l c Nguyn Thy Hng tn tnh gip v hng dn chng em trong sut thi gian thc hin n. Mc d chng em c gng ht sc nhng n chc chn khng th trnh khi nhng thiu st, rt mong s ch bo gp t Qu thy c v kin xy dng t cc bn n c hon thin hn na. TP. H Ch Minh, ngy 30 thng 06 nm 2010 Nhm SVTH: V TH NGC AN NGUYN KIU OANH NGUYN TH MINH TM
http://www.ebook.edu.vn
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MC LC
LI CM N ........................................................................................................................i MC LC ............................................................................................................................ii DANH MC BNG ...........................................................................................................ix DANH MC HNH ......................................................................................................... xiii DANH MC T VIT TT ......................................................................................... xviii LI NI U ...................................................................................................................xix
PHN 1. TNG QUAN V THC PHM CHC NNG........................................... 1 CHNG 1. KHI NIM THC PHM CHC NNG ............................................. 2 1.1. Khi nim thc phm chc nng ....................................................................... 2 1.2. Phn bit thc phm chc nng vi mt s thc phm khc .............................. 4 1.2.1 1.2.2 1.2.3 1.2.4 Thc phm thng thng (Ordinary food ingredients) ................................ 4 Thc phm chc nng (Functional Foods) ................................................... 4 Thc phm thuc (Medical Foods)............................................................... 4 Thuc v dc liu (Drug) ........................................................................... 5
1.3. Lch s nghin cu thc phm chc nng trong nc v trn th gii ............... 6 1.3.1. Trn th gii .................................................................................................. 6 1.3.2. Tnh hnh s dng thc phm chc nng Vit Nam.................................. 6 CHNG 2. PHN LOI THC PHM CHC NNG - VAI TR SINH HC...... 8 2.1. Phn loi thc phm chc nng ........................................................................... 8 2.1.1. Cc cht x chc nng trong dinh dng ..................................................... 8 2.1.2. Cc loi ng a phn t chc nng (Oligosaccharides) ........................... 9 http://www.ebook.edu.vn - ii -
2.1.3. Acid amin, peptide v protein chc nng ..................................................... 9 2.1.4. Vitamin v khong cht ................................................................................ 9 2.1.5. Vi khun sinh acid lactic, acid butyric........................................................ 10 2.1.6. Acid bo cha no ........................................................................................ 10 2.1.7. Cc loi sc t thc vt ............................................................................... 11 2.2. Phn loi da theo nguyn liu thc phm chc nng ...................................... 11 2.2.1. Thc phm chc nng c ngun gc thc vt ............................................ 11 2.2.2. Thc phm chc nng c ngun gc ng vt ........................................... 13 2.3. Vai tr sinh hc ca mt s loi thc phm chc nng ti sc khe ................ 16 2.3.1. Thc phm chc nng ngun gc thc vt ................................................. 16 2.3.2. Thc phm chc nng t ngun nguyn liu sinh vt bin v ng vt .... 22 2.3.3. Thc phm chc nng t ngun nguyn liu nm...................................... 27 CHNG 3. PHNG PHP LM GIU CC CHT DINH DNG CHC NNG ............................................................................................................................. 33 3.1. Chn ging cy trng vt nui giu cht dinh dng chc nng ...................... 33 3.2. Lm giu cht dinh dng thng qua con ng ch bin thc phm ............. 34 3.3. Lm giu cht dinh dng thng qua k thut, chn nui ................................. 35 CHNG 4. NHNG QUY NH CHUNG V QUN L THC PHM CHC NNG TRN TH TRNG ........................................................................................ 36 4.1. Qui nh v s cng nhn tc dng cc cht dinh dng chc nng ................ 36 4.1.1. Qun l tiu chun v mt khoa hc cc cht dinh dng chc nng........ 36 4.1.2. Yu cu chp nhn ca c quan qun l nh nc v an ton thc phm v dc phm trc khi a ra th trng tiu th ...................................................... 37 4.2. Qui nh v tn gi v dn nhn, lu hnh trn th trng................................ 37 4.1.1. Mc tiu ca dn nhn thc phm thng thng v TPCN ....................... 37
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4.1.2. C quan qun l dn nhn thc phm ........................................................ 37 PHN 2. NG DNG CNG NGH DI TRUYN TRONG SN XUT THC PHM CHC NNG ...................................................................................................... 39 CHNG 1.THNH TU PHT TRIN CA THC PHM CHUYN GEN ...... 40 1.1. Tnh hnh pht trin ca thc phm chuyn gen trn th gii v Vit Nam ..... 40 1.1.1. Trn th gii .................................................................................................. 40 1.1.2. Tnh hnh pht trin thc phm chuyn gen Vit Nam .............................. 47 1.2. Nhng c tnh mi ca sinh vt chuyn gen c dng trong thc phm chc nng ............................................................................................................................. 48 1.2.1. Thc vt ......................................................................................................... 48 1.2.2. ng vt ........................................................................................................ 51 CHNG 2. CC PHNG PHP CHUYN GEN .................................................. 57 2. 1.Thc vt................................................................................................................ 57 2. 1.1. Phng php chung ...................................................................................... 57 2.1.2. K thut c bn ............................................................................................. 58 2.2. ng vt ............................................................................................................... 71 2.2.1. Nguyn tc ..................................................................................................... 71 2.2.2. Cc phng php ........................................................................................... 76 CHNG 3. TNG CNG FOLATE TRONG CC LOI CY THC PHM .. 81 3.1. Folate .................................................................................................................... 82 3.1.1. Gii thiu v folate ........................................................................................ 82 3.1.2. Cc dng ca folate ....................................................................................... 83 3.1.3. S thiu ht folate v sc khe ..................................................................... 84 3.1.4. Nhu cu folate ............................................................................................... 85
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3.2. C chua tng cng folate th h 1 ...................................................................... 86 3.2.1. Nguyn l ...................................................................................................... 86 3.2.2. Vt liu v phng php ............................................................................... 88 3.2.3. Kt qu........................................................................................................... 92 3.2.4. Bn lun ....................................................................................................... 100 3.3. C chua tng cng folate th h 2 .................................................................... 102 3.3.1. Vt liu v phng php ............................................................................. 102 3.3.2. Kt qu......................................................................................................... 103 3.4. Go tng cng folate ........................................................................................ 105 3.4.1. Vt liu v phng php ............................................................................. 105 3.4.2. Kt qu......................................................................................................... 106 3.5. Nhng thnh tu v hng pht trin ................................................................ 112 3.5.1. Thnh tu..................................................................................................... 112 3.5.2. Hng pht trin .......................................................................................... 114 3.6. Kt lun v cc loi cy trng chuyn gen c lm giu folate ...................... 117 CHNG 4. KT LUN V CC LOI CY CHUYN GEN GIU CHT DINH DNG CHC NNG ............................................................................................... 118 PHN 3. NG DNG CNG NGH VI SINH TRONG SN XUT THC PHM CHC NNG ................................................................................................................. 120 CHNG 1. TO SPIRULINA ................................................................................... 121 1.1. Gii thiu chung .............................................................................................. 121 1.2. Tng quan v to Spirulina ............................................................................. 123 1.2.1. Lch s pht hin....................................................................................... 123 1.2.2. Phn loi ................................................................................................... 123 1.2.3. c im sinh hc ca to Spirulina ........................................................ 123 http://www.ebook.edu.vn -v-
1.2.4. Thnh phn ha hc ca Spirulina ........................................................... 126 1.2.5. Tnh hnh nui trng v pht trin Spirulina ............................................ 129 1.2.6. Tnh hnh nghin cu v ng dng Spirulina ........................................... 130 1.2.7. Cng ngh nui trng to Spirulina.......................................................... 136 1.2.8. Cc phng php ph v t bo S.platensis ............................................. 140 1.3. Nghin cu tnh hung .................................................................................... 142 1.3.1. Nghin cu x l to Spirulina................................................................. 142 1.3.2. Nghin cu chit sut cc hp cht chng oxy ha t S.platensis ........... 154 1.3.3. Nghin cu nh hng ca sinh khi Spirulina platensis ln h vi khun ca sa ln men ABT trong sut thi gian bo qun ............................................ 167 1.4. Gii thiu mt s sn phm Spirulina hin nay .............................................. 180 1.5. Kt lun............................................................................................................ 182 CHNG 2. FRUCTOOLIGOSACCHARIDE (FOS) ............................................... 184 2.1. Tng quan v FOS ........................................................................................... 184 2.1.1. Khi nim FOS ......................................................................................... 184 2.1.2. Ngun gc FOS Cu to ........................................................................ 185 2.1.3. Tnh cht ca FOS: ................................................................................... 189 2.1.4. nh hng ca FOS ln c th v sc khe con ngi ........................... 191 2.1.5. Tnh an ton v ng dng ca FOS .......................................................... 195 2.1.6. Tnh hnh nghin cu v sn xut FOS trn th gii ................................ 197 2.1.7. Gii thiu cc enzyme trong sn xut FOS .............................................. 204 2.1.8. Tim nng cho sn xut FOS ti Vit Nam .............................................. 209 2.2. Thu nhn v tinh sch enzyme -fructofuranosidase t chng nm mc Aspergillus niger IMI303386 .................................................................................... 211 2.2.1. Tm tt nghin cu ................................................................................... 211 2.2.2. Nguyn liu v phng php .................................................................... 211 http://www.ebook.edu.vn - vi -
2.2.3. Kt qu v bn lun .................................................................................. 215 2.3. Ln men ti s dng nhiu chu k chng Aspergillus oryzae CFR 202 thu nhn enzyme fructosyltransferase v ng dng sn xut ng FOS ............................... 222 2.3.1. Tm tt nghin cu ................................................................................... 222 2.3.2. Nguyn liu v phng php .................................................................... 223 2.3.3. Kt qu v bn lun .................................................................................. 224 2.4. C nh t bo nm mc Aspergillus japonicus vo cht mang gluten v ng dng sn xut ng fructooligosaccharide ............................................................. 226 2.4.1. Tm tt nghin cu ................................................................................... 226 2.4.2. Nguyn liu v phng php .................................................................... 227 2.4.3. Kt qu ...................................................................................................... 228 2.5. Sn xut FOS cao t sucrose s dng h enzyme -fructofuranosidase v glucose oxidase ......................................................................................................... 233 2.5.1. Tm tt nghin cu ................................................................................... 233 2.5.2. Nguyn liu v phng php .................................................................... 233 2.5.3. Kt qu v bn lun .................................................................................. 235 2.6. ng dng cng ngh enzyme thu nhn ng chc nng FOS t dch ma Vit Nam ................................................................................................................... 244 2.6.1. Tm tt nghin cu ................................................................................... 244 2.6.2. Vt liu v phng php nghin cu ........................................................ 244 2.6.3. Kt qu nghin cu v tho lun: ............................................................. 245 2.7. Nghin cu s dng ng FOS trong sn xut mt s thc phm chc nng Vit Nam ................................................................................................................... 252 2.7.1. Sn xut bt dinh dng tr em ................................................................ 252 2.7.2. Sn xut bnh bch quy ............................................................................. 254
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2.7.3. Sn xut ko .............................................................................................. 257 2.8. Kt lun chung v sn xut FOS v ng dng cho n nay ............................ 259 KT LUN CHUNG TI ....................................................................................... 261 TI LIU THAM KHO.............................................................................................. 262
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DANH MC BNG
PHN 1: TNG QUAN V THC PHM CHC NNG Bng 1.1: Phm tr gia thc phm v thuc ...................................................................... 5 Bng 2.1: H thng phn loi FOSHU ............................................................................... 15 Bng 2.2: Hm lng lycopene trong mt s sn phm c chua ....................................... 19 Bng 2.3: Kh nng c ch cc gc t do ca cc hp cht trong tr xanh ....................... 21
PHN 2: NG DNG CNG NGH DI TRUYN TRONG SN XUT THC PHM CHC NNG Bng 1.1: Din tch cy trng CNSH nm 2007 trn th gii............................................ 41 Bng 1.2: Trang tri nui ging ng vt sn xut protein dc phm ............................. 51 Bng 1.3: Nhng protein c tc dng cha bnh trong sa c sn xut ra bi th cho sa chuyn gen ................................................................................................................... 53 Bng 1.4 : Mt vi thng s lin quan n vic tit sa ng vt c v......................... 53 Bng 1.5 : c tnh sn lng protein sinh phm trong sa v qui m n th chuyn gen trn th gii ......................................................................................................................... 54 Bng 1.6: Mt s thay i cc thnh phn ca sa c xut ...................................... 56 Bng 2.1: Cc phng php chuyn gen khng s dng cc yu t virus ......................... 77 Bng 3.1: Nhng cy lng thc c folate......................................................................... 83 Bng 3.2: Nhu cu folate ngh cho tr em v ngi ln ................................................ 85
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PHN 3: NG DNG CNG NGH VI SINH TRONG SN XUT THC PHM CHC NNG Bng 1.1: Thnh phn ha hc ca Spirulina .................................................................. 126 Bng 1.2: Thnh phn vitamin ca Spirulina ................................................................... 127 Bng 1.3: Thnh phn khong ca to Spirulina ............................................................. 127 Bng 1.4: Thnh phn acid amin ca to Spirulina ......................................................... 128 Bng 1.5: Cc cht mu trong Spirulina .......................................................................... 128 Bng 1.6: Cc loi thc phm c ch bin t to Spirulina ......................................... 131 Bng 1.7: Mi trng c bn( mi trng Zarrouk) ........................................................ 137 Bng 1.8: Mi trng b sung 1....................................................................................... 138 Bng 1.9: Mi trng b sung 2....................................................................................... 138 Bng 1.10: So snh mu th nghim t 1 n 10 vi mu chun .................................... 151 Bng 1.11: Cc mu so snh khng b sung ng c so snh vi mu chun .......... 152 Bng1.12: So snh hm lng phycocyanin gia cc mu .............................................. 153 Bng 1.13: Mc m ha th nghim ca cc yu t dng trong thit k Box-Behnken .......................................................................................................................................... 160 Bng 1.14: Ma trn thit k bi Box-Behnken cng vi th nghim v d on gi tr hiu sut ca dch chit. ........................................................................................................... 163 Bng 1.15: Thnh phn v hm lng tng i ca acid bo trong dch chit t ......... 166 Bng 1.16: Nhng thnh phn c th ng gp trong cht hot ng chng oxy ha t S.platensis ......................................................................................................................... 166 Bng 1.17: S lng tn ti (log CFU/ml) ca Streptococcus thermophilus, Lactobacillus acidophilus, v Bidobacterium spp. trong sa ln men b sung Spirulina v ln men thng thng trong thi gian bo qun 15C. ............................................................... 174
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Bng 1.18: T l % tn ti ca Streptococcus thermophilus, Lactobacillus acidophilus, v Bidobacterium spp. trong sa ln men b sung Spirulina v ln men thng thng trong thi gian bo qun 15C. ............................................................................................... 174 Bng 1.19: pH v chua ca sn phm sa ln men b sung Spirulina v ln men thng thng trong thi gian lu tr 150C. ............................................................................. 176 Bng 1.20: S lng tn ti (logCFU/ml) ca Streptococcus thermophilus, Lactobacillus acidophilus, v Bidobacterium spp. trong sa ln men b sung Spirulina v ln men thng thng trong thi gian bo qun 4C. ................................................................. 177 Bng 1.21: T l % tn ti ca Streptococcus thermophilus, Lactobacillus acidophilus, v Bidobacterium spp. trong sa ln men b sung Spirulina v ln men thng thng trong thi gian bo qun 4C. ................................................................................................. 177 Bng 1.22: pH v chua ca sn phm sa ln men b sung Spirulina v ln men thng thng trong thi gian lu tr 40C. ............................................................................... 179 Bng 2.1: Hm lng FOS ca mt s loi cy qu (mg/g cht kh) .............................. 186 Bng 2.2: Ngun enzyme tng hp FOS t thc vt ....................................................... 188 Bng 2.3: Vi sinh vt sn xut FOS ................................................................................. 189 Bng 2.4: c tnh ca FOS so vi mt s loi ng khc ........................................... 190 Bng 2.5: Din bin tng trng ca ngnh cng nghip ma ng trn ton quc ..... 210 Bng 2.6: Kt qu tinh sch -fructofuranosidase t A. niger IMI 303386 ..................... 215 Bng 2.7: nh hng ca cc ion kim loi v hp cht ln hot tnh enzyme fructofuranosidase ca A. niger ........................................................................................ 221 Bng 2.8: Thnh phn ng so snh gia FOS v FOS cao ..................................... 243 Bng 2.9: Thnh phn ng trong dch ma ................................................................... 245 Bng 2.10: nh hng ca t l Enzyme/c cht ti s bin i thnh phn ng ....... 249 Bng 2.11: Thnh phn v hm lng ng c trong sn phm FOS ........................... 250 http://www.ebook.edu.vn - xi -
Bng 2.12: Thnh phn ca bt dinh dng tr em ......................................................... 253 Bng 2.13: Kt qu nh gi cm quan bt dinh dng tr em ....................................... 254 Bng 2.14: Thnh phn ca bnh bch quy ...................................................................... 256 Bng 2.15: Kt qu nh gi cm quan bnh bch quy .................................................... 257 Bng 2.16: Thnh phn ca hai loi ko .......................................................................... 259 Bng 2.17: Kt qu nh gi cm quan ca hai loi ko.................................................. 259
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DANH MC HNH
PHN 1: TNG QUAN V THC PHM CHC NNG Hnh 2.1: Cng thc cu to Soyasaponin I IV .............................................................. 17 Hnh 2.2: Cng thc cu to isoflavone v dn xut ......................................................... 17 Hnh 2.3: Cng thc ha hc ca lycopene ........................................................................ 18 Hnh 2.4: Cng thc cu to cc hp cht catechin trong tr xanh ................................... 20 Hnh 2.5: Cc hp cht curcumin trong c ngh ................................................................ 22 Hnh 2.6: Gii thiu hnh nh mt s loi nm .................................................................. 32 PHN 2: NG DNG CNG NGH DI TRUYN TRONG SN XUT THC PHM CHC NNG Hnh 1.1 : Din tch cy trng CNSH trn th gii t 1996 n 2007 ............................... 42 Hnh 2.1: Vi khun Agrobacterium tumefaciens nhn di knh hin vi. .......................... 59 Hnh 2.2: Khi u thc vt do Agrobacterium tumefaciens gy ra................................... 59 Hnh 2.3: Cng thc cu to ca opine (octopin, nopalin) ................................................ 60 Hnh 2.4: S gen ca Ti-plasmid trong vi khun A. tumefaciens ................................. 64 Hnh 2.5: S plasmid ti t hp da trn nguyn tc ca Ti-plasmid .......................... 64 Hnh 2.6: Qu trnh to cy chuyn gen nh A.tumefaciens. ............................................. 65 Hnh 2.7 : Sng bn gen (Hng Biorad) ............................................................................. 66 Hnh 2.8: S nguyn l hot ng ca sng bn gen ................................................... 67 Hnh 2.9 : S mng phospholipid kp .......................................................................... 68 Hnh 2.10 : Cuvette nha c in cc ................................................................................ 68 Hnh 2.11: S b tr mch c bn ca my xung in .................................................. 69 http://www.ebook.edu.vn - xiii -
Hnh 2.12 : S plasmid cha DNA ngoi lai i qua cc l tm thi trn mng bo cht ............................................................................................................................................ 70 Hnh 2.13: Vi tim gen ngoi lai vo nhn ca protoplast ................................................. 71 Hnh 2.14 : S to ng vt chuyn gen ....................................................................... 72 Hnh 2.15: Trng c chch (Misgurnus anguillicaudatus) trc v sau khi kh mng th cp (chorion) ....................................................................................................................... 75 Hnh 2.16 : Phc hp DNA-calcium phosphat .................................................................. 78 Hnh 2.17: S kt hp gia DEAE-dextran v DNA ......................................................... 78 Hnh 3.1: Cu trc ha hc ca folate ................................................................................ 82 Hnh 3.2: Con ng sinh tng hp folate ......................................................................... 87 Hnh 3.3: Cu trc vector pMON10086 ............................................................................. 88 Hnh 3.4: Vng 5 ca cDNA GCHI tng hp ................................................................... 89 Hnh 3.5: Nguyn l to cy c chua chuyn gen th h 1................................................. 89 Hnh 3.6: Phn tch HPLC hunh quang nhng pteridine oxi ha trong qu chn (Ct Ultramex C18 IP) ................................................................................................................. 93 Hnh 3.7: S thay i trong lng pteridine tng ca qu i chng (V2) v qu GCHI+ trong sut qu trnh chn ..................................................................................................... 94 Hnh 3.8 : Cc giai on chn ca c chua ........................................................................ 94 Hnh 3.9: nh lng cc pteridine chnh trong qu chn ca 3 th bin np GCHI+ i din ..................................................................................................................................... 95 Hnh 3.10: Phn tch HPLC hunh quang peak 1 v 2 cha bit trc v sau khi x l vi HCl, -glucosidase hoc -glucosidase ............................................................................. 96 Hnh 3.11: Hm lng folate tng trong qu chn ca 12 th bin np GCHI+ v 10 th bin np i chng ............................................................................................................. 97
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Hnh 3.12: Phn tch folate trong qu GCHI+ v qu i chng ...................................... 98 Hnh 3.13: nh hng ca PABA ngoi sinh n hm lng folate ................................ 99 Hnh 3.14: Nguyn l to cy c chua chuyn gen th h 2 ............................................ 103 Hnh 3.15: Hm lng folate, PABA, v pteridine tch ly trong cc dng AtADCS+, GCHI+ v GCHI+/AtADCS+ ......................................................................................... 104 Hnh 3.16: Gin i din ca T-DNA trong cc vector dng chuyn gen ............ 105 Hnh 3.17: Mc PABA v folate tng trong nhng dng A ........................................... 107 Hnh 3.18: Mc pterin v folate tng trong nhng dng G ............................................. 108 Hnh 3.19: Mc PABA, pterin v folate tng trong nhng dng GA ............................. 108 Hnh 3.20: So snh thnh phn cc loi folate chnh trong dng GA so vi dng WT ... 110 Hnh 3.21: T l tng i ca polyglutamate v monoglutamate folate ca dng GA v WT ................................................................................................................................... 111 Hnh 3.22: S thay i mc folate tng trong ht ca dng GA khi nu ........................ 112
PHN 3: NG DNG CNG NGH VI SINH TRONG SN XUT THC PHM CHC NNG Hnh 1.1: Hnh dng to Chlorella, Scenedesmus, Spirulina .......................................... 121 Hnh 1.2: Hnh dng ca to Spirulina ............................................................................ 124 Hnh 1.3: S vng i ca Spirulina .......................................................................... 125 Hnh 1.4: S ca qu trnh chit SC-CO2 ................................................................... 161 Hnh 1.5: Hot cht chng oxy ha c nh gi bng phng php c ch qu trnh peroxide ha ca acid linoleic ......................................................................................... 165 Hnh 2.1: Cng thc cu to ca FOS ............................................................................. 187 Hnh 2.2: S thay i nng insulin trong mu ........................................................... 191
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Hnh 2.3: S thay i nng glucose trong mu .......................................................... 191 Hnh 2.4: S thay i nng fructose trong mu ......................................................... 192 Hnh 2.5: Quy trnh sn xut FOS t FTS vi sinh vt ..................................................... 201 Hnh 2.6: S quy trnh sn xut FOS lin tc v khng lin tc ................................. 202 Hnh 2.7: C ch chuyn ha to FOS t sucrose ca FTS A.pullulans ......................... 206 Hnh 2.8: Qu trnh oxy ha glucose di tc dng ca enzyme GOD .......................... 208 Hnh 2.9: Phn ng oxy ha glucose di tc dng ca h 2 enzyme GOD v CAT ..... 209 Hnh 2.10: Sc k trao i ion DEAE-Sepharose ........................................................... 216 Hnh 2.11: Sc k lc gel Ultrogel AcA44 ca -fructofuranosidase ............................. 217 Hnh 2.12: Kt qu in di SDS-PAGE ca -fructofuranosidase ................................. 218 Hnh 2.13: nh hng ca pH ln hot tnh v n nh ca -fructofuranosidase .... 219 Hnh 2.14: nh hng ca nhit ln hot tnh v n nh ca -fructofuranosidase .......................................................................................................................................... 220 Hnh 2.15: S thay i pH v hot tnh FTS trong qu trnh nui cy nhiu chu k A. oryzae CFR 202 ................................................................................................................ 225 Hnh 2.16: Hm lng FOS sinh ra bi FTS qua cc chu k nui cy A. oryzae ............ 225 Hnh 2.17: Nng v phn khi lng ca cc loi ng theo thi gian ................... 229 Hnh 2.18: Sn xut FOS s dng t bo c nh v t bo khng c nh ..................... 230 Hnh 2.19: nh hng ca lng t bo c nh trong mng gluten ............................... 231 Hnh 2.20: Phn khi lng FOS v nng sut to FOS theo tc dng chy ............. 232 Hnh 2.21: nh hng ca nhit (A) v pH (B) ln hn hp enzyme ........................ 236 Hnh 2.22: nh hng ca nhit n t l FOS to thnh .......................................... 237 Hnh 2.23: nh hng ca tc khuy trn n t l FOS to thnh ............................ 238
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Hnh 2.24: nh hng ca lu lng oxy n sn xut FOS ......................................... 239 Hnh 2.25: nh hng ca nng sucrose n t l to FOS ....................................... 240 Hnh 2.26: nh hng ca nng glucose oxidase ln t l to FOS .......................... 241 Hnh 2.27: T l cc loi ng trong hn hp phn ng iu kin ti u .................. 242 Hnh 2.28: Sc k HPLC ca FOS v FOS cao .................................................... 242 Hnh 2.29: nh hng ca nhit n kh nng vn chuyn Fructose ........................ 246 Hnh 2.30: nh hng ca pH n kh nng vn chuyn Fructose ................................ 247 Hnh 2.31: nh hng ca thi gian n kh nng vn chuyn Fructose ...................... 247 Hnh 2.32: Sc k ca ng FOS v cc loi ng trong mu nghin cu ........... 250 Hnh 2.33: Sc k ca ng FOS chun .................................................................. 250 Hnh 2.34: S quy trnh sn xut siro FOS t nc ma s dng Pectinex Ultra SP-L .......................................................................................................................................... 251 Hnh 2.35: S quy trnh sn xut bt dinh dng tr em c b sung FOS ................ 253 Hnh 2.36: S quy trnh sn xut bnh bch quy s dng ng FOS ....................... 255 Hnh 2.37: S quy trnh sn xut ng cng s dng ng FOS .......................... 258
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DANH MC T VIT TT
AAPH : 2,2-azobis (2-amidinopropane) hydrochloride. ABT : H vi khun Lactobacillus acidophilus (A), Bifidobacteria (B), v Streptococcus thermophilus (T) ADCS : enzyme aminodeoxychorismate synthase BHT DFE FDA FOS FTS : Butylated hydroxytoluene. : dietary folate equivalents: hm lng folate tng ng trong ba n : Food and Drug Administration: t chc Thc phm v Thuc Hoa K : Fructooligosaccharide : Fructosyltransferase DALY : disability adjusted life years: t l khuyt tt c iu chnh hng nm FAME : Fat Acid Methyl Esters.
GC-MS : Gas Chromatography-Mass Spectometry Sc k kh-khi ph. GCHI :enzyme GTP cyclohydrolase I GOD NTD RSM THF WT : Glucose oxidase : neural tube defects: d tt ng thn kinh : Response Surface Method Phng php b mt p ng. : tetrahydrofolate : wild type: chng hoang di
PAPA : p-aminobenzoate SC-CO2: Supercritical carbon dioxide CO2 siu ti hn. Trolox : 6-hydroxyl-2,5,7,8-tetramethylchroman-2-carboxylicacid.
- xviii -
LI NI U
Trong hn 20 nm qua, ngi dn cng nh gii khoa hc c thm mt ci nhn na v thc phm. Thc phm khng ch l duy tr s sng m cn thm kh nng tng cng sc khe, gim thiu cc bnh mn tnh do thiu cn bng dinh dng. T khi ngun cho s tm hiu v ch bin loi thc phm trong ngoi vic cung cp nhu cu dinh dng c bn m cc thnh phn cu to cn c tc dng tch cc vo nhng nhim v khc nhau ca c th. l Thc phm chc nng. Cc nh khoa hc trn th gii d bo rng: thc n ca con ngi trong th k XXI s l thc phm chc nng. Cc hot cht m thc phm chc nng mang li cho con ngi chnh l nhng v thuc qu, gip con ngi tng cng min dch, chng lo ha, ko di tui th, phng v cha cc bnh mn tnh, k c ung th. Loi thc phm chc nng u tin c k n l nhng thc phm m khi dng t nhin c nhng hot cht c li cho con ngi. Tip l nhm thc phm c t hot cht hn, phi b sung hoc tinh ch, c c li dng d s dng, hay bin i gen tng hm lng mt s cht c li. Ngy nay vi s pht trin mnh m ca cng ngh sinh hc, vic ch bin v sn xut thc phm chc nng tr nn d dng hn. Con ngi to ra cc thc phm chc nng rt a dng v th loi v phong ph v hot tnh sinh hc. i vi nc ta, y l lnh vc c nhiu trin vng, bi ngun ti nguyn thin nhin phong ph v a dng, cng vi s u t vo cng ngh sinh hc, bc u t c mt s thnh tu ng ghi nhn.
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Phn 1: Tng quan v thc phm chc nng
PHN 1 TNG QUAN V THC PHM CHC NNG
-1-
Chng 1: Khi nim thc phm chc nng
CHNG 1 KHI NIM THC PHM CHC NNG
1.1.
Khi nim thc phm chc nng Khi nim v tn gi v thc phm chc nng bt ngun t Nht Bn. Vo nm 1980
B Y t v Sc khe ca nc ny bt u xy dng h thng t chc trong B, t chc ny c nhim v iu chnh v cng nhn nhng loi thc phm c hiu qu ci thin sc khe ca cng ng dn c. H cho php ghi trn nhn hiu hng ha thc phm s dng cho sc khe con ngi, c vit tt t cm t ting Anh l FOSHU (Foods for Specified Health Use). Sau hn 20 nm hot ng trn lnh vc ny, n thng 9 nm 2001 c trn 271 sn phm thc phm mang nhn hiu FOSHU. Nht, thc phm chc nng c nh ngha nh sau : Nhng loi thc phm c hiu qu ln sc khe bi cc cht dinh dng truyn thng v cc hot cht sinh hc c cha trong n, ngi ta gi l thc phm chc nng. Sau , thc phm chc nng xut hin trn nhiu nc khc. M quan im v thc phm chc nng ca ADA (The American Dietetic Association): Thc phm chc nng l bao gm tt c cc thnh phn c trong n v cng l thc phm c lm mnh them, lm giu thm hoc nng cao thm yu t no , c hiu qu tim nng n sc khe khi tiu th mt phn n trong khu phn c nhiu loi mt cch thng xuyn vi mc c tc dng. Theo FDA th : Thc phm chc nng l loi thc phm cung cp cc cht dinh dng c bn c ch cho sc khe. Thc phm chc nng l thc phm m nu n n, th sc khe s tt hn khi khng n n. V d nh rau xanh v tri cy c cha cht lm tng cng sc khe. Nhng cht c hot tnh sinh hc trong thc phm chc nng c ch cho sc khe hoc c nh hng sinh l theo hng mong mun. Theo IFIC (The International Food Information Council) th thc phm chc nng l thc phm c li cho sc khe bi cc cht dinh dng c bn. -2-
Theo ILSI (The International Life Sciences Institute of North America) th thc phm chc nng l loi thc phm c cha hot tnh sinh hc c ch cho sc khe trn c s cc cht dinh dng c bn. Vin nghin cu Y hc ca Vin hn lm khoa hc M cho rng : thc phm chc nng l thc phm c cha mt hay nhiu hn nhng nguyn liu thc phm c sa i nng cao hiu qu cho sc khe. Trung Quc th thc phm chc nng c coi l thc phm bao gm cc cht dinh dng nh thc phm bnh thng, nhng c bit c cha yu t th hai hay th ba c tc dng phng chng bnh nh l dc liu. Ti Vit Nam t 1990 1991, Vin Dinh dng xc nh : Thc phm chc nng l thc phm c cha cc cht c hot tnh sinh hc cn thit cho sc khe bao gm c thc phm ch bin ci tin, thc n c truyn dn tc v cc thnh phn khng dinh dng khc c tc ng c bit v cn thit cho sc khe. Thuc tnh chc nng ni ln vai tr sinh hc ca mt hay nhiu cht dinh dng chc nng c trong thc phm truyn thng, n c pht hin ra vi nhng thnh phn cc cht c bit c hu ch cho sc khe. Cn phi c s kt hp nghin cu ym tr xc nh hiu qu sc khe cng nh nguy c ca thc phm chc nng khi n n iu chng vi nhng thnh phn c hot tnh sinh l mnh trong thc phm chc nng. Chuyn ngnh dinh dng s tip tc cng vi cng ngh thc phm, nh nc, hi ng khoa hc, v cc c quan truyn thong phi c nhng ch dn chnh xc r rng v mt khoa hc ca thc phm v dinh dng ngi tiu th bit cch p dng. Khi nghin cu v thc phm chc nng v thuc tr bnh, ngi ta thy n nh l vng giao thoa gia thc phm v thuc. N va cha cc cht dinh dng nh l thc phm truyn thng, li va c hot cht sinh hc c tc dng phng tr bnh nh l thuc [3].
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1.2.
Phn bit thc phm chc nng vi mt s thc phm khc S khc bit gia Thc phm chc nng, Thc phm thuc v Thc phm thng
thng c th hin qua s sau :
Thc phm Thc phm Thc phm thng thng chc nng thuc
Thuc v dc liu
1.2.1 Thc phm thng thng (Ordinary food ingredients) L thc phm vi cc thnh phn v gi tr dinh dng c bn c ghi trn nhn v phi m bo yu cu v sinh an ton thc phm c cng ng v c quan qun l thc phm chp nhn. Khi n loi thc phm ny th c th pht trin mt cch bnh thng trong iu kin bnh thng [3]. 1.2.2 Thc phm chc nng (Functional Foods) L thc phm ngoi cht dinh dng c bn ra, cn cha mt s hot cht chc nng c bit mc cao, c tc dng phng chng bnh tt, m bo cho sc khe bn vng. Thc phm chc nng phi c cng b thnh phn cc cht dinh dng chc nng trong thc phm v c hng dn s dng phng chng nhng bnh tt g. Thc phm chc nng khng cn phi c s k n hoc qun l theo di iu tr trong phng v cha tr bnh, thc phm phi c ng k theo qui nh ca iu l V sinh an ton thc phm [3]. 1.2.3 Thc phm thuc (Medical Foods) L thc phm phi t yu cu cao trong ch bin v phi trn cc thnh phn dinh dng cng vi cc hot cht c tc dng dc l theo tiu chun k thut ca ngnh dc v ngnh thc phm, phi t tiu chun thuc ung v v sinh an ton thc phm, phi ng k theo tiu chun thuc v thc phm ca B Y t. Theo FDA M, t nm 1938 th -4-
khi s dng thc phm thuc phi c Bc s k n, chn on dinh dng c ch n thch hp cho bnh nhn. V d nh thc n qua xng h tr sc lc cho bnh nhn trong qu trnh iu tr [3]. 1.2.4 Thuc v dc liu (drug) L sn phm bo ch phi c tun th nghim ngt ca ngnh dc, m bo an ton v hiu qu trong vic s dng phng v tr bnh, thuc phi c th nghim hon chnh trn lm sang. Thuc phi ng k theo tiu chun thuc v phi c s k n ca Bc s. Trong bng di y tm lc cc c im ca cc loi thc phm v thuc, cc yu cu s dng v dn nhn trn bao b. Bng 1.1: Phm tr gia thc phm v thuc Phm tr Thuc v dc liu Sn phm Dc phm c qui nh s dng, Bc s k n. Dc phm khng c qui nh k n ca Bc s, c hng dn. Sn xut theo qui trnh sn xut thuc, thc phm. c Bc s chn on dinh dng v k n. Th h I: Chn la thc phm chc nng c trong t nhin. Th h II: Thc phm c b sung tng cng hot cht chc nng. Th h III: Thc phm c phi hp cc hot cht chc nng. Thc phm mi Thc phm dinh dng c bit. Thc phm thng thng [3]
Thc phm thuc Thc phm chc nng
Thc phm thng thng
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1.3.
Lch s nghin cu thc phm chc nng trong nc v trn th gii
1.3.1. Trn th gii T nm 1900 c khi nim s dng thc phm phng bnh v tng cng cho sc khe. Lc by gi ngi ta bit d dng mui giu iod phng v cha bnh bu c, ngy nay chng ta coi l thc phm chc nng. Trc ngi ta cn bit s dng c c rt cha bnh qung g, ngy nay ta coi l bnh thiu vitamin A v c rt cung cp tin cht vitamin A cha bnh. Thi k c i Hypocrat coi thc n cng l phng tin iu tr bnh. Nhng lc by gi, ngi ta cha hiu mt cch r rng cht no trong thc phm c gi tr phng v cha bnh, phn ln h dng thc phm phng v cha bnh theo kinh nghim. V vy t th k th 19 tr v trc, ngi ta c quan nim khi c b phn no ca c th b bnh th tm thc n tng ng n vo s cha c bnh, ni khc i l au ci g n ci ny. iu ny khng ng vi cc bnh truyn nhim. Sau ny khi ngnh ha hc hu c, ha phn tch, ha sinh pht trin, ngi ta mi lm r ra vai tr sinh hc ca mi loi cht dinh dng trong thc n i vi c th. nh ngha thc phm chc nng ngy cng sng t hn, ngy cng c s giao thoa gia ngnh Y Dc vi ngnh ch bin thc phm. Ni khc i ngi thy thuc cng l ngi u bp, ngi u bp cng l ngi thy thuc. Hin nay Trung Quc v mt s quc gia khc pht trin thc phm chc nng rt mnh. Theo Zonglian Jin v Bodi Hui, trng i hc Khoa hc Bc Kinh (2003) th n cui nm 2002 c n 3799 sn phm thc phm chc nng c B Y t Trung Quc chun y. Trong s ny c n 71,5% thc phm chc nng thuc nhm gip cho c th tng cng sc khng khng bnh, gip cho c th iu chnh lng m mu v gii thot mt mi, th gin [3]. 1.3.2. Tnh hnh s dng thc phm chc nng Vit Nam i vi nc ta, vic nghin cu to ra cc ch phm thc phm chc nng mang phng chm cng ngh cao, bn sc c truyn l hng nghin cu rt l th v c li th, bi v chng ta c th mnh v ti nguyn sinh hc nhit i v c kho tng kinh -6-
nghim phong ph ca nn y hc dn tc. T lu i, nhn dn ta bit s dng bt cc chng bnh ci xng cho tr em, s dng cn trng v cc ng vt rng vi mc ch b dng v lm thuc cha bnh. Ngoi ra, cn nhiu loi sn phm bin c gi tr dinh dng cao, dc liu qu nh yn so, bo ng, hi sm, vi c, cc loi nhuyn th binphc v cc ba yn tic cung nh. Kho tng kinh nghim ny khng ngng c b sung t th h ny qua th h khc qua qu trnh lao ng chinh phc thin nhin v ang c nn y hc hin i soi sng, chng minh. Phng Ha sinh protein, thuc Vin Cng ngh sinh hc tin hnh phn tch sinh ha 4 loi hi sm n c, 4 loi rn bin n c v loi cu gai. T pht hin c mt s hot cht sinh hc quan trng c trong tht ca chng. Cc pht hin ny to c s khoa hc cho vic nghin cu v sn xut thc phm chc nng, cc ch phm tng lc u tin cho vn ng vin Vit Nam. Bc u a vo s dng gip cho cc vn ng vin t c mt s ci thin v th lc, thi u thnh cng. Nhng kt qu nghin cu bc u v thc phm chc nng trong nc c ngha to ln, c trin vng ng gp cao cho ngnh cng nghip dc ca nc ta. Trong tng lai s pht trin nhng loi thc phm cung cp nng lng cao, c th tch nh, thun li cho vic vn chuyn, khng phi nu nng v khu v phi a dng. Cc yu t tr nh v nng cao sc khng, sc chng chu ca c ths c a vo thc phm chc nng dng cho nhn dn, ngi lao ng tr c, c bit cho b i trong cc cuc hnh qun thn tc v trong chin tranh tnh hnh mi[14].
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Chng 2: Phn loi thc phm chc nng Vai tr sinh hc
CHNG 2
PHN LOI THC PHM CHC NNG - VAI TR SINH HC
2.1. Phn loi thc phm chc nng C nhiu cch phn loi: Da vo c tnh cu to ha hc, ngi ta chia cc cht dinh dng chc nng theo cc nhm sau: 2.1.1. Cc cht x chc nng trong dinh dng Cht x l cc polysaccharide khng phi l tinh bt, l b khung, gi ca cc m, t bo thc vt v c sc chng vi cc men tiu ha ca ngi. Cht x c nhiu trong rau, qu, phn cm ca ht go. Thc phm c nhiu cht x c tc dng lm khi phn tr nn ln, xp, kch thch nhu ng rut, lm gim thi gian lu phn trong rut gi, chng to bn. Ngoi ra cht x cn hp ph c t trong rut, khng cho hp thu vo c th, thi chng ra ngoi theo phn. Ngi ta theo di thy, khi lng phn nh hn 100g mi ngy d lm tng nguy c ung th i trng. Do cn c khi lng phn ln hn 132g mi ngy. iu cn lng cht x cn thit l 17,9g/ngy. Thc phm c nhiu cht x cn lm chm hp thu ng, c tc dng tt cho ngi bnh tiu ng, bo ph. Cht x cn ngn cn s ti hp thu cholesterol, c tc dng tt cho ngi bnh tim mch. Mt s cht x tan c kh nng ln men sinh acid hu c bi vi sinh vt rut gi, to mi trng acid, c ch vi khun ln men thi c hi, ng thi cc acid hu c ny cng c hp thu, cung cp nng lng khng phi ng cho c th.
-8-
2.1.2. Cc loi ng a phn t chc nng (Oligosaccharides) C nhiu loi ng a phn t (3 9 phn t ng n), nh ng a fructooligosaccharide c nhiu trong rau qu, lactooligosaccharide c trong sa, nhng oligosaccharide khc c nhiu trong ht u nnh Cc loi ng ny c th khng c kh nng tiu ha hp thu on trn ca rut do lm chm hp thu ng, gim bt gnh nng sn xut insulin ca tuyn ty, v th c tc dng tt cho ngi mc bnh tiu ng. Loi ng ny cng c nhiu trong rau qu. 2.1.3. Acid amin, peptide v protein chc nng Acid amin, peptide v protein l thnh phn rt quan trng duy tr sc khe, s sng. Gn y chng cn c bit thm nh l mt thc phm chc nng. Acid amin cn thit cho vic iu tr, phc hi sc khe cho ngi bnh tt, chn thng, iu ha hot ng ca h thn kinh trung ng. Peptid v protein c vai tr quan trng trong vic tng cng hot ng ca h thng min dch nh protein khng th trong sa u, protein khng th chng bnh ng rut trong lng trng c sn xut bng cch tim vaccine vi khun gy bnh ng rut cho g mi trng. Ngoi ra, protein cn c tc dng trong vic iu ha hp thu vitamin, cht khong, hp thu v nc. Mt s loi protein nh gelatin, casein , nhng cht ny c ch chuyn dng t angiotensin I thnh angiotensin II, do lm gim huyt p, phng chng bnh cao huyt p. Lng peptide chng nng c trong thc phm t nhin nhn chung khng cao. V vy, ngi ta to ra cc loi peptide ny bng phng php ha sinh, cng ngh gen hay vi sinh vt trong cng ngh enzyme thu mt s lng ln tng cng vo thc phm ch bin. 2.1.4. Vitamin v khong cht Ngoi nhng tc dng thng thng, vitamin v khong cht cn c mt s tc dng khc trong vic phng chng bnh tt.
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Carotene, vitamin A, E, C, glutathion, tocopherol, st, km, selenium c kh nng chng oxy ha nn c kh nng phng c nhng bnh mn tnh, c bit l bnh tim mch, ung th v lo ha. Vitamin B6, B12 v acid folic cng lm gim nguy c mc bnh tim mch, c tc dng tt cho s pht trin bo thai v cho sc khe b m mang thai. Km v vitamin A lm tng cng hot ng ca h thng min dch, phng nga cc bnh nhim trng, c bit l tiu chy v nhim trng h hp tr nh. 2.1.5. Vi khun sinh acid lactic, acid butyric y l nhm vi khun c ch tiu biu cho nhng vi sinh vt ng rut, c tc dng lm gim hi chng khng dung np lactose, d phng v iu tr tiu chy. Ngoi ra n cn lm gim cholesterol trong mu, phng chng bnh ung th rut kt, nht l acid butyric. Nhng vi khun ny c cung cp qua ng ming nh l mt probiotic, c tc dng tng cng min dch, hn ch to bn, iu tr nhim trng tit niu sinh dc. 2.1.6. Acid bo cha no Vai tr phng bnh ca acid bo cha no mt ni i (MUFA) v nhiu ni i (PUFA), omega-6 v omega-3 c nghin cu. Mt s acid bo thuc nhng loi ny c kh nng phng nga mt s bnh mn tnh nh: phng nga hnh thnh huyt khi, x va ng mch, phng bnh tng huyt p, gim m mu, lon nhp tim. Ngoi ra cn c kh nng chng vim khp, bnh vy nn, ung th. Lecithin l mt phospholipid c tc dng cng vi omega-3 lm gim cholesterol mu, qua gim nguy c mc bnh tim mch. Lecithin c nhiu trong lng trng g, u nnh, rau qu.
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2.1.7. Cc loi sc t thc vt Thc vt c rt nhiu loi sc t khc nhau, da trn cu trc ha hc v mu sc ca n, sc t thc vt c chia lm 3 nhm chnh: nhm sc t c mu xanh (Chlorophyll), nhm sc t mu vng (Carotenoid) v nhm sc t mu tm (Anthocyanin). Cc sc t thc vt, nht l nhm carotenoid c vai tr chng oxy ha rt mnh, v vy c tc dng ph hy cc gc t do, ngn chn s hnh thnh gc t do trong c th, t bo v tt t bo, trnh tn thng do cc yu t vt l, ha hc gy ra. Nh c tnh ny m sc t thc vt c vai tr phng nga bnh ung th. Ngoi ra, cc sc t thc vt cn c s dng lm mu thc phm t nhin rt an ton cho c th.
2.2. Phn loi da theo nguyn liu thc phm chc nng 2.2.1. Thc phm chc nng c ngun gc thc vt Mt s thc phm chc nng c ngun gc thc vt ph bin nh: u nnh: L thc phm truyn thng ca nhiu nc trn th gii k c Vit Nam. Ngy nay, ngoi quan tm n gi tr dinh dng protein ca n, t thp k 90 n nay, ngi ta cn ch nhiu n cc cht dinh dng chc nng trong u nnh. N l loi thc phm c kh nng phng chng cc bnh tim mch, ung th, bnh long xng v nhng biu hin ca hi chng tin mn kinh. C chua: Mt s nghin cu gn y cho thy n nhiu c chua lm gim ng k nguy c ung th tin lit tuyn, ung th v, ung th tuyn tiu ha, ung th c t cung, ung th bng quang, ung th da v phi. C chua cn lm gim nguy c nhi mu c tim. Kh nng phng chng bnh ung th v tim mch ca c chua c cho l nh lycopene, mt dng ca carotene c kh nng chng oxy ha mnh. - 11 -
Ti (Allinum sativum): Ti l loi thc phm chc nng thng c s dng nht, c vai tr rt quan trng trong vic nng cao sc khe con ngi. N c kh nng phng bnh ung th, l cht khng sinh thc vt t nhin, chng tng huyt p v gim cholesterol mu. Trong ti c nhiu hp cht cha lu hunh tan trong nc v tinh du to nn mi v rt r v c trng, nh vy gip cho ti c c nhng tc dng y hc trong vic phng chng bnh tt. Ti cn c tc dng lm gim nguy c mc cc chng bnh tim mch v huyt p. Cc loi rau ci (Broccoli v Cruciferous Vegetables): Nhiu nghin cu dch t hc cho thy nhng ngi tiu th nhiu rau h ci, c bit l ci bp, sp l (c bit bng xanh), ci brussel gim nguy c mc bnh ung th. Nhng loi rau ci ny cha hp cht glucosinolate, mt loi glycoside c cha lu hunh chng li cht gy ung th, c bit l ung th v v n c ch receptor estrogene. Cam qut: Cc loi qu thuc nhm ny gm cam, qut, chanh, qut, bi Mt s nghin cu dch t hc ch ra rng cc loi qu thuc nhm ny c kh nng phng chng nhiu loi ung th ngi nh hm lng vitamin C, acid folic v lng cht x kh cao trong n. Ru vang v nho : Ru vang, c bit l vang c th lm gim nguy c mc bnh tim mch. T l mc v t vong do bnh tim mch c nam v n gim r rt nhng ngi c s dng thng xuyn ru vang. vng sn xut v s dng nhiu ru vang Php, mc d ngi dn y n nhiu m heo tuy nhin t l mc bnh tim mch li rt thp v h ung ru vang hng ngy. Trong ru vang c flavonoid, c bit ru vang c nhiu phenolic c tc dng ngn nga qu trnh oxy ha ht m LDL trong mu, t lm gim s tch ng cholesterol trn thnh mch, trnh x va ng mch. Ngoi ra trong ru vang cn c nhng cht ngn nga bnh ung th.
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Tr: Tr l mt trong nhng thc ung ph bin nht trn th gii. Trong tr c hp cht polyphenolic, c nhiu trong tr xanh, c vai tr chng ung th, c bit l ung th v. Hp cht polyphenolic c nhiu dn xut khc nhau, c tc dng chng oxy ha mnh , bo v t bo trnh t bin gen, v vy m n phng chng c ung th. Mt s bng chng khc cng cho thy vic tiu th tr xanh cn lm gim nguy c mc bnh tim mch, v trong tr xanh cha nhiu hp cht flavonoid. 2.2.2. Thc phm chc nng c ngun gc ng vt Nhng loi thc phm chc nng c ngun gc t ng vt ng k bao gm: c, sa v sn phm t sa, tht b C v du c: Trong c, c bit l c bin c nhiu acid bo cha no Omega-3. y l loi acid bo cha no c nhiu ni i (PUFA). Acid bo Omega-3, c bit l DHA rt cn cho s pht trin no b a tr, ngoi ra n c tc dng lm gim chlesterol xu LDL v lm tng cholesterol tt HDL, v vy c tc dng bo v tim mch, trnh cao huyt p v x va ng mch. Sa v cc sn phm t sa: Sa m l thc n tt nht cho s pht trin ca em b v n c thnh phn dinh dng rt y v cn i, d tiu ha. Ngoi ra trong sa m cn c mt lng khng th ng k ph hp vi c th tr chng li s xm nhim vi trng gy bnh. Theo ti liu ca H Huy Khi (2004) th trong sa m cn c yu t bifidus m bn cht ca n l lactooligosaccharide, c tc dng kch thch nhm vi sinh vt hu ch trong rut gi nh Bifidobacterium pht trin, km hm s pht trin ca vi khun gy hi. Nhng vi khun c ch trong ng rut c coi l probiotic bao gm: Bifidobacterium, Lactobacillus, Enterobacteriaceae Ngy nay ngi ta s dng n ch bin sa chua yoghurt.
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Sn phm sa iu tr giu cht x: Sa iu tr l loi sa c thay th cht bo bi cht x tan, t rau qu. y l loi sa c hm lng cholesterol rt thp, c s dng ph bin ti Hoa k v Nht bn. Cht oligosaccharide b sung vo sa c tc dng kch thch vi khun Bifidobacterium rut gi pht trin, c ch ln men thi. Sa giu -globulin: Ngi ta s dng nhng chng vi sinh vt c bit (bioincubator) sn xut to nhiu -globulin trong sa vi mc ch iu tr bnh. Sa chua probiotics: S dng chng vi khun hu ch ng rut Bifidobacterium cy vo trong sa chua h tr vi sinh vt ng rut cnh tranh c ch vi sinh vt gy bnh v c hi cho ng tiu ha. T loi sa chua probiotics ny c tc dng phng chng bnh tiu chy do vi khun gy ra. Lactoferrin: Mt dng protein c cha st c trong sa ng vt c chc nng khng s pht trin ca vi sinh vt gy bnh. N c coi l mt ph gia thc phm thin nhin thay th nhiu ph gia ha hc khc trong ch bin tht hp, lp xng, xc xch. N khng ch c tc dng sinh hc cao m cn lm gim lng vi sinh vt c hi gy bnh trong ng rut v d dy. N cn c ph bin trong thc n qua ng tiu ha. Phomai ci tin: S dng chng vi sinh vt kt hp vi quy trnh ch bin lm gim cholesterol, gim lng bo v natrium trong phomai, rt tt cho ngi bnh tim mch. Chng vi sinh vt c bit ny lm thay i thnh phn cht lng dinh dng c li cho sc khe, c tc dng phng v iu tr bnh.
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Ngoi cch phn loi nh trn, mt s nc cn c cc cch phn loi khc nhau. V d Nht Bn, bng phn loi h thng FOSHU (Food for Specific Health Use) nh sau: Bng 2.1: H thng phn loi FOSHU Tuyn b v sc kho Yu t chc nng Prebiotics: oligosaccharides, rafftinose, lactulose, arabinose. Probiotics:lactocillus, bifidobacterium. m u nnh, alginate, chitosan, sitosterol ester Chui acid amin Diaglycerol v sitosterol Casein, calcium citrate isoflavone Manitol, polyphenols, paltinose, xylytol Bt m albumin, tiu ho globin, polyphenol S sn phm Loi thc phm trn th trng Nc gii kht, yaourt, bnh biscuit, ng vin, u nnh ng, dm, chocolate, soup bt, sa ln men, miso soup, ng cc Nc gii kht, tht vin, xc xch, sa u nnh, bnh biscuit, magarin. Nc gii kht, soup, acid lactic, nc ung ln men, u nnh. Du n Nc gii kht, u nnh ln men (natto), mt. Chocolate, chewing gum. Ko, soup, nc gii kht. [73]
Thc phm ci thin ng tiu ho
336
Thc phm cho ngi c cholesterol mu cao Thc phm cho ngi c huyt p cao Thc phm cho ngi c triacyglycerol huyt thanh cao Thc phm lin quan hp th v chuyn ch khong cht Thc phm Noncaloriogenic Thc phm cho nhng ngi quan tm n ng huyt
28
42
17 6 4
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2.3. Vai tr sinh hc ca mt s loi thc phm chc nng ti sc khe con ngi 2.3.1. Thc phm chc nng ngun gc thc vt u nnh Tn khoa hc: Glycine max (L.) Merr., Glycine soja Sieb., et Zucc, G. hispida Moench. H: u (Fabaceae) Thnh phn ha hc: ht u nnh cha: nc 9.42%, protein 29.6%, cht bo 13.524.2%, cht x 2.84-6.27%, carbohydrate 14.07-23.88%. Protein u nnh tng i cn i acid amin. Cht bo trong u nnh cha nhiu acid bo cha no omega-3 v omega-6. Trong v ht u nnh c rt nhiu cht fructooligosaccharide, l nhng cht x tan c tc dng nh l mt prebiotic (khng tiu ha on trn rut non, nhng tiu ha tt rut gi do vi khun cng sinh Bifidobacterium ln men sinh ra acid hu c cung cp cho vt ch). Ngoi ra trong u nnh cn c cha carotene (100UI/100g) v cc vitamin khc. u nnh sng c cha cc enzyme amylase, urease, lipoxidase, carboxylase, catalase v cc cht kt dnh, cc cht khng men tiu ha protein (anti-trypsine). c bit u nnh c cha cc dn xut flavonoid, l nhng cht chng oxy ha, v isoflavone (c cc dn xut l genistein v daidzein), c tnh cht ging estrogen gi l phytoestrogen. Trong u nnh cn c cha cc cht saponin gi l soyasaponin I IV. Tc dng dc l phng chng bnh tt: Bo v gan: soyasaponin III v IV (c 2 n v ng) c tc dng bo v gan mnh hn soyasaponin I v II (c 3 n v ng). c ch monoaminoxydase A (MAO: mt loi enzyme oxy ha acid amin): flavonoid trong u nnh c tc dng c ch MAO, t bo v acid amin c 1 amin. Chng oxy ha (antioxydant): isoflavone c tc dng chng oxy ha mnh hn tocopherol gp 80 100 ln.
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Lm gim cholesterol mu: nhm cholesterol cao va phi, u nnh lm gim cholesterol ton phn 9.3% (23.2mg/100ml mu); Cholesterol t trng thp (LDLcholesterol) 12.9%; triglycerid mu gim 10.5% (13.3mg/100ml). Tc dng kiu estrogen: phytoestrogen (genistein v daidzein) c tc dng lm gim cn bc ha ro rc, kh chu nhng ph n tin mn kinh v mn kinh.
Hnh 2.1: Cng thc cu to Soyasaponin I IV [3]
Hnh 2.2: Cng thc cu to isoflavone v dn xut [3] - 17 -
C chua Tn khoa hc: Solanum lycopersicum L., hoc Lycopersicum esculentum Mill. H: c (Solanaceae) Thnh phn ha hc: qu c cha trigonelin, stachydrin, cholin. V qu cha nasunin, shisonin, delphinidin-3-monoglucoside. L cha 1,2,3,4-tetrahydroxynortropan, 4-ethyl-1,2benzendiol. Ngoi ra cn c melongosid F, melongosid H, melongosid K, melongosid M, solasonin, arigininglycoside, nasunin. c bit trong qu c chua chn c nhiu lycoxanthin, monohydroxylycopene. C chua cn cha trigonelin, -amino-4ethylglycoxalin, acid cafeic, acid clorogenic, acid neoclorogenic, 5-nucleotid-5hydroxytryptamin, acid cyanhydric (vt), delphinidin. Qu c cha nhiu pectin 11%, acid oxalic. Ht c chua cha du bo 21.2%, trong c nhiu acid linoleic. Tc dng dc l: Cao chit vi cn ethylic v aceton t l c chua c tc dng gim au, an thn v gy t. C chua c tc dng chng i tho ng trn ng vt th nghim. L c chua c cha tomatin, mt cht khng khun chng nm. Tomatin cn c dng bn tng hp steroid. C chua giu lycopene, cht c kh nng chng oxy ha rt tt. Bng con ng chn ging v cng ngh sinh hc bin i gen, cc nh khoa hc to ra loi c chua mi c hm lng lycopene cao gp 3.5 ln c chua thng, c kh nng chng ung th rt mnh.
Hnh 2.3: Cng thc ha hc ca lycopene [65] - 18 -
Bng 2.2: Hm lng lycopene trong mt s sn phm c chua Loi sn phm c chua Sp c chua, 1 chn M s Spaghetti xt c chua, chn C chua ng hp, chn Xt c chua nm, 2 mung canh 1 tri c chua ti chn, trung bnh S lng lycopene (mg) 24.8 19.4 11.8 5.1 3.7 [3] Tr xanh Tn khoa hc: Camellia sinensis (L.) O.Ktze, Thea sinensis Seem. H: tr (Theaceae) Thnh phn ha hc: trong l tr ti c polyphenol 22.2%, protein 17.2%, cafein 4.3%, x 27%, tinh bt 0.5%, ng kh 3.5%, pectin 6.5%, tro 5-6%. Trong l ti c carotene, riboflavin, cc acid nicotinic (PP), pantothenic v ascorbic. Cht tannin trong tr xanh c rt nhiu hot cht sinh hc nh: epicatechin, galocatechin, ester galonyl ca epicatechin tinh du tr xanh ch yu cha jasmon, fufuryl alcol, -muurolen, geranial, methyl phenyl carbinol. Tc dng dc l: Chng i tho ng: th nghim trn chut gy o tho ng nhn to bng alloxan. Nhm chut dng tr 10g/kg th trng lng ng huyt khng tng, nhm i chng khng dng tr ng huyt tng. Tng s dng thiamin (vitamin B1): dng tr lm tng chuyn thiamin thnh thiamin pyrophosphat. Kt qu lng thiamin trong c th gim, v vy dng nhiu tr s gy thiu thiamin trm trng. Chng oxy ha: nh vo cc hot cht trong polyphenol.
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Lm se nim mc ng tiu ha, chng tiu chy. Tuy nhin lm gim tiu ha hp thu cc dng cht, c bit l Fe. Cafein, theophylin, theobromin trong tr c tc dng kch thch thn kinh, tng cng sc lm vic ca tr c v c, tng h hp, tng iu ha nhp tim, li tiu v kch thch n ngon. Hm lng Fluor kh cao trong tr xanh lm tt cho rng, chng su rng. Cc hp cht catechin trong tr xanh: catechin l mt hn hp dng flavan-3-ols (cha CH2- ti vng C), l mt trong cc nhm ca flavonoids. C 4 loi catechin tng ng vi 4 cu trc khc nhau:
Hnh 2.4: Cng thc cu to cc hp cht catechin trong tr xanh [3] Catechin c ch hu hiu cc gc t do, chng bo ph, c tc dng dit khun E.coli O-157. EGCG chng li tn thng DNA v cc bu trong phi do gc t do gy ra, ngn chn s pht trin ca t bo ung th, ngn nga bnh tiu ng pht trin, bo v tim, gim t bin gen, gim nguy c vim khp
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Bng 2.3: Kh nng c ch cc gc t do ca cc hp cht trong tr xanh Tn hp cht trong tr xanh EGCG EGC ECG EC C ngh Tn khoa hc: Curcuma domestica Valet., Curcuma longa L. H: gng (Zingiberaceae) Thnh phn ha hc: c ngh n : nc 13.1%, protein 6.3%, cht bo 5.1%, x 2.6%, carbohydrate 69.4%, cht khong 3.5%. Carotenoid quy ra theo vitamin A l 50UI/100g. Cc hot cht trong c ngh c: cc cht mu phenolic ch yu l cc dn xut ca diarylheptan. Trong c 3 cht ch yu l curcumin I, II, III. Cht mu curcumin 0.3% tan c trong nc. Cc hp cht tinh du ngh khong 1 5% trong c tumeron, zingibren, cineol. Tinh du ngh c xc nh l nhng sesquiterpen ceton arturmeron. Tc dng dc l: Chng vim cp tnh v mn tnh thng qua c ch chng oxy ha ti ch vim do sc t curcumin, bo v m t bo, phng chng bnh thn, gim hi chng Azheimers, chng x va ng mch, ngn nga ung th, bo v tim do lm gim lng cholesterol v lipid mu. % c ch gc t do DPPH 74.8 59.3 36.1 32.0 % c ch gc Superoxide 69.4 39.9 23.9 11.2 c ch men Lipoxygenase IC (g/ml) 4.6 7.7 6.1 40.6 [3]
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Hnh 2.5: Cc hp cht curcumin trong c ngh [65] 2.3.2. Thc phm chc nng t ngun nguyn liu sinh vt bin v ng vt 2.3.2.1. Ngun nguyn liu t rong bin Thnh phn ha hc ca rong bin Sc t: Thnh phn sc t khc nhau ty tng loi rong: dip lc t, sc t , sc t vng, sc t nu, sc t xanh lam Glucid: Bao gm nhiu loi: monosaccharide, diosaccharide, polysaccharide. Monov Dio-saccharide: Mannitol (rong nu); Galacetose, mannose (rong ). Disaccharide: i vi rong nu thng c cc cht: Alginic, Acid fucxinic, Fuccoidin, Laminarin, Cellulose. i vi rong gm: Agar, Carrageenan, Furcellaran, Xilan, Itridophican, cellulose, tinh bt rong . Protein: Hm lng protein ty tng loi rong, thng protein trong rong nu khng cao bng rong nhng n kh hon ho nn c th s dng lm thc phm. Ngoi ra cn c cht khong, nc, lipid
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Vai tr ca rong bin L ngun thc phm ca con ngi: Rong bin c coi l thc phm cho sc khe. Nht c nhng mn n c trng: Nori (t loi rong Phorphyra), Kombu (t loi rong Laminaria), Wakame (t loi rong Undaria pinnatifida)Ngoi ra cn nhiu mn n c ch bin kt hp vi to b, ph tai nh ph tai nu vi c, tht, nu cho vi go hay mn sp ph tai. Gi tr dc liu v phng chng bnh tt ca rong bin: Rong bin c xem nh l thuc tr hen suyn, d dy, thuc nhun trng, nga sn ti mt, chng nhim trng ng tit niu, kin ton kh nng sinh dc, thuc tr phong tht li thm tnh cht an thn v cng hu dng vi bnh ngoi da. Rong bin c tc dng phng v tr cc bnh: Ung th, tim mch, h cht m, lm long mu ngn nga s ng cc tc ngn mch mu, bnh bu c do thiu iod Ngoi ra, theo nghin cu ca trng i hc New South Wales, c th trong rong bin c c cha hp cht furanone, n c tc dng ngn chn v ph v khu tr ca vi khun, ni m vi khun t tp li thnh tng m c bo v bi cht nhy c cu trc bng polysaccharide gi l biofilm, chnh biofilm bo v cho vi khun trnh c tc ng khng sinh, khng th, nh m vi khun khng b tiu dit, tn ti c trong c th. Cht Frucosterol mi tm ra gn y c tc dng ngn nga vic to ra cc cc mu ng trong mch mu. Mt s nguyn t vi lng trong rong v cng cn thit cho c th con ngi nh Selenium c cha trong rong kh cao: 0,3-0,4 mg/kg vt cht kh, cht ny tham gia cu trc trong enzyme Glutathion peroxidase c tc dng ph hy cc gc t do trong c th. Rong bin gip h thng min dch hot ng tt v gia tng hot ng tng th ca con ngi. N cng gip qu trnh trao i cht hiu qu hn v chng li s lo ha. Rong bin c cha nhiu cht khong nh km, thic, selen, crom, antimon, bimut, nhng cht t tm thy trong cc loi thc phm ngy nay. Cc loi vitamin, bao gm - 23 -
vitamin E, A, C v B12 cng c mt hm lng kh ln trong rong bin, chng em li cho con ngi y dng cht. 2.3.2.2. Ngun nguyn liu t ng vt
Chitosan Oligosaccharide: cht x trong ng vt Chitosan Oligosaccharide l mt ng amin chc nng, khng c. N c ly trch t v tm, cua. Chitosan oligosaccharide l mt hn hp oligomers ca D-glucosamine. Trng lng phn t trung bnh ca n khong 2000. Chitosan Oligosaccharide b phn hy bi s thy phn do enzyme chitosanase. Sn phm thy phn chitosan c hai dn xut l chitin v chitosan. Hn hp chitin v chitosan c gi l chitosan v chitosan chim t l cao hn nhiu so vi chitin. Chitosan Oligosaccharide i qua d dy v rut non m khng b phn hy v cng khng hp thu. V vy, Chitosan Oligosaccharide xung kt trng, y n b enzyme ca vi sinh vt c ch trong rut gi phn gii ln men sinh acid c tc dng nh mt prebiotic, c ch vi khun ln men thi gy bnh ng rut. Nh vy Chitosan Oligosaccharide c hiu qu ln sc khe th hin qua cc chc nng ni bc: Gn vi cht bo v c ch s hp thu cht bo nn gp phn chng bo ph, gim thp cholesterol xu LDL-cholesterol, tng cholesterol tt HDL-cholesterol, chng ung th, chng nhim khun, thc y s sn xut khng th, h thp ng huyt, kim sot huyt p, phng nga chng to bn, lm tng s hp thu Calcium, phng nga bnh tim mch, lm gim mc acid uric trong mu. Sa ong cha Tc dng ca sa ong cha: Tng cng nng lng cho hot ng cc c quan quan trng trong c th nh: no b, c tim, tuyn thng thnthng qua vic cung cp nng lng v thc y qu trnh hp thu cc cht sinh nng lng v bin dng n.
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Sa ong cha thc y ti to c quan b h tn, thc y hnh thnh t bo mi. Mc khc sa ong cha cng ngn chn hin tng thoi bin ca t bo hin hu. V vy sa ong cha p dng rt tt cho tr em, ngi gi, thai ph, ngi bnh phc hi sau cn au nng. Gip gii c gan, duy tr tnh n hi ca mch mu, phng tr chng x cng mch mu. Phc hi huyt cu i vi ngi b thiu mu do: st rt, x tr hay quang tuyn X trong trng hp tr bnh nhn ung th. iu ha lng ng trong mu, ng thi ci thin trnh trng tng qut ca ngi bnh qua nh hng trn h thn kinh trung ng v mng li tun hon vi mch, t c tc dng tt i vi ngi b bnh tiu ng. Ngoi ra cn nhiu tc dng khc t sa ong cha. Tuy nhin khng nn lm dng hay cng iu vai tr ca sa ong cha, sa ong cha c nh hng tch ly do khng nn dng thng xuyn. Sa chua y l thc phm b dng cao, ng thi cng l loi thuc ngn nga bnh vim rut, tiu chy. Sa chua lm tng tui th: kt qu thng k trn nhiu quc gia cho thy s ngi th trn 100 tui cao r rt cc a phng c tp qun dng sa chua. Mt cng trnh nghin cu cho thy nhng con b c nui bng sa chua c kh nng min nhim vi trng salmonella. Vi khun Lactobacillus acidophilus, mt trong nhng loi vi sinh thng dng ln men lm sa chua, c tc dng chng nhim trng ng rut mt cch r rt. Nm 1963, bc s Shahani ly trch 1 cht khng sinh trong sa chua t tn l Acidophylline c tc dng hu hiu khng thua km Penicilline. n nay ngi ta pht hin
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c ti thiu 10 loi khng sinh trong sa chua khng c hi, d phn hy trong mi trng, khng tn d trong c th. Khm ph gn y nht ca , Nht Bn v Thy S cho thy sa chua cn l tc cht hng phn v kin ton h thng min nhim, sa chua lm tng hm lng khng th chng vi khun. Gn y nht ngi ta cn tm thy hm lng interferol c th c gia tng gp 3 ln trong mu ca ngi c thi quen dng sa chua thng ngy. Bo co u tin t Bulgarie v kh nng cha lnh ung th trn sinh vt th nghim nui bng sa chua. Sau vin Ung th New York chng minh l sa chua c th ngn chn s pht trin ca t bo ung th, vi khun lactic trong sa chua c kh nng c ch hot ng ca cc yu t gy ung th, ngn chn c cc c t rut sinh ung th. Nh vy, theo Metschnikow cho sa chua tr thnh dc phm cn phi bin sa ti thnh sa chua vi thnh phn vi sinh hu ch, sau ng dng sa chua nh l dc phm c hiu. C v cht du Omega trong c Tc dng ca c v du c bin: nh hng trn h thng tim mch: Ngi ta nhn thy bnh ngnh tc mch c tim l nguyn nhn hng u cc quc gia c mnh danh l vn minh, hin i, tiu th rt nhiu tht ng vt nui cng nghip. Trong khi bnh ny rt xa l vi dn tc Esquimo vn c thi quen n c bin mi ngy. Mt cng trnh nghin cu ti Nauy cho thy ngi b chng mu c ch cn n mi ngy 100g c bin th sau 6 tun thnh phn ca mu tr li bnh thng m khng cn dng thuc, cht m trong mu thm ch cn tt hn ngi n chay kin tht. Tc dng ngn nga bnh ung th v: Cc chuyn gia trng i hc Rutgers, Hoa K nhn thy kch t Prostaglandine c xu hng tng cao trong mu ca ngi sng trong vng ang c ung bu. Du gan c vi cht bo Omega c kh nng ngn chn s hnh thnh thoi qu ca kch t ny, t ngn nga c bnh ung th v.
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Du c cn dng tr thp khp, c kh nng tr thin u thng v cn c hiu qu iu tr phc hi cho ngi b vim thn mn. C bin v du c Omega cn l sn phm dinh dng thch hp cho ngi cao huyt p. Loi nhuyn th c v: Trai, nghu, s, c Trai l thc phm v cng qu gi nh vo hm lng km trong tht trai rt cao. C th con ngi ch cn 15mg Zn mi ngy m trong 100g tht trai c n 70mg Zn. Cc loi hi sn c v cng nh: nghu, s, tm, c cng c tc dng tng t nh trai. Cht bo trong cc hi sn ny c cha nhiu acid bo thit yu nn n c tc dng lm gim lng m no (LDL), nhng n c tc dng ci thin hm lng cht bo cha no (HDL) trong mu, HDL gip ch cho vic vn chuyn cholesterol trn thnh mch, trong m bo v gan ri thi ra ngoi theo dch mt. Ngi ta cn pht hin trong m loi nhuyn th c nhiu acid bo cha no rt quan trng nh Docosahexenoic acid (DHA) rt cn pht trin mng t bo no. Nghu, s c nghin cu massachussets, cc nh khoa hc chng minh cc loi thc phm ny cung cp cho no b nhiu hot cht hng phn s bi tit kch t ni sinh y mnh hot ng tm thn, thc y kh nng sng to 2.3.3. Thc phm chc nng t ngun nguyn liu nm Nm mo, Mc nh Tn khoa hc: Auricularia polytricha L loi nm c m ging tai mo, mp nhn nheo cun vo trong, mc t nhin trn cc gi th l cy g mc. Thnh phn ha hc: Trong 100g mc nh cha: 10,6g protein; 0,2g lipid; 65g glucid; 7,0g cellulose. Tc dng dc l v gi tr phng chng bnh tt:
- 27 -
Nm mo l thc phm c gi tr v cng ln phng chng bnh tim mch vi kh nng thay th cc loi thuc lm long mu vn c nhiu phn ng ph. Cc chuyn gia i hc Washington ly trch t nm mo mt hot cht chng ng mu c tn gi l Adenosine vi c ch tc ng tng ng vi Aspirine. Nm mo c tc dng ko di tui th, dng tr thin u thng hoc ngn nga bin chng vim tnh mch hu sn. Nm mo cn c kh nng phng chng vi khun v ngn chn s pht trin ca t bo ung th. Tuy nhin pht huy kh nng lm long mu, khng cn phi n nhiu nm mo, ch cn n 10g nm mo mi ngy l . Tuyt i khng nn n sng nm mo, tt nht nn nu canh, nu ming, hm vi thuc bc Nm ng c, nm hng Tn khoa hc: Lentinula edodes Thnh phn cc hot cht dc phm: Ni bt nht l cc dn sut mch vng c cha lu hunh nh cyclohexasulphur. Hot tnh dc phm ca nm ng c: Hot tnh khng sinh, khng khun, khng virus ca nm: lentinan (mt ch phm ca nm hng) c tc dng khng khun, khng virus, khng nm bnh v k sinh trng, chng li s vim nhim ca virus vim no, chng bi nhim khun cc bnh nhn b AIDS. Gn y hot tnh chng HIV-1, gim c tnh ca AZT bi lentinan v cc dn xut cng c chng minh, c bit sulphat lentinan c ch rt mnh hot tnh ca reverse transcriptase (enzyme sao chp ngc ca HIV). Hot ng lm gim cholesterol: Cht Eritidenin gm Lentysin v Lentinacin do Mizuno (1990,1993) xc nh trong qu th nm hng nui trng, cht ny c kh nng lm gim mc cholesterol v cc lipid trung tnh trong mu, v vy n c tc dng tt vi nhng ngi c bnh tim mch v huyt p. - 28 -
Nm s, nm bo ng Tn khoa hc: Pleurotus ostreatus Nm phn b Bc M, chu , chu u v mt s ni khc.Chng sng trn thc vt hoi sinh, ng vai tr cha kha trong vic phn hy cy g cht gi cn bng h sinh thi rng. Tc dng dc l ca dch chit t nm s: Dch chit nm s c kh nng kim sot c hiu qu bnh tim mch v cholesterol mu. Nhng nghin cu gn y cho thy nm s h thp mc cholesterol v triglycerol trong mu rt tt. Nm s c cha mevinolin v mt s hp cht tng i t, c tc dng cnh tranh c ch enzyme HMG CoA reductase, y l enzyme rt quan trng tng hp cholesterol. Chnh v vy khi s dng nm ny n hoc chit xut hot cht cha bnh c cng dng: chng ung th, tng cng p ng min dch, chng li s vim nhim, chng li virus gy bnh, ng thi n cng c tnh cht nh mt khng sinh. Nm Linh chi Tn khoa hc: Ganoderma lucidus Nm Linh chi thng sng hoi sinh trn g mc nh: g lim, lim xt, mung en, me Thnh phn ha hc: Sterol: Ergosterol 0,3-0,4%, enzyme: Lysozyme, protease acid v mt s enzyme khc, protid: protein ha tan, polypeptide, acid amin, ng: trehalose, manitol, amin: betain, alkan: tetracosan, hentricotan, cc acid bo, polysaccharide v nhiu nguyn t khong khc. Tc dng dc l ca nm Linh chi:
- 29 -
Tc dng ln ng huyt: Cao nc Linh chi lm gim ng mu chut nht trng. Cc glucan A, B v C c tc dng h ng mu r rt. i vi ngi Linh chi c tc dng lm n nh ng huyt, nht l nhng ngi b bnh i tho ng. Tc dng ln tim mch: Linh chi tc dng tt ln bnh au tht ngc, bnh ng mch vnh, iu ha v n nh huyt p. i vi bnh nhim m, x mch, dng nm Linh chi c tc dng gim cholesterol ton phn, lm tng nhm lipoprotein t trng cao trong mu, lm gim h s sinh bnh. Nm Linh chi lm gim xu th kt b ca tiu cu, gim nng m trong mu, gim co tc mch, gii ta cn au tht tim. Tc dng chng vim: Vim ph qun, hen, vim gan mn tnh, thp khp, bnh ng tiu ha. i vi bnh v h hp, nm Linh chi em li kt qu tt, nht l vi nhng ca iu tr vim ph qun d ng, hen ph qun ti 80% c tc dng gim v lm nh bnh theo hng khi hn. i vi bnh ung th: Nm Linh chi lm tng cng v khi phc h min dch, v vy khi iu tr ung th cho kt qu tt, khi u tiu bin hon ton. Nm ng trng h tho Bn cht l dng k sinh ca loi nm Cordyceps sinensic thuc nhm Ascomycetes trn c th u trng ca loi Hepialus Fabricius. Thnh phn ha hc: Protein: cc dng protein, peptide, c mt y cc loi acid amin; cacbohydrate: cc dn xut bt ngun t D-mannitol, cc dng ng n Oligosaccharide; ng a Polysaccharide; sterol, acid bo, mt s acid hu c khc; Cc loi vitamin (B1, B2, B12, E, K) v cc loi khong cht. Thnh phn hot cht v dc l ca n: Adenosine: Chen v Chu (1996) nghin cu c tnh cht ca 2deoxyadenosine p dng trong phng php cng hng t ht nhn v quang ph hng ngoi. Cordycepin: Cordycepin (3deoxyadenosine) v cordycepic acid (D-mannitol) l nhng hp cht u tin loi Cordyceps c vai tr p ng khng khi u bn cnh chc - 30 -
nng h tr h thng min dch. C ch ny c thc hin khi c s thay i trong cu trc nucleoside Adenosine bnh thng. Adenosine b mt oxy, khng hnh thnh cu ni gia cc nucleoside gn nhau trong thang xon DNA, khng gi c cu trc bn vng na. Cc tin hnh sao chp k tip ca DNA cng khng th thc hin khi c s thay th Adenosine bng Cordycepin trong phn t DNA, dn n s tng hp mi DNA khng th xy ra, khng c s phn chia hay hnh thnh t bo mi. iu ny t nh hng n s sng ca t bo ng vt c v v mc phn t t bo c c ch sa sai DNA. Tuy nhin s thay th ny rt c ngha i vi hu ht t bo vi khun v virus (bao gm c virus HIV) v chng khng c c ch sa sai. Cc loi ng: Nhm vng 5-Cacbon bao gm Cyclofurans vi chc nng cha r. Nhm beta-Glucan v Beta-Mannan to lien kt cho to poly beta-mannan. Nhm cc ng polymer 5-6 Cacbon tham gia lin kt to chui trong cu ni alpha v beta. Hydroxyethyladenosine, HEAA: l nhm him thy v hu nh khng tm thy ngun no khc trong t nhin. Nhm c ch min dch, chu trch nhim chng s thi loi c th: Cyclosporin, mt vi hp cht khc gn h Cordyceps Cc polysaccharide: Nhng polysaccharide bt ngun t Cordycepic acid c gi tr cao trong iu tr bnh tng Glucose huyt, chng khun, chng khi u. Cc protein v nhng hp cht cha Nito, nhm sterol v nhng thnh phn khc. [3]
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Hnh 2.6: Gii thiu hnh nh mt s loi nm [65]
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Chng 3: Phng php lm giu cc cht dinh dng chc nng
CHNG 3
PHNG PHP LM GIU CC CHT DINH DNG CHC NNG
3.1. Chn ging cy trng vt nui giu cht dinh dng chc nng T trc n nay, khi tuyn chn ging cy trng, vt nui ngi ta thng ch n ch tiu nng sut v th hiu v hnh thc sn phm ca ngi tiu dng, t khi la chn da trn ch tiu hm lng cc cht dinh dng chc nng. iu ny dn ti gi tr cc hot cht chc nng dn dn suy gim. V vy gi tr phng bnh ca loi thc phm khng cn nh gc ban u ca chng. Con ngi n nhiu cht dinh dng sinh nng lng dn n bnh bo ph ngy cng nhiu. Cn c s nhn thc ng n hn v mc tiu chn ging, t to ra thc phm ngy cng b dng hn, c ngha phng bnh bo v sc khe cho con ngi ngy cng tt hn. Sau y l nhng gi trong cng tc chn ging theo hng ny:
i vi ging rau ci: V d gia cc ging ci bp v ci b ngi ta nhn thy loi
trng thng c hm lng carotene rt thp hoc khng c. Tri li nhng ging c mu xanh th bao gi cng c hm lng caroten cao hn, c tc dng ngn nga nguy c cc loi ung th nim mc.
i vi cc loi c qu th loi c mu vng, mu nh khoai lang ngh, b , c
chua, gc, thanh long rut vng, i rut hng bao gi cng c hm lng carotene, lycopen, xantophyllcao hn loi c rut trng. y l nhng cht chng oxy ha kh mnh nn n c tc dng bo v mng t bo, t cng c tc dng ngn nga nguy c ung th.
i vi cc loi ht cc th loi c mu vng c cha nhiu carotenoid hn so vi
loi trng. Ngy nay n , cc nh khoa hc to ra c ging la c rut vng rt giu caroten, c th l mt ng gp rt ln trong vic phng nga c hiu qu bnh thiu vitamin A tr em thuc cc vng ngho.
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Vi nhng k thut hin i trong cng ngh sinh hc, con ngi c th nhanh chng t c thnh tu a cc gen iu khin tng hp ra nhiu cc hot cht chc nng trong cc loi thc phm thng thng bin n thnh thc phm chc nng c tc dng phng nga bnh tt, bo v sc khe cho nhn loi ngy cng tt hn. 3.2. Lm giu cc cht dinh dng chc nng thng qua con ng ch bin thc phm Trc y, mc tiu chnh ca cng ngh sau thu hoch l ch bin thc phm, bo v thc phm c lu hn, lm cho thc phm tr nn ngon hn, hp dn hn, quyn r hn i vi ngi tiu dng. Ngy nay, tuy cc mc tiu vn cn tn ti, song ngi tiu dng cn i hi thc phm ch bin phi c gi tr phng nga bnh tt, bo v sc khe tt hn, an ton hn khi tiu th chng. lm c iu ny cn phi:
Ci tin cng ngh ch bin c lm h hi cc hot cht sinh hc chc nng trong
nguyn liu thc phm nh: hp kh trng nhit cao, sy kh v bo qun lu di trong khng kh, phi nng ko di di tia bc x mt tri sang cng ngh mi, t lm h hng vitamin v cc hot cht sinh hc khc nh: kh trng nhanh bng tia cobalt kt hp vi tit trng bng phng php pasteur, sy chn khng nhit thp, s dng phng php bao b ht chn khng cch ly thc phm vi khng kh.
Thng qua con ng ch bin c th b sung, tng cng cc cht dinh dng v
hot cht chc nng. V d nh: sa cho ngi cao tui ngi ta b sung nhiu calcium hn, rt ra nhiu cht bo hn so vi bnh thng ngn bnh long xng. Sa cho tr em th nn giu cht bo, c nhiu acid bo thit yu, c bit l DHA tham gia cu to mng t bo thn kinh, gip cho s pht trin no b ca tr c thun li, lm tng ch s thng minh. Mui n cho ngi vng cao xa bin v vng c nguy c bnh bu c nn b sung Iod (KI) chng li bnh tuyn gip do thiu Iod v cn nhiu v d khc na v vic lm giu cht dinh dng chc nng qua con ng ch bin thc phm. Ngnh ch bin thc phm Vit Nam tuy cn rt tr so vi Chu u, nhng quan im n phng bnh v cha bnh c t th k 17 (thi k ca nh Bc hc Y hc phng ng Hi
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Thng Lng ng), chng ta c rt nhiu nguyn liu nhit i ch bin thnh thc phm chc nng, cn c nhng con ngi nhit tm v thc tin bin tng tr thnh hin thc trong thi i ngy nay. 3.3. Thng qua k thut, chn nui lm gia tng hm lng cc hot cht sinh hc chc nng trong thc phm ca ngi i vi cc ging cy trng c nng sut cao, ngn ngy thng s tch ly cc nguyn t vi lng cn thit trong sn phm t hn ging cy trng nng sut thp, thi gian sinh trng ko di. Mt s vng t ngho mt vi nguyn t vi lng no th hm lng ca n cng thp trong sn phm cy trng. T nguyn liu thc n gia sc thiu yu t dinh dng vi lng s gy cho th trng thi thiu cn i. Con ngi n cc sn phm cy trng vt nui nh th cng s b bnh thiu vi cht dinh dng. khc phc s khim khuyt ny, ngi ta gii quyt vn bng hai con ng: B sung vo thc n nhng vi cht dinh dng thiu hoc thng qua con ng bn phn lm giu yu t vi lng thiu ht trong sn phm cy trng. Trong thc n chn nui nu b sung y cc yu t dinh dng chc nng v c gii php k thut cc hot cht chc nng hp thu nhiu v t b bin i th s tch ly cao trong sn phm chn nui. T cung cp thc phm c gi tr phng bnh cho ngi. V d ngi ta b sung Selen hu c (Se-Plex) vo thc n lm tng lng Se trong tht chng bnh thoi ha c do thiu Se trong thc phm ngi. Nam Triu Tin v mt s quc gia pht trin trn th gii, tht heo nui bng thc n c b sung Sel-Plex bn gi cao hn bnh thng. B sung acid Omega-3 vo thc n g lm giu acid bo thit yu, lm gim lng cholesterol trong trng c tc dng phng nga bnh tim mch. [3]
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Chng 4: Nhng quy nh chung v qun l thc phm chc nng
CHNG 4 NHNG QUY NH CHUNG V QUN L THC PHM CHC NNG TRN TH TRNG
4.1. Qui nh v s cng nhn tc dng cc cht dinh dng chc nng ln sc khe Theo quan im ca ADA, M th cc loi thc phm chc nng phi c lm r rng bn cht khoa hc ca n i vi sc khe. Khi c kt qu th nghim lm sng r rng trn c th, lm sng t tc dng ca cc hot cht sinh hc trong thc phm chc nng th mi c s chng nhn ca FDA (Food and Drug Administration). Sau y l cc vn ca thc phm chc nng cn phi c c quan qun l nh nc v thc phm v thuc cng nhn: 4.1.1. Qun l tiu chun v mt khoa hc ca cc cht dinh dng chc nng C th da vo cc c quan khoa hc nh: Vin hn lm, Vin nghin cu khoa hc k thut, Vin nghin cu tiu chun cht lng xy dng tiu ch chng nhn mt loi thc phm chc nng mi cho tng lai. M c c quan chuyn trch l FDA lm vic ny. H c quyn hn qun l v iu hnh cp Quc gia. C 5 yu cu cn phi c chng nhn cho mt loi hot cht chc nng no trong thc phm chc nng: Yu cu v hm lng dinh dng: Cho bit r s hin din ca cht dinh dng chc nng c bit mc chc chn. Yu cu v cu trc ha hc v chc nng ca cht dinh dng chc nng: Phi c lm r trong thc phm v nh hng ca n n chc nng sinh l c th. Yu cu tc dng ln sc khe: Phi c pht trin trong mi lin h gia ch n v nhng nguy c bnh tt. Xc nhn sc khe: Mi lin quan gia nhng thnh phn trong khu phn n v nguy c bnh tt.
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4.1.2. Thc phm chc nng yu cu phi c chp nhn ca c quan qun l nh nc v an ton thc phm v dc phm trc khi a ra th trng tiu th Bt c thc phm no, d l thc phm thng thng khi a ra th trng mua bn u phi chu s qun l nh nc v an ton thc phm. Ty theo mi nc m c quan qun l c tn gi khc nhau. Vit Nam l cc v sinh an ton thc phm trong B Y t. M l Hip hi thc phm v thuc FDA. Ring thc phm chc nng ngoi s qun l ca thc phm thng thng, cn phi chu s qun l v thnh phn v tc dng ca cc dc cht chc nng phng chng bnh tt trong thc phm. Tc dng lm sng ca thc phm phi c minh chng bng nhng kt qu th nghim khoa hc chnh xc, khi mi c c quan qun l an ton thc phm cho php ghi trn nhn bao b. Ngoi ra, cc cht c chc nng sinh hc trong thc phm cn cn phi t n mt liu lng thch hp th chng mi pht huy c chc nng sinh hc, v vy cn c khuyn co liu lng s dng hp l. 4.2. Qui nh v tn gi v dn nhn, lu hnh trn th trng 4.1.1. Mc tiu ca dn nhn thc phm thng thng v thc phm chc nng Dn nhn sn phm l giy cam kt ca nh sn xut vi khch hng c s cng nhn ca c quan qun l nh nc, l c s qun l v sinh an ton thc phm. Dn nhn i vi thc phm chc nng rt cn thit hng dn ngi tiu dng bit cch s dng thc phm chc nng ph hp vi tnh trng sc khe ca mnh, phng chng bnh tt trong trng hp c th. Dn nhn cn mang tnh cht qung co, nhng phi hon ton ng vi tc dng tht m thc phm chc nng mang li, khng nn ni qu mc kh nng ca n. 4.1.2. C quan qun l dn nhn thc phm Dn nhn thc phm mi quc gia, v d nh M phi da trn cc c quan qun l nh nc v thc phm nh: FDA (Food and Drug Administration) v c quan thanh tra an ton thc phm FSIS (Food Safety and Inspection Serviec). FDA qui nh dn nhn cho - 37 -
tt c cc thc phm, ngoi tr tht gia sc v gia cm phi c thm mt c quan qun l thanh tra an ton thc phm FSIS v s qun l ca B Nng nghip M USDA. Vit Nam cha c t chc ny, nhng c Cc qun l v sinh an ton thc phm, Cc th y, B Nng nghip. C th coi y l nhng c quan chu trch nhim qun l dn nhn thc phm hng dn ngi tiu dng bit cch chn la thc phm. Hot ng gio dc v dn nhn dinh dng cho thc phm l hot ng phi tin hnh song song. Ngi tiu dng c bit c tc dng dinh dng v an ton thc phm th mi tm hiu trn nhn thc phm chn mua. trnh s gian ln ca nh sn xut thc phm v s nhm ln cho ngi tiu dng, nht thit phi c c quan kim tra v qun l cc ch tiu ng k trn mt hng thc phm hay ni khc i l nhn hiu thc phm. i vi nh sn xut thc phm th dn nhn thc phm l t ng k cht lng hng ha ca h vi c quan qun l thc phm nh nc. i vi ngi tiu dng th dn nhn thc phm l s cam kt ca nh sn xut i vi khch hng, l s hng dn s dng cho khch hng. Trong thi k cng nghip ha, hin i ha t nc, ngy cng c nhiu hng ha thc phm ch bin sn, ngy cng giao lu nhiu nc trn th gii. Hng ha m khng c nhn mc s khng c giao lu mua bn khng nhng trn th gii m ngay c trong nc cng vy. Hng ha nhn mc lung tung, khng theo khun mu s rt kh qun l, tt yu s khng c lu thng mua bn trn th trng. V vy, qui nh kch thc v cc ch tiu cng b trn nhn mc nh th no cn c s thng nht chung khng nhng trong phm vi khu vc m c trn th gii. [3]
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Phn 2: ng dng cng ngh di truyn trong sn xut thc phm chc nng
PHN 2
NG DNG CNG NGH DI TRUYN TRONG SN XUT THC PHM CHC NNG
SVTH: Nguyn Kiu Oanh
39
Chng 1: Thnh tu pht trin ca thc phm chuyn gen
CHNG 1
THNH TU PHT TRIN CA THC PHM CHUYN GEN
1.1. Tnh hnh pht trin ca thc phm chuyn gen trn th gii v Vit Nam 1.1.1. Trn th gii 1.1.1.1 Thc vt Tnh hnh chung : T nm 1996 n nm 2007, sau 12 nm c a vo canh tc i tr, mang li li ch n nh v bn vng, cy trng CNSH ang c trng ngy cng nhiu trn ton th gii. Nm 2007 l nm th 12 lin tip din tch cy trng CNSH tip tc c m rng. ng ch , din tch trng tip tc tng 2 con s, t 12% tng ng vi 12,3 triu hc-ta (30 triu mu) mc tng cao th nh trong vng 5 nm tr li y. Din tch t canh tc cy CNSH ln ti 114, 3 triu hc-ta. Trong 12 nm u c a vo canh tc, cy trng CNSH mang li nhiu li ch v kinh t v mi trng cho nng dn c cc nc cng nghip cng nh cc nc ang pht trin, ni hng triu ngi nng dn ngho cng c hng nhng li ch v mt x hi v nhn o, gp phn gip h xa b ngho i [1]. Nm 2007, Hoa K, Argebtina, Brazil, Canada, n v Trung Quc l cc nc a cy trng CNSH vo canh tc nhiu nht. Hoa K vn dn u th gii vi 57,7 triu hc-ta (chim 50% din tch t trng cy CNSH trn th gii). ng ch l 63% ng chuyn gen, 78% bng chuyn gen v 37% cc loi cy chuyn gen khc Hoa K l cc sn phm mang nhiu gen kt hp (cc sn phm kt hp nhiu c tnh) c cha hai hay ba c tnh v em li nhiu li ch trn mt cy trng. Xu th ca tng lai l s dng nhng loi cy trng cng ngh sinh hc mang kiu gen kt hp ny nhm p ng nhu cu ca nng dn v ngi tiu dng [1]. Trong 12 nm va qua, cc nh khoa hc n lc to ra ging cy trng mang tnh chng chu tt hn i vi cc yu t sinh hc gy ra bi cn trng c di v bnh cy. SVTH: Nguyn Kiu Oanh
40
Hin nay cc nh khoa hc ang tp trung nghin cu cc loi cy nh sn, khoai lang, cy la min v rau gip a dng ho v cn bng chng trnh pht trin cy trng cng ngh sinh hc. Bng 1.1: Din tch cy trng Cng ngh sinh hc nm 2007 trn th gii Th t 1* Nc USA* Din tch (triu hc-ta) 57.7 Cy trng CNSH
u tng, ng, bng, ci canola, b, u , c alfalfa 2* Argentina* 19.1 u tng, ng, bng 3* Brazil* 15.0 u tng, bng 4* Canada* 7.0 Ci canola, ng, u tng 5* India* 6.2 Bng 6* China* 3.8 Bng, c chua, cy dng, thuc l, u , ht tiu 7* Paraguay* 2.6 u tng 8* South Africa* 1.8 Ng, u tng, bng 9* Uruguay* 0.5 u tng, ng 10* Philippines* 0.3 Ng 11* Australia* 0.1 Bng 12* Spain* 0.1 Ng 13* Mexico* 0.1 Bng, u tng 14 Colombia <0.1 Bng, cm chng 15 Chile <0.1 Ng, u tng, ci canola 16 France <0.1 Ng 17 Honduras <0.1 Ng 18 Czech Republic <0.1 Ng 19 Portugal <0.1 Ng 20 Germany <0.1 Ng 21 Slovakia <0.1 Ng (*) 13 nc c xem l c din tch trng ln, t 50,000 ha tr ln. [1]
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Hnh 1.1 : Din tch cy trng CNSH trn th gii qua cc nm t 1996 n 2007 [1] Thnh tu trong lnh vc thc phm chc nng : Cy la Cc nh khoa hc to ra ging la c ht go vng giu carotene bng con ng chuyn gen. Go ht vng c tn gi bng ting Anh l Golden rice. N c to ra bi s chuyn gen vo ging la truyn thng, lm cho ging la truyn thng c kh nng tng hp beta-carotene. C 3 gen iu khin tng hp beta-carotene sau y: 1. psy (phytoene synthase) t cy thy tin hoa vng (Narcissus pseudonarcissus) 2. lyc (lycopene cyclase) t cy thy tin hoa vng (Narcissus pseudonarcissus). 3. crt1 t loi vi khun trong t l Erwinia uredovora Gen psy, lyc v crt1 c chuyn vo h gen trong nhn cy la v tr pha sau on promoter c bit trong ni nh. Cng trnh ny do Gio s Ingo Potrykus, ngi
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Thy S v Gio s Peter Beyer c sng to ra v cng b vo nm 2000. Hin nay ging go ht vng ny ang c nhn ging sn xut n . C chua Nm 1994, ging c chua bin i gen u tin c FDA chp nhn v a ra th trng. Tn thng mi ging c chua ny l Flavr Savr tomato. Trong c chua ny ngi ta loi tr gen tng hp ra enzyme polygalacturonase (enzyme ny phn gii pectin lm cho qu chn tr nn mau h, kh bo qun), gip cho vic bo qun c chua c lu hn, tt hn. Ngoi ra ngi ta cn ci thin mu ca c chua hn, giu lycopene hn, to c ng u v chn cng lc, rt tin cho c gii ha thu hoch v chuyn ch. Cc nh khoa hc cng to c ging c chua chuyn gen giu lycopene, khng su bnh. Thng qua con ng chn ging hay bin i gen, ngi ta c th lm cho hm lng lycopene trong ging c chua mi cao gp 3-4 ln so vi ging c chua bnh thng. FDA th nghim v chp nhn l ging c chua chuyn gen ny khng tng hp ra protein gy d ng, n c tiu ha nhanh trong d dy, rut. Ging c chua GMO ny c th lm tng hm lng lycopene v beta-carotene, nu c nhn gen iu khin tng hp cht ny cao. Ngoi ra, ging c chua ny cn c nhng u im hn v mi, v v kh nng chng nm gy bnh tn cng. u tng u tng l mt loi cy trng lu i. y l loi cy cha du em li li ch kinh t to ln nht trn th gii. Ht u tng c cha t l amino acid khng thay th nhiu hn c tht. u tng c bin i gen mang cc tnh trng nh kh nng chng thuc dit c v c hm lng oleic acid cao. Cy ci du Cy ci du c bin i gen vi mc ch ci thin cht lng dinh dng, c bit l hm lng cht bo ha tan ca loi cy ny. Cy ci du c bin i gen mang cc tnh trng chng chu thuc dit c, c hm lng laurate v oleic acid cao.
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Khoai ty Khoai ty c xem l cy lng thc quan trng th t trn th gii. Khoai ty bin i gen mang cc tnh trng nh kh nng khng cn trng v khng virus. Gn y, ngi ta nghin cu cho ra i mt loi khoai ty chuyn gen cha vaccine nga vim gan B. Loi khoai ty ny c kh nng khng virus vim gan B bng cch to ra khng nguyn virus [24]. u u l mt loi cy trng quan trng khu vc ng Nam . y l loi thc phm cha nhiu carotenoid, c cha khong 7.2mg trong mt qu. u cn c nh gi cao trn lnh vc thc phm chc nng nh cc enzyme nh papain, c tc dng ging nh cc enzyme do d dy tit ra nn rt cn thit cho vic tiu ha thc phm. Enzyme ny t ra ht sc hiu qu trong vic phn hy cc hn hp protein, c tc dng h tr trong cc bnh x ha ti mt, tiu ha kh khn v nng rt d dy, thiu nng tuyn ty, gim hm lng enzyme pancreatin tit ra. Trong y hc c truyn Nam M, u c nh gi c hiu lc tr bnh tiu ng, hen suyn v chng k sinh trng ng rut, iu tr hiu qu bnh ho lao nu dng u n trong thi gian di. Hin nay ging u chuyn gen rut giu lycopene, chng ung th, li khng c virus c pht trin cc nc thuc khu vc ng Nam . Chui Trong s cc loi cy trng nhit i, chui rt c a thch do hng v hp dn ca n. Chui khng c cht bo, giu cht dinh dng, cha hm lng kali v cht x rt cao, v l ngun cung cp vitamin C chng oxy ha. Chui ang c nghin cu bin i gen mang cc tnh trng nh khng virus, chn chm. Chui cng l loi cy d kin c dng lm vaccine thc phm (edible vaccine) phng chng nhiu loi bnh dch khng khip cc nc ang pht trin [2, 4].
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2.1.1.2. ng vt Th chuyn gen Th l i tng chuyn gen nhm mc ch to ra protein qu s dng trong y dc thng qua tuyn sa bi cc l do sau y : - Gi phi th thp nn c th to ra mt lng ln th chuyn gen. - Thi gian mang thai ngn v thnh thc sinh dc nhanh. - Gi sn xut thp. - V mt di truyn, th gn vi ngi hn bt k sinh vt cho sa no khc. Do vy n l m hnh c chn cho vic sn xut cc protein cha bnh cho ngi c bit l cc protein phc tp. - Khng truyn cc bnh nghim trng do virus gy ra cho ngi. - Mt th ci c th tit mt lng sa ln n 250ml sa mi ngy. Trong qui trnh chun, mi ngy ch c 100-150ml sa c ly t mt th ci in hnh, tng ng vi 15 lt mi nm i vi mt th ci. - Lng protein ti t hp trong sa th chuyn gen bin i t 1-10g trong mt lt. Cc loi protein c th c sn xut trong sa th l cc khng th n dng (monoclonal antibody), vaccine... D chuyn gen Mt th nghim cng c thc hin to ra d chuyn gen bng k thut vi tim vo hp t ly tm (Amstrong v cng s, 1987; Fabricant v cng s, 1987). T l d con cho sa chuyn gen sinh ra l 5-10%. Mt s protein dc phm c biu hin sa d chuyn gen. B chuyn gen Cc nh khoa hc trng i hc Vermont sn xut dng thun v tnh b Jersey cung cp gen chng bnh vim v cho ngnh Cng ngh sinh hc to ging b sa c kh nng chng li bnh vim v do vi khun. SVTH: Nguyn Kiu Oanh
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G chuyn gen G chuyn gen c pht trin nhm mc ch: sn xut dc phm v cc protein khc trong trng s dng trong y hc cho ngi v vt nui. Trong lnh vc ny, g mi v trng ca chng ang c s dng nh l cc nh my sn xut protein v khng th. Cc nh khoa hc to ra g chuyn gen c th sn xut protein trong trng. y l mt bc quan trng tin n mc tiu nhn ging g mi c th nhng qu trng vng m thuc c ng gi trong . Sn xut thuc trong lng trng trng c nhiu li th : - G c th nhiu th h trong mt thi gian ngn. Mt th h ca g l khong 6 thng, ngn hn rt nhiu so vi cc ng vt c v ln nh d chng hn phi cn 18 thng sinh trng t phi chuyn gen mi c kh nng cho sa. - G cng sn xut c nhiu protein, hng nm mi g mi c th xp x khong 330 qu trng cha 6,5 gam cc protein khc nhau. - V mt sinh hc th lng trng trng t phc tp hn sa v c nhiu phng php tch chit cc protein khc nhau t trng mt cch d dng. Nghin cu g chuyn gen ang c s dng : - Ch to vaccine. - Sn xut khng th trong trng. Cc khng th ny c thm vo trong thc n chng nhim khun (nh E. coli). - Sn xut khng th thc y s sinh trng trong non hong cung cp nguyn liu cho vt nui nhm mc ch tng tc sinh trng ca chng. - Sn xut protein ti t hp lactoferrin v lysozym. y l cc cht b sung vi thuc khng sinh thc y s sinh trng hoc khng th trong khu phn thc n gia cm. - Sn xut khng th chng ung th ngi. - Tit ra hormone sinh trng ngi cha bnh ln.
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- Sn xut trng c hm lng cholesterol thp hn phc v cho con ngi. - Sn xut isoflavon u nnh trong trng bn cho ngi tiu dng. - Sn xut cc khng th nh immoglobulin chim hoc IgY mt cch c bit, thay th cho vic s dng cc ng vt th nghim. - To dng sn xut g tht (broiler). Mt nhm cc nh khoa hc Anh pht trin thnh cng loi g Chuyn trng bin i gen c kh nng ra nhng qu trng cha cc loi protein chng ung th. Trung tm sng to ra cu Dolly bng sinh sn v tnh nm 1997, lai to thnh cng khong 500 con g bin i gen c kh nng trng chng ung th. Loi protein cha bnh nm trong lng trng trng, l protein miR24 (mt loi khng th cho php cha tr bnh ung th da) c kh nng ngn chn s tng sinh ca virus trong t bo. C th chit xut sn xut thuc cha ung th c hiu qu v t tn chi ph. Tuy nhin cc nh nghin cu vn cn phi mt khong 10 nm na cung ng i tr cho th trng mt loi protein chng ung th gi r ny [2]. 1.1.2. Tnh hnh pht trin thc phm chuyn gen Vit Nam V k thut cng ngh gen, Vit Nam ta cn rt non tr ch mi bt u nghin cu trong nhng nm gn y ch yu trn i tng l thc vt. L do d thy nht l do ng vt c b gen ln phc tp v c ch iu ha biu hin gen cht ch. Trong nhng nm qua, cc phng th nghim trong nc tp trung nghin cu chuyn gen vo mt s cy trng quan trng nh : la, ng, u tng, bng, ci du, ci bp, hoa, thuc l v cc cy lm nghip khc. Cc gen khng thuc dit c v khng bnh kh vn c chuyn vo hai ging la DT10 v DT13, gen khng bnh bc l vo ging la VL902, gen khng su t vo ging ci bp CB26. Cc gen CryIA(c) (khng su), GNA (khng ry), Xa-21 khng bc l) c chuyn vo loi ph la indica, gen -caroten (tin cht vitamin A) vo cc ging la MTL250, IR64, KDML c ging la giu vitamin A, gen Bt vo cy bng, gen anti-ACO (gen qu chn chm v hoa lu tn) vo hoa cc, gen BT vo ng, gen v bc (protein coat) khng m vng vo
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u . Mt s dng bch n c bin np gen thay i hm lng v tnh cht ca lignin cng ang c th nghim. Do nc ta cha c lut an ton sinh hc, c bit l i vi cy trng bin i gen nn tt c cc nghin cu mi ch dng quy m phng th nghim v nh knh. Bn cnh vic nghin cu v chn to ging cy trng bin i gen, chng ta cng ang xy dng nhng quy trnh xc nh cy trng v sn phm bin i gen v nh gi kh nng gy hi ca chng. thc hin nhim v ny, cc thng tin v cc gen hin ang c s dng chuyn vo cy trng c thu thp v phn tch nh Xa-21 (khng bnh bc l la), gen CryIA (a, b, c, d) (khng su), gen chitinaza (khng nm), cc gen P5C5, OAT, TPS, nhaA, HAL (chu hn v mn), cc gen CgS, SA (tng hm lng acid amin), cc gen CP, replicaza (khng bnh m vng), gen anti-ACO (gen qu chn chm v hoa lu tn), gen bar (khng thuc tr c). Quy trnh xc nh s c mt ca mi loi gen trong cy trng, sn phm thc n cho ngi v cho vt nui ang c xy dng. Mt s quy trnh c ng dng trong thc t xc nh cy trng v sn phm bin i gen nh quy trnh xc nh gen bar v gen Bt [6].
1.2. Nhng c tnh mi ca sinh vt chuyn gen c dng trong thc phm chc nng 1.2.1. Thc vt Sau y l mt s hng nghin cu chnh trong cng ngh chuyn gen thc vt to ra ging mi giu cht dinh dng chc nng, nng cao cht lng sn phm. 1.2.1.1. Carbohydrate v acid bo Carbohydrate ng vai tr quan trng v c nhiu chc nng i vi thc vt v con ngi. Ngi ta c th thay i thnh phn carbohydrate ca thc vt bng vic biu hin nhng gen mi hoc bt hot nhng gen hin c. V d: ngi ta thnh cng trong vic chuyn gen m ha cho RNA antisense ca enzyme tng hp cc ht tinh bt di s iu khin ca promoter 35s RNA (enzyme
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tng hp amylose). Khoai ty c to ra bng cch ny ch cha amylopectin m khng c amylose. Acid bo gm mt chui carbon di vi u cui l nhm carboxyl. Acid bo khc nhau v di ca chui carbon v bo ha. Nhng c im ny nh hng n c tnh ha hc ca acid bo. C th thay i cht bo theo hai hng sau: - Th nht l thay i t l acid bo bo ha v cha bo ha. - Th hai l to ra nhng acid bo c mch carbon di, cha bo ha, v chng c coi l thc phm c gi tr. V d: mt gen m ha cho enzyme thioesterase c a vo cy ci du, tng hm lng lauric acid (CH3(CH2)10COO-), thun li cho vic sn xut b. Gn y cc nh nghin cu ca i hc Bristol (Anh) thng bo v vic sn xut hai chui di acid bo khng sn sinh ra cholesterol vi s lng ln thc vt bc cao. Vic sn xut ra cc loi du thit yu cy Arabidopsis thaliana cho thy thc vt chuyn gen c th tr thnh ngun cung cp cc acid bo quan trng dng trong n ung m chng ta thng ch nhn c t c. Ngi ta cng p dng thnh cng k thut gen i vi cy Arabidopsis thaliana to ra cc acid bo thit yu khc nh arachidonic acid v eiconsapentaenoic acid [4]. 1.2.1.2. Hm lng protein v amino acid khng thay th Hm lng protein v thnh phn amino acid thay i rt nhiu trong thc phm thc vt v thng thiu cc amino acid khng thay th nh lysine, methionine, threonine v tryptophan Trong tng lai iu ny s c gii quyt bng vic to cc dng cy u tng hoc ng mang gen m ha cho protein giu nhng amino acid ny. Mt trong nhng thnh cng u tin l to dng ng bin i gen c cn bng amino acid tt hn (hm lng protein cao hn) c tn l Opaquez, c hm lng lysine cao hn (tng 20% so vi i chng).
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1.2.1.3. Vitamin, cht khong v cc nguyn t vi lng Thc vt l ngun cung cp vitamin, cht khong v cc nguyn t vi lng (tr vitamin B12 v D). Cc thc vt khc nhau c lng vitamin v cht khong khc nhau. Tuy nhin go l lng thc chnh cho con ngi nhng hu nh khng cha vitamin A. Cy la ch sn sinh ra hp cht carotenoid c chuyn thnh vitamin A trong nhng b phn c mu xanh ca cy, tuy nhin trong ht go m con ngi vn dng li khng c hp cht ny. Nu ch s dng go trong cc ba n m khng thm cc loi thc n c cha vitamine A s dn n cc bnh v mt do thiu viatamine A. V vy mt dng la chuyn gen ra i p ng cho nhu cu ny, l dng la to ra nhng ht go mu vng c gi l go vng Golden rice. Dng la ny c chuyn gen m ha cho - carotene mt tin cht ca vitamine A. Gen ny c biu hin ni nh v vy tin vitamin A khng b mt i khi xay xt. Ni nh by gi c mu vng l do -carotene. 300g go ny cha lng -carotene, p ng nhu cu hng ngy ca con ngi. Ngoi ra, cn c nhng dng la chuyn gen giu vitamine E, st p ng nhu cu v vitamin v khong cht cho con ngi. 1.2.1.4. Vaccine thc phm Gn y, cc nh khoa hc coi cy trng nh mt ngun cung cp cc loi vaccine phng bnh, bi v nhng loi vaccine thng thng i hi phi c lu gi trong mi trng lnh, iu v cng kh khn nhng ni xa xi ca cc nc ang pht trin. Mt nghin cu gn y cng b mt bc t ph trong lnh vc sn xut vaccine t thc vt, l loi khoai ty chuyn gen cha vaccine nga vim gan B. Loi khoai ty ny c kh nng khng virus vim gan B bng cch to ra khng nguyn virus. Cc nh nghin cu hy vng rng khi n loi khoai ty ny, cht khng nguyn s gy ra mt phn ng min dch nh trong c th ngi. T , c th ngi s to ra cht min dch c th i vi bnh ly nhim vim gan B [24].
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1.2.2. ng vt Hin nay trn th gii c nhiu phng th nghim v ang tp trung nghin cu to ra ng vt chuyn gen giu cht dinh dng chc nng, tuy nhin ch yu tp trung vo nhng hng sau: 1.2.2.1.To ra ng vt chuyn sn xut protein qu dng trong y dc y l hng c nhiu trin vng nht bi v nhiu protein dc phm qu khng th sn xut qua con ng vi sinh hoc sinh vt bc thp, do nhng sinh vt ny khng c h enzyme to ra nhng protein c cu to phc tp. Bng 1.2: Trang tri nui ging ng vt sn xut protein dc phm Loi Protein dc phm -1-antitrypsin Anti-thrombin III Collagen Fibrinogen Human hemoglobin Human serum albumin Lactoferrin LAtPA (Tissue Plasminogen Activator) Monoclonal antibodies (Mab) Anti-TNF IgG1 (monoclonal) Chc nng cha bnh C chc nng chng vim Chng s nhim trng v x cng nghn mch, cc mu ng. Cha bng, gy xng Cha bng, phu thut, trong ha tr liu, trong dc phm Thay th mu ngi, truyn mu Cha bng, shock, chn thng, phu thut Tr vi khun gy bnh ng rut Cha lot do mu tnh mch Chng ung th kt trng Chng li cn au tim Loi th D, Cu D B Heo, Cu Heo D, B B D D D [66] nh s dng tuyn sa ca ng vt bc cao sn xut ra protein qu ln u tin c Clark (1987) xut. Ni dung ca k thut ny l gn gen cu trc vi lactoglobulin (l promoter iu khin s biu hin ca gen tuyn sa). Khi a t hp
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c cha promoter -lactoglobulin vo cu, chut, Clark thy chng biu hin rt cao tuyn sa. Sn xut protein thng qua vic sn xut sa c nhiu li th: - Tuyn sa ca ng vt c v l mt c quan sn xut sinh hc thch nghi cao cho s bi tit. - Tuyn sa l mt h thng sn xut khng l c kh nng to ra t 23g (b sa) n 205g (chut) protein/kg c th trong thi k tit sa ti a. - Nng t bo trong tuyn sa ca ng vt c v ln hn trong nui cy t bo thng thng t 100 n 1000 ln. - Nhiu protein c sn xut tuyn sa ca ng vt c v c hot tnh dc cao do c quan ny c iu kin thc hin chn ha (maturation) protein. - Sa l dch tit ca c th c th c thu nhn mt cch d dng nht, c bit l t ng vt nhai li. - S biu hin ca gen tuyn sa ca ng vt c v l chnh xc v thi gian. - Sn lng sa tit ra ng vt c v l kh ln: d ln n 800 lt/nm, cu 400 lt/nm, b 8000 lt/nm, chut l 1,5ml/ln... Cho n nay rt nhiu protein dc phm qu v ang c nghin cu sn xut qua tuyn sa ca ng vt nh: - 1- antitripsin v yu t lm ng mu IX (blood clotting factor IX) ca ngi c tit ra trong sa chut, sa cu vi nng tng ng l 5mg/ml v 25mg/ml. - Cht hot ha plasminogen m ngi (human tissue plasminogen activator) lm tng ng mu c tit ra sa d v sa chut. - Gen urokinase ngi c a thnh cng vo ln v tit ra tuyn sa nh gen khi ng alpha-casein b. - Protein C ngi c to ra t sa chut v sa ln chuyn gen...
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Bng 1.3: Nhng protein c tc dng cha bnh trong sa c sn xut ra bi th cho sa chuyn gen Tn protein Antithrombin III Factor VIII, Factor IX CFTR (cystic fibrosis transmembrane conductance regulator) Lactoferrin -1- antitrypsin Lysostaphin Spider silk protein ng vt D D, Heo, Cu Cu ng dng trong iu tr bnh Lm gim s lng mu cn thit trong mt s trng hp phu thut Cha bnh a chy mu (hemophilia) Cha bnh x u nang (cystic fibrosis)
B Cu B D
Protein khng sinh t nhin c s dng trong phu thut tim Cha bnh x u nang v kh thng Hp cht khng khun phng bnh vim v b Sn xut ch Y khoa siu nh, siu chc, siu mnh, t tiu [67]
Bng 1.4 : Mt vi thng s lin quan n vic tit sa ng vt c v ng vt Thi gian mang thai (thng) 0.75 1 4 5 5 9 Thi gian thnh thc (thng) 1 6 8 6 6 15 S con trong mt la 10 8 9 2 2 1 Sn lng sa tit ra 1.5ml 1 1.5l 200 400l 200 400l 600 800l 8000l S thng tit sa 3-6 7-8 15 - 16 16 - 18 16 - 18 30 - 33 [2] Hin ti c 2 protein c sn xut bng con ng ny l 1-antitripsin ngi v cht hot ho plasminogen m ngi. Cht u c sn xut qua sa cu vi nng 35g/l, cn cht sau sn xut qua sa d. Nhng sn phm ny hng nm mang li cho hng Genetech (M) hng trm triu USD. Hin ti cc nh khoa hc M mun gim gi SVTH: Nguyn Kiu Oanh
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Chut Th Ln Cu D B
thnh ca sn phm ny bng cch sn xut qua sa th. Ngi ta d on gi thnh sn xut hormone ny qua sa th ch bng 1/3 gi thnh hin ti sn xut nh vi sinh. L do l chu k sinh sn ca th ngn v lng protein sa ca th li cao. Trong mt nm lng protein sa ca 6 con th bng ca mt con b. Hin ti chut chuyn gen hormone sinh trng tit ra protein ny vi nng 0,5g/l. Bng 1.5 : c tnh sn lng protein sinh phm trong sa v qui m n th chuyn gen trn th gii Loi protein sinh phm Protein C tPA -1-antitrypsin Factor IX Human serum albumin c tnh hng nm cn (kg) 100 75 5,000 2 1,000 c tnh qui m th 33 1,100 600 33,000 42,000 13 12 300 8,300 Loi th B Heo D Cu Cu D Cu Heo B D [66] Mt khc, cc protein dc phm mong mun cng c to ra trong dch c th khng thuc m v nh mu. Cho n nay phng php ny ch mi c s dng biu hin hemoglobin ngi vi mc cao ln chuyn gen (Sharma, 1994). Bn cnh hai phng php trn, cc nh khoa hc pht trin ng vt chuyn gen sn xut ra dc phm trong bng quang ca chng. Kh nng s dng nc tiu ca ng vt sn xut protein tng ln vo nm 1995, khi Tung-Tien Sung i hc New York chng minh rng c nhng gen ch hot ng bng quang. Cc gen ny m ho cho protein uroplakins. Protein ny l mt thnh phn tham gia hnh thnh nn mng bng quang. Kerr (1998) nghin cu to ra chut chuyn gen sn xut hormone sinh trng ngi t nc tiu. Gen hormone sinh trng ngi c ni vi promoter urolapkin. Promoter ny kim sot v tr v thi gian hot ng ca gen. Chut mang gen ngoi lai to ra
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500 nanogam hormone sinh trng ngi trong mt mili lt nc tiu thi ra. Mc d sn phm ca chut chuyn gen ch l mt lng nh nhng chng cho thy rng trong tng lai nc tiu ca vt nui c th s c la chn. Nc tiu c nhng u th vt tri so vi sa. C ng vt c v ci u tit nc tiu, c bt u ngay sau khi sinh ra. Nc tiu ca cc i gia sc cha nhiu protein hn trong sa ca chng. Mt khc, trn thc t chi ph cho vic tinh ch thuc t nc tiu thp hn so vi sa. Mt vi protein c th khng thch hp i vi vic khai thc t sa bi v chng lm tn thng m v. 1.2.2.2. Nng cao nng sut, cht lng ng vt bng cch thay i cc con ng chuyn ha trong c th ng vt Nhiu phng php c xut nng cao cht lng dinh dng ca sa. Trong hng ny ni bt l nhng nghin cu nng cao cht lng sa b, sa cu bng cch chuyn gen lactose vo cc i tng quan tm. S biu hin ca gen ny c iu khin bi promoter ca tuyn sa. Trong sa ca nhng ng vt chuyn gen ny, ng lactose b thy phn thnh ng galactose v ng glucose. Do vy nhng ngi khng quen ung sa cng c th s dng c sa ny m khng cn qu trnh ln men. Mi y, cc nh khoa hc (Brigid Brophy, 2003) chuyn thm cc gen m ho -casein (CSN2) v kappa-casein (CSN3) b vo cc nguyn bo si ca b v to ra b chuyn gen cho sa c mc -casein v kappacasein cao hn bnh thng: hm lng casein tng ln 8-20%, hm lng kappa-casein tng gp 2 ln v t l kappa-casein so vi casein tng s thay i mt cch ng k. Hai loi casein l protein ch yu trong sa v l thnh phn chnh ca sa ng, cha kho ca s sn xut ph-mt v sa chua. Cc protein ny rt quan trng, chng lm cho sa c hm lng protein cao nhng cha nhiu nc.
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Bng 1.6: Mt s thay i cc thnh phn ca sa c xut S thay i Tng -CN v -CN Tng v tr phosphoryl ho trong casein Tit -LG Gim -LA Thm lactoferin ngi Gim s biu hin ca acetyl-CoA cacboxylase Biu hin gen Ig Thay th cc gen protein sa b bng cc gen protein sa ngi Tng nng kappa-CN Kt qu Ci tin tnh bn vng i vi nhit v tng hm lng calcium Tng hm lng calcium Ci tin tnh tiu ho, gim d ng v gim ngun cystein s cp trong sa. Gim lactose Tng cng s hp thu st v bo v chng li s nhim trng rut Gim hm lng m, ci tin cht lng dinh dng Bo v chng li cc bnh nh Salmonella v Listeria To ra sa ging nh sa ngi Tng tnh n nh ca s kt t casein, gim kch thc mixen v gim ng keo (gelation) v ng t (coagulation) [2]
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Chng 2: Cc phng php chuyn gen
CHNG 2 CC PHNG PHP CHUYN GEN
2. 1.Thc vt 2. 1.1. Phng php chung Mt s vn sinh hc nh hng n qu trnh chuyn gen Khng phi ton b t bo u th hin tnh ton nng (totipotency). Cc cy khc nhau c phn ng khng ging nhau vi s xm nhp ca mt gen ngoi lai. Cy bin np ch c th ti sinh t cc t bo c kh nng ti sinh v kh nng thu nhn gen bin np vo genome. Thnh phn ca cc qun th t bo c xc nh bi loi, kiu gen, tng c quan, tng giai on pht trin ca m v c quan. Thnh t bo ngn cn s xm nhp ca DNA ngoi lai. V th, cho n nay ch c th chuyn gen vo t bo c thnh cellulose thng qua Agrobacterium, virus v bn gen hoc phi ph b thnh t bo chuyn gen bng phng php xung in, siu m v vi tim. Kh nng xm nhp n nh ca gen vo genome khng t l vi s biu hin tm thi ca gen. Cc DNA (tr virus) khi xm nhp vo genome ca t bo vt ch cha m bo l lin kt n nh vi genome. Cc DNA (tr virus) khng chuyn t t bo ny sang t bo kia, n ch ni m n c a vo. Cc bc c bn ca qu trnh chuyn gen: Xc nh gen lin quan n tnh trng cn quan tm. Phn lp gen (dng phng php PCR hoc sng lc t th vin cDNA hoc t th vin genomic DNA). SVTH: Nguyn Kiu Oanh
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Gn gen vo vector biu hin (expression vector) bin np. Bin np vo E. coli. Tch chit DNA plasmid. Bin np vo m hoc t bo thc vt bng mt trong cc phng php khc nhau. Chn lc cc th bin np trn mi trng chn lc. Ti sinh cy bin np. Phn tch xc nhn c th chuyn gen (PCR hoc Southern blot) v nh gi mc biu hin ca chng (Northern blot, Western blot, ELISA hoc cc th nghim in vivo khc...). Nguyn liu thc hin s bin np l cc t bo thc vt ring l, cc m hoc cy hon chnh.
2.1.2. K thut c bn 2.1.2.1. Phng php chuyn gen gin tip nh Agrobacterium Nguyn tc: Phng php ny a DNA vo t bo thc vt qua trung gian ca vi khun Agrobacterium tumefaciens (y l loi c dng ph bin). Sau khi xm nhim, chng gn mt on DNA (T-DNA) vo b my di truyn ca t bo thc vt, dn n ri lon cc cht sinh trng ni sinh, to ra khi u. Vi khun A.tumefaciens l vi khun gram m trong t mang c tnh. A.tumefaciens v mt s loi h hng c kh nng chuyn mt phn nh DNA ca n vo t bo thc vt (nhiu loi thc vt ht kn hai l mm v mt s t thc vt mt l mm) v qua kch thch to khi u (gi l crown-gal). Nhng khi u ny l khng gian sng ca vi khun. Mt s cht dinh dng (opine) c li cho vi khun cng c to ra trong nhng khi u ny. Nhng opine ph bin nht l nopalin v octopin. V ha hc opine l nhng sn phm ngng t ca mt amino acid vi mt cetoacid hoc mt amino acid vi ng. Octopin c to nn t amino acid l arginine v pyruvate, cn nopalin c to nn t arginine v -cetoglutaraldehyd.
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Hnh 2.1: Vi khun Agrobacterium tumefaciens nhn di knh hin vi [2].
Hnh 2.2: Khi u thc vt do Agrobacterium tumefaciens gy ra [2]
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Hnh 2.3: Cng thc cu to ca opine (octopin, nopalin) [2]. Vic gy ra khi u v tng hp opine lin quan vi s c mt ca mt loi plasmid c kch thc ln l Ti-plasmid trong t bo vi khun A. tumefaciens. Ti-plasmid ca A. tumefaciens: Ti-plasmid (tumor inducing plasmid cm ng to khi u) c tm thy trong tt c cc dng A.tumefaciens gy c, c kch thc khong 200-800 kb. Chng c duy tr n nh trong Agrobacterium nhit di 30oC. Qua nghin cu cho thy trong Tiplasmid c hai vng quan trng l: vng T-DNA (transferred DNA) v vng gy c (virulence vir gen) lin quan n s hnh thnh khi u, s tip hp v s ti bn ca plasmid trong Agrobacterium. Ngoi ra, cn c vng ng vai tr phn gii opine (vng T-DNA c kh nng tng hp loi opine no th vng ny c kh nng phn gii opine cung cp nng lng cho t bo vi khun). - T-DNA: l mt phn ca Ti-plasmid (vng T-DNA ny c kch thc khong 12- 24 kb), c chuyn vo thc vt (transfer-DNA). Trn nh v nhng gen m ha cho sinh tng hp auxin, cytokinin, opine v cc gen to khi u. Trong Ti-plasmid, T-DNA c gii hn bi hai vng, vai tri v vai phi (LB: left border v RB: right border). Cc vai ny gm mt trnh t lp li ca 25 bp, l trnh t nhn bit cho vic ct T-DNA. Vai tri 5 -TGNCAGGATATATATNNNNNNGTNANN- 3 Vai phi 5 -TGGCAGGATATATATNNNNNNGTAAAN- 3 SVTH: Nguyn Kiu Oanh
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(Trnh t chung ca vai tri v vai phi ca T-DNA trn Ti-plasmid. N l mt nucleotide bt k trong 4 nucleotide. Trnh t ny c th khng ging nhau cc chng vi khun). Mc d vai tri cng cha on lp li 25 bp tng t vai phi, nhng nhng nghin cu cho thy vng ny khng tham gia vo qu trnh bin np. - Vng vir: nm ngoi vng T-DNA ( di khong 25 kb), c khong 25 gen c nhn bit trong 7 n v phin m l vir A, vir B, vir C, vir D, vir E, vir F, vir G. Nhng gen vir ny gi cc vai tr nh: nhn ra t bo thc vt, tn cng vo t bo thc vt, ct b on, chuyn gen v c l c c vai tr trong s xm nhp ca T-DNA vo t bo k ch. Trong , vir A, B, D, G cn thit cho vic to khi u; vir C, E nh hng n hiu qu chuyn gen trn bin k ch. C ch bin np T-DNA vo t bo k ch: xy ra qu trnh bin np t bo thc vt phi b thng. V lc ny t bo thc vt tit ra cc hp cht lm tn hiu cm ng cho vi khun tn cng v xm nhp. Cc hp cht cm ng c chia lm hai loi: -Cc monoccharide: to ra s hp dn trn mt qung ng di i vi nhng chng vi khun Agrobacterium c v khng c n v tr vt thng r. - Dng tin phenol v cc cht trung gian ca s sinh tng hp lignin c vai tr rt c bit. nng thp (<10-7M i vi acetosyngone) cc phenolic ny hot ng nh cht li ko chuyn bit ch i vi chng Agrobacterium c. Sn phm ca vir A v vir G lm trung gian cho s li ko theo cc ha cht ny. Tuy nhin nng cao hn th cc hp cht phenolic cng loi ny c vai tr nhng cht cm ng chuyn bit s iu ha gen vir thng qua du hiu kp (vir A v G) ca c ch ti np tng t vi cc h thng iu ha ca cc loi vi khun khc. Qu trnh bin np T-DNA vo t bo k ch gm 10 bc nh sau: (1) Vi khun nhn ra v tn cng vo t bo k ch. (2) Cc hp cht cm ng ca t bo k ch tit ra cm ng vi sn phm ca h thng tn hiu kp (vir A v G).
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(3) Vng gen vir c hot ha. (4) Bn sao ca T-DNA c to ra di s c mt phc hp protein ca vir D1,D2. (5) Bn sao ny kt hp vi protein ca vir D2, to thnh phc hp T-DNA protein v vi mt s protein ca cc vir khc i vo t bo cht ca t bo k ch thng qua knh vir B/D4T4SS. (6) Phc hp ny li kt hp vi sn phm ca vir E, ri i vo t bo cht ca t bo k ch. Protein ca vir D2 v sn phm ca vir E gn vi bn sao T-DNA c tc dng bo v si T-DNA khng b phn ct bi cc enzyme exonuclease. (7) Phc hp T-DNA protein i vo nhn t bo k ch. (8) Khi vo trong nhn, T-DNA xc nh im xen vo. (9) T-DNA tch ra khi cc protein bo v. (10) T-DNA gn vo b gen ca t bo k ch mt cch ngu nhin. ng dng vi khun Agrobacterium tumefaciens trong chuyn gen thc vt: Trn thc t, A. tumefaciens ch gy hi cy hai l mm, v vy ngi ta cho rng chng ch c th bin np T-DNA vo b gen ca cy hai l mm. Gn y, nhiu tc gi chng minh khi nhim vi khun Agrobacterium, cc cy mt l mm cng c th sn xut opine v c th khai thc vic chuyn gen ca Agrobacterium cy mt l mm. Mc d, Ti-plasmid ca A.tumefaciens l vector t nhin nhng chng li c nhc im so vi vect nhn dng: - S sn xut phytohormon ca t bo thc vt chuyn gen (c nui cy trong mi trng) ngn cn chng ti sinh thnh cy trng thnh hon chnh v khe mnh. Do , gen m ha cho enzyme tham gia tng hp auxin v cytokinin cn phi c loi b khi Ti-plasmid. - Gen m ha cho cc enzyme tham gia tng hp opine khng c vai tr i vi k thut to thc vt chuyn gen v c th lm gim sc sng do tiu hao nng lng vo vic tng hp opine. Do , cc gen ny phi loi b khi plasmid. SVTH: Nguyn Kiu Oanh
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- Ti-plasmid c kch thc ln (200-800 kb) gy kh khn trong qu trnh thao tc to DNA ti t hp. V vy, cc on DNA khng cn thit cho qu trnh to dng v biu hin gen cn c loi b. - Ti-plasmid khng sao chp trong E.coli nn vic nhn dng plasmid ti t hp trong E.coli khng th thc hin c. Do , khi xy dng vect plasmid phi thm on ori ca plasmid E.coli c th sao chp trong t bo E.coli. khc phc nhng nhc im, trn cc nh khoa hc to ra vect ti t hp da trn nguyn tc ca Ti-plasmid. Nhng vect ny gm c cc thnh phn sau: - Cc gen chn lc (a selectable maker gene) nh: neomycin phosphotransferase, gen to kh nng khng kanamycin ca t bo thc vt chuyn gen. - Trnh t ori cn thit cho s sao chp v nhn i ca plasmid trong E.coli. - Trnh t vai phi ca vng T-DNA: vng ny tuyt i cn thit cho qu trnh chuyn gen xy ra. Mt s vect c th c trnh t c hai vai. - Vng polylinker (multiple cloning site) thun tin cho vic ct v chn gen vo plasmid . Kh nng chuyn gen t nhin c khai thc chuyn gen ngoi lai vo b my di truyn ca t bo thc vt theo mun, bng cch nui mu cy trong mi trng thch hp c cha Agrobacterium mang vect ti t hp cn chuyn. Agrobacterium s xm nhp vo m mu cy t cc vt thng mp v chuyn plasmid mang gen cn chuyn vo t bo thc vt. Sau , nhng t bo ny c ti sinh trn mi trng thch hp to thnh cy hon chnh mang gen mong mun.
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Hnh 2.4: S gen ca Ti-plasmid trong vi khun A. tumefaciens [2].
Hnh 2.5: S plasmid ti t hp da trn nguyn tc ca Ti-plasmid [2]
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Hnh 2.6: Qu trnh to cy chuyn gen nh A.tumefaciens [2].
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2.1.2.2. Phng php chuyn gen trc tip Chuyn gen bng sng bn gen Sng bn gen (Gene gun) l mt thit b s dng a thng tin di truyn vo t bo, c thit k u tin cho bin np DNA ngoi lai vo t bo thc vt v c pht trin vo u thp nin 1980 do cc nh thc vt hc i hc Corrnell cng vi cc nh nghin cu Corrnell Nanofabrication Facility, Newyork, USA. Mc d c nhiu thit k k thut khc nhau nhng nguyn l chung ca phng php ny l s dng p lc xung ca kh helium gia tc cc ht.
Hnh 2.7 : Sng bn gen (Hng Biorad )[2] Sng bn gen bao gm hai bung bng thp khng g, kch thc 6x7x10 ni vi hai bm chn khng. DNA ngoi lai c gn vo cc ht tungsten c ng knh rt nh, khong 1m (cc kim loi nng khc nh vng v bc cng c s dng nhng khng thng xuyn do gi c t). Cc ht ny c t trn mt ci a mt bn trong ca sng. S bng n kh helium 1000psi lm cho ci a bn v pha trc vi tc 1300 feet/s, tng ng vi tc khi mt vin n ri khi nng sng. Mt tm chn lm dng a li v cc ht vng hay tungsten c phng v pha cc t bo ch. Chng xuyn qua vch t bo v phng thch cc phn t DNA. Sng bn gen s dng k thut DNA ti t hp hp nht s biu hin cc gen a phn phi. Cc t bo bin i di truyn c th c s dng to thc vt bao gm c s sa i di truyn mong mun trong tt c cc t bo ca chng (Voiland, 1999) [2].
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Hnh 2.8: S nguyn l hot ng ca sng bn gen [2]
Chuyn gen trc tip vo t bo trn (protoplast) DNA d xm nhp c vo t bo thc vt, phi loi b vch t bo to protoplast. Protoplast c th c duy tr trong mi trng nui cy nh cc t bo sinh trng mt cch c lp hoc vi mt mi trng c hiu, vch t bo c th c to thnh v ton b cc cy c th c ti sinh t cc t bo ny. Qu trnh chuyn gen nh th ny c thc hin mt cch trc tip bng mt c ch vt l n gin, khng cn c vector. nng cao hiu qu bin np, ngi ta x l protoplast vi PEG (polyethylene glycol) hoc bng xung in. Phng php chuyn gen ny rt c hiu qu, c bit i vi nhng loi thc vt m phng php chuyn gen gin tip nh Agrobacterium khng th thc hin c. Tuy nhin, vic to protoplast cng nh ti sinh cy t protoplast khng n gin, tn nhiu cng sc, b nh hng ca nhiu yu t mi trng. SVTH: Nguyn Kiu Oanh
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Vi phng php ny, cc nh khoa hc chuyn gen thnh cng vo mt s loi cy mt l mm nh loi la ph Japonica (Datta, 1990), ng (Doon, 1990), la m (Vassil, 1992) [2]. Chuyn gen bng k thut xung in K thut xung in (electroporation) l mt phng php c hc c s dng a cc phn t phn cc vo trong t bo ch qua mng t bo. Trong phng php ny, mt xung in cao th trong khonh khc (vi phn nghn giy) c kh nng lm ri lon cu trc mng kp phospholipid, to ra cc l thng tm thi cho php cc phn t DNA ngoi lai t mi trng xm nhp vo bn trong t bo.
Hnh 2.9 : S mng phospholipid kp [2] K thut xung in da trn trng thi tng i yu ca cc tng tc k nc ca phospholipid kp v kh nng tp hp li mt cch t ng ca n sau khi b ri lon (Purves, 2001). Cc t bo ch v DNA ngoi lai c to thnh dch huyn ph v cho vo trong mt cuvette nha c in cc.
Hnh 2.10 : Cuvette nha c in cc [2] SVTH: Nguyn Kiu Oanh

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to ra xung in cao th trong mt thi gian ngn ngi ta s dng mt thit b gi l my xung gen (gene pulser). Qu trnh c bn din ra bn trong my ny c th c trnh by bng s .
Hnh 2.11: S b tr mch c bn ca my xung in [2] S ny cho thy mch in c bn cung cp in cho k thut xung in. Khi cng tc th nht ng, t in np in vo v tch mt in p cao. Khi cng tc th hai ng, in p ny phng qua dch huyn ph t bo. Mt xung in cn thit cho k thut ny thng l khong 10.000-100.000 v/cm (thay i ty theo kch thc ca t bo) trong vi phn triu giy n mt phn ngn giy. Xung in ny lm ri lon phospholipid kp ca mng t bo v to ra cc l tm thi. Kh nng in qua mng t bo cng lc tng ln 0,5-1,0 v. V vy cc phn t c np in ny i qua mng t bo thng qua cc l bng cch thc tng t nh in di.
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Hnh 2.12 : S plasmid cha DNA ngoi lai i qua cc l tm thi trn mng bo cht [2] Khi cc ion np in v cc phn t i qua cc l, mng t bo phng in v cc l ny ng li mt cch nhanh chng v phospholipid kp phc hi li cu trc c (Weaver, 1995). Lc ny cc phn t mong mun trong t bo v chng c s dng cho cc nghin cu tip theo. Phng php ny c th s dng i vi gn nh tt c cc loi t bo ca cc loi. Lc u phng php ny c s dng chuyn gen vo cc t bo ng vt c v, v sau cho c t bo thc vt dng protoplast... Cc ng dng ca k thut xung in bao gm: - Bin np DNA - Chuyn plasmid trc tip gia cc t bo - Dung hp t bo a kch thch - Phn phi thuc qua da - Liu php ha in khi u ung th - Liu php gen Chuyn gen bng phng php vi tim Nguyn tc ca phng php vi tim l mt lng nh DNA c tim trc tip vo nhn t bo trn mt cch c hc di knh hin vi.
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Theo phng php ny, t bo c gi trong ng thy tinh (kim gi) bng lc ht yu v DNA c bm trc tip vo nhn t bo qua pipet thy tinh rt nh (kim tim). Qu trnh tim c theo di bng knh hin vi v s thnh cng c nhn ra bi mt cht pht hunh quang c tim cng vo t bo.
Hnh 2.13: Vi tim gen ngoi lai vo nhn ca protoplast [2].
2.2. ng vt 2.2.1. Nguyn tc Gm cc bc chnh sau: 1. Tch chit, phn lp gen mong mun v to t hp gen biu hin trong t bo ng vt. 2. To c s vt liu bin np gen. 3. Bin np gen vo phi ng vt 4. Nui cy phi v cy truyn hp t (i vi ng vt bc cao) 5. Phn tch nh gi tnh n nh v s biu hin ca gen l 6.To ra dng ng vt chuyn gen gc mt cch lin tc sn xut ng vt chuyn gen.
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Hnh 2.14 : S to ng vt chuyn gen [2]. 2.2.1.1. Tch chit, phn lp gen mong mun v to t hp gen biu hin trong t bo ng vt Tch chit, phn lp gen mong mun Gen ngoi lai trc khi c chuyn vo genome ca t bo vt ch phi c phn lp v tinh ch. Cc cng c s dng to dng bao gm cc enzyme c bit c hot tnh ct v ni DNA (enzyme ct gii hn v ligase), cc mu (probe), vector v t bo vt ch. T bo vt ch thng c s dng l t bo vi khun E.coli v vector thng c s dng l plasmid. DNA mu cha gen mong mun v vector plasmid phi c ct bi cng mt loi enzyme ct gii hn. Cc on DNA mu sau khi c ct c mang gen mong mun s c xen vo vector plasmid v cc u ca cc on DNA mu ny v cc u ca vector plasmid s c ni vi nhau nh ligase to thnh plasmid ti t hp. Sau cc plasmid ti t hp c bin np vo cc t bo vi khun E.coli v cc t bo vi khun
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tin hnh sinh trng. Vo thi im ny, cc t bo vi khun cha plasmid mang gen mong mun s c pht hin bng mu d. Chng c nui cy trong mi trng thch hp sinh trng pht trin to ra hng triu bn sao ca vector cha gen ny. Vector cha gen ny s c tch ra khi t bo vi khun v gen mong mun s c tch chit. C th phn lp c gen mong mun t mRNA hoc protein. T mRNA di tc ng ca enzyme sao chp ngc s tng hp ra DNA b sung mch n (single strand complement DNA-ss cDNA), tip theo l DNA b sung mch kp (double strand complement DNA- ds cDNA). DNA b sung (complement DNA- cDNA) ny khc vi DNA gc l khng cha cc intron m ch bao gm cc exon. Hoc t sn phm protein, c th suy ra trnh t nucleotid ca gen cu trc trn c s trnh t cc axit amin trong phn t protein. Sau c th thit k cp mi (primer) d tm on gen mong mun. To t hp gen chuyn biu hin trong t bo ng vt cc u ca cu trc c th c sa i bng cch b sung cc trnh t polylinker cha mt s v tr nhn bit cc enzyme ct gii hn khc nhau. Trnh t polylinker cho php c th xen vo cu trc ny mt vector kim tra v to dng. Yu t iu ho gn u 5 ca gen l promoter, c vai tr quyt nh trong vic iu ho s biu hin ca gen. Promoter t bo ng vt c ngun gc hoc t ng vt nh methallothionein (MT), thymidine kinase, actin, amylase, insulin, -lactoglobulin, adiposite P2 ... hoc t virus ng vt nh Simian virus (SV40), Rous sarcoma virus (RSV)... Mt yu t iu ho khc l yu t tng cng (enhancer), c chc nng tng cng s biu hin ca gen, khng ph thuc vo v tr v s nh hng i vi gen. u 3 ca gen cng phi mang mt trnh t poly-A m bo thch hp cho qu trnh phin m v dch m.
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2.2.1.2. To c s vt liu bin np gen ng vt c v th giai on bin np gen thch hp nht l trng giai on tin nhn (pronucleus), giai on m nhn ca tinh trng v trng cha dung hp (fusion) vi nhau. i vi ng vt c v, trng chn c thu nhn bng phng php s dng kch dc t theo chng trnh c xy dng cho mi loi hoc bng phng php nui cy trng trong ng nghim. Sau th tinh nhn to to ra trng tin nhn. to ra mt ng vt chuyn gen, yu cu cn phi s dng hng trm thm ch hng ngn trng th tinh. V d: b t c mt lng phi ln nh th, n phi c kch thch rng mt lng ln trng (siu rng trng) thnh thc v c th tinh nhn to. Ngi ta c ch gen tng hp hormone inhibin (inhibin c xem l hormone c ch s rng trng) t lm tng t l rng trng tru b. S th tinh nhn to ch xy ra khi mt tinh trng gp mt trng trng thi thnh thc ti u. i vi c, giai on bin np thch hp l phi giai on 1-4 t bo. Giai an ny c to ra bng thu nhn trng, tinh dch bng phng php s dng kch dc t (kch thch t trong nhau thai ca ngi (HCG) hoc no thu th c chp...), ri th tinh nhn to. 2.2.1.3. Chuyn gen vo ng vt Vic t c mc ch ca vic to ra ng vt chuyn gen i hi s pht trin ca cc phng php c hiu qu chuyn gen mong mun vo phi. Hin nay c nhiu phng php khc chuyn gen nhau ang c s dng to ng vt chuyn gen: vi tim, chuyn gen bng cch s dng cc t bo gc phi, chuyn gen bng cch s dng vector virus... 2.2.1.4. Nui cy phi trong ng nghim (i vi ng vt bc cao) T bo trng tin nhn sau khi vi tim c nui cy trong ng nghim pht trin n giai on phi du (morula) hoc ti phi (blastocyst). giai on ny mng
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trong (pellucida) b bong ra v phi c th lm t c d con. Nhng phi ny c cy chuyn vo con nhn c gy cha gi (pseudopregnant) pht trin thnh c th con. i vi ng vt bc thp nh c khng cn giai on ny. Tuy nhin c, trng sau khi th tinh mng th cp (chorion) dy ln, rt dai v dnh gy tr ngi cho vic nh v chnh xc mi kim tim vo v tr mong mun c th a c DNA vo trng. Mt khc giai on phi mt t bo c rt ngn trong khi vic vi tim i hi nhiu thao tc t m v chnh xc. khc phc cc nhc im ny, ngi ta c th tin hnh loi mng th cp (Hnh 2.15), ko di giai on phi 1-4 t bo v p nhn to phi trn to c bt.
Hnh 2.15: Trng c chch (Misgurnus anguillicaudatus) trc v sau khi kh mng th cp (chorion) [2] A. Trng c chch cha kh mng th cp B. Trng c chch c kh mng th cp 2.2.1.5. Kim tra ng vt c sinh ra t phi chuyn gen khng nh ng vt c c chuyn gen l vo hay khng, ngi ta phi kim tra xem gen l c xm nhp c vo b my di truyn ca ng vt trng thnh hay khng v sn phm ca gen l c c tng hp ra hay khng.
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i vi vn th nht ngi ta s dng phng php lai phn t trn pha rn (Southern blot, Northern blot...) hoc PCR. i vi vn th hai, sn phm ca gen l c nh gi hai mc : phin m v dch m. Sn phm phin m c nh gi bng phng php RT-PCR, sn phm dch m c nh gi bng phng php Western blot, ELISA hoc k thut min dch phng x (RIA) pht hin protein l trong ng vt. Theo di cc th h sau ca ng vt chuyn gen (F1, F2, F3, ...) xc nh gen l c di truyn hay khng. 2.2.1.6. To ngun ng vt chuyn gen mt cch lin tc Sau khi kim tra thy gen ngoi lai a c di truyn n nh, tin hnh lai to v chn lc to dng ng vt chuyn gen.
2.2.2. Cc phng php 2.2.2.1. Cc phng php chuyn gen khng s dng virus Cc phng php chuyn gen vo t bo ng vt c v khng s dng virus da trn nhng nguyn tc sau: - DNA c chuyn nm dng kt ta hay trong cc phc hp tch in (kt ta calcium phosphate, chuyn nhim DEAE-dextran). - Thng tin di truyn c chuyn vo t bo ch sau khi c gn kt hay gi vo trong cc ti m hay ti mng sao cho c th dung hp vi mng nguyn sinh cht ca t bo ch (dung hp t bo trn, lipofection, erythrocyte ghost transfer). - Vic chuyn cc acid nucleic c thc hin hon ho thng qua vic m t bo bng cc phng php vt l (vi tim, laserporing, electroporation). chn phng php ti u cho tng trng hp c th, cn phi xem xt cc yu t khc nhau nh: vector, loi t bo chuyn nhim, s lng mu th nghim, thi gian biu hin, phng php phn tch phc v chuyn gen v s lng DNA cn phi c duy tr trong t bo ch sau chuyn nhim.
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Bng 2.1: Cc phng php chuyn gen khng s dng cc yu t virus Phng php Kt ta calcium phosphate DEAE-dextran Lipofection To l bng dng in Dung hp t bo trn Dung hp vi cc erythrocyte ghost To l bng tia laser Vi tim Mt s im cn lu Cho cc t bo c tnh kt dnh, lng ln DNA/t bo c chuyn . L phng php thng c s dng nht Thng s dng cho dch huyn ph cc t bo. Lng ln DNA c chuyn. L phng php hiu qu ; RNA v protein cng c chuyn. Tnh c ca lipid c gii hn. Lng nh DNA hay s hp nht tng bn sao mt c chuyn n nh vo trong tng t bo ring bit ; c bit thun li vi cc t bo lymphoid. Thng c s dng cho cc t bo lyphoid. Vic chuyn ton b cc hp cht trong t bo trn c th khng c li. Acid nucleic v protein c th c chuyn. Hiu qu cao, thit b my mc phc tp v t tin. Cng vic chun b rt tn km v phc tp. Ch mt s gii hn cc t bo c th c tim, tuy nhin hiu sut l 100%. Nhn hay thnh phn ca t bo cht u c th l i tng c tim. [12]
Phng php chuyn nhim calcium phosphate K thut calcium phosphate (calcium phosphate technique) c pht trin u tin l xc nh s ly nhim ca DNA virus (Graham,1973) v hin nay c s dng rng ri th nghim hot ng bin np ca DNA virus cng nh DNA tch chit t cc t bo eukaryote (Wigler, 1978; Graham, 1979; Pellicer, 1980). Nguyn l : cc t bo nhn vi cc cht ng kt ta DNA v calcium phosphat (Hnh 2.16). Kt ta ny bm vo t bo v sau s hp th vo t bo qua qu trnh m bo (Loyter, 1982). Trong t bo, cc phn t DNA ngoi lai nm trong khng bo c to thnh do m bo v lysosome th hai nhng rt t DNA i n nhn v hp nht vo genome ch.
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Hnh 2.16 : Phc hp DNA-calcium phosphat [2] Phng php chuyn nhim DEAE-dextran y l tc nhn ha hc u tin c s dng chuyn DNA vo t bo ng vt nui cy. Nguyn l : DNA ngoi lai c cho vo mt trng nui cy vi s c mt ca DEAE-dextran. DEAE-dextran s kt hp vi DNA tch in m (Hnh 2.17). S d tha in tch dng trong phc hp DNA-polycation do s ng gp ca polycation, cho php phc hp ny i n kt hp cht ch hn vi mng t bo tch in m. S xm nhp ca phc hp c th t c nh s nhp bo (endocytosis).
Hnh 2.17: S kt hp gia DEAE-dextran v DNA [2] SVTH: Nguyn Kiu Oanh
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Dung hp t bo trn Trc tip dung ha t bo trn ca E.coli vo t bo ng vt c v. Electroporation (to l bng dng in): trnh by phn 2.1.2.2. Lipofection : Cc ti lipid mng (lyposomes) ni vi DNA tip xc vi t bo, s gn dnh xy ra v tip l s dung hp ca cc ti mang in tch dng vo cc t bo mang in tch m. S s dng cc lipid cation tng hp lm tng r rt hiu sut ca lipofection. Tuy nhin phng php ny ph thuc nhiu vo loi v thnh phn lipid s dng cng nh loi t bo nhn. Phng php vi tim y l phng php hiu qu nht a DNA ngoi bo vo cc b phn c th ca t bo. Thao tc c thc hin trn cc t bo phi (embryonal cell), l iu kin tin quyt cho vic sn xut cc ng vt chuyn gen.
2.2.2.2. Phng php chuyn gen nh vector virus V nguyn tc, bt k loi virus no cng c th c s dng lm vector chuyn vt liu di truyn vo trong t bo. Nhiu nhm trong s , cc papovavirus, adenovirus, retrovirus...c s dng vo nhng mc ch chuyn bit. Cc phn khc nhau ca genome virus c thay th bng gen cu trc quan tm. Virus c th c s dng ly nhim vo t bo giai on sm ca phi trc khi c chuyn ghp vo con m. Gen chuyn vi vector retrovirus xm nhp mt cch hiu qu vo h gen ca vt ch nhng virus s dng phi l virus an ton, khng gy bnh. Cc c th chuyn gen sinh ra t phng php ny l dng khm. Gen chuyn ch c th di truyn c nu retrovirus hp nht vo mt s t bo sinh dc
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Th h (F1) cn kim tra s biu hin ca gen chuyn. Khi gen chuyn hp nht trong cc t bo sinh dc th c gi l th khm dng mm. Sau chng c lai cng dng khong 10-20 th h cho n khi thu c cc ng vt chuyn gen ng hp t v gen chuyn c mt trong tt c mi t bo. giai on ny, phi mang gen chuyn c th c ng lnh v c bo qun cho cc qu trnh cy chuyn v sau.
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Chng 3: Tng cng folate trong cc loi cy thc phm
CHNG 3
TNG CNG FOLATE TRONG CC LOI CY THC PHM
M u Tetrahydrofolate (THF) v cc dn xut ca n (gi chung l folates) l nhng cofactor thit yu cho chu trnh chuyn ha mt carbon cn thit hnh thnh methionine, serine, purines, v thymidylate [31]. Trong khi thc vt v vi sinh vt c th tng hp folates, con ngi v ng vt khc thiu con ng tng hp folate hon chnh. V vy con ngi i hi mt ngun cung cp t thc phm. i vi con ngi, ngun cung cp ny ch yu l t thc vt. Cc loi rau xanh v cc loi ht h u giu folate, nhng cc loi thc phm ch yu nh: ng cc, c, qu li tng i ngho folate. Lng folate trong cc ba n tht s khng cc nc ngho v di mc ti u ngay c nhng nc giu [36]. Cc hu qu ca thiu ht folate bao gm khuyt tt bm sinh, thiu mu, v tng nguy c mc cc bnh v mch v mt s bnh ung th [22]. Do tm quan trng ca cht folate c bit l i vi ph n mang thai, nn cc chng trnh tng cng dng cht trong thc phm c trin khai nhiu nc [69]. Ch n ung thiu folate c th c ci thin bng cch thm acid folic tng hp vo thc phm ch yu (fortification), hoc bng cch ung vin acid folic (supplement), nhng cc gii php ny c chi ph thng xuyn cao v rt kh thc hin cc nc ang pht trin. Lng acid folic thm vo phi tng ng vi mc tiu th trung bnh t sn phm thc phm lm giu, c ngha l c 100 g thc phm lm giu, s tng ng vi 170 g ch n ung folate tng ng (DFE) bi v acid folic c cho rng c mc kh dng sinh hc nhiu hn folate t nhin 1.7 ln.
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Tuy nhin, tiu th qu nhiu axit folic (> 1mg/ngy) c th che giu tnh trng thiu vitamin B12, do vn lm giu thc phm vn cn l mt vn gy tranh ci trong Lin minh Chu u. cc nc th gii th ba, lm giu sinh hc (biofortification) cho cc loi cy trng ch yu phi hp vi thc hnh y t cng cng thng thng s gip ch trong vic t c cc ch n ung folate ngh, c bit l cc nc ang pht trin. Tng cng hm lng folate trong thc n bng k thut chuyn ha (metabolic engineering) l mt chin lc thay th y ha hn [22]. 3.1. Folate 3.1.1. Gii thiu Folate l mt trong nhng vitamin B c t nhin trong thc phm. Folate thuc nhm vitamin tan trong nc [16] Folic acid (vitamin B9) l dng folate trong thuc b vitamin (supplement) v c thm vo cc loi thc phm tng cng (fortification) [68].
Hnh 3.1: Cu trc ha hc ca folate [22]
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Phn t folate cha pteridine, p-ABA v glutamate. Cu trc folate trn (hnh 3.1) l dng monoglutamyl ca tetrahydrofolate (THF). Vng pteridine ca folate c th tn ti dng tetrahydro, dihydro hoc oxi ha hon ton [22]. 3.1.2. Cc dng ca folate Folate l mt coenzyme tham gia vo phn ng chuyn ha 1 nguyn t carbon [16]. Tetrahydrofolate v cc dn xut 5,10-methylenetetrahydrofolate; 5,10-
methenyltetrahydrofolate; 10-formyltetrahydrofolate v 5-methyltetrahydrofolate l nhng dng hot ng ca folic acid. Chc nng ch yu ca tetrahydrofolate l iu ha duy tr nng formaldehyde v formate trong c th trng thi cn bng va khng gy c lm hi t bo va sn sng tham gia qu trnh chuyn ha cn thit. Ngoi ra n cn l ngun cung cp nhm methyl CH3 [16]. Nhng ngun c nhiu folate l u , cy h u, thc phm cam qut, rau n l mu xanh m, bnh m v ng cc c tng cng, cc loi hnh v ht [3]. Bng 3.1: Nhng cy lng thc c folate Loi cy Go (ht trng, cha nu) La m (ht cng, trng, cha nu) Ng (ht vng, cha nu) C chua (qu) u Ha Lan (xanh, cha nu) Rau spinach (l, cha nu) u (hng, ht trng thnh, cha nu ) Hm lng folate (nmol/g phn n c) 0.13-0.18 0.84-0.95 0.42 0.2-0.64 1.45 4.31 10.28 [53]
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3.1.3. S thiu ht folate v sc khe Hin nay, FDA chp nhn folate nh mt cht dinh dng chc nng [3] Folate rt cn thit cho vic sn sinh ra t bo hng cu khe mnh [53]. Nu c th khng c folate, c th b thiu mu, cm thy mt mi hoc yu t, hoc khng th tp trung t tng. Ngoi ra, folate cn cn thit c th to ra cht di truyn (DNA) mi, chng hn nh trong thi gian thai nghn. Nu ph n d nh c thai, folate rt quan trng v folate gip nga mt s khuyt tt bm sinh [68], c th gim nguy c sinh con thot dch no, ty sng, qui thai [3, 54]. Kt qu ca vic thiu folate dn n cc ri lon nghim trng, bao gm c cc khuyt tt ng thn kinh (NTD) nh tt nt t sng tr s sinh v bnh thiu mu. Ch n ung y folate c th ngn nga s khi u ca nhng tnh trng ny [53]. Ngi ta cho rng cc ng thn kinh c hnh thnh gia ngy 21 v 27 sau khi th thai (trc khi hu ht ph n nhn ra h ang mang thai), nguy c NTD ch c th c gim thiu nu ph n c lng folate cao t giai on gn th thai n tun mang thai th 12. Nghin cu gn y cho thy rng t l NTD cc vng ngho nht khu vc n v Trung Quc c th gp 10 ln so vi phng Ty [22]. Tnh trng thiu folate cng lin quan vi s xut hin ca mt s ri lon thoi ha thn kinh (nh bnh Alzheimer), nguy c bnh tim mch cao hn, v s pht trin ca mt lot cc bnh ung thu, mc d khng c mi quan h nhn qu no c xc minh cho n ngy hm nay. Tiu dng ngun thc phm c hm lng folate cao rt quan trng trong thi k phn bo nhanh v tng trng, c bit trong giai on thai k. Thiu folate s gy ra hin tng thiu mt phn no (anencephaly) hoc hin tng nt t sng (spina bifida) hoc cc dy thn kinh khng ng li c tr em, v hin tng thiu mu hng cu to (megaloblastic anemia) ngi trng thnh. Nhiu nghin cu cho thy b sung thm folic acid s lm gim c ngha cc hin tng khim khuyt ng thn kinh, t qu (stroke) v mt vi bnh ung th [53]. SVTH: Nguyn Kiu Oanh
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3.1.4. Nhu cu Di y l nhu cu folate ngh c biu din di n v DFE (lng folate tng ng trong thc phm) Bng 3.2: Nhu cu folate ngh cho tr em v ngi ln tui 1-3 4-8 9-13 14-18 19+ Nam v N (g DFE/ngy) 150 200 300 400 400 Ph n c thai Ph n cho con b (g DFE/ngy) (g DFE/ngy) 600 500 600 500 [70]
C khc bit v mc hp th folate trong thc phm t nhin v folic acid trong thuc b hoc thm vo thc phm lm giu. 1 g DFE (1 g folate trong thc phm) tng ng vi 0.6 g folic acid t thc phm tng cng hoc thuc b ung trong ba n, v tng ng 0.5 g thuc b ung khi bng i [68]. Theo nh bng 3.2 th ngi ln cn 400 g lng folate tng ng trong thc phm (DFE) mi ngy. Ph n trong tui mang thai t 14 tui tr i, d c k hoch c thai hay khng, cn 400 g DFE mi ngy. Ph n c thai v cho con b sa m cn nhiu folate hn. Lng folate c ngh l 600 g DFE cho ph n c thai v 500 g DFE cho ph n cho con b sa m Ung hn 1 milligram mi ngy phi c ch nh ca bc s [70].
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3.2. C chua tng cng folate th h 1 [27] Cc nh khoa hc chn c chua iu khin con ng tng hp folate, bi v chng c tm quan trng trn ton th gii nh mt cy lng thc, v mt di truyn hc phn t c hiu bit r rng, v chng cha hm lng folate tng i thp [28]. Cc nh nghin cu ti i hc Florida tm ra mt cch tng hm lng cc vi cht dinh dng trong c chua s dng vic thay i bin dng. Nhm nghin cu ca Roco Daz de la Garza to ra mt loi c chua chuyn gen sn sinh ra hm lng cht folate tng ng vi na hm lng xut cn cho ngi ln trong mt khu phn n chun [27, 69]. 3.2.1. Nguyn l Folates l phn t gm ba phn bao gm pteridine, p-aminobenzoate (PABA), v mt hoc nhiu glutamate. thc vt, phn pteridine c sn xut trong t bo cht, PABA c hnh thnh trong lp th, c hai hp li vi nhau v c glutamyl ha trong ty th [27]. Trong qu c chua, cc enzyme u tin ca qu trnh tng hp pteridine, GTP cyclohydrolase I (GCHI), bin mt lc qu bt u chn, ct t ngun cung cp pteridine. Bi v c chua chn cha mt lng ln glutamate v mt lng trung bnh PABA [27, 28] Bc u tin hp l nng cao qu trnh tng hp folate l duy tr lin tc hot ng ca GCHI. Mt cch tim nng t c iu ny l s dng mt gen tng hp da trn GCHI ca ng vt c v v enzyme ny dng nh khng chu tc ng km hm ngc (retroinhibition) in planta. C 2 l do. Trc tin, s km hm ngc GCHI ca ng vt c v c qua trung gian bi mt protein iu ha phn hi, m thc vt khng xut hin [21]. Th hai, sn phm c ch cui ca GCHI ng vt c v l tetrahydrobiopterin, cht ny dng nh thiu thc vt [35]. Cc nh khoa hc a ra cc k thut thnh cng tc ng vo con ng folate c chua bng cch s dng mt gen GCHI tng hp.
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Hnh 3.2: Con ng sinh tng hp folate [22] Cc hp cht vit tt: ADC, aminodeoxychorismate; DHF, dihydrofolate; DHM, dihydromonapterin; DHN, dihydroneopterin; DHP, dihydropteroate; Glc, glucose ester; Glun, polyglutamate; HMDHP, hydroxymethyldihydropterin; P, phosphate; P2, diphosphate; P3, triphosphate; THF, tetrahydrofolate. Cc enzyme: 1, GTP cyclohydrolase I; 2, DHN-P3 pyrophosphatase; 3, non-specific phosphatase; 4, dihydroneopterin aldolase; 5, aminodeoxychorismate synthase; 6, aminodeoxychorismate lyase; 7, hydroxymethyldihydropterin pyrophosphokinase; 8, dihydropteroate synthase; 9, dihydrofolate synthase; 10, dihydrofolate reductase; 11, folylpolyglutamate synthase; 12, p-ABA glucosyltransferase. SVTH: Nguyn Kiu Oanh
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3.2.2. Vt liu v phng php 3.2.2.1. Cu trc vector biu hin Vector pMON10086 [34] cha promoter E8 ca c chua, terminator u Rubisco, v gen khng kanamycin npt II c iu chnh bng cch ct v tr NotI v ni mt polylinker (5-GGATCCGCGGCCGCGAATTCAGGCCTGGTACCGGCGCGCCAGATCT-3) vo v tr BamHI duy nht gia promoter E8 v terminator. cDNA GCHI ca ng vt c v (GenBank accession no. BE136861) c iu chnh bng cch s dng mi PCR thay i trnh t ca codon khi u sang trnh t tng ng ca thc vt TAAACAATG v thay th 17 codon him (Hnh 3.4). Nhng nucleotide c m ha li c in m v nghing. cDNA tng hp ny c chn vo gia vng NotI v AscI ca vector iu chnh v a vo chng Agrobacterium tumefaciens bng phng php in bin np.
Hnh 3.3: Cu trc vector pMON10086 [34] SVTH: Nguyn Kiu Oanh
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Hnh 3.4: Vng 5 ca cDNA GCHI tng hp [27]
Hnh 3.5: Nguyn l to cy c chua chuyn gen th h 1 [27] 3.2.2.2. Cy chuyn gen Cu trc trnh t GCHI v vector c iu chnh c dng bin i c chua (Lycopersicon esculentum Mill., cv. MicroTom). Th bin np c chn v ti to trn mi trng cha 100g.ml-1 kanamycin. Cy non khng kanamycin c sng lc tm ra cu trc GCHI bng phng php PCR vi mi mong mun nm trong promoter E8 (5-CTTTCTTGTTCCCATTTCTC-3) v mi o ngc (reverse primer) t vng m ha GCHI (5-ATGCACATGTGTGTCGCTTC-3); th bin np vector rng c kim tra li bng cch s dng mi cho gen npt II. Cy dng tnh vi cu trc GCHI c chuyn qua t v pht trin n trng thnh trong bung tng trng (16-h ngy; 23C; mt dng quang hp, 200 mol photon mi m2 mi s mi 8-h m; 20C) v ti vi dung dch dinh dng. Tri cy c thu hoch cc giai on nu trong vn bn. thng nht, cc giai on chn v c quy nh tng ng l 3 v 7 ngy sau giai on breaker. Trong SVTH: Nguyn Kiu Oanh
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th nghim b sung PABA vo, tri cy giai on breaker c ct ti gc ca cc cung, b sung PABA thng qua cung vi 2mol PABA trong 100 l nc hoc nc trong 1 ngy (i vi mu i chng), sau kh cung (destalked) v cho chn thm trong hn 6 ngy 23C. 3.2.2.3. Phn tch Western Blot bng khng th sn xut khng nguyn ti t hp, cc cDNA GCHI tng hp c nhn dng vo vng EcoRI v XhoI ca plasmid pET28b (Novagen), c thm cc on mu hexahistidine cho c hai u (termini). Cu trc ny c in bin np vo t bo Escherichia coli BL21 (DE3) CodonPlus-RIL (Stratagene), v protein ti t hp c phn lp t cc t bo cm ng isopropyl -D-thiogalactoside bng sc k i lc Ni2+ di iu kin bin tnh, tip theo l chun b SDS/PAGE. Khng th th c chun b bi Cocalico Biologicals (Reamstown, PA). hin th biu hin GCHI, protein c chit xut t m v qu bng cch nghin trong 0.1M Tris HCl (pH 8,0) c cha ascorbate 15 mM, DTT 2 mM, v Polyvinylpolypyrrolidone 3% (wt/vol). Sau khi c lm sch bng cch ly tm, cc mu (80 g protein) c tch trn gel SDS-polyacrylamide, sau c chuyn qua mng nitrocellulose, v kho st vi huyt thanh min dch pha long 1:2,000. 3.2.2.4. Phn tch pteridine v PABA. Nhng mi qu i din (0,70g) c nghin thnh bt trong N2 lng v ng nht vi 7 ml methanol. i vi phn tch pteridine, mt phn chia 600l ca cht ng nht (homogenate) c trn vi 250l ca CHCl3 v 50l nc, lc trong 40 pht. Sau thm 225l CHCl3 v 340l nc. Sau khi lc trong 20 pht v ly tm ph v nh tng, pha dung dch nc c ly ra, sy in vacuo, v ha tan li trong 200l nc. Mu c oxi ha bng cch thm 0,1 th tch ca mt dung dch gm I2 1% v KI 2% (wt/vol) trong HCl 1
90
M v trong bng ti trong 1h, I2 tha sau c ly ra bng cch thm 10l Naascorbate 5% (wt/vol). Cc mu b xi ha c tch bng HPLC vi ct Ultremex C18 IP (Phenomenex, Belmont, CA) 5 - m, 250 x 4,6-mm, hoc ct Synergi Fusion-RP 80 (Phenomenex) 4 m, 4,6 x 250-mm tch ra vi Na-phosphate 10 mM (pH 6,0) 1,5 ml/min. Cc peak c pht hin bng mu d hunh quang Waters 2.475 (350-450 nm) v xc nh c bng cch tham chiu n cc thang chun v tnh cht quang ph. kim tra cc trng thi xi ha pteridine, 2-mercaptoethanol 10 mM v Naascorbate 2% (wt /vol) c thm vo mi trng dch chit methanol, v bc oxi ha c b qua. nh pteridine lin hp c thu hi t phase ng nh phng php trao i ion v c x l bng HCl (1M, 100C, 1 h) hoc vi -glucosidase nm men hoc almond -glucosidase (2 n v trong 40 l dung dch Na-phosphate 10mM, pH 6,0, 37C, 5-120 pht). Cc nh ca 6-carboxypterin, neopterin, monapterin, v 6-hydroxymethylpterin c nh lng tng i so vi thang chun pteridines; nh lin hp c nh lng tng i tng ng vi pteridine t do v qu trnh thy phn khng lm thay i nng sut hunh quang. Thang chun Pteridine t phng th nghim Schircks (Jona, Thy S). Mt phn 5-ml dch chit methanol trn c s dng cho phn tch PABA tng (v d nh, PABA t do cng vi PABA glucose ester) bng thy phn acid, sc k trao i cation, s phn ct ethyl acetate, v HPLC hunh quang. 3.2.2.5. Phn tch Folate Folates c chit xut t nhng phn qu i din (0,5-1,0 g) bng cch ng nht polytron trong 10 ml Na-Hepes 50 mM / 2 (N-cyclohexylamino) ethanesulfonic acid 50 mM iu chnh n pH 7,9 vi HCl c cha CaCl2 1 mM, Na-ascorbate 2% (Wt /vol), v 2-mercaptoethanol 10 mM. Sau un si trong 10 pht v ly tm (13.000 x g) trong 10 pht. Cc pellet sau c chit li cng mt cch nh vy.
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Cc dch chit kt hp c x l bng 1ml huyt thanh chut thm tch 37C trong 2 h kh glutamyl ha folate. Mu sau un si trong 15 pht, ly tm nh trn, lc qua si thu tinh, v t vo ct i lc folate. Sau khi c ra sch vi 5 ml K-phosphate 25mM (pH 7,0)/ Naascorbate 1% (m 1) c cha NaCl 1M, sau vi 5 ml ch dung dch m 1, cc ct c tch ra vi 5 ml HPLC phase ng A (xem bn di) c cha acid ascorbic 1%. Mu tch ra (400 l) c a phn tch HPLC vi pht hin in ha [20], s dng mt ct Prodigy ODS2 (Phenomenex) 5 m, 150 x 3,2 mm v mt my d bn-knh (CoulArray Model 5600A, ESA, Chelmsford, MA) vi in th t 0, 300, 500, v 600 mV. Phase ng l mt hn hp 2 cu t ca K2HPO4 28 mM v H3PO4 0,59 mM (pH 2,5) (m A) v hn hp ca m A 75% (vol/vol) v CH3CN 25% (m B) vi mt chng trnh tch ra phi tuyn trong 55-pht t 90% m A n 100% m B mc 1 ml/min. Kt qu my d c hiu chnh bng cch s dng THF, 5-methyl-THF, 5,10 methenyl-THF, 5-formyl-THF, v acid folic chun t Schircks. phn tch ca chiu di ui polyglutamyl [20], qu trnh x l huyt thanh chut c b qua. Chit xut folate t hng cu ngi c s dng xc nh thi gian duy tr ca 5 methyl-THF polyglutamates [45]. 3.2.3. Kt qu 3.2.3.1. Biu hin vt mc GCHI trong qu chn lm tng mc Pteridine Mt gen GCHI tng hp c xy dng bng cch m ha li mt phn cDNA GCHI ng vt c v (Hnh 3.4) v bng cch iu chnh cc trnh t xung quanh codon u tin ph hp vi s ng nht thc vt [37]. Gen tng hp ny c t sau E8 promoter chn c hiu [26] trong vector kp Agrobacterium v c a vo cy c chua. Kim tra bng phn tch Western xc nh th bin np biu hin protein c kch thc ng 27kDa trong kiu chn c hiu.
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12 th bin np GCHI+ nh th, to thnh 1 dy cc mc biu hin protein, c chn cho nhng phn tch xa hn vi 10 vector i chng rng. Cc cy GCHI+ v tri ca chng khng thy c khc bit so vi nhng cy i chng. Pteridines c phn tch bi HPLC hunh quang sau khi chuyn i sang dng oxi ha y , dng hunh quang. Kt qu pteridine ca qu GCHI+ khc rt nhiu so vi pteridine ca nhng vector i chng (Hnh 3.6). o giai on chn , lng pteridine tng ca qu GCHI+ cao hn 3 n 140 ln hn mc trung bnh ca nhng mu i chng. Theo di pteridines trong qu trnh chn cho thy qu GCHI+ bt u khc so vi nhng mu i chng sau giai on chn xanh v hm lng pteridine ca chng cao nht giai on chn (Hnh 3.7). Mu tch ly pteridine ph hp vi cc hot ng d kin ca promoter E8 c s dng iu khin biu hin GCHI [26].
Pht Hnh 3.6: Phn tch HPLC hunh quang nhng pteridine oxi ha trong qu chn (Ct Ultramex C18 IP) [27] M102: Th bin np GCHI+ i din; V6: Vector i chng CPt: 6-carboxypterin; NPt: neopterin; MPt: monapterin; U1: unknown 1; U2: unknown 2; HMPt: 6-hydroxymethylpterin.
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Pteridien (nmol/g trng lng ti)
Hnh 3.7: S thay i trong lng pteridine tng ca qu i chng (V2) v qu GCHI+ trong sut qu trnh chn [27] MG: giai on chn mu xanh l cy (mature green stage); Br: giai on breaker (breaker stage); R: giai on qu mu (red stage); RR: giai on chn (red-ripe stage)
Hnh 3.8 : Cc giai on chn ca c chua [27]
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3.2.3.2. S hnh thnh Pteridine Loi pteridine chnh tm ra trong tri cy GCHI+ bao gm neopterin, monapterin, 6hydroxymethylpterin, dng oxi ha ca cc cht trung gian tng hp folate, v 6carboxypterin, mt cht d ha ca pteridines v folates (Hnh 3.6). S nhn dng ca 6-hydroxymethylpterin c xc nhn bng cch s dng HPLC MS kim tra khi lng ca n ([M+H]+= m/z 194) v m hnh phn mnh lin quan n mt tiu chun ng tin cy. Qu GCHI+ cng cho thy nh 7,5 v 9,5 pht khng ph hp bt k cc tiu chun no (Hnh 3.6). Acid thy phn chuyn ha nhng cht khng xc nh ny (unknowns) v dng neopterin v monapterin, tng ng, cng nh x l vi almond -glucosidase nhng khng phi -glucosidase nm men (Hnh 3.10). Kt qu ny ch ra rng c hai nh cha bit c chnh l -D-glycoside. Cc t l tng i ca nhiu pteridines tng t nhau trong tt c cc qu GCHI+, bt k mc tuyt i (Hnh 3.9). Qu i chng c nh nh vi cng s ln retention ging nh hu ht cc pteridines trong qu GCHI+ (d liu khng c hin th) nhng c qu t vt liu xc nh quang ph.
Pteridien (nmol/g trng lng ti)
Hnh 3.9: nh lng cc pteridine chnh trong qu chn ca 3 th bin np GCHI+ i din [27] CPt: 6-carboxypterin; NPt: neopterin; Mpt: monapterin; NPt-G: neopterin glycoside; MPt-G: monapterin glycoside; HMPt: 6-hydroxymethylpterin SVTH: Nguyn Kiu Oanh
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Peak 1 cha bit i chng
Peak 2 cha bit i chng
Pht
Pht
Hnh 3.10: Phn tch HPLC hunh quang peak 1 v 2 cha bit trc v sau khi x l vi HCl, -glucosidase hoc -glucosidase [27]
Cc trng thi xi ha in vivo ca pteridines trong cc qu GCHI+ i din c nghin cu bng cch so snh cc mu chit xut vi s hin din ca mercaptoethanol-2 v ascorbate ( duy tr trng thi oxi ha t nhin) v khng c x l oxy ha, vi cc mu tng t c chit xut v xi ha nh bnh thng.
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V pteridines dng kh khng pht hunh quang, s khc bit trong kch thc gia cc nh gia x l oxy ha v khng oxy ha c o bng pteridines dng kh. Phng php ny ch ra rng vn pteridine b kh c trng 50-90% giai on breaker v giai on mu , nhng 20% b kh giai on chn . Cc pteridines dng kh xut hin l cc dng dihydro, khng phi tetrahydro, bi v x l alkalinke I2 khng sinh ra bt c pterin no, mt sn phm theo gi nh c phn ra t tetrahydro pteridines trong cc iu kin ny [30]. 3.2.3.3. Tch ly pteridine lm tng hm lng Folate. Hm lng folate tng ca qu i chng gim trong khong t 0.8 n 2.3 nmol/g trng lng ti (FW), trong khi hu ht tt c cc qu GCHI+ vt qu khong ny (Hnh 3.11) v ngha ca phn b GCHI+ gp i nhng mu i chng (2.99 so vi 1.47nmol/g FW; khc bit ngha mc P < 0.001 bng t test). S tng folate c tch ly ch yu bi 5-methyl-THF v 5,10-methenyl-THF, c 2 u l dng ch yu trong qu i chng ch cha vector (hnh 3.12).
Folate (nmol/g trng lng ti)
Hnh 3.11: Hm lng folate tng trong qu chn ca 12 th bin np GCHI+ v 10 th bin np i chng [27]
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Hnh 3.12: Phn tch folate trong qu GCHI+ v qu i chng [27] (Phase ng c tnh axt c s dng trong phn tch HPLC gy ra chuyn i nh lng ca 10-formyl-THF sang 5,10-methenyl-THF, cho nn cc nh 5,10 -methenylTHF bao gm c 10-formyl-THF v 5,10-methenyl-THF bt k c th tn ti t trc) Phn tch cc dng polyglutamyl ca 5-methyl-THF cho thy mt phn b tng t trong GCHI+ v qu i chng, vi hexaglutamate chi phi trong c hai loi qu ny. Cc dng folate khc cng tn ti ch yu nh polyglutamates trong c GCHI+ v qu i chng (d liu khng c hin th). Kt hp vi nhau, nhng d liu ny cho thy folate tng ln trong qu GCHI+ tt c u dng bnh thng. Vng phn b ca mc pteridine tng so vi hm lng folate cho thy mc folate ti a t c vi mc pteridien tng s 25nmol mi g FW v khi tng pteridine nhiu hn khng mang li li ch g. Trong thc t, phn tch hi quy ch ra s tng quan tiu cc ng k gia hm lng folate v mc pteridine cho tri cy vi >25 nmol pteridine mi g FW. Tng quan tiu cc tng t cng c tm thy gia folate v mi pteridine ring l ngoi tr hydroxylmethylpterin. Xu hng tiu cc nh vy s lm tng kh nng nu pteridines SVTH: Nguyn Kiu Oanh
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vt qu gii hn, vt ra ngoi s khng cho hm lng folate cao, m thc s cn c th ngn cn hnh thnh folate. 3.2.3.4. S tch ly folate c lin quan n s suy gim ca PABA Bi v vn PABA tng trong c chua khng c tc ng l khong cng ln (1-2 nmol/g FW) [46] vi s tng folate trong qu GCHI+, cc nh khoa hc kim tra mi quan h gia mc folate v PABA. Mt mi quan h tiu cc r rng c chng minh, v qu GCHI+ c hm lng folate cao nht c rt t PABA. Hn na, c th tnh ton c t d kin th nghim rng trong 5 qu cha nhiu folate nht, 90-97% phn PABA hin din trong qu dng lin kt cht ch vi folate. 3.2.3.5. PABA ngoi sinh tng s tch ly folate kim tra s suy gim ca PABA c gii hn s tch ly folate hay khng, qu GCHI+ c thu hoch giai on breaker, cung cp PABA qua cung (hoc nc vi mu i chng), v cho chn. M qu sau c phn tch kim tra PABA v folate. Gia cc qu t 7 th bin np khc nhau, nhng qu c cho PABA vo c mc PABA cao hn nhng qu i chng. (5726 so vi 1.80.5 nmol mi g FW) v c hm lng folate cao hn t 2.5 n 10 ln (hnh 3.13). Cho PABA vo qu cha vector rng khng lm tng ng k mc folate (d liu khng c hin th). Mc folate trong mu i chng cho nc vo qu GCHI+ (hnh 3.13), chn ngoi cy, th tng t qu chn trn cy (hnh 3.11).
Nc PABA
Hnh 3.13: nh hng ca PABA ngoi sinh n hm lng folate [27] SVTH: Nguyn Kiu Oanh
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3.2.4. Bn lun t mt enzyme khng cm ng ngc (feedback-insensitive) u con ng tng hp l k thut c bn gia tng dng chuyn ha, v nh kt qu c trnh by, n lm vic trn nhnh pteridine ca qu trnh tng hp folate trn qu c chua. GCHI ng vt c v c chuyn vo c d kin s khng chu km hm ngc in planta v th hin hiu qu cao trong vic tng dng pteridines, c th t n mc 140 ln gi tr trung bnh trong qu i chng. Vi mt k thut c bn tng t, nhng s dng GCHI vi khun, cng t c thnh cng trong gia tng hm lng pteridine trong l Arabidopsis [33]. Nhng cht trung gian tng hp folate dihydroneopterin, dihydromonapterin, v hydroxymethyldihydropterin nm gia pteridines c tch ly trong c chua GCHI+, cho thy rng ba enzyme trc tip xui dng ca GCHI tt c vn hot ng trong qu trnh chn ca tri cy v c th lm dng tng ln. Acid-khng bn cng gc ca neopterin v monapterin cng trong s cc pteridines tm thy trong qu GCHI+ v, ging nh cc pteridines khc, xut hin rng ri dng dihydro in vivo. Da trn tnh nhy cm ca chng thy phn bi almond -glucosidase, chng c xc nh l -D-glycosides. Mc d glycoside ca pteridines nhiu loi, bao gm c neopterin v monapterin, c bit n t vi khun lam v cc sinh vt nhn s khc, dng nh khng c bo co trc v s xut hin ca chng trong thc vt. Bi v qu c vector i chng cho thy nhng nh nh vi s ln duy tr tng t nh glycosides, nhng hp cht ny c th c vai tr nh l mt vn d tr t nhin cho tin cht ca folate. Nu nh vy, mt s tng ng c th c suy ra vi tin cht PABA, m tn ti ch yu in vivo nh glucose lin hp. So vi lng d tha ln ca pteridines trong qu GCHI+, s gia tng folate l khim tn (2 ln trong hu ht qu GCHI+, v gn bng 3 ln trong trng hp cao nht)
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Do , r rng c mt s kim ch v dng trong con ng tng hp folate trong qu c chua mt vi im h ngun t hydroxymethyldihydropterin (Hnh 3.2). Cc kim hm tng t p dng cho cc con ng tng hp folate trong l Arabidopsis, bi v gn 1.000 ln tng pteridines ch tng 3 ln folates [33]. Mt ng c vin mnh m cho s kim hm ny l vic cung cp PABA (hnh 3.2). Cc mi tng quan ngc gia PABA v mc folate, nhng s suy gim nghim trng ca PABA trong qu GCHI+ vi hm lng folate cao, v s gia tng folate trong khi PABA c cung cp (hnh 3.13) tt c ch ra rng PABA tr thnh gii hn thng lng folates tng trong qu chn. Cch l gii ny tip tc c ng h bng vic tm thy biu hin ca gen u tin ca qu trnh tng hp PABA, aminodeoxychorismate synthase, chm dt t ngt sau khi giai on breaker. Mc tiu k thut tip theo l tng cng sinh tng hp ca PABA trong qu c chua c thit k sn xut vt qu hm lng pteridines. Tht th v, vic cung cp PABA cng hn ch tng hp folate trong vi khun lactic acid. S tch ly qu nhiu cc cht trung gian din ra khi qu trnh km hm ngc c hy b bng k thut, i khi c th phn tc dng. c mt s bng chng v iu ny trong nghin cu ca chng ti, bi v trong qu giu pteridine nht, mc folate vn c tng quan ngc vi mc pteridine tng v vi hu ht cc mc pteridine ring l. C th hnh dung l pteridine tch ly c vai tr nh l cht nh hng tiu cc n con ng folate, v d nh enzyme km hm cnh tranh hoc cht vn chuyn. D trong trng hp no i na, vic tch t qu nhiu pteridine r rng khng cn thit tng cng mc folate. Hn na, mc pteridine bn ngoi phm vi t nhin cho cy l khng mong mun t quan im dinh dng v nh hng ca lng pteridine cao l cha bit. So snh vi phm vi t nhin ca mc pteridine bo co trong cy trng thc phm, mc pteridine tng trong qu GCHI+ cao hn 10 ln, v mc trong l Arabidopsis qua k thut l nhiu hn gp 1,000 ln.
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Qu GCHI+ chuyn gen l mt bc quan trng u tin hng ti mt sn phm lm giu sinh hc hu hiu, bi v mc folate cao nht t c cho n nay (4nmol mi g FW) l tng ng vi 180g mi 100g tiu chun. Mc ny s cung cp cho ton b ch n ung ngh i vi mt a tr v gn mt na cho ngi ln. Tuy nhin, cc mc folate cao c quan st trong qu GCHI+ chn trn cy, v qu tng ng chn sau khi ct cung giai on breaker cha t folate hn. nh hng tiu cc ny ca s ct cung c th quy cho mng li ln folate gim xung trong qu ct cung [21] v c th gii thch v sao hm lng folate ca c chua tr qu thp, bi v chng chn sau khi thu hoch. Trong mi trng hp, s mt mt folate sau thu hoch cho thy rng s quan tm k thut phi c t ln trn s gim ht folate cng nh s tng hp folate. 3.3. C chua tng cng folate th h 2 [28] 3.3.1. Vt liu v phng php 3.3.1.1. Cu trc vector biu hin Trong th nghim ny cc nh khoa hc s dng vector pMON10086 cha promoter E8 c chua, trnh t m ha AtADCS, At2g28880. Trnh t tng ng ca thc vt TAAACA c thm vo trc codon bt u. cDNA tng hp ny c chn vo gia vng NotI v AscI ca vector iu chnh v a vo chng Agrobacterium tumefaciens bng phng php in bin np [28]. 3.3.1.2. Cy c chua chuyn gen C chua chuyn gen bi A. tumefaciens cha cu trc AtADCS, s c chn lc v ti to tng t nh c chua chuyn gen th h 1.
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Hnh 3.14: Nguyn l to cy c chua chuyn gen th h 2 [28] 3.3.1.3. Lai cc cy chuyn gen th nghim ny ngi ta tin hnh lai cc cy chuyn gen GCHI+ v AtADCS+ vi nhau. Qu ca cc dng AtADCS+ v GCHI+/AtADCS+ s c thu hoch giai on chn , bo qun trong N2 lng, tr -80oC cho n khi c em ra phn tch. 3.3.2. Kt qu Qu chuyn gen AtADCS+ cha mc trung bnh gp 19 ln lng PABA, so vi mu i chng thuc chng hoang di, khng c s gia tng hm lng folate. Qu chuyn gen GCHI+/AtADCS+ tch ly lng folate trung bnh gp 19 ln mu i chng, mc folate t c (840 g mi 100g phn n c) c th cung cp nhu cu folate hng ngy ca mt ngi trng thnh.
103
Hnh 3.15: Hm lng folate, PABA, v pteridine tch ly trong cc dng AtADCS+, GCHI+ v GCHI+/AtADCS+ [28]
104
3.4. Go tng cng folate [53] Mc d go l loi cy lng thc chnh, cung cp 80% lng calo hng ngy cho 3 t ngi, n li ngho ngun vi cht dinh dng thit yu, bao gm folates (vitamin B9) (xem bng 3.1). Do , thiu folate rt ph bin, c bit l cc nc ang pht trin. Cc nh khoa hc tng cng mc folate trong go bng cch biu hin vt mc gen A. thaliana m ha cho GTPCHI v ADCS, t mt locus T-DNA n. Gen km hm ngc GTPCHI ca cy c la chn trnh s tch ly qu cao khng mong mun ca cht trung gian. 3.4.1. Vt liu v phng php so snh hiu qu ca k thut tng hp sinh hc folate t 2 nhnh, ba vector bin i gen c xy dng (Hnh 3.16). Cu trc G cha mt cDNA m ha A. thaliana GTPCHI, di s kim sot ca cc promoter globulin (glb-1) c hiu ca ni nh go. Cu trc A cha cDNA m ha A. thaliana ACDS di s kim sot ca promoter glutelin B1 (gluB1) c hiu ca ni nh go. Cu trc GA xy dng kt hp c hai caset biu hin G v A trn mt T-DNA n.
Cu trc G
Cu trc A
Cu trc GA
Hnh 3.16: Gin i din ca T-DNA trong cc vector dng chuyn gen [53] SVTH: Nguyn Kiu Oanh
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Thanh m miu t cc promoter, mi tn en miu t vng cDNA m ha, thanh mu xm cho bit terminators phin m. LB v RB, cnh T-DNA tri v phi, T35S v Tn, terminators phin m ca bn sao 35S ca cauliflower mosaic virus (virus gy bnh TV) v gen nopaline synthase ca Agrobacterium tumefaciens( l vi khun c dng hnh cy gy do chen 1 phn on nh ca DNA gi l T-DNA), 35S, Glb-1 v GluB1, phn li cauliflower mosaic virus 35S promoter vi trnh t tng cng nhn i, promoter globulin la v promoter glutelin B1 la, GTPCHI, m ha trnh t A. thaliana GTP cyclohydrolase I; ADCS, m ha trnh t ca ADC synthase t A. thaliana; hptII, gen m ha hygromycin phosphotransferase Tt c cc vect c chuyn vo trong b gen ca la Nippon Bare japonica bng vi khun Agrobacterium tumefaciens. Cy chuyn gen c kiu hnh khng th phn bit c vi chng hoang di (WT) trong sut qu trnh pht trin v cng c kh nng to ht tng t. 3.4.2. Kt qu 3.4.2.1. Phn tch dng chuyn gen A Mc PABA tng trung bnh gp 49 ln so vi cy i chng (15,3 7,6 so vi 0,32 0,03 nmol / g, tng ng) (hnh 3.17), th hin s nng cao hiu qu ca nhnh PABA trong qu trnh sinh tng hp folate.
106
(nmol/g trng lng ti)
Hnh 3.17: Mc PABA v folate tng trong nhng dng A [53] Tuy nhin, hm lng folate tng trong tt c cc dng A c phn tch b hn 6 ln so vi cc ht WT hoc ht ca cc cy i chng c chuyn gen nhng cha vector rng (hnh 3.17), bc l mt s km hm cho n nay cha c pht hin ln qu trnh sinh tng hp folate bi nng PABA cao trong khi khng c mt mc dihyroneopterin cao. Mc PABA trong c chua chuyn gen cng cao (ln n 140 nmol/g) biu hin vt mc ADCS A. thaliana, khng c s gim ng thi mc folate [28], c th phn nh s khc bit ch yu trong vic iu ha sinh tng hp ca folate trong c chua v go. 3.4.2.2. Phn tch dng chuyn gen G Nhng ht bin i gen dng G khng cho thy mt s khc bit ng k trong hm lng folate so vi nhng cy i chng. Tuy nhin, phn tch nhng cht trung gian sinh tng hp pterin v dng oxy ha ca chng (hydroxymethyldihydropterin (HMDHP), dihydroneopterin (DHN), hydroxymethylpterin (HMP) v neopterin) cho thy lng tch ly pterin trung bnh cao
107
gp 25 ln. Pterin phong ph nht l neopterin (69,9% 12,6%), tip theo HMP (25,9% 11,6%) v DHN (5,4% 1,8%), trong khi HMDHP khng c pht hin (Hnh 3.18).
Hnh 3.18: Mc pterin v folate tng trong nhng dng G [53] 3.4.2.3. Phn tch dng chuyn gen GA
Hnh 3.19: Mc PABA, pterin v folate tng trong nhng dng GA [53]
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Ngc li vi 2 dng A v G, ht ca cc dng GA chuyn gen th hin s tch ly lng folate ln, t 6,0 n 38,3 nmol / g, cao hn 15-100 ln so vi WT hoc trong cc cy chuyn gen vi vector rng (Hnh 3.19). ng ch , tt c cc dng GA tch ly c lng PABA tng i cao, mc d thc t l lng tha PABA c lin quan trc tip n vic sinh tng hp folate. Tuy nhin, s lng PABA trung bnh thp hn 2 ln so vi dng A (7,8 2,8 v 15,3 7,6 nmol / g, tng ng) v thp hn 4,5-10 ln trong c chua c tng cng folate [28]. S gia tng gii hn ca lng PABA trong go tng cng khng nn quan tm nhiu v PABA c lit k trong hp cht GRAS (thng c coi l an ton) [71] vi mt lng gii hn 30 mg / ngy. Lng ny tng ng vi 17 kg ht ca dng GA ng hp t 17.8 vi mc PABA cao nht (12,8 2,7 nmol / g). Mc pterins trung bnh trong nhng ht ca dng GA (0,23 0,12 nmol / g) thp hn 5,5 ln so vi dng G (1,23 0,54 nmol / g), mc d vn cn cao hn 4 ln so vi ht i chng. Tuy nhin, cc mc ny vn thp hn 80 ln so vi c chua c tng cng folate [28] v cng mc so vi qu c chua WT, do v tnh an ton ca go tng cng sinh hc, v hm lng pterin vt mc, c th gy hu qu i vi sc khe con ngi, do vai tr quan trng ca pterins trong sinh l con ngi [44]. Ht la tng cng folate cha PABA v pterin t hn nhiu so vi c chua tng cng folate [28]. T l mol ca folates / PABA / pterins trong c chua tng cng folate cao xp x 1/2.5/0.75 [28], so vi 1/0.5/0.013 go tng cng. Cc phn folate chnh trong thc vt chuyn gen GA c i din bi 5methyltetrahydrofolate, m trung bnh chim n 89% ca tng lng folate (Hnh 3.20). iu ny lm cho go tng cng sinh hc vt tri hn so vi go tng cng cht acid folic, m l bt buc mt s nc. Tiu th lng folic acid t tng cng cng nghip cao c th che y cc triu chng thiu ht vitamin B12, nhng iu ny khng phi l vn khi s dng 5-methyltetrahydrofolate nh mt cht b sung. V vy, go tng
109
cng folate l mt thay th tuyt vi cho cc khu vc, ni tng cng acid folic cng nghip l khng th.
H4PteGlu: tetrahydrofolate; 5-CH3-H4PteGlu:5methyltetrahydrofolate; PteGlu: folic acid; 5-CHO-H4PteGlu: 5formyltetrahydrofolate; 10-CHO-PteGlu: 10-formylfolic acid; 5,10-CH=H4PteGlu: 5,10methenyltetrahydrofolate
Hnh 3.20: So snh thnh phn nhng loi folate chnh trong dng GA so vi dng WT [53] Hn na, bi v polyglutamylation c bit nh l cht nh hng tiu cc n sinh hc kh dng folate, chng ti xc nh t l phn trm ca polyglutamylated folates trong lng folate tng ca 2 dng chuyn gen so vi WT. Khong 2,6-14% folates l polyglutamated trong dng chuyn gen so vi 50% trong WT (Hnh 3.21), cho thy kh dng sinh hc ca folates trong cc dng tng cng cao hn.
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Hnh 3.21: T l tng i ca polyglutamate v monoglutamate folate ca dng GA v WT [53]. Mc ti a ca folate tng cng c trong ht go, c th l 38,3 nmol / g, cao hn trong c chua tng cng folate (25 nmol / g) [28] v tng ng vi 1.723 g/100 g khi lng ti, l lng folate cao nht c bo co trong cc loi thc vt cho n nay. N vt qu ngng folate ngh hng ngy cho ngi ln (400 g) nhiu hn gp 4 ln v cng trn 2,5 ln lng folate cho ph n mang thai (600 g) [70]. Th nghim nu chng minh, sau 30 pht c un si th 45% folate tht thot (Hnh 3.22). Gi s rng mc kh dng sinh hc trung bnh ca folate t nhin trong mt ch n ung hn hp khong 50%, 100 g cc ht go tng cng folate c kh nng p ng yu cu folate hng ngy cho mt ngi trng thnh hoc t nht l cung cp hu ht yu cu hng ngy.
111
Cha nu nu (nmol/ g trng lng ti)
Hnh 3.22: S thay i mc folate tng trong ht ca dng GA khi nu [53] 3.5. Nhng thnh tu v hng pht trin 3.5.1. Thnh tu 3.5.1.1. Biu hin vt mc GTP cyclohydrolase I (GTPCHI) Nhng nghin cu u tin trn qu c chua v Arabidopsis thaliana [27, 33] nhm biu hin vt mc enzyme u tin trong nhnh pteridine ca con ng folate, enzyme GTP cyclohydrolase I (GTPCH I, Hnh 3.2). Trong c 2 trng hp u s dng gen khng c ngun gc t thc vt (gen c ngun gc t ng vt hu nh v vi khun (GenBank accessions BE136861 v AE000304). Tuy nhin, hm lng folate ch tng ln 2 n 4 ln, cho thy s cn thit ca nhng k thut xa hn na ca con ng ny.
112
Phn tch PABA tng (PABA cng vi glucose ester ca n) trong qu c chua chuyn gen cho thy s suy gim rt ln ca PABA. Cung cp PABA ngoi sinh cho vic biu hin vt mc GTPCHI c chua chuyn gen lm tng hm lng folate nhiu hn t 2.5 n 10 ln. iu ny ch ra rng khng nhng cn thit phi tng ng thi c 2 tin cht ca folate (pterin v PABA) m cn phi chng t tim nng c tht ca vic tng nng folate trong t bo thc vt. 3.5.1.2. Kt hp biu hin vt mc GTPCHI vi aminodeoxychorismate synthase (ADCS) Nhng pht hin y ha hn ny thc y mt vng khc trong qu c chua, trong , enzyme u tin ca nhnh p-ABA ca con ng folate, aminodeoxychorismate synthase (ADCS, hnh 3.2) s c biu hin vt mc, s dng gen t Arabidopsis (At2g28880; GenBank NP_850127) [28]. Kt qu l qu chuyn gen cha mc trung bnh gp 19 ln lng p-ABA, so vi mu i chng thuc chng hoang di, khng c s gia tng hm lng folate. Khi c im ny c kt hp vi lng pteridine cng c biu hin vt mc bng cch lai, qu chuyn i gen 2 ln tch ly lng folate trung bnh gp 19 ln mu i chng, mc folate t c (840 g mi 100g phn n c) c th cung cp nhu cu folate hng ngy ca mt ngi trng thnh. Tuy nhin, k thut ny cng dn n s tch ly pteridines v p-ABA gp 20 ln so vi chng hoang di i chng. Mc d mc p-ABA trong c chua chuyn gen v hi vi sc khe con ngi, trng thi ca pteridines vn cha c lm r v cn c kho st [28]. Con ngi v ng vt tng hp pterin quan trng, tetrahydrobiopterin (H4B), tham gia vo qu trnh tng hp nitric oxide v cc cht dn truyn thn kinh, chng hn nh dopamine [41]. Ngoi ra, dihydroneopterin v dng xi ha ca n, neopterin, cng c l cht nh du c bit n trong s kch thch h min dch v c s dng rng ri trong chn on nhiu bnh [41]. Chng cng tham gia vo phn ng stress [41]. Do tnh trng xo trn ca pteridine c th c nh hng n sc khe con ngi, tuy nhin
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vai tr v tng tc ca tng hp ni sinh vi ch n ung pteridines trong qu trnh chuyn ha ng vt c v vn cha r rng. Trong phng php to ging la c lm giu folate, gene Arabidopsis m ha GTPCHI (GenBank AF489530) v ADCS c biu hin vt mc di s kim sot ca promoter mnh trn mt locus gene duy nht. Ht go chuyn gen biu hin vt mc c 2 gen ca Arabidopsis cha hm lng folate cao gp 100 ln so vi chng hoang di (1723 so vi 17g/100g khi lng ti). Th nghim nu chn cng chng minh rng 100g go chuyn gen c th cung cp nhu cu folate hng ngy ca mt ngi trng thnh hay t nht lng cung cp nhiu nht ca n [53]. Hn na, mc ca nhng cht trung gian c tng hp, pterin v p-ABA, thp hn ng k so vi qu c chua c lm giu folate. 3.5.2. Hng pht trin 3.5.2.1. Li ch trc tip ca vic lm giu sinh hc S dng mt cy lng thc chnh c lm giu folate s gip chin u vi s thiu ht folate. iu ny mang li hai li ch ngay lp tc. Trc tin, vic lm giu sinh hc cung cp s bn vng khi so snh vi lm giu cng nghip (b sung acid folic tng hp cho cc loi thc phm c ngun gc t ng cc) v dc phm b sung. Vic pht trin ca mt loi cy trng lm giu sinh hc trn quy m ln u t mt ln m c th mang li li ch cho sc khe ca hng triu ngi v do c th to ra mt hiu ng cp s nhn [25]. Th hai, cy trng lm giu sinh hc c th ci thin lng folate cho mt b phn dn s nng thn suy dinh dng khng c kh nng hng li t thc phm tng cng cng nghip hoc cc loi thc phm b sung, thng ch c sn trong thnh ph. V vy, tng cng sinh hc l b sung cho cc k hoch can thip khc.
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3.5.2.2. S thch nghi theo vng m bo nng dn c th thng qua thc phm tng cng sinh hc, iu quan trng l nng sut cy trng v/ hoc li nhun phi ng thi tng. Vic ny c th t c bng cch chuyn c im tng cng sinh hc vo kiu gen cho nng sut cao. V vy, s c nhng dng tng cng sinh hc a vo chng trnh gy ging kt hp vi cc ging a phng thch nghi vi mt h sinh thi nng nghip. iu ny r rng s i hi mt mc nht nh ca nng lc nghin cu quc gia. Tuy nhin, trong khu vc nh ng Nam , cc h thng hiu qu cho vic ph bin ging cy trng ci thin c ti ch, lm cho vic thc hin ch tn chi ph ti thiu. 3.5.2.3. S sinh li ca vic tng cng sinh hc Vic tng cng sinh hc d kin s sinh li da trn cc nghin cu nh lng li ch sc khe tim nng cho vitamin A, st v km c trong cy trng tng cng s dng phng php tip cn DALY [42]. M hnh ny tnh ton vic gim gnh nng bnh tt hin ti lin kt vi tnh trng thiu vitamin t tng cng sinh hc m ra, c tnh n lng tiu th hin ti cc cy trng lng thc, vic tiu th d kin ca cc vi cht dinh dng sau khi tng cng sinh hc cho cy trng, v t l dn s d kin s tiu th cy trng tng cng sinh hc. p dng phng php DALY cho tc ng ca go vng 2 (mt phin bn ci tin ca tng cng -carotene go vng) n c tnh gim i gnh nng ca thiu vitamin A (VAD) t 9% (tc ng yu) n 59% (tc ng mnh) [52]. Ngay c vic gim 9% chi ph c th chuyn sang cu hng ngn ngi t m hoc t vong do bnh truyn nhim. Hn na, go vng 2 ha hn s c chi ph hiu qu, bi v ngi ta c tnh rng, ngay c di tc ng thp, chi ph tit kim mt DALY l t hn 20 la M so vi mt chi ph ti thiu l 134 la M thc hin bng cch dng vitamin A b sung [52].
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Rt t kh nng thc phm thc vt c lm giu folate s gy nn bt k thay i cm quan no, ging nh trng hp ca go vng, cho nn t quan im ny, ngi tiu dng c th chp nhn nhng sn phm ny. Tuy nhin, mi quan tm v kh nng tc ng gy hi mi trng (v d nh: mt a dng sinh hc) v y t (v d: bnh d ng) l khng th trnh khi, c bit l EU, nu tng cng sinh hc t c bng k thut trao i cht. iu ny khng c ngha l phng php tip cn k thut cn c t b, bi v chp nhn cy trng 'bin i gen' pht trin cng ging nh l chp nhn nhng li ch ca n, nh xy ra cho nhng t ph khoa hc v y t khc trong hai th k trc (v d nh nhng tranh ci rng ri xung quanh vic tim phng i tr u tin, mt s can thip xa bnh u ma, vo thi gian mt cn bnh cht ngi). Cc ri ro sc khe v li ch ca thc phm chuyn gen hu nh ch ph thuc vo thnh phn ha hc (cc cht dinh dng, cht chuyn ha, cc yu t antinutritional, ) ca sn phm, ch khng phi vo cng ngh s dng t c s thay i. Tht vy, c th ni rng k thut di truyn cung cp nhiu s thay i c mc ch, c hiu v d on c trn thnh phn cy lng thc hn so vi nhiu phng php tip cn "thng thng" (Bao gm chn nui thng thng, ging t bin, lai soma, ). Hng pht trin chung Vic lm giu folate trn hai loi mt l mm v hai l mm thnh cng khng nh rng vic tng ng thi trn 2 nhnh pterin v p-ABA c th c s dng nh l mt bc tin chung c th ng dng vo nhng cy khc. S ti u ha dng chuyn ha c th t c bng cch tc ng vo nhng enzyme khc. Cng vic trong tng lai s bao gm vic to ra cc dng c folate cao bng cch lai ging hoc chuyn gen trc tip. y l thun li ln bi thc t l c hai gen chuyn u nm trn mt T-DNA n. K thut trao i cht cng c th c p dng cho cc loi cy thn c v khng phi thn c quan trng vi kinh t quc dn. Nghin cu su hn s l cn thit nh gi kh dng sinh hc v hiu lc sinh hc ca cy
116
tng cng cht folate cng nh s n nh folate trong thi gian lu tr lu di in hnh cho cc sn phm ng cc. 3.6. Kt lun v cc loi cy trng chuyn gen c lm giu folate Go, c chua v cy Arabidopsis tng cng folate c pht trin bng cch s dng k thut trao i cht n gin. Cc nh khoa hc hiu bit v ha sinh folate trong cy trng d tnh cc loi cy trng tng cng khc, chng hn nh la m, chui v khoai ty. Hin nay, s thiu ht folate vn cn, v s tip tc nh vy cho n khi trin khai cc k hoch trn c s nng nghip gip gim gnh nng ton cu ny. Tng cng folate trn cy lng thc nn l nhng can thip c gi tr, b sung v hiu qu trong cuc chin chng thiu ht folate ton cu, trn tt c cc nc ngho.
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Chng 4: Kt lun
CHNG 4
KT LUN V CY CHUYN GEN GIU CHT DINH DNG CHC NNG
Cc ging cy trng chuyn gen ngy cng c pht trin nh vo cc cng c ca Cng ngh sinh hc hin i. Chnh v vy m bn cnh cc thun li m thc phm chuyn gen mang li, nhiu ngi cng lo ngi rng liu cc loi thc phm ny c an ton bng cc loi thc phm c c nh s dng cc bin php nng nghip truyn thng hay khng. Di y l tm tt cc li ch tim tng v nguy c tim n ca cc cy chuyn gen. Li ch tim tng 1. Cy trng bin i gen tng nng sut, gim bnh tt v su bnh, t gim s dng thuc tr su, gim thiu nhim mi trng. 2. Thm vo cht dinh dng v mi v c ci thin, c hiu qu cho sc khe ngi tiu th. 3. Thc phm bin i gen c th lm tng hot cht thuc, nht l nhng thc phm c chc nng vaccine s lm tng cng kh nng min dch ca con ngi i vi bnh tt nan y nh vim gan siu vi B,, m thc phm thng thng khng th c c. 4. Rau sn xut khng th, c th n tr bnh nhim trng. Mt s v d nh: la go giu vitamin A v st, khoai ty tng hm lng tinh bt, vaccine n c ng v khoai ty, du n c li cho sc khe hn t u nnh v ci du Nguy c tim n 1. Hin ti ngi ta cha bit c mt cch chc chn thc phm bin i gen gy hi cho con ngi v mi trng nh th no?
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Chng 4: Kt lun
2. Do thn trng nn thc phm GMO kh c c hi pht trin nhanh c. Ngi ta s rng thc phm chuyn gen tng hp ra mt loi protein gy d ng c hi n sc khe lu di ca con ngi. 3. Mt khc thc phm chuyn gen c th l nguyn nhn lm mt tnh a dng sinh hc m thin nhin to ha ra qua mt qu trnh thch ng lu di, s mt i kh nng thch nghi ca cc sinh vt. 4. T cy thc phm GM, cc gen GM th phn cho ln c di s ra sao? Cui cng, v tm quan trng ca lng thc thc phm cho con ngi, nn cc chnh sch lin quan ti cy chuyn gen s phi da trn nhng cuc tranh lun ci m v trung thc c s tham gia ca mi thnh phn trong x hi.
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Phn 3: ng dng cng ngh vi sinh trong sn xut thc phm chc nng
PHN 3 NG DNG CNG NGH VI SINH TRONG SN XUT THC PHM CHC NNG
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Chng 1: To Spirulina
CHNG 1 TO SPIRULINA
1.1. Gii thiu chung u nhng nm 70 ca th k XX, nhng nh khoa hc ngi Php pht hin ra to c kh nng pht trin nhanh v c hm lng protein rt cao. To (agae) l mt nhm vi sinh vt, nhng chng khc vi vi khun v nm men ch chng c dip lc v c kh nng tng hp c cc cht hu c t cc cht v c di tc dng ca nh sng mt tri. To chia lm 9 ngnh: to lam (Cyanophyta); to lc (Chorophyta); to silic (Diatomea); to vng nh (Chysophyta); to gip (Pynophyta); to mt (Euglenophyta); to roi lch (Hererocontac); to (Rhodophyta); to nu. Trn th gii hin nay c nhiu loi to. Tuy nhin, cho n nay ch c ba loi to n bo c sn xut theo qui m ln v c li ch kinh t cao: Chlorella, Spirulina, Scenedesmus, trong hai loi Chlorella v Spirulina c sn xut nhiu hn.
Hnh 1.1: Hnh dng to Chlorella, Scenedesmus, Spirulina [65] Nhng u im c bn ca to n bo c cc nh khoa hc v cc nh sn xut quan tm bao gm:
SVTH: Nguyn Th Minh Tm
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To n bo c hm lng protein rt cao (chim khong 70-75% cht kh), protein ca to thuc loi protein hon ho v c cht lng cao. Cho n nay ngi ta cha tm thy c t nguy him tn ti trong sinh khi to. To l loi sinh vt t dng, chng hon ton c th t tng hp cht hu c cho s pht trin ca mnh t cc cht v c, CO2, nh sng nh thc vt. To c kch thc t bo ln, chng hon ton p ng tt mi yu cu k thut, c bit rt thun li trong giai on thu nhn. Chng khng b virus tn cng, sng trong nhng iu kin n gin. Hin nay, to Spirulina c nui trng nhiu hn to Chlorella, v gia hai loi to ny, to Spirulina c rt nhiu u im hn hn to Chlorella: To Spirulina c hm lng protein cao hn hn Chlorella. Protein trong t bo Spirulina l 60-70%, Chlorella l 40-50%. Kch thc to Spirulina ln hn kch thc to Chlorella. Mt khc, to Spirulina trong qu trnh pht trin c xu hng ni trn b mt, trong khi to Chlorella c kch thc nh li c xu hng lng chm khi khng khuy trn. Thu hoch to Spirulina bng nhng phng php rt n gin, trong khi thu hoch to Chlorella phc tp ging nh thu hoch sinh khi nm men hoc sinh khi vi khun. To Spirulina cha rt nhiu loi vitamin, trong khi to Chlorella cha t loi vitamin hn. Thnh t bo to Spirulina mng, thnh t bo Chlorella dy. Do h s tiu ha khi ta dng to Spirulina cao hn to Chlorella. To Spirulina pht trin trong mi trng kim cn to Chlorella pht trin trong mi trng acid yu. Khi dng CO2 nh ngun carbon, m ngun carbon ny trong iu kin kim t d chuyn ha sang dng d hp th theo phn ng sau:
Spirulina hp th CO2 theo chiu hng ny tt hn to Chlorella [13]. V vy, ngy nay to Spirulina c nghin cu v ng dng rt nhiu trong cc lnh vc ca i sng. SVTH: Nguyn Th Minh Tm
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1.2. Tng quan v to Spirulina 1.2.1. Lch s pht hin Spirulina l tn gi do nh to hc ngi c Deurben t vo nm 1827 da trn hnh thi c trng nht l dng si xon c vi khong 5-7 vng u nhau khng phn nhnh. Cng vo nm 1827, Turpin ln u tin phn lp c Spirulina t ngun nc t nhin. Nm 1963, gio s Clement (ngi Php) nghin cu thnh cng vic nui Spirulina qui m cng nghip. Nm 1973, T chc Nng lng Quc t (FAO) v T chc Y t Th gii (WHO) chnh thc cng nhn Spirulina l ngun dinh dng v dc liu qu, c bit trong chng suy dinh dng v chng lo ha. Nm 1977, Vin sinh vt hc l ni tin phong trong vic nui trng Spirulina Vit Nam theo m hnh ngoi tri, khng mi che, c sc kh CO2 ti x nghip nc sui Vnh Ho (Bnh Thun) [3]. 1.2.2. Phn loi

Ngnh: Cyanophyta Lp: Cyanophyceae B: Oscillatoriales H: Oscillatoriaceae Ging: Spirulina [13]
1.2.3. c im sinh hc ca to Spirulina To Spirulina c dng xon l xo khong 5-7 vng u nhau khng phn nhnh. ng knh xon khong 35-50 m, bc xon 60 m, chiu di thay i c th t 0,25mm. Nhiu trng hp to Spirulina c kch thc ln hn. To l trung gian gia vi khun v to nhn thc. Ngi ta cho rng to Spirulina ging vi vi khun hn, do to Spirulina cn c tn l vi khun lam. To c kh nng vn chuyn theo hnh thc trt xung quanh trc ca chng. Vn tc vn chuyn ca chng c th t 5 micron/giy [13].
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1.2.3.1. Cu to
Hnh 1.2: Hnh dng ca to Spirulina [65] L to lam a bo dng si, gm nhiu hnh tr xp khng phn nhnh. Mi t bo ca si c chiu rng 5 m, di 2mm. Khng c lc lp m ch cha thylacoid phn b u trong t bo. Khng c khng bo. Khng c nhn in hnh, vng nhn khng r, trong c cha DNA (Hedeskog v Hifsten A.1980) [13]. Thnh t bo c cu trc nhiu lp cha mucopolymer, pectin, v cc loi polysaccharide khc. Mng t bo nm st ngay di thnh t bo v ni vi mng quang hp thylacoid ti mt vi im. B my quang hp ca Spirulina:[5]
+
Phycobilisome: cha phycobiliprotein v protein lin kt c gn vo b mt
ngoi ca thylacoid. Phycobilisome c khi lng khong 7 triu dalton v c th tch nguyn vn nghin cu. i vi phycobilisome c c phycoerythin v phycocyanin th lp ngoi cng l phycoerythin, tip theo l phycocyanin v phn trong cng l allophycocyanin.
+
Phycobilisome hot ng nh mt anten thu nhn nng lng mt tri chuyn
vo PS II. Con ng truyn nng lng bt u t phycoerythin sang phycocyanin v cui cng n allophycocyanin trc khi t ti PS II.
+
C khong 50% nng lng nh sng mt tri Spirulina nhn c nh Cc sc t quang hp gm chlorophylla, carotenoid, phycocyanin, allophycocyanin
phycobilisome.
+
v thng c carotenoid-glycoside nh myxoxanthophyll, oscillaxanthin. SVTH: Nguyn Th Minh Tm

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Spirulina c cha 3 nhm sc t chnh: Chlorophyll hp th nh sng lam v . Carotenoid hp th nh sng lam v lc. Phycobillin hp th nh sng lc, vng v da cam. 1.2.3.2. Sinh sn ca Spirulina To lam l vi sinh vt xut hin cng lc vi vi khun trn tri t (Cifferi O, Tiboni O, 1985). Hin nay ngi ta bit khong 2500 loi. To lam phn b rt rng v c kh nng chu nhit rt cao. Ngi ta pht hin chng sng nhng sui nc nng n 690C [13]. To Spirulina c phng thc sinh sn v tnh, t mt c th m trng thnh (gi l trichome), t phn chia thnh nhiu mnh, mi mnh gm mt s vng xon (2-4 t bo, gi l hormogonia). to thnh cc hormogonia, si Spirulina s hnh thnh cc t bo chuyn bit cho s sinh sn (gi l on necridia). Cc necridia hnh thnh cc a lm 2 mt v to ra hormogonia bi s chia ct ti v tr cc a. Khi pht trin, dn dn phn u hormogonia b tiu gim v tr nn trn nhng vch t bo vn c chiu dy khng i. Cc hormogonia pht trin, trng thnh v chu k sinh sn lp li m bo vng i ca Spirulina.
Hnh 1.3: S vng i ca Spirulina [13] SVTH: Nguyn Th Minh Tm

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Thng thng Spirulina sinh sn bng cch gy ra tng khc. Trong trng hp gp iu kin khng thun li, Spirulina cng c kh nng to bo t ging nh vi khun. Chu k pht trin ca Spirulina rt ngn. Chu k ny thng din ra trong 24h nh ca to Chlorella [13]. 1.2.4. Thnh phn ha hc ca Spirulina Spirulina cha hm lng protein rt cao v cha y cc vitamin. Bng 1.1: Thnh phn ha hc ca Spirulina STT 1 2 3 4 5 6 7 Thnh phn Protein tng s Glucid Lipid Acid nucleic Dip lc Carotene Tro S lng (% cht kh) 60 70 13 16 78 4,29 0,76 0,23 45 [13]
Spirulina c gi tr dinh dng cao v cha hm lng protein cao v cc cht c hot tnh sinh hc khc. Gi tr protein trung bnh ca Spirulina l 65%, cao hn so vi nhiu loi thc phm. V d, hm lng protein ca c v tht l 15-20%, nc tng l 35%, sa c c l 35%, trng l 12% v ng cc l 8-14% [22]. Spirulina l ngun giu vitamin B12 nht. Nu hng ngy ta s dng 1g Spirulina th s p ng nhu cu vitamin B12 hng ngy. Ngoi ra, Spirulina cn cha cc vitamin khc nh A, B1, B3, B6, E v H (Bng 1.2). Spirulina giu st v calcium, h tr tt cho mu, cho xng v rng. Lng calcium ca Spirulina cao hn trong sa. Lng st trong Spirulina cao hn 12 ln so vi trong cc loi thc phm khc. Ngoi ra, Spirulina cng giu magnesium, potassium. Nhng khong a lng bao gm sodium, calcium, magnesium, potassium, chlorine, sulfur v phosphorous. Cc khong vi lng gm iodine, calcium, magnesium, fluoride, manganese, boron, nickel v cobalt (Bng 1.2).
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Bng 1.2: Thnh phn vitamin ca Spirulina Vitamin Trn 10g Nhu cu hng ngy cho php 5000 1,5 1,7 20 2,0 6,0 30 400 10 % so vi nhu cu hng ngy cho php 460 21 21 7 4 533 3 0,04 1 [32] Bng 1.3: Thnh phn khong ca to Spirulina Khong Trn 10g Nhu cu hng ngy cho php 1000 mg 18 mg 1000 mg 400 mg 15 mg 3500 mg 2 mg 2 mg 120 g 2400 mg 70 g % so vi nhu cu hng ngy cho php 7 55 8 10 2 4 6 25 21 4 14 [32] SVTH: Nguyn Th Minh Tm
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Vitamin A (-caroten) Vitamin B1 (Thiamine) Vitamin B2 (Riboflavin) Vitamin B3 (Niacin) Vitamin B6 (Pyridoxine) Vitamin B12 (Cyanocobalamine) Vitamin E (-tocopherol) Folacin Phanthothenic acid Biotin Inositol
23000IU 0,31g 0,35 g 1,46 g 80 g 32 g 1IU 1 g 10 g 0,50 g 6,40
Calcium Iron Phosphorus Magnesium Zinc Potassium Copper Manganese Chromium Sodium Selenium
70 mg 10 mg 80 mg 40 mg 300 g 140 mg 120 g 500 g 25 g 90 mg 10 g
Spirulina cha 18 trong s 20 loi amino acid c bit. Mt s amino acid c hm lng cao trong Spirulina nh glutamic acid (14,6%); aspartic acid (9,8%); leucine (8,7%); aniline (7,6%) (Bng 1.4). Bng 1.4: Thnh phn acid amin ca to Spirulina STT Thnh phn Isoleucin Leucin Lysin Methionin Phenilalanin Theonin Tryptophan Valin Alanin g/10g S lng (% tng cht kh) 5,6 8,7 4,7 2,3 4,5 5,2 1,5 6,5 7,6 STT Thnh phn Arginin A.aspartic Cystin A.glutamic Glycin Histidin Prolin Serin Tyrosin g/10g S lng (% tng cht kh) 6,9 9,8 1,0 14,6 5,2 1,6 4,3 5,2 4,8 [13]
1 2 3 4 5 6 7 8 9
350 540 290 140 280 320 90 400 470
10 11 12 13 14 15 16 17 18
430 610 60 910 320 100 270 320 300
Cc cht mu trong Spirulina: Spirulina c mu xanh lam-lc l do Spirulina cha nhiu sc t vi hm lng cao nh chlorophyll, phycocyanin, beta-caroten, xanthophylls (Bng 1.5). Bng 1.5: Cc cht mu trong Spirulina Cht mu Phycocyanin Chlorophyll Carotenoids Mu sc Xanh da tri Xanh l cy Mu vng cam Hm lng trong 10g % Spirulina 14% 1400g 1,0% 100 g 0,37% 37 g [32]
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1.2.5. Tnh hnh nui trng v pht trin Spirulina 1.2.5.1. Trn th gii Spirulina c trng i tr cc nc trn th gii t nhng nm 1972, cc nc sn xut vi to ch yu tp trung Chu v vnh ai Thi Bnh Dng. Nhng khu vc v vng lnh th c sn lng vi to ln l Trung Quc, Nht Bn, i Loan, Hn Quc, Hoa K, Mehico Khi u l vo nhng nm 1970, mt doanh nghip to u tin ca Hoa K bt tay vo nui th nghim m hnh pilot trn cc b nhn to. H chn thung lng hoang mc Imperial thuc bang California v ni y c nhit trung bnh cao nh nh nng mt tri v trnh xa vng nhim th. n nm 1981, mt s hp tc u tin gia doanh nhn California v thng nhn Nht bn hnh thnh nn Earthrise Farms v chnh thc i vo sn xut n nh nm 1982. Ngy nay, Earthrise Farms cung cp sn phm cho hn 40 quc gia v ngun Spirulina y c xem l tt nht [32]. Ngoi ra, trn th gii cn c cc trang tri nui trng to Spirulina vi quy m ln, cht lng cao nh:

Trang tri Twin Tauong (Myanmar) Trang tri Sosa Texcoco (Mehico) Cng ty to Siam (Thi Lan) Trang tri Chenhai (Trung Quc) Nng tri Hawai ( Hoa K)
1.2.5.2 Vit Nam Vit Nam, t nm 1972 cc nh khoa hc bt u t vn nghin cu to Spirulina do GS.TS Nguyn Hu Phc ch tr. Nm 1976, vic th nghim nui trng to Spirulina c tin hnh trong thi gian 4-5 thng ti Ngha , H Ni thu c kt qu kh kh quan.
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Vo nm 1985, S Y T thnh ph H Ch Minh tip nhn ging to Spirulina u tin do ng b R.D.Fox tng. Sau , to ging c giao cho Trm nghin cu dc liu (nay l Trung tm dinh dng thnh ph H Ch Minh) gi ging v nui trng [3]. Hin nay, c 2 ni nui trng to Spirulina ln nc ta, l:

Cng ty c phn nc khong Vnh Ho (Bnh Thun) V mt c s Bnh Chnh, thnh ph H Ch Minh.
C th ni, Vnh Ho l n v tin phong trong vic nui trng v sn xut to Spirulina ln nht nc ta. Vic nui trng Spirulina ti thnh ph H Ch Minh li l ngun nguyn liu sn xut thc n ch yu cho g, tmSau mt thi gian khng tm c u ra v gi thnh cha hp l nn cc c s trn khng th tip tc vic nui trng trn c na. Nhn chung, lch s nghin cu v nui trng to Spirulina nc ta thu c nhiu kt qu ban u ng kch l. Tuy nhin, cho n nay vic nui trng a s vn mang tnh nh l, lc hu khng p ng c nhu cu s dng to ngy cng cao. V vy, trc nhng gi tr v mi mt m to Spirulina mang li, cn phi tin hnh ci thin, thc y ngnh cng nghip nui trng to nhm p ng nhu cu trong nc v xut khu ra th trng nc ngoi. 1.2.6. Tnh hnh nghin cu v ng dng Spirulina 1.2.6.1. Trn th gii Nghin cu ng dng Spirulina trong thc phm Spirulina c nghin cu sn xut v c ng dng trong nhiu lnh vc khc nhau cc nc trn th gii. Hin nay, Spirulina c nghin cu ng dng nhiu trong thc phm v m phm. Spirulina c nghin cu b sung vo rt nhiu sn phm thc phm nh: m si, yaourt, ko, tr xanh, bnh quy, bnh m, biaCc sn phm ny c bn siu th ca nhiu nc nh: Chi L, an Mch, H Lan, M, c, New Zealand (Bng 1.6) [19]. Nghin cu ng dng Spirulina trong m phm
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Cc sn phm m phm c b sung Spirulina cng xut hin cc siu th nh: Sn phm bo v da, du gi, kemCc thnh phn chit xut t to Spirulina nh protein, polysaccharide, vitamin v khong c dng sn xut cc m phm lm p cho ph n nh: m phm sn sc bo v da u, bo v tc, bo v da, lm lnh so mau chng, chng mn nht v lm trng da [59]. Bng 1.6: Cc loi thc phm c ch bin t to Spirulina STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Loi thc phm Die (nc st to Spirulina) Bnh m trng M gi Bnh bch quy Bnh ngt Cousorus (tht bm) Sa chua Pastas (bt sa) Chocolate Ru, bia Kem caramel Thch gelatine Nc mt hoa qu Mt nh Atories (ru t ng ca Mehico) Hn hp ng cc, bt m v to Spirulina) Bt ng c x l bng nc vi nng c bt go v to Spirulina) Bt ng v to Spirulina Xc xch T l Spirulina c b sung (%) 9,1 3 14 4 36 2 25 36 35 510 510 510 510 510 5,58 1214 10,4 Nh ch bin Orstrom Tchard Nestle INIA Sosa Lauce Nestle Dijon Dijon Sosy Dunal Sasa E.N.C.B Sasa E.N.C.B -
18 20
1014 1030
Bnh vin Bichaf [9]
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Nghin cu ng dng Spirulina trong dc phm c ch kh nng ti to ca s sinh sn HIV-1 bng nc chit xut Spirulina: Nc chit xut Spirulina ngn nga s sinh sn HIV-1 con ngi nh cc bch cu lumpo T v bch cu n nhn ca h min dch c gia tng trong mu ngoi bin. Chit xut c c 5-10g/ml cho thy lm gim s sn sinh virus khong 50%, v chit xut c c 100 g/ml cho thy c ch 90-100% m khng c tnh i vi t bo thng [32]. Calcium Spirulan t to xanh Spirulina, c ch s ti to mng bao virus: Vic phn ct trc tip cc hot tnh sinh hc ca chit xut t to Spirulina dn n vic c lp cc cht polysaccharide sulfate mi c tn gi l Calcium Spirulina (Ca-SP) nh mt cht chng virus chnh yu. Polysaccharide c tng hp bi ribose, mannose, fructose, galactose, xylose, glucose, acid galacturonic, sulfate v calcium. Ca-SP c tng hp ngn chn s ti to nhiu loi virus pht trin bao gm virus Herpes n bo dng 1, virus si, quai b, cm A v HIV-1. Ngi ta khm ph ra rng Ca-SP ngn chn c qu trnh thm nhp ca virus vo trong cc t bo ng thc vt. Chit xut to Spirulina-Dunaliella c kh nng ngn nga ung th ming: Chit xut ca to Spirulina-Dunaliella cho thy l ngn nga c s pht trin ca khi u trong ming chut ti khi tim dch Spirulina vo chng in hnh 3 ln mi tun trong 28 tun. Nhng ng vt khng c iu tr, tt c u c nhng khi u ni chung bn phi ming ti. Nhng con c nui bng canthanxanthin theo thng k cho thy gim xung ng k v s lng v kch c khi u so vi nhng con ch c kim sot. Nhng ng vt c nui dng vi -carotene cng c chng minh l gim ng k mt lng nh hn v c s lng v kch c khi u. Tuy nhin, mt phn nh nhm chut ti c n to c du hiu ca chng lon sn v ung th biu m sm l nguyn nhn hy dit. Cc nghin cu khc: Hc vin y khoa qun s Trung Hoa, vin y hc v vin bc x Suzhou thuc b cng nghip ht nhn nghin cu nh hng ca Spirulina, kt qu cho thy vic b SVTH: Nguyn Th Minh Tm
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sung Spirulina lm tng kh nng min dch v tng t l sng st ca chut khi chiu cc tia phng x gy cht ngi. Mt s bnh vin thnh ph Kumming, tnh Yuan, dng Spirulina nh mt loi thuc c tc dng gim lng lipid trong mu. i hc Beijing chit xut thnh cng phn t c hot tnh sinh hc t Spirulina ngn chn nh hng ca vic nhim cc kim loi nng, cng nh ngn chn s pht trin ca cc khi u. Nhiu c quan Trung Quc tp trung vo cc nghin cu sinh hc phn t ngn chn khi u bu, chng li s lo ha v chng cc tia phng x [9]. Trn th gii c rt nhiu sn phm Spirulina c bn di dng thuc vi nhiu tn gi khc nhau nh: Linagreen, Heilina, Spirulina kayaky, Spirulian C, Light Force Spirulina. Spirulina thng sn xut di dng vin nn, mi vin c trng lng 500mg trong cha khong 200-300mg to kh. Loi ny c dng cha tr mt s bnh nh vim gan, vim khp, ung th, tng cng sc khe, gim cn v phng chng suy dinh dng tr em. Nghin cu ng dng Spirulina trong chn nui Trung Quc s dng Spirulina lm thc n thay th quan trng cho tm kch thch kh nng tng trng nhanh, tng kh nng min dch v sng st ca tm. Thc n cho tm c b sung Spirulina gip gim thi gian nui cng nh t l t vong. Vin nghin cu t v phn bn ca Hc vin khoa hc nng nghip Trung Hoa hp tc vi Vin nghin cu nui trng Jianxi nghin cu vic b sung vo thc n cho tm Tangxian, tnh Hebei. Chiu di u trng tm tng ln 18% sau khi dng sn phm b sung Spirulina. T l sng st ca tm con tng n 23,8%. Gi ca thc n cng r hn 48% so vi cc thc n thng. Thc n c cha Spirulina gip tng sc khng ca cc loi c c gi tr cao, tng kh nng sng st t 15% ln 30%. Khi thm Spirulina vo thc n gia sc, gia cm, tc sinh trng ca chng tng ln. Spirulina cng c s dng lm thc n cho c cnh, loi thc n ny c sn xut bi cng ty Guangdong Jiande, ph bin Nht Bn v cc nc ng Nam .
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Nghin cu ng dng Spirulina trong x l mi trng T nm 1975, Oswald v cng s ti trng i hc Tng Hp Califonia th nghim dng Spirulina trong x l nc thi cng nghip v i n kt lun: trong h x l nc thi, Spirulina c vai tr to O2, tng kt lng, loi tr kim loi v cc cht hu c c hi. 1.2.6.2. Vit Nam T nm 1977, Nh nc ch trng vo vic nghin cu v nui trng th nghim vi to Spirulina, bc u thnh cng mt s ni nh Vnh Ho, c Lc, ng NaiT ngun nguyn liu Spirulina t cht lng cao v n nh, cc nh khoa hc sn xut thnh cng mt s loi thuc nh Linavina, Lactogil (x nghip Mekophar); Cm b, bt dinh dng Enalac (Trung tm dinh dng tr em thnh ph H Ch Minh), Gelule Spilina (Lebo, Helvinam, Trng i Hc Y Dc) Trung tm dinh dng tr em Tp. H Ch Minh:[3], [15] Theo bo co thng 05 nm 1997 ca Trung tm dinh dng tr em th t nm 1989, Trung tm dinh dng c thnh ph giao cho chc nng nghin cu v pht trin Spirulina. Tuy nhin, vic tiu th vi to Spirulina trong vi nm gn y gp kh khn v ngi tiu dng cha quen do mu sc v mi l. V vy trung tm nghin cu v a Spirulina vo thc n, v khi a Spirulina vo c th bng con ng ny s thun li hn v t chu nh hng ca yu t cm quan, ng thi gp phn hi phc nhanh chng sc khe cho bnh nhn. Ngoi ra, trung tm cn sn xut bt dinh dng Enalac c b sung Spirulina gii quyt vn suy dinh dng tr em, phc hi dinh dng cho ngi gi, bc u t c nhiu thnh qu ng khch l. sn xut 50-100 tn bt dinh dng/ thng, cn cung cp s lng Spirulina kh l 750-1500 kg. iu ny cho thy nhu cu cung cp Spirulina hin nay l rt ln. Kt qu tin hnh th nghim s dng bt dinh dng Enalac cc bnh vin cho mi i tng ca Trung tm dinh dng t c nhiu thnh qu tt p nh:
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Bt dinh dng Enalac d s dng, cung cp nhiu nng lng v cc yu t vi
lng, d tiu ha, hp thu rt tt trong vic h tr cho cc bnh nhn nng khng t n c hay cn b sung dinh dng.
Trong vic iu tr suy dinh dng ngi gi v tr em th s ngi tng cn sau Enalac an ton cho ngi s dng v c gi thnh ph hp trong iu kin kinh t Tuy nhin, c th khng nh chc chn v pht huy c tm nng ca loi siu
t dng Enalac l trn 70%.
nc ta hin nay.
thc phm ny chng ta cn thc hin nghin cu lm sn su rng hn trn mi i tng v ko di trong thi gian cn thit. Vin Nghin cu ng Dng Cng Ngh (B Khoa Hc Cng Ngh v Mi Trng): Bng cc phng php cng ngh sinh hc, cn b ca Vin Nghin Cu ng Dng Cng Ngh (B Khoa Hc Cng Ngh v Mi Trng) chit xut c mt s cht c hot tnh sinh hc cao nh phycocyanin. Vic kt hp phycocyanin v tia x Cobalt 60 trong iu tr bnh ung th vm hng. Kt qu l hn ch 70-80% s pht trin ca t bo ung th, bnh nhn phc hi v tng th trng sau . Nhiu loi vitamin, khong v cc hp phn dinh dng khc trong Spirulina c tc dng bi dng sc khe, chng suy dinh dng, bo v c th khi tc hi ca cht phng x v chng suy mn do nhim hi c. Vin Sinh Vt Hc: Gio s ng nh Kim (Vin Sinh Vt Hc) v Y.Lemoine (i Hc S Phm Pari) tin hnh nui trng Spirulina trong bnh tam gic cha mi trng Zarrouk trong iu kin kim sot v nhit , nh sng, thi gian chiu sng. Sau khi vi to Spirulina t yu cu v sinh trng v pht trin, gio s v cng s tin hnh xc nh protein tng (bng phng php Bio Rad), hm lng phycobiliprotein, xc nh acid tng s v cc acyl lipid (sc k trn my Girdel Chromatographie Serie 300).
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Ngoi ra, vic th nghim nui trng Spirulina bng nc thi hm biogas khng ch l bin php m rng sn xut v h gi thnh sn phm m cn gii quyt mi trng sinh thi cho nng thn. Loi vi to ny cn c s dng x l nc thi giu NH4 t nh my sn xut ure thuc x nghip Lin Hip Phn m Ha Cht H Bc, kt qu cho thy: nc thi sau khi pha long v b sung thm mt s cht khong cn thit ri dng nui Spirulina mang li nng sut cao v c tc dng bo v mi trng. 1.2.7. Cng ngh nui trng to Spirulina 1.2.7.1. Gii thiu cc h thng nui to Spirulina Cng ngh nui trng Spirulina theo h thng h (O.E.S): Spirulina sng trong mi trng dinh dng ng trong bnh, chu, b c vn ng bng khuy trn theo kiu tnh tin 2 chiu v to hp thu nh sng mt tri pht trin. Kiu nui ny ph thuc vo thi tit cn c gii php khc phc. Cng ngh nui trng to Spirulina theo h thng kn (C.E.S): Spirulina c nui trong cc b ln men vi sinh khi (bioreactor) vn ng bng my khuy trn theo 3 chiu, to hp thu nh sng nhn to hay t nhin. Nhiu kiu CES c thit k nh thng ln men c in hoc kiu ng xon c[7]. 1.2.7.2. Tiu chun chn ging Spirulina Chn ging theo mc ch s dng: lm thc phm (chn ging giu protein, vitamin, khng c hoc cha t mi kh chu khi s dng), lm dc phm (chn ging chit xut c cht mong mun vi liu lng cao), lm m phm ( chn ging chit xut ra c nhiu cht dng da, chng lo ha da nh vitamin E- chng oxy ha) Chn ging t hp ph, tch t cc cht c ca mi trng nui cy nh: Pb, arsenic. Ging Spirulina cht lng tt l ging hp ph t nht cc cht c trong cng iu kin th nghim. Chn ging cho nng sut cao, d thu hoch, d thch nghi, sc chng chu tt. Ging spirulina phi c mua nhng c s uy tn. ng thi ni nui trng Spirulina cng nn c trang b nhng phng th nghim phc v cho cng tc gi v nhn ging phc v sn xut. nc ta c bo tng ging to Vit Nam l ni cung cp
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ging v t vn xy dng qui trnh nui to - do gio s Dng c Tin thnh lp t nm 1982 [7]. 1.2.7.3. iu kin nui to Spirulina cn mi trng nui kim (mui natri cacbonat v pH cao). Nhit nc dao ng t 25 400C, nhit ti thch l 350C. Cn nh sng tin hnh qu trnh quang hp to ra sinh khi. Mi trng nc cn c khuy u lin tc. Cc cht dinh dng cn c cung cp l phosphor, nit, st cng vi cc cht khong khc, cc cht khong ny cn c cung cp thng qua dng ha tan trong dung dch. Di iu kin ti u, Spirulina c th tng gp i sinh khi v th tch sau 24h nui. C th xy dng cc trang tri gia nh vi quy m t 10 50m2, cc trang tri th cng c th ti 300m2, trang tri thng mi c th ti 1hecta v cc trang tri quy m cng nghip ln vi sn lng thu c t 100 500kg hoc 1 tn sinh khi to trn ngy [3]. 1.2.7.4. Mi trng nui to Thnh phn chnh l NaHCO3 pha trn vi cc mi trng di y: Bng 1.7: Mi trng c bn( mi trng Zarrouk) Ha cht K2HPO4 K2SO4 MgSO4.7H2O CaCl2.2 H2O Hm lng 0,1g/200ml 0,2g/200ml 0,04g/200ml 0,008g/200ml Ha cht NaNO3 NaCl FeSO4 EDTA Hm lng 0,5g/200ml 0,2g/200ml 0,0002g/200ml 0,016g/200ml [13]
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Bng 1.8: Mi trng b sung 1 Ha cht H3BO3 ZnSO4.7 H2O MoC3 Hm lng 2,86g/l 0,22g/l 0,01g/l [13] Bng 1.9: Mi trng b sung 2 Ha cht K2Cr2(SO4)3.24 H2O Ti2(SO4)3 Hm lng 960g/l 400g/l Ha cht NiSO4.7 H2O Co(NO3)2.6 H2O Hm lng 478g/l 44g/l [13] 1.2.7.5. Qui trnh nui to u tin phi la chn v tr cho trang tri. Kch thc v kiu trang tri cng cn c xc nh theo c th v kh hu, ngun nc, kh nng ti chnh. Xy dng b, lp t cc cnh khuy v m t. Chun b cc chng to v nhn ging chng t trong phng th nghim. Phi chun b t nht 100 lt chng ging. Chn lng mi trng nui cy thch hp, t tn km nht v cc ngun ha cht phi d nhp. Nu b c kch thc ln hn 10m2, cn ro bng cht liu nilon, plastic hoc b tng v lp t cc my bm khuy. i vi cc b nui rt ln, phi tin hnh nui cy lin tc v sau khi thu hoch phi bt li t nht 1/5 th tch b. Ngoi ra, trnh sc pH v s thay i p sut thm thu qu t ngt, ta phi b sung mi trng nui cy mi vi tng lng nh theo chu k 12h mt ln. Cng thc n gin nht tin hnh xy dng b th cng l: sn bng b tng, khung g v che chn bng vt liu plastic. Ha cht MnCl2.4 H2O CuSO4.5 H2O Hm lng 1,81g/l 0,08g/l
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Nhit nc cn kim tra 2 ln trong ngy. Tt nht l ghi li nhit tng gi nu c thit b ghi t ng. Nhit phi duy tr khng cao hn 410C v khng thp hn 210C trong sut c ngy. Gi tr pH ti u l 9,5 song thng trong cc b nui dao ng t 10 10,5. Vo bui chiu do quang hp mnh nn pH c th tng ln n 11,5. Nhng vo ban m do qu trnh h hp, chuyn ha carbohydrate thnh protein,v gii phng CO2 vo trong nc nn pH li tr v mc 10 10,5 vo sng hm sau [3]. 1.2.7.6. Thu hoch to Thu hoch c thc hin bng cch lc qua mng polyester, ng knh mt li 30m. Thit b lc c t nghing cht t c th tin hnh lc c lin tc ng thi ra v vt. Sau chuyn chng qua giai on vt nc bng my vt, p hoc nh mng rung cho nc chy bt xung. Bnh to sau c ct ra thnh tng ming, khc nh dao. Sau giai on ny nc vn chim 70 80%. Trong giai on ny to Spirulina do cha nhiu m nn chng d b vi khun tn cng v ln men to sn phm khng mong mun ch trong vi gi ty thuc vo nhit . l l do ti sao ngi Aztecs v Kanembous ngh cch sinh khi vo cc b ct, chng b ct ht nc v lm xung ct. Nh c ct nng di nh sng mt tri chng s tr nn kh ch sau 4 5h, nh vy c th chng li s xm nhp ca vi khun. Tuy nhin, vic phi trc tip di nh sng mt tri li c th gy ra vic ph hy dip lc t, lm gim hm lng carotene v vitamin nhm B ca sn phm. V vy cc trang tri nui trng Spirulina th cng, nh l thng phi bng cch cho dch to vo trong cc hp kim loi ri em phi ngoi nng t s dng hi nng v gi thi qua lm bay hi nc. Ngi ta cn s dng thit b n gin hnh xylanh, mt u c chm cc l nh ng knh 2mm, ri cho rong to vo trong. Sau p mnh mt u, to s chy ra thnh cc si nh si m, tip theo tri nh ln cc khung bng kim loi hoc bng g ri a vo trong cc hp lm kh. Hp lm kh c kch thc cc l vo v ra bng nhau cho php khng kh lu thng c d dng [3].
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1.2.8. Cc phng php ph v t bo S.platensis 1.2.8.1. Phng php vt l Phng php sc nhit: Phng php ny c thc hin bng cch a huyn ph t bo xung nhit thp, sau ngay lp tc nng nhit ln nhanh n 400C, khi thnh t bo b ph v v ta thu c huyn ph t bo v. Dch huyn ph c pha long, lc qua giy thu dch chit. Phng php ph v t bo bng sng cc ngn (microware): L k thut da trn c tnh gia nhit ca vi sng (microware). Nhng sn phm ni bo ca vi khun, c bit l cc phn t nh c trch ly d dng sau khi t bo c x l vi vi sng. Phng php sc thm thu: Trong phng php ny, u tin to ra p sut thm thu cn bng gia bn trong v bn ngoi t bo trong mi trng giu saccharose. Sau nhanh chng pha long dung dch saccharose. S chnh lch p sut t ngt s lm tn thng mng t bo. Phng php ng lnh v r ng: Huyn ph t bo c ng lnh trong t ng, sau c r ng nhit phng. Cc t bo b trng ln v cui cng b v ra do s hnh thnh v tan ra ca cc tinh th . 1.2.8.2. Phng php sinh hc S dng enzyme Lysozyme: iu kin ti u cho hot ng ca enzyme:

Nhit 450C Nng Lysozyme: 5000g/ml, pH 6.0 Nng c cht: 0,1g Spirulina/10ml Thi gian : 60 pht
S dng enzyme Viscozym L: iu kin ti u cho hot ng ca enzyme:
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Chng 1: To Spirulina Nhit 450C Hm lng Viscozym: 10% V huyn ph Nng c cht: 1g Spirulina/50ml Thi gian : 60 pht
Theo nghin cu ca Lng nh Qut (2007), Nghin cu cc phng php x l sinh khi vi khun lam S.platensis sn xut mt s loi nc ung giu dinh dng, Lun vn thc s. tin hnh th nghim cc phng php x l trn v thu c kt qu: Phng php sc nhit: Cho hiu sut thp, tuy nhin hot tnh protein ha tan trong dch chit c m bo. Phng php sc thm thu: Cho hiu sut 55,1% nng ng saccharose 50% sau thi gian l 1h v protein khng b bin tnh. Phng php ny thch hp vi sn phm cha hm lng ng cao. Phng php x l bng sng cc ngn: Cho hiu sut cao hn 3 phng php trn, t hiu sut 66,1% 3 chu k. Tuy nhin, trong phng php ny protein b bin tnh theo chu k. X l bng Lysozyme: Cho hiu sut 69,3% nng Lysozyme 5000g/ml X l bng Viscozym L: Cho hiu sut 73,6% hm lng 10% Viscozym so vi dch huyn ph. Nh vy, nhn chung phng php x l bng enzyme cho hiu qu tt hn so vi cc phng php vt l thng thng. Sau khi ph v t bo, hn hp c em i nghin, sau tch vch v cc mnh v t bo ra khi dch bng cc phng php lc, ly tm[8].
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1.3. Nghin cu tnh hung 1.3.1. Nghin cu x l to Spirulina: [48] 1.3.1.1. Lnh vc nghin cu Spirulina l sn phm thc phm giu cht dinh dng, n bao gm c c tnh ca rau xanh v thnh phn cht dinh dng ca chnh n. iu ny cho php biu din cc cht dinh dng ca Spirulina di dng thc n thng thng d hp th. Spirulina thng c cung cp di dng bt kh. S lng ln bt Spirulina thng c sn xut bi vic thu hoch to t t cc qu trnh sn xut quy m ln trong cc h nhn to ngoi tri. Tuy nhin, do bt Spirulina sn xut bng phng php ny c mi v c trng ca to nn n c s dng rt hn ch trong ngnh cng nghip thc phm, ch yu s dng trong thc phm b dng v thc n cho ng vt. c mt vi phng php c p dng gim mi v c trng ca Spirulina nh thm dch chit t l tr vo dch huyn ph Spirulina, sau lm kh v to bt. Tuy nhin, bt Spirulina thu c bng phng php ny vn cn gi mt phn mi v c trng ca Spirulina. ng thi, trong cng nghip sn xut thc phm ni chung, iu cn thit l gim thiu lng vi khun tp nhim. Thng thng hay s dng phng php x l nhit 1300C, sau a vo h ln men v thc hin qu trnh ln men chua nh trc, sn phm sau ln men c lm mt v nhit m qu trnh ln men khng cn xy ra. Phng php ny c th lm gim s lng vi khun tp nhim nhng v x l nhit cao nn cc cu t hot ng mong mun trong Spirulina cng b mt. V vy, yu cu t ra l x l Spirulina sao cho c th gim thiu mi v c trng, gim s vi khun tp nhim nhng vn gi li c cc cu t hot ng mong mun trong Spirulina. 1.3.1.2. Tm tt nghin cu Khi thc hin s pha trn Spirulina vi vi khun lactic v nui cy vi khun lactic, ngi ta nhn thy n c th lm gim mi v c trng ca Spirulina. S gim mi v c trng ca Spirulina v gim lng vi khun tp nhim l nh hot ng khng khun
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ca cc acid hu c nh acid acetic, acid lacticv cc cht khng khun to ra trong qu trnh nui cy ca vi khun lactic. Trong khi vn gi c cc cu t hot ng trong Spirulina. Spirulina c x l theo cch ny s d dng s dng trong cc thc phm sc khe v cc thc phm lin quan khc. Theo cch ny, cc nghin cu hin nay a ra mt quy trnh x l Spirulina, quy trnh ny bao gm nghin cu Spirulina khng qua giai on un nng v tit trng, vi khun lactic v ng, sau kho st qu trnh nui cy vi khun lactic. Spirulina c s dng khong 1-20g/100g khi lng ca tng Spirulina, vi khun lactic v nc kt hp. S lng vi khun lactic khi bt u nui cy khong 106 109 t bo/g Spirulina (cht rn c s). Trong trng hp ny vi khun lactic thng c nui pH t 6-8 trong khong thi gian 24h. Spirulina c dng c th dng bt hoc dng ti sng. Vi khun lactic s dng thuc loi Pediococcus hoc Lactobacillus. ng s dng l loi galactooligosaccharide. 1.3.1.3. M t chi tit nghin cu Spirulina: To Spirulina s dng thuc lp Cyanophyceae, h Oscillatoriaceae, ging Spirulina. V d nh S.platenis, S.maxima, S.geitleri, S.siamese, S.major, S.subsalsa, S.princeps, S.laxissima, S.curta, S. spirulinoides. Spirulina xut khu di dng cc hnh thc: spirulina sng, spirulina kh, spirulina qua x l bng my mc c hc hoc cc phng php x l khc. Spirulina sng c th thu c bng phng php tch ly tm v lc sau khi thu hoch t b nui. Spirulina kh to ra t Spirulina sng thu c bng phng php trn c ng lnh, sau sy kh hoc phun kh. Spirulina qua x l bng my mc c hc hoc cc phng php khc bao gm i tng l Spirulina sng c a vo cc my c hc ng nht hoc c th c x l sau khi sy kh. Spirulina c dng trong qu trnh nghin cu x l tt nht l dng Spirulina sng v n gi li nhiu hn cc cu t hot ng ca Spirulina.
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Ty mc khc nhau m nc c loi b trong qu trnh thu hoch. Spirulina sng nhn chung u dng huyn ph trong nc. Cng c th dng paste c hm lng nc thp hn huyn ph, hoc dng bnh c hm lng nc thp hn dng paste. Spirulina c th c dng di bt c hnh thc no nhng c l thch hp nht l dng Spirulina dng huyn ph. Nu Spirulina kh hoc Spirulina qua x l c dng trong qui trnh nghin cu th c th dng trng thi kh hoc thm nc vo n thnh dng huyn ph, paste hoc dng bnh nh trng hp ca Spirulina sng. trnh s mt mt cc cu t hot ng trong Spirulina, Spirulina c dng trong nghin cu phi cha qua giai on x l tit trng ban u. Spirulina cha cc thnh phn duy tr sc khe bao gm: Phycocianin c tc dng chng vim gan v bo v hot ng ca gan, chlorophyll (cht dip lc) c tc dng khng khun, chng d ng, thc y s trao i cht, ngn nga ung th, lm sch muVic x l tit trng s tiu hy cc cu t hot ng. Vi khun lactic: Vi khun lactic c dng t thi c i trong vic x l nhiu loi thc phm, bao gm cc sn phm nh sa ln men, ung qua qu trnh , rau da v tri cy, vi mc ch bo qun v to hng liu thc phm. Vi khun lactic v cht chuyn ha ca chng cng c nhiu tc ng sinh l mong mun nh lm gim cholesterol mu, hot ng h huyt p. V th chng c s dng nh cc sn phm thc phm thc y sc khe. Vi khun lactic c phn loi theo mi trng n sng nh vi khun lactic trong sa, vi khun lactic t thc vt, vi khun lactic ng rut, vi khun lactic c xut x trong cc h mui t nhin ni Spirulina pht trin. Vi khun lactic cng c th c phn loi theo nhit ti u cho s pht trin ca n nh vi khun lactic a m, vi khun lactic a nhit Vi khun lactic s dng trong nghin cu c th thuc cc chi Lactobacillus, Pediococcus, Tetragenococcus, Carnobacterium, Vagococcus, Leuconostoc, Weissella,
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Oenococcus,
Atopobium,
Streptococcus,
Enterococus,
Lactococcus,
Aerococcus,
Alloiococcus, Melissococcus v Bifidobacterium. Vi khun lactic thuc chi Pediococcus thch hp s dng hn v n c th lm gim mi v c trng ca Spirulina. Cc vi khun lactic c s dng c th c pht trin trn mi trng thch hoc mi trng lng ri c bo qun bng cch lm lnh hoc sy kh. Thch hp nht i vi bo qun vi khun lactic l c cy chuyn v nui dng trong mi trng lng v vi khun lactic c t l tng trng nhanh chng v hot cao, c kh nng to ra hng v (acetaldehyde, diacetyl) v axit hu c. Mi trng lng m vi khun lactic c nui cy l mi trng MRS c thit k bi Man, Rogosa and Sharpe, mi trng whey cha cc thnh phn ca sa, mi trng sa khng bo. Chun b mi trng nui cy bao gm vic a vi khun lactic vo mi trng lng v duy tr mi trng trong tnh trng hiu kh hoc k kh thch hp vi vi khun lactic c nui cy. Thc hin 8-36h ch tnh hoc khuy trn nhit 20 400C. Qu trnh x l: Hn hp Spirulina v vi khun lactic c duy tr trng thi m t hoc c gi trong nc. Thng thng hay gi hn hp trong nc. Khi phi trn hn hp, tng hiu qu nui cy vi khun lactic v tng kh nng to hng, th thch hp hn c l dng Spirulina dng huyn ph ca Spirulina sng v vi khun lactic c nui cy trong mi trng lng. Nu lng nc khng c th thm nc vo ha trn. Nc c s dng l nc qua tit trng. t hiu qu tt khi thu hoch v lm kh sau qu trnh nui cy th Spirulina cha trong hn hp c duy tr trong nc vi lng tt nht t 1-20g/100g hn hp. Hn hp c t trong trng thi nui cy tnh hoc khuy trn nh cnh khuy, nui cy hiu kh hoc k kh ty loi s dng. c th ngn chn s tp nhim ca vi khun bn ngoi, s vi khun lactic thch hp bt u cho qu trnh nui cy vi khun lactic l t 106-109 t bo/1g Spirulina.
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pH khi hn hp Spirulina v vi khun lactic c gi trong nc thay i theo tng giai on v thch hp trong khong pH t 6-8. Nhit nui cy c th l bt c nhit no m ti vi khun lactic c th pht trin. Tuy nhin, nhit tt nht cho s pht trin ca vi khun lactic v trnh s tn tht cc cu t hot ng trong Spirulina, nhit thch hp l t 20 400C. Thi gian nui cy tt nht l t 8 -24h. ngn chn hon ton s pht trin ca vi khun tp nhim, ng thi gim c mi v c trng ca Spirulina, s vi khun lactic sau nui cy thch hp l tng t 80 300 ln so vi lng vi khun bt u nui cy. duy tr mt pH ti u cho s pht trin ca vi khun lactic, pH c th c iu chnh bng cch thm mt hp cht c bn nh amonium hydroxide. Tuy nhin, nh s hin din ca acid lactic v mt s cht c to bi vi khun lactic, pH ca huyn ph Spirulina c a v 4, y l iu mong mun gim cc vi khun tp nhim. gim thiu n mc ti a mi v c trng ca Spirulina, ngi ta nghin cu v b sung thm ng vo hn hp nui cy c ch mi v. ng c th l monosaccharide, oligosaccharide, polysaccharide. C th monosaccharides bao gm glucose, galactose, mannose, frucrose, ribose and xylose. Oligosaccharides bao gm disaccharide nh sucrose and maltose, fructooligosaccharide, xylooligosaccharide v raflinose. Polysaccharides bao gm amylose, amylopectin, cellulose, glycogen, -glucan v mucopolysaccharide. ng thch hp s dng l oligosaccharide v tt nht l galactooligosaccharide. Vic pha trn ng vo hn hp theo nhiu cch, c th Spirulina c a vo hn hp m ng c ha trn vi vi khun lactic trc , hoc c th vi khun lactic c a vo hn hp m ng pha trn vi Spirulina trc . Thch hp hn l dng ng dng rn, mc d c th dng dng dung dch bng cch ha tan ng vo nc hoc cc dng tng t. Lng ng c s dng thch hp l t 3-10g/100g hn hp. Tc dng khng khun ca acid hu c (acid lactic, acid acetic) v cc cht khng khun c sinh ra trong sut thi gian tng trng ca vi khun lm gim vi khun tp
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nhim trong huyn ph Spirulina, lm cho vi khun lactic chim u th. Hn na, hng v c sinh ra t vi khun lactic nh acetaldehyde hay diacetyl lm gim mi v c trng ca to Spirulina, cung cp mt hng v mi trong sn phm thc phm. Spirulina thu c c th c s dng trc tip m khng cn loi b bt vi khun lactic. Nu cn thit to Spirulina thu c sau qu trnh x l c th c sy kh hoc lm thnh bt. Qu trnh x l kh c thc hin n m 4-7%. V vic x l nn gi sao cho duy tr c hm lng vi khun lactic a thch. V d phng php lm kh thch hp nh sy kh, phun kh, phng php phun kh c li th hn v mt chi ph. Trong sut thi gian x l kh, hiu qu sn xut tng ln khi tng nhit ln ti a. Tuy nhin, m bo cht lng Spirulina tt v trnh lm gim s lng vi khun lactic th nhit sy thch hp l 30-700C v tt nht l 40-600C. Kt lun: Spirulina thu c sau qu trnh x l c kh nng gim thiu s lng vi khun tp nhim khng mong mun, gim mi v c trng ca to Spirulina. Hn na, to Spirulina c th c kt hp vi vi khun lactic to hn hp vi nhiu thnh phn c ch cho sc khe. Do , Spirulina nh vy c th c s dng khng ch trong thc phm y dc, m cn trong cc sn phm thc phm ni chung, thc ung, sn phm b sung dinh dng 1.3.1.4. Gii thiu cc v d minh ha nghin cu Cc v d c s (nui cy ging vi khun), cc v d nghin cu (thc hin theo iu kin nghin cu) v cc v d so snh c a ra minh ha. V d c s: V d c s 1: Chun b ging vi khun lactic nui cy: Vi khun lactic Lactobacillus plantarum bo qun ng lnh c cy vo 10ml mi trng MRS v nui cy 300C trong 18h cung cp ging vi khun lactic. y gi l vi khun lactic nui cy 1.
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V d c s 2 n 5: Tng t nh v d c s 1, ch thay th vi khun lactic. V d 2: S dng Lactobacillus lactis Vi khun lactic nui cy 2 Vi khun lactic nui cy 3
V d 3: S dng Lactobacillus acidophilus V d 4: S dng Leuconostoc V d 5: S dng Pediococcus V d nghin cu: V d 1:
Vi khun lactic nui cy 4 Vi khun lactic nui cy 5
Spirulina c nui cy v thu hoch trong b nui cy trong 7 ngy k t ngy chiu sng lin tc bng cch s dng nh sng nhn to, sau thu hoch. Sau khi c thu hoch, Spirulina c ra bng nc mui sinh l, 20g Spirulina sau khi ra c to huyn ph trong 180ml nc mui sinh l c b sung 10g galactooligosaccharide. Vi khun lactic nui cy 1 chun b v d c s c tim vo dch huyn ph mt lng khong 5108 t bo/g trng lng kh ca Spirulina. iu chnh v pH 7.0 vi dung dch NaOH 0,1M v nui cy c lc 300C trong 24h. Sau , dch nui cy c a vo my sy phun nh v phun kh n nhit thnh phm 550C v thu c bt Spirulina. y c coi l bt Spirulina 1. V d 2 n 5: Thc hin tng t nh v d 1, ch thay th vi khun lactic. V d 2: S dng vi khun lactic nui cy 2 V d 3: S dng vi khun lactic nui cy 3 V d 4: S dng vi khun lactic nui cy 4 V d 5: S dng vi khun lactic nui cy 5 Bt Spirulina 2 Bt Spirulina 3 Bt Spirulina 4 Bt Spirulina 5
Khi thc hin v d nghin cu 1-5, s lng vi khun lactic sau khi nui cy tng ln 10-80 ln so vi s lng vi khun lactic khi bt u nui cy. V d 6: 10g bt kh Spirulina c to huyn ph trong 190ml nc mui sinh l to thnh dung dch huyn ph. Huyn ph ny c b sung 10g galactooligosaccharide . Sau dch huyn ph c tim vo dung dch vi khun lactic nui cy 1 c chun b
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Chng 1: To Spirulina trc vi hm lng 2108 t bo/g Spirulina. Dung dch c chnh pH n 7.0 bng NaOH 0,1M, qu trnh nui cy c thc hin 300C trong 24h. Sau , dch nui cy c a vo my sy phun nh v phun kh n nhit thnh phm 550C v thu c bt Spirulina. y c coi l bt Spirulina 6. V d 7 n 10: Tng t v d 6, ch thay th vi khun lactic. V d 7: S dng vi khun lactic nui cy 2 V d 8: S dng vi khun lactic nui cy 3 V d 9: S dng vi khun lactic nui cy 4 V d 10: S dng vi khun lactic nui cy 5 Bt Spirulina 7 Bt Spirulina 8 Bt Spirulina 9 Bt Spirulina 10
Khi thc hin v d nghin cu 6-10, s lng vi khun lactic sau khi nui cy tng 50-200 ln so vi s lng vi khun lactic bt u nui cy. V d so snh: V d so snh 1: Spirulina c nui cy v thu hoch trong b nui cy trong 7 ngy k t ngy chiu sng lin tc bng nh sng nhn to, sau thu hoch. Sau khi thu hoch Spirulina c ra sch bng nc mui sinh l, 20g Spirulina c to huyn ph trong 180ml nc mui sinh l. Huyn ph sau c a vo my sy phun nh v sy kh n nhit thnh phm 550C, bt Spirulina ny c dng lm mu i chng, y gi l bt Spirulina A. Kim tra mu bt t 1 n 10 v so snh vi mu bt A: Tng t bo vi khun khc vi khun lactic trong bt Spirulina t 1 n 10 v bt Spirulina A c xc nh, v v mi cng c nh gi bng cch kim tra cm quan. Cc kt qu c hin th trong bng 1.10. Xc nh tng t bo v kim tra cm quan c thc hin bng cch dng nhm 10 ngi. Xc nh s lng vi khun: Dch huyn ph c chun b bng cch dng 1g bt Spirulina trong 19ml nc mui sinh l, huyn ph c pha long thm 1 ln, 10 ln, 102 ln, 103 ln, 104 ln. Mi
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ml ca mi mu pha long c trn vi 10ml mi trng agar trn a petri, ng rn, sau nui cy 350C trong 48h. S dng mt mi trng thch xc nh c 30-300 khun lc pht trin, tng cc khun lc trn mi trng agar c xc nh. S ny c nhn ln vi pha long. nh gi v v: Bt Spirulina A c s dng nh mu i chng. Trong mi trng hp, 0,1g Spirulina c t trn li v nm th. Cch chm im v da trn cc tiu ch bn di v cng v c xc nh bng cch s dng cng thc (1.1). Gi tr cng thp hn chng t mi v c trng ca Spirulina yu hn. +3: V c trng Spirulina ging nh mu chun +2: V c trng ca Spirulina yu +1: V c trng ca Spirulina rt yu 0: Khng c v c trng ca Spirulina. Cng = (0 N0 + 1 N1 + 2 N2 + 3 N3 )/N (1.1) Trong : N l tng s chuyn gia th, N0 l s chuyn gia cho im 0, N1 l s chuyn gia cho im 1, N2 l s chuyn gia cho im 2, N3 l s chuyn gia cho im 3. nh gi mi: Bt Spirulina A c dng nh mu i chng. Trong mi trng hp, 2g bt Spirulina c t trong mt ti polyethylene, ming ti c ng v chng c rung trong 10s, sau mi trong ti c ngi, cc chuyn gia chm im da trn cc tiu ch di y, v cng mi c xc nh bng cng thc (1.1). Gi tr cng thp hn chng t mi Spirulina yu hn. +3: Mi c trng Spirulina ging nh mu chun +2: Mi c trng ca Spirulina yu +1: Mi c trng ca Spirulina rt yu 0: Khng c mi c trng ca Spirulina.
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Bng 1.10: So snh mu th nghim t 1 n 10 vi mu chun Mu 1 2 3 4 5 6 7 8 9 10 i chng STT bt Spirulina 1 2 3 4 5 6 7 8 9 10 A STT vi khun lactic nui cy 1 2 3 4 5 1 2 3 4 5 Khng S lng vi khun 0 0 0 0 0 0 0 0 0 0 4.0 104 V + 0.3 + 0.4 + 0.4 + 0.3 + 0.2 + 0.8 + 0.7 + 0.7 + 0.7 + 0.4 + 3.0 Mi + 0.3 + 0.3 + 0.3 + 0.3 + 0.2 + 0.7 + 0.7 + 0.7 + 0.6 + 0.5 + 3.0 [48] V d so snh 2: Khng b sung galactooligosaccharide. Huyn ph c chun b bng cch ly 10g bt kh Spirulina ha trong 190ml nc mui sinh l. Huyn ph c tim vo vi khun lactic nui cy 1 c chun b trc vi mt lng khong 2 108 t bo/g Spirulina. pH c chnh v 7 bng dung dch NaOH 0,1M v nui cy trong 24h 300C. Dch thu c c a vo my sy phun nh v phun kh n nhit thnh phm 550C thu c bt Spirulina, bt ny c gi l bt Spirulina B. V d so snh 3 n 5: Tng t v d 2, ch thay th vi khun lactic. V d 3: S dng vi khun lactic nui cy 2 V d 4: S dng vi khun lactic nui cy 3 V d 5: S dng vi khun lactic nui cy 4 So snh th nghim t 2 n 5: Bt Spirulina C Bt Spirulina D Bt Spirulina E
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Tng t bo vi khun khc vi khun lactic (c gi l s t bo sng) c xc nh trong bt Spirulina B n E thu c trong cc v d so snh 2 n 5, mi v v cng c nh gi bng phng php cm quan. Kt qu c hin th trong bng 1.11. Tng t nh phng php dng nh gi mi v v trong cc th nghim 1 n 10. Bng 1.11: Cc mu so snh khng b sung ng c so snh vi mu chun Mu STT STT vi khun lactic bt nui cy Spirulina 1 B 2 C 3 D 4 E Khng A ng S lng vi khun 2.0 102 3.0 102 8.0 103 1.0 103 4.0 104 V Mi
Mu so snh 2 Mu so snh 3 Mu so snh 4 Mu so snh 5 Mu chun
Khng Khng Khng Khng Khng
+ 0.8 + 0.9 + 1.2 + 1.1 + 3.0
+ 0.6 + 0.9 + 1.2 + 1.2 + 3.0 [48]
R rng rng t cc v d so snh v cc v d nghin cu 1 n 10, vic b sung ng c ch s pht trin ca t bo sng v gim bt mi v c trng. V d so snh 6: Hm lng phycocyanin c xc nh trong bt Spirulina F (v d so snh 6) thu c bng cch tit trng bt nhit cao, b sung ng galactooligosaccharide v nui cy. So snh vi bt Spirulina 6 thu c trong v d nghin cu 6 (s dng Spirulina bt m khng tit trng nhit cao) v bt Spirulina ban u khng c tit trng nhit cao. Bt Spirulina F c sn xut nh sau: 10g bt Spirulina trong 190ml nc mui sinh l v huyn ph c tit trng trong 3s 1300C, sau huyn ph c lm lnh nhanh chng n nhit phng. Huyn ph ny v sau c dng sn xut bt Spirulina bng phng php nh v d nghin cu 6. Sn phm to thnh c gi l bt Spirulina F. SVTH: Nguyn Th Minh Tm
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Hm lng phycocyanin c phn tch theo phng php ca Hip hi thc phm dinh dng v sc khe ca Nht Bn. l, 0,5g bt Spirulina c b sung vo 25ml m sodium phosphate (pH 6), to huyn ph v chit xut. Dch chit sau c ly tm v lc qua giy lc, thu supernatant. Tip theo 2ml ca supernatant c pha long vi nc thnh 50ml v o ph bc sng 560nm, 618nm v 650 nm. Hm lng phycocyanin c xc nh bng cng thc 1.2: Phycocyanin (%) = [(0,198 A618 0,0019 A560 0,133 A650) D 100]/W (1.2) Trong : A560 l hp thu 560nm, A618 l hp thu 618nm, A650 l hp thu 650nm, D l mc pha long (trong trng hp ny, 25 x 25 = 625), v W l khi lng mu (mg). Bng1.12: So snh hm lng phycocyanin gia cc mu STT bt Spirulina F 6 A Hm lng phycocyanin (%) 0.8 7.8 9.0 [48] V d so snh ny chng t bt Spirulina nui cy t to Spirulina c tit trng nhit cao c cha hm lng phycocyanin nh hn nhiu bt Spirulina thu c theo quy trnh nghin cu. Nh vy, vi cc th nghim trn ta thy vic t to trong mi trng nui cy vi khun lactic c b sung ng c tc dng gim ng k lng vi khun tp nhim v mi v c trng ca to.
V d so snh 8 V d nghin cu 6 i chng
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1.3.2. Nghin cu chit sut cc hp cht chng oxy ha t S.platensis 1.3.2.1. Gii thiu mt vi hp cht chng oxy ha v cc acid thit yu thu c t nghin cu Flavonoids: Flavonoid l nhm hp cht c nhiu tc dng sinh hc. Cc dn cht flavonoid l cht chng oxy ha do c kh nng dp tt cc gc t do nh OH, ROO (l cc yu t gy bin d, hy ha t bo, ung th, tng nhanh s lo ha). ng thi, flavonoid to phc vi cc ion kim loi nn ngn cn cc phn ng oxy ha m nhng ion l enzyme xc tc. Do , cc cht flavonoid c tc dng bo v c th, ngn nga x va ng mch, tai bin, lo ha, thoi ha gan, tn thng do bc x. Flavonoid lm bn thnh mch, c dng trong cc trng hp ri lon chc nng tnh mch, tr, ri lon tun hon vng mc Trn h tim mch, nhiu flavonoid nh quercetin, rutin, myciretinc tc dng lm tng bin co bp tim [64]. -caroten: -caroten t nhin t cc loi tri cy, rau ci c tc dng gim nguy c ca nhiu bnh ung th. c bit -caroten t Spirulina c hm lng cao gp 20 ln trong c rt. Ch cn s dng 1g Spirulina/ ngy c th gim chng bnh v mt i vi tr sp ti tui n trng. 1g Spirulina/ngy gip gim nguy c ung th vm ming ca 44% n ng c thi quen nhai tru thuc. Ngoi ra, -caroten cn c hng lot tc dng c li cho sc khe. Li ch ng k nht l kh nng hot ha mt s loi t bo min dch ca c th, c kh nng lm tng dung tch phi gip ht th su hn, tc dng gim tn thng DNA, bo v da trnh tc hi ca nh nng mt tri, h thp nguy c mc mt s loi ung th, gp phn gim nng cholesterol mu cng nh nguy c mt s bnh tim mch. -caroten cn l ngun cung cp vitamin A an ton (vitamin A liu cao c th gy ng c) v c th chuyn -caroten thnh vitamin A chm hn [60]. Vitamin A: (retinol, axerophthol...) Vitamin A c nhiu chc nng quan trng i vi c th con ngi: SVTH: Nguyn Th Minh Tm
154
Mt c cu to bi cc sc t c cha vitamin A. V vy s c mt ca vitamin A l mt phn khng th thiu i vi vic m bo th gic ca con ngi. Vitamin A kch thch qu trnh pht trin ca cc biu m nh m sng, rut v cc con ng h hp. N cng nh hng c bit n da, kch thch s lin so v phng nga cc chng bnh ca da nh trng c. Vitamin A c vai tr quan trng trong s pht trin t bo ca con ngi, l yu t khng th thiu i vi s pht trin ca phi thai v tr em. Vitamin A cn c vai tr i vi s pht trin ca xng, thiu vitamin A lm xng mm v mnh hn bnh thng. Vitamin A ko di qu trnh lo ha do lm ngn chn s pht trin ca cc gc t do. Hot ng km hm ca n vi cc gc t do cng dn n ngn chn c mt s bnh ung th [58]. -tocopherol: L hp cht c hot tnh vitamin E. C tc dng ngn cn qu trnh oxy ha cc thnh phn thit yu trong t bo, ngn cn to thnh cc sn phm oxy ha c hi, tng hp thu vitamin A qua rut, bo v vitamin A khi b thoi ha do oxy ha lm cho nng vitamin A trong t bo tng ln, chng li tc dng ca chng tha vitamin A. Ngoi ra cn c tc dng kim hm tc lo ha da [61]. Acid palmitic, acid linoleic, acid linolenic: Acid palmitic l acid bo bo ha, khng c ni i, gm 16 carbon. Acid linoleic, acid linolenic c cng nhn l acid bo thit yu. Acid linoleic (LA) gm 18 carbon, 2 ni i, thuc nhm -6 v acid linolenic 18 carbon, 3 ni i, thuc nhm -3. Cc acid bo ny l tin cht ca cc acid bo dng -3 v -6 nh acid arachidonic gm 20 carbon, 4 ni i, thuc nhm -6 v EPA gm 20 carbon, 5 ni i, thuc nhm -6; DHA gm 22 carbon, 6 ni i, thuc nhm -3 v t to ra mt lot cht c hot tnh sinh hc cao nh cc loi protaglandines, leukotrienes, thromboxanes [62].
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1.3.2.2. Gii thiu phng php chit xut ti u s dng trong nghin cu Trong nhng nm gn y, phng php chit xut CO2 siu ti hn nhn c s quan tm ngy cng tng nh mt cch thay th quan trng cc phng php chit xut dung mi truyn thng. CO2 l mt dung mi l tng chit xut mt vi loi hp cht t nhin s dng trong thc phm v n khng c hi, khng n, lun sn c v d dng loi b sau khi chit xut. Do , cht lng chit xut CO2 siu ti hn l cao hn so vi chit xut lng-lng vi dung mi hu c hoc bng hi nc chng ct v nhng phng php ny c th gy ra s bin cht do nhit hoc li dung mi c hi trong sn phm. Khi nim chit sut siu ti hn: L qu trnh phn tch mt hay mt s cht t hn hp (dc liu, hn hp nguyn liu) bng cch s dng cht lng CO2 siu ti hn nh mt dung mi. Khi trng thi ny CO2 c c tnh v tan tng t nh mt cht lng ng thi c kh nng khuch tn v nht gn vi cht kh, nh vy chng c kh nng khuch tn v ha tan nhanh cc hot cht trong nguyn liu. Gore (1891) l ngi u tin pht hin ra kh nng ha tan tt ca Naphtalen v Camphor trong CO2 lng. Sau Andrews (1875) nghin cu v c tnh ca CO2 trng thi siu ti hn. Tuy nhin, ti u nhng nm 1970 cng ngh chit xut cc hp cht t nhin bng dung mi CO2 siu ti hn (SC-CO2) mi thc s pht trin v i vo ng dng trong cng nghip thc phm, m phm, dc phm nh: loi cafein trong c ph v ch xanh, chit xut du vng en, chit polyphenol t ch xanh, loi b cholesterol trong thc phm, loi alcol trong ung, chit xut phm mu, chit xut cc hot cht chng oxy ha, chit xut tinh du, hng liu t thc vt s dng trong m phm, thc phm u im ca SC-CO2 trong chit xut hot cht t nhin: CO2 c im ti hn thp (nhit gn nh nhit phng, p sut thp) v vy cc hot cht t b oxy ho hay phn hu bi nhit v oxy ho tan, ngoi ra vn thit k h thng chit xut m bo p lc siu ti hn cng d dng hn nn kh nng ng dng cho quy m sn xut cng nghip cng thun li.
156
Sn phm sau khi chit khng cn tn d dung mi v CO2 d dng chuyn sang trng thi kh v bay hi ton b sau khi gim p sut, nhit xung di im ti hn. V vy, chit xut theo phng php ny rt ph hp i vi cc sn phm dng lm thc phm, thuc, m phm. Kh nng chit xut chn lc do:
tan ca SC-CO2 thay i khi p sut v nhit t siu ti hn thay i vy
n s ho tan chn lc cc cht khc nhau nhit , p sut tng ng. Thng thng cc tinh du d bay hi c th c chit p sut di 100bar, trong khi cc cht bo c chit p sut cao hn.
Dung mi SC-CO2 s phn cc hn khi c ho trn vi cc dung mi ph tr
phn cc nh: methanol, ethanol v vy kh nng ho tan cc hp cht s a dng hn. Tuy nhin, cc dung mi ph tr c th lm thay i im ti hn ca CO2 do trong thc nghim cn phi kho st t l dung mi ph tr thch hp t nh hng n im ti hn. Thi gian chit xut ngn: Do cht lng siu gii hn c h s khuch tn cao hn cht lng, trong khi nht thp, sc cng b mt nh nn kh nng khuch tn ca dung mi vo trong t bo nhanh hn v vy thi gian chit c rt ngn hn cht lng thng thng. Khng thay i hoc mt hng thm, mu sc t nhin ban u ca hot cht. Khng to ra mi ,v l do SC-CO2 l cht tr, khng mi v v bay hi hon ton khi thay i trng thi siu ti hn. CO2 khng n mn thit b, khng gy chy n trong qu trnh vn hnh, an ton, thn thin vi mi trng, gi thnh r, d kim, ngoi ra c th ti s dng trong thi gian di [63].
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1.3.2.3. Nghin cu m hnh ti u ha qu trnh chit tch hp cht chng oxy ha t S.platensis bng phng php CO2 siu ti hn: [56] Tm tt: Qu trnh chit tch CO2 siu ti hn ca nhng cht chng oxy ha t Spirulina platensis c ti u ha bng cch s dng phng php b mt p ng RSM (Response Surface Method). Khong 10,26g/kg dch chit t Spirulina c th thu c di iu kin ti u 480C, 20MPa trong khong 4h. Cc hot cht chng oxy ha c xc nh di iu kin ti u, xc nh bng phng php c ch qu trnh peroxide ha acid linoleic, kt qu thu c cho thy kh nng hot ng thp hn so vi BHT v Trolox nhng cao hn ng k so vi -tocopherol trong 300 pht v tr thnh tng t -tocopherol sau . Cc thnh phn ca dch chit c phn tch r hn v kt qu cho thy dch chit cha 85,1g/kg flavonoids, 77,8g/kg -caroten, 113,2g/kg vitamin A v 3,4g/kg -tocopherol. Cc acid bo ch yu cha trong dch chit bao gm acid palmitic (35,32%), acid linolenic (21,66%), v acid linoleic (20,58%). Gii thiu: Spirulina platensis l mt loi thc phm sc khe c gi tr cao vi hm lng cao v protein, vitamin, khong cht, cc acid bo cha no, zeaxanthin, myxoxanthophyll, v cng c bo co v tim nng dc phm (Li, Guo, & Li, 2003; Morist, Montesinos, Cusido, & Godia, 2001). Vic thu hi cc thnh phn hot tnh t Spirulina platensis t ra mt vn do s hin din ca dung mi hu c cn st li v s mt n nh ca dch chit. Chit xut CO2 siu ti hn (SC- CO2) l mt la chn hp dn chit tch cht lng thng thng do thn thin vi mi trng v khng d lng dung mi c hi. Hn na, qu trnh chit tch SC- CO2 c thc hin trong qu trnh chit xut cht chng oxy ha t l hng tho, cy x thm, v cc loi tho mc. Cht hot ng chng oxy ha c x l bi SC- CO2 cao hn ng k so vi cc phng php thng thng. V vy, qu trnh chit tch cht lng siu ti hn c xem l mt qu trnh rt c trin vng trong tng lai.
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Trong bi bo ny, phng php b mt p ng RSM c thng qua ti u ha iu kin v nhit , p sut, thi gian cho qu trnh chit tch SC- CO2 v t c phng trnh hi qui mong mun. Trong , cht hot ng chng oxy ha ca dch chit di iu kin ti u c xc nh bng cch c ch qu trnh peroxide ha acid linoleic. Hn na, cc thnh phn ca dch chit ny c phn tch c th gp phn rt ln vo hot ng chng oxy ha. V vy, iu ny d kin s tm c cht chng oxy ha t nhin vi hot lc rt ln t Spirulina. Nguyn liu v phng php: Nguyn liu: Spirulina platensis c cung cp bi Jiangsu Academic of Agricultural Sciences (Nanjing, Trung Quc). Butylated hydroxytoluene (BHT) t khu cng nghip ha cht Nanjing (Nanjing, Trung Quc). Linoleic acid, 2,2-azobis (2-amidinopropane) hydrochloride (AAPH) thu c t cng ty TNHH Ha cht cng nghip Wako (Osaka, Nht Bn). Rutin, vitamin A, -caroten, -tocopherol, 6-hydroxyl-2,5,7,8tetramethylchroman-2-carboxylicacid (Trolox) c mua t cng ty ha cht Sigma (ST.Louis, MO, USA). Ti u ha qui trnh: Qui trnh tch chit c ti u ha bi Box-Behnken thu c hiu sut cht chng oxy ha t Spirulina cao hn. Cc yu t v mc kho st trong nghin cu c th hin trn bng 1.13. Thit k th nghim, phn tch d liu, xy dng m hnh bc 2 c tin hnh bng cch s dng phn mm Design Expert (Phin bn 6.0.5, StatEase Inc., Minneapolis, MN, USA).
159
Bng 1.13: Mc m ha th nghim ca cc yu t dng trong thit k BoxBehnken Yu t Nhit ( C) p sut (MPa) Thi gian (h)
0
K hiu A B C -1 32 20 2
Mc m ha 0 40 30 3
1 48 40 4 [56]
Chit sut cht chng oxy ha: Chit xut cht lng siu ti hn c thc hin trn bnh chit cht lng siu ti hn Huaan (cng ty TNHH Huaan, Nantong, Trung Quc) vi dung tch bnh chit 1lt. Biu dng chy ca qu trnh chit cht lng siu ti hn c th hin Hnh 1.4. Cc b phn chnh ca thit b bao gm bnh chit p sut cao v 2 bnh tch. T l dng chy ca kh CO2, nhit v p sut chit tch c gim st bi cc ng h trn mt trc, thi gian tch chit c thit lp bng cch hn gi. CO2 c cung cp t mt xylanh kh. Trc khi n c a vo b cha dch chit c y mu bi my bm, CO2 c iu p n p sut mong mun v un nng n nhit qui nh t c trng thi siu ti hn. Ethanol tuyt i hot ng nh mt cht ng dung mi c b sung vo mt bnh phun hi v ha trn vi mu trong bnh chit bi mt my bm khc. H thng ny c n nh trong 2h di iu kin trn trc khi bt u th nghim. Dch chit chit xut di diu kin ti u c thu nhn phn tch hot tnh chng oxy ha v thnh phn.
160
Hnh 1.4: S ca qu trnh chit SC-CO2 [56] 1.Xylanh kh t; 2. B phn lc; 3. B phn lm mt; 4. My nn; 5. B phn to nhit trc; 6. My chit; 7,8. B phn phn tch; 9. Lu lng k; 10. p k o p sut dng chy; 11. ng h ch th p sut. Xc nh hot cht chng oxy ha: Hot cht chng oxy ha trong dch chit t S.platensis c xc nh bi h thng acid linoleic (Huang, Mendis, & Kim, 2005; Takeshi, Mizuho, Reiji, Hachiro, & Nobutaka, 2001). Mu gm: 1ml ca 5mg cht chit ha tan trong ethanol nguyn cht, 4ml dung dch m Natri phosphate 0,05M (pH = 7). Mu i chng gm: 1ml ethanol nguyn cht v 2ml nc ct c trn vi 2ml acid linoleic 2,5%(v/v) trong ethanol. Qu trnh peroxide ha c bt u bng cch b sung 0,4ml AAPH 0,1M v c thc hin 370C trong bng ti. Cc mc oxy ha c o bng cch c mc hp thu bc sng 500nm sau khi nhum mu vi FeCl2 v ammonium thiocyanate (NH4SCN) (Kikuzaki & Nakatani, 1993). BHT, -tocopherol, Trolox c dng nh cht chun so snh.
161
Phn tch cc thnh phn ca dch chit: Xc nh hm lng flavonoids: Hm lng flavonoids trong dch chit t S.platensis c o bng phng php to phc mu vi AlCl3 da trn s hnh thnh phc hp Al-flavonoids (Djeridane et al., 2006). Dch chit c pha long vi ethanol nguyn cht ti mt nng thch hp. Sau 1ml ca mu sau pha long c trn vi 1ml ca dung dch methanolic 2% (w/v) ca AlCl3. Sau khi nhit phng 15 pht, o hp thu ca hn hp bc sng 430nm bng my quang ph. Rutin c s dng nh mt cht chun. nh lng -caroten, vitamin A v -tocopherol: -caroten trong dch chit c kim sot bng h thng HPLC (Cng ty Shimadzu, Kyoto, Nht Bn). c trang b vi DGU-14 A Degasser, LC-10AT quaternary pump, v SPD-10AV UVVisible pht hin 450 nm. Qu trnh phn tch c thc hin trn ct Zorbax SB-C18, 150mm 4,6mm, kch thc ht 5m (Agilent Technologies, Wilmington, DE, USA) (Zhang & Omaye, 2001). Pha ng bao gm acetonitrile/methylene chloride vi t l 7:3 (v/v), tc chy 1ml/pht. Vitamin A v -tocopherol trong dch chit ng thi cng c xc nh bng h thng phn tch HPLC (Zhang & Omaye, 2001). Pha ng s dng dung mi methanol, tc 1ml/pht. Vitamin A v -tocopherol c pht hin bc sng tng ng 325 v 292 nm. Phn tch mu acid bo: Mu acid bo trong dch chit c phn tch bng phng php GCMS (Hartvigsen, Hansen, Lund, Bukhave, & Hlmer, 2000). u tin, mu c to FAME (Fat Acid Methyl Esters) vi MeOH, xc tc acid sunfuric m c. Tip , mu c tim vo h thng GC 2500C. Nhit l c ci t 1800C trong 1 pht sau tng ln 2300C vi 100C/1 pht. Nhit cui cng c duy tr trong 3 pht. Sau , cc thnh phn c xc nh bng cch phn tch ph khi lng. Vic phn tch cc thnh phn c so snh vi tiu chun ca vin Quc Gia M v th vin cng ngh (NIST).
162
Kt qu v tho lun: Ti u ha qui trnh: Box-Behnken thit k ma trn ca cc yu t c a ra trong bng 1.14 cng vi cc th nghim v d on gi tr hiu sut ca dch chit t S.platensis. Bng cch p dng nhiu phng php phn tch hi quy, phng trnh hi quy c th thu c v a ra nh phng trnh 1.3. Y = 7.02900 0.26063A 0.011275B 1.14775C + 0.0032266A2 + 0.00059000B2 + 0.13400C2 0.00065625AB + 0.011563AC 0.0010000BC Trong Y l sn lng d on ca dch chit t S.platensis. Bng 1.14: Ma trn thit k bi Box-Behnken cng vi th nghim v d on gi tr hiu sut ca dch chit. STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Nhit (0C) -1 1 -1 1 -1 1 -1 1 0 0 0 0 0 0 0 0 0 psut (MPa) -1 -1 1 1 0 0 0 0 -1 1 -1 1 0 0 0 0 0 Thi gian (h) 0 0 0 0 -1 -1 1 1 -1 -1 1 1 0 0 0 0 0 Sn lng (g/kg) Thc nghim D on 3.96 4.00 6.98 6.68 3.99 4.28 4.93 4.89 4.79 4.23 4.89 4.66 4.67 4.90 8.53 9.09 3.74 4.26 2.97 3.23 5.74 5.48 4.49 3.97 2.31 2.33 2.31 2.06 2.31 1.98 2.31 2.50 2.31 2.69 [56]
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(1.3)
Bng vic gii quyt cc ma trn nghch o vi phn mm Design Expert, mc ti u ca cc yu t th nghim l 480C, 20MPa, v hn 4h, di nhng iu kin ny d on sn lng ti a ca dch chit t S.platensis l khong 10,26g/kg. Tnh ph hp ca m hnh c kim chng bng thc nghim. Cc th nghim xc minh c thc hin di s kt hp khc nhau ca cc tham s trong qui trnh c to ra bi phn mm Design Expert. Cc h s tng quan (R) gia thc nghim v d on gi tr l 0,9882, iu ny cho thy rng gi tr th nghim v gi tr d on l tng thch v cng cho thy rng m hnh l tha ng v chnh xc. Hot cht chng oxy ha ca dch chit: Hot cht chng oxy ha ca dch chit t S. platensis c kho nghim bng phng php c ch qu trnh peroxide ha ca acid linolenic v kt qu c th hin trong hnh 1.5. Kh nng hp th cao l th hin mc cao ca qu trnh peroxide ha acid linoleic v hot ng chng oxy ha thp ca cc cht chng oxy ha. Hot ng ca cht chit t S.platensis c so snh l thp hn so vi BHT v Trolox nhng cao hn ng k so vi -tocopherol trong 300 pht v tr thnh tng t sau . Hot ng chng oxy ha ca cht chit xut t S.platensis v cht chng oxy ha chun gim theo th t: Trolox > BHT > cht chit > -tocopherol. Tuy nhin, phn tch thng k ch ra khng c s khc bit ng k gia Trolox v BHT. V m hnh ny c xem nh l tng t vi BHT > Trolox > -tocopherol c bo co bi Dubuisson, Gulcin v Hu (Dubuisson et al., 2001; Gulcin, Mshvildadze, Gepdiremen, & Elias, 2006; Hu, Xu, Chen, & Yang, 2004).
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Hnh 1.5: Hot cht chng oxy ha c nh gi bng phng php c ch qu trnh peroxide ha ca acid linoleic [56]
Cc thnh phn ca dch chit: Acid bo c phn tch bng GC-MS, acid palmitic (35,32%), acid linolenic (21,66%), v acid linoleic (20,58%) l nhng thnh phn chnh hin din (Bng 1.15). Hn na, cc thnh phn m c th ng gp to ln cho hot ng chng oxy ha cng c thm d phn tch, kt qu l 85,1g/kg flavonoids, 77,8 g/kg -caroten, 113,2g/kg vitamin A, 3,4g/kg -tocopherol cha trong dch chit t S.platensis. Bng 1.16.
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Bng 1.15: Thnh phn v hm lng tng i ca acid bo trong dch chit t S. platensis Thi gian gi li Thnh phn Cng thc phn Hm lng ca GC-MS (min) t tng i (%) C12H18O 2,6-Diisopropylphenol 1.71 4.13 Pentadecanoic acid 1.16 4.97 C15H30O2 Palmitic acid 35.52 6.75 C16H30O2 1.57 6.87 Zoomaric acid 1.74 7.06 C16H30O2 Zoomaric acid 5.96 7.15 C16H30O2 0.83 9.05 Stearic acid 2.02 9.84 C18H36O2 Oleic acid 2.81 10.21 C18H34O2 Oleic acid 2.70 10.32 C18H34O2 Linoleic acid 1.28 10.65 C18H32O2 Linoleic acid 18.40 10.99 C18H32O2 Linolenic acid 21.66 11.50 C18H30O2 Linoleic acid 0.90 12.39 C18H32O2 0.69 12.86 1.06 16.88 ' - ' Thnh phn cha c nghin cu trong th vin NIST [56]
Bng 1.16: Nhng thnh phn c th ng gp trong cht hot ng chng oxy ha t S.platensis Thnh phn Flavonoids -caroten Vitamin A -Tocopherol Hm lng (g/kg) 85.1 7.3 77.8 6.8 113.2 2.7 3.4 0.3 [56]
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Kt lun: Dch chit t Spirulina platensis c nghin cu cha nhiu thnh phn hot ng c ch cho sc khe, ngn nga hoc c ch ung th, thc y min dch... V vy, vic nghin cu chit tch v ti u ha quy trnh chit tch nng cao hiu sut ang c nhiu nh khoa hc quan tm. Ngoi cc cht chng oxy ha v acid thit yu cha trong to, mt s nghin cu khc cng tm thy hot tnh khng khun ca dch chit, chng c th nghim v nhn thy c th chng li bn loi vi khun khc nhau: vi khun gram dng (Staphylococcus aureus), vi khun gram m (E.coli), nm men (Candida albicans) v nm mc (Aspergillus niger) [47]. Nh vy, dch chit t to Spirulina ngy cng c ng dng rng ri trong ngnh thc phm. 1.3.3. Nghin cu nh hng ca sinh khi Spirulina platensis ln h vi khun ca sa ln men ABT trong sut thi gian bo qun: [55] Tm tt: Mc tiu ca nghin cu ny l nghin cu tc ng ca sinh khi vi khun lam (S.platensis) ln sn phm sa ln men probiotic trong thi gian lu tr hai nhit khc nhau. Sa ln men ABT b sung Spirulina c sn xut bng cch s dng qu trnh ln men nhanh chng vi mi (ABT) l h vi khun Lactobacillus acidophilus (A), Bifidobacteria (B), v Streptococcus thermophilus (T). Nui cy trong 6h 400C. i vi cc sn phm b sung vi to lam, sinh khi S.platensis c thm vo sa v khuy trn u pH 4,5 4,6. Sau sa ln men ABT c lm lnh n 250C ri y vo cc ng ly tm y kn np c v trng, tip tc c lm lnh 40C trong 24h, sau c lu tr 150C trong 18 ngy hoc 40C trong 42 ngy. Phn tch vi sinh v o chua c thc hin nh k. Sinh khi to S.platensis tc ng c li n s tn ti ca vi khun mi ABT bt k nhit lu tr. S acid ha xy ra 150C, trong khi pH vn gi n nh trong sut qu trnh lu tr 40C. S phong ph ca cc cht c hot tnh trong S.platensis c tm SVTH: Nguyn Th Minh Tm
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quan trng rt ln v gi tr dinh dng, do cc sinh khi ca vi to cung cp mt c hi mi trong sn xut cc sn phm sa chc nng. Gii thiu: Bidobacteria v mt s loi Lactobacillus gn y nhn c s ch nh nhng vi khun probiotic, duy tr s cn bng lnh mnh gia h vi sinh vt c li v c hi trong ng tiu ha. Chng lin kt v tc ng tch cc n sc khe, do , c kt hp vo mt lot cc sn phm sa. Thuc tnh probiotic ca Bifidobacterium spp. v Lactobacillus acidophilus bao gm ngn nga v gim bt tiu chy (Alm, 1997), trnh s ly nhim rota virus tr s sinh (Saavedra et al., 1994), phng chng to bn ngi cao tui (Alm, 1991), ci thin s dung np lactose (Lin et al, 1991), gim mc cholesterol trong mu (Agerbaek et al.,1995), kch thch h thng min dch (Schiffirin et al.,1995) v ci thin trong vic bo v chng li ung th (Krishnakumar v Gordon, 2001). Cc nh khoa hc tin tng rng ngi tiu dng s khng cn phi tiu th nhiu thuc, vitamin nhn to, khong b sung nu qu trnh ln men sa c lm giu vi cc vitamin, protein, acid bo thit yu v nguyn t vi lng c ngun gc t nhin. Mt cch n gin t c iu ny l s dng vi khun lam trong sn xut cc sn phm sa (Varge Szigeti, 1998). Vi khun lam thuc to prokaryote, c s lin quan cht ch n vi khun hn cc loi to eurkaryote. S.platensis l mt vi to ph du hnh thnh cc qun th ln trong cc b nc. Sinh khi kh ca S.platensis c m t 3-7%, 55-60% protein, 6-8% lipid, 12-20% carbohydrate, 7-10% cht tro, 8-10% cht x, 1-1,5% cht dip lc a v nhiu loi vitamin. S.platensis c bit giu protein. Protein c tim nng kinh t cao nht l biliprotein (v d c-phycocyanin, allophycocyanin), l sc t xanh khi ha tan trong nc. Protein c cha hm lng phycocyanin ln n 20%. Thnh phn acid bo chu nh hng ch yu ca iu kin mi trng. S.platensis c c trng bi 45-50% acid bo
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bo ha v 50-55% acid bo cha bo ha, cha n 30% ca acid bo l acid -linolenic, mt acid bo cha no rt tt v c xem l c thuc tnh ca thuc. T nhng nm 1970, S.platensis c bn trn th trng v tiu th nh l mt ngun thc phm an ton cho con ngi v c chp nhn l ngun dinh dng cho con ngi bi chnh ph, c quan y t v hip hi 80 nc gm c M v Hungary. Da trn cc tiu ch nghin cu v an ton v cht lng, hn 70% th trng hin nay, Spirulina l dnh cho con ngi, ch yu l thc phm sc khe. Tng s sn phm sn xut hng nm ca sn phm b sung sinh khi Spirulina c c tnh khong 10001500 tn (Belay, 1997). Nhiu t chc trn th gii ang nghin cu m bo mt sn phm probiotic c th cung cp s lng ln vi khun c ch nhm m bo li ch cho khch hng. Nng t nht ti thi im tiu th 106-107 CFU/g. Quy nh thc phm Hungary quy nh rng sa ln men probiotic phi c v cho n ht hn s dng, s lng vi khun c ch phi trong khong 107CFU/g sn phm. Mc ch ca nghin cu ny l iu tra tc ng ca sinh khi vi khun lam ln c tnh ca h vi khun v vi sinh vt gy h hng sa ln men ABT trong sut thi gian lu tr 150C v 40C. Nguyn liu v phng php: Nguyn liu: Sa tit trng thng mi (4lt), mi lt cha 36g cht bo, 34g protein, 47g lactose v 7g cht tro, trong b sung thm 10g bt sa tch bo. Nguyn liu th ny c o lng sang 2 l (2lt), c un nng n 900C v trong 10 pht trc khi lm lnh v nhit cy mm nhm m bo bin tnh protein trong dch sa (Kessler, 1988).
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Nui cy vi khun mi: ABT c nui cy trong b ng kh DVS c thit lp bi hc vin nghin cu ngnh sa ca Hungary. N dng cy mm trc tip trong ch bin sa v nui cy DVS khng cn phi hot ha hoc x l trc khi s dng. Sinh khi S.platensis: Sinh khi S.platensis c thu t vin ch bin ng cc (Bergholz-Rehbrucke, c). Mi kg cha 576g protein, 111g lipid tng, 114g cht tro. Nghin cu trc y, 3g/l sinh khi vi khun lam c b sung l tt nht v thuc tnh cm quan v chi ph. Sn xut v lu tr ca sa ln men ABT: ABT sau nui cy c b sung vo sa x l nhit c lm lnh xung 420C vi t l 0,2 U/L, tng ng vi 2% (v/v). Nui cy trong 6h 400C. Trong trng hp sn phm cha vi khun lam, sinh khi S.platensis c thm vo sa (2lt) v khuy trn lin tc pH 4,5 4,6. Sau , hn hp sa ln men ABT c b sung Spirulina c lm lnh xung 250C trong nc ri y vo 40 ng ly tm (50ml) y kn np c v trng. Do , c tt c 80 n v sn phm. Sau 24h lm lnh 40C, mt na s mu c t vo ni lm mt v si m 150C, mt na khc lu tr trong iu kin lnh 40C. Quy trnh th nghim c lp li 3 ln. Phn tch vi sinh: Ba mu cha vi khun lam v 3 mu thng thng c thc hin ti mi thi im ly mu, tc l, sau 0, 3, 6, 9, 12, 15 v 18 ngy lu tr 150C v sau 0, 7, 14, 21, 28, 35, 42 ngy lu tr 40C. Mu c v trng loi b khi ng ly tm, pha long mu bng cch trn 10ml mu vi 90ml dung dch peptone 0,1%. Mu c th pha long thm nu cn thit. Phng php a c s dng m vi sinh vt, ngoi tr coliforms v E.coli, phng php nh lng MPN c s dng xc nh hai loi ny.
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Streptococcus thermophilus: Thch M17 c dng m S.thermophilus. pH mi trng l 6,9 0,1. Cc a cy c 370C trong 48h iu kin hiu kh. S.thermophilus hnh thnh cc khun lc vi ng knh 1-2mm, cc n v hnh thnh khun lc (CFU), c th hin di dng log CFU/ml bo co s tn ti ca Streptococcus. Lactobacillus acidophilus: Mi trng thch MRS-maltose vi pH 6,2 0,1 c s dng m Lactobacillus acidophilus. Cc a c 370C trong 72h. Bnh nui cy k kh 2,5 lt c s dng to iu kin k kh, oxy kh quyn c hp th bi cc ti AnaeroGen AN 25. Tng s c th hin di dng log CFU/ml. Lactobacilli c xc nh trn cc loi khun lc c s c xc nhn bng knh hin vi. Lactobacillus acidophilus l vi khun gram dng, dng que vi ui trn. Bidobacterium spp: Acid nalidixic (0,030g), neomycin sulfate (0.200g), lithium chloride (0,600g), paromomycin sulfate (0,250g). Tt c c thu t Sigma Chemical Co. (St. Louis, MO), c to huyn ph trong nc ct (100ml), sau c v trng bng cch lc qua thit b lc Milipore (Millipore Corporation, Bedford, MA) vi ng knh l 0,22m. pH ca dung dch c iu chnh n 7,3 0,1 bng NaOH 0,1M trc khi kh trng. 5ml ca dch khng sinh ny c b sung vo 100ml MRS- agar (pH 6,2 0,1) trc khi s dng. Cc mi trng nui cy nh vy c s dng m Bifidobacteria. Cc a c 370C trong 5 ngy. iu kin k kh c to ra bng cch dng bnh nui cy k kh vi cc ti AnaeroGen AN 25. Tng s c th hin di dng log CFU/ml. Cc khun lc Bifidobacteria c nhn dng vi hnh dng bt thng dng ch V, Y hoc hnh ht u v c chng thc bng cch quan st di knh hin vi.
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Nm men v nm mc: YGC agar (Merck KGaA,DarmStadt, c), c s dng m nm men v nm mc. pH ca mi trng 6,6 0,1. Cc a nui cy c hiu kh 250C trong 5 ngy. Coliforms v E.coli: Phng php MPM c dng xc nh coliforms v E.coli. Mi trng Brilliant Green Bile 2% c b sung vi tryptophane v 4-methylumbelliferyl-beta-Dglucuronide c phn phi vo ng nghim ca Durham. pH ca dch 7,2 0,1. Cc ng th nghim c 370C trong 24-48h di iu kin hiu kh. Trong th nghim ny, s hin din ca coliforms c pht hin bi s hnh thnh bong bng kh trong cc ng Durman. Cc ng dng tnh vi coliforms c kim tra di nh sng UV (366nm). Thuc th Kovacs indole c b sung vo ng nghim pht hunh quang di nh sng UV v s hin din ca E.coli c xc nhn bi s xut hin cc vng trn sau 1 n 2 pht. Cc mu nui cy th nghim c so snh vi mu i chng. o acid: Gi tr pH c o nhit phng vi dng c o pH HI 8521 v kt hp vi cc in cc (Hanna Instruments Deutschland GmbH, Karlsruhe, c) c chun ha vi dung dch m pH chun 4 v 7. Chun chua: Mu 20ml c chun vi NaOH 0,1N, dng phenolphtalein l cht ch th, chua th hin di dng Soxhlet-Henkel (0SH). Kt qu c th hin di dng % ca acid lactic. Phn tch thng k: Tc ng ca sinh khi S.platensis trn h vi khun c trng, pH v chua ca sa ln men ABT trong thi gian lu tr c phn tch bi phn mm STATISTICA 4.5. Kt qu ca s khc bit c thnh lp P < 0,05 trong mi trng hp.
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Kt qu v tho lun: Lu tr 150C: S tn ti ca h vi khun c trng trong mu sa ln men ABT lu tr 150C c minh ha trong bng 1.17 v 1.18. Streptococcus thermophilus l thnh phn c s lng nhiu nht, tng gi tr ca n vt qu 109 CFU/ml vo u thi gian lu tr. Pht hin ny l ph hp vi c tnh c a ra bi nh sn xut cho nui cy mi ABT. Mu 0 ngy, tng s S. thermophilus trong sa ln men ABT b sung sinh khi S.platensis cao hn so vi mu thng thng, iu chng t tc dng kch thch ca sinh khi vi khun lam ln s pht trin vi khun mi hnh cu (Varga et al, 1999). Sau 3 ngy nhn thy c s tng ln trong tng s S. thermophilus trong c sn phm thng thng v sn phm b sung Spirulina nhng cng v sau quan st thy c xu hng gim xung. Tuy nhin, tng Streptococci c tm thy trong phm vi 109 CFU/ml sau 18 ngy bo qun 150C. S khc bit gia Streptococci trong mu cha vi khun lam v mu thng thng l c s tng ln ca Streptococci trong mu cha vi khun lam sut thi gian lu tr. T l phn trm kh nng tn ti ca L. acidophilus trong sut thi gian bo qun 150C cng tng t nh S. thermophilus. Tuy nhin, cn lu rng tng s L. acidophilus ban u thp hn ca Streptococcus. Sau khi tng nh tng s t bo tn ti trong sut 3 ngy lu tr u tin, tip tc quan st thy c s gim dn. Medina v Jordano (1995) thy rng mt sinh vt tn ti trong sa chua probiotic ban u tng ln trong qu trnh sn xut, t ti a v ri gim xung trong sn phm trong sut thi gian lu tr lnh. Qui nh thc phm Hungari yu cu cc sn phm sa ln men c cha vi khun lactic c nng ban u t nht l 107 CFU/g.
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Bng 1.17: S lng tn ti (log CFU/ml) ca Streptococcus thermophilus, Lactobacillus acidophilus, v Bidobacterium spp. trong sa ln men b sung Spirulina v ln men thng thng trong thi gian bo qun 15C. Thi gian lu tr (ngy) 0 3 6 9 12 15 18 S. thermophilus B sung Bnh Spirulina thng 9.170.10 9.030.06 9.450.15 9.260.18 9.350.12 9.150.11 9.300.13 9.020.03 9.280.11 9.060.14 9.230.13 9.050.11 9.270.08 8.990.15 L. acidophilus B sung Bnh Spirulina thng 7.380.07 7.110.08 7.490.07 7.270.17 7.350.08 7.200.14 7.340.12 7.190.15 7.390.19 7.180.13 7.320.11 7.150.13 7.300.06 7.090.16 Bifidobacterium spp. B sung Bnh Spirulina thng 6.190.09 6.360.08 5.640.15 5.950.12 5.360.14 5.650.09 5.170.03 5.410.12 5.220.07 5.370.04 5.150.03 5.310.08 5.130.08 5.330.14 [55]
Bng 1.18: T l % tn ti ca Streptococcus thermophilus, Lactobacillus acidophilus, v Bidobacterium spp. trong sa ln men b sung Spirulina v ln men thng thng trong thi gian bo qun 15C. Thi gian lu tr (ngy) 0 3 6 9 12 15 18 S. thermophilus B sung Bnh Spirulina thng 100.00 100.00 169.82 190.55 131.83 151.36 97.72 134.90 107.15 128.82 104.71 114.82 91.20 125.89 L. acidophilus B sung Bnh Spirulina thng 100.00 100.00 144.54 128.82 123.03 93.33 120.23 91.20 117.49 102.33 109.65 87.10 95.50 83.18 Bifidobacterium spp. B sung Bnh Spirulina thng 100.00 100.00 28.18 38.90 14.79 19.50 9.55 11.22 10.72 10.23 9.12 8.91 8.71 9.33 [55]
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Theo nhng bo co trc y (Chr.Hansen A/S, 1995; Medina and Jordano, 1995). Ban u tng s Bidobacterium spp. dao ng trong khong 1106 n 5106 CFU/ml. Gim s lng tn ti ca Bifidobacteria l r rt hn so vi hai vi khun lactic kia. Ch c 10% c gi li sau 9 ngy bo qun, sau khng c s gim thm ng k mt Bifidobacteria trong 9 ngy tip theo. Mu cha vi khun lam c tng s Bifidobacteria cao hn so vi mu thng thng cui thi gian bo qun. S tn ti ca Bifidobacteria trong cc sn phm sa ln men l mt iu ng quan tm. S tn ti ca Bidobacterium spp. iu kin pH thp (3,7 4,3) c nghin cu bi Lankaputhra et al.(1996). Hn 50% cc chng c kim tra mt kh nng tn ti sau 6 ngy lu tr pH 4,3, tuy nhin mt s chng sng tt sau 6 tun lu tr tt c gi tr pH nghin cu. S tn ti ban u cao ca Bifidobacteria (>108 CFU/g) c tc dng bo v chng li nhng acid gy hi n vi sinh vt. Sa ln men c c trng bi mc oxy thp, chua cao, v sn xut ra cc hp cht khng sinh nh vi khun mi. V vy, mt s vi sinh vt gy bnh hay gy h hng khng th tn ti trong sn phm ny (Northolt, 1983). Khng c s tng trng ca nm men, nm mc, vi khun coliform hay E.coli trong sa ln men ABT b sung Spirulina hoc trong sa thng thng trong sut thi gian lu tr, mc d cc sn phm c sn xut di iu kin phng th nghim v mu c lu tr ti mt nhit tng i cao. pH ca sn phm ln men c th gim ng k trong sut thi gian lu tr, c th nh hng n s sinh trng v s sng cn ca L. acidophilus v Bidobacterium spp., s tng trng ca Bidobacteria chm ng k pH di 5,0, y l iu cn ch i vi tng s Bidobacteria so vi 2 vi khun mi kia. Bng 1.19 th hin s thay i pH v chua ca sn phm sa ln men ABT trong thi gian lu tr 150C.
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Bng 1.19: pH v chua ca sn phm sa ln men b sung Spirulina v ln men thng thng trong thi gian lu tr 150C. Thi gian lu tr (ngy) 0 3 6 9 12 15 18 pH B sung Spirulina 4.33 0.01 4.20 0.01 4.13 0.01 4.07 0.02 4.06 0.01 4.05 0.01 4.04 0.01 Bnh thng 4.28 0.01 4.18 0.01 4.11 0.01 4.10 0.01 4.08 0.01 4.07 0.01 4.06 0.01 chua B sung Bnh thng Spirulina 0.96 0.01 0.98 0.02 1.17 0.01 1.22 0.01 1.23 0.01 1.26 0.01 1.25 0.01 1.28 0.01 1.26 0.01 1.30 0.01 1.26 0.01 1.31 0.01 1.27 0.01 1.31 0.02 [55] pH ca mu vi khun lam cao hn so vi mu thng thng vo lc bt u lu tr. Thc t sinh khi S.platensis c tnh kim v n ch b sung vo sa trong giai on u v khuy trn lin tc pH 4,5 4,6. Tuy nhin, s acid ha xy ra v nhit lu tr cao v v sinh khi vi khun lam c tc ng kch thch s sn sinh acid. Khong 0,3% acid lactic v acid acetic c hnh thnh trong 18 ngy lu tr 150C v hm lng trong mu b sung Spirulina nhiu hn so vi mu thng thng. Do , pH ca sn phm ln men c b sung Spirulina thp hn sa ln men thng thng vo cui thi gian lu tr. Lu tr 40C: Bng 1.20 v 1.21 minh ha cho s tn ti ca h vi sinh vt c trng trong sa ln men ABT trong sut thi gian lu tr 40C. Hu nh khng c hin tng gim s lng tn ti ca S. thermophilus trong 4 tun u tin khi lu tr 40C, nhng sau quan st thy s st gim ng k ca S. thermophilus, mu vi khun lam b tc ng t hn so vi mu thng thng. Sn phm b sung Spirulina c tng s S. thermophilus cao hn so vi sa ln men thng thng sau 6 tun bo qun lnh.
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Bng 1.20: S lng tn ti (logCFU/ml) ca Streptococcus thermophilus, Lactobacillus acidophilus, v Bidobacterium spp. trong sa ln men b sung Spirulina v ln men thng thng trong thi gian bo qun 4C. Thi gian lu tr (ngy) 0 7 14 21 28 35 42 S. thermophilus B sung Bnh Spirulina thng 9.170.10 9.030.06 9.280.06 9.120.16 9.210.12 9.130.08 9.210.12 9.110.03 9.110.07 9.010.06 9.050.18 8.850.14 8.860.16 8.640.18 L. acidophilus B sung Bnh Spirulina thng 7.380.07 7.110.08 7.480.08 7.250.11 7.420.10 7.280.11 7.400.11 7.250.13 7.380.15 7.200.09 7.350.07 7.010.14 7.310.13 7.000.17 Bifidobacterium spp. B sung Bnh Spirulina thng 6.360.08 6.190.09 6.420.06 6.300.06 6.340.03 6.170.08 5.880.05 5.820.06 5.910.09 5.730.10 5.230.09 5.070.03 4.860.05 4.690.08 [55]
Bng 1.21: T l % tn ti ca Streptococcus thermophilus, Lactobacillus acidophilus, v Bidobacterium spp. trong sa ln men b sung Spirulina v ln men thng thng trong thi gian bo qun 4C. Thi gian lu tr (ngy) 0 7 14 21 28 35 42 S. thermophilus B sung Bnh Spirulina thng 100.00 100.00 123.03 128.82 125.89 109.65 120.23 109.65 95.50 87.10 66.07 75.86 40.74 49.98 L. acidophilus B sung Bnh Spirulina thng 100.00 100.00 138.04 125.89 147.91 109.65 138.04 104.71 123.03 100.00 79.43 93.33 77.62 85.11 Bifidobacterium spp. B sung Bnh Spirulina thng 100.00 100.00 128.82 114.82 95.50 95.50 42.66 33.11 34.67 35.48 7.59 7.41 3.16 3.16 [55]
177
Robinson (1987) bo co v s tn ti ca L. acidophilus trong yogurt acidophilus. Cc sn phm c kim tra cho cc gi tr 9,5 106; 7,6 106 v 4,0 106 CFU/ml tng ng gi tr th nghim ban u v sau 7, 14 ngy lu tr lnh. Trong nghin cu ny, tng t vi nhng g th nghim vi S. thermophilus, khng c s tn tht v kh nng tn ti ca Lactobacilli xy ra trong 28 ngy u tin bo qun lnh. Sau , tip tc quan st thy c s st gim. S lng tn ti ca Lactobacilli c gi li trong sn phm b sung Spirulina l cao hn sn phm thng thng trong cng giai on. Tng s L. acidophilus t gi tr yu cu 107 CFU/ml mi thi gian ly mu, do , c tim nng cung cp y li ch sc khe cho n 42 ngy lu tr lnh. Reuter (1989) ni rng, chua thch hp, s nh hng ca oxy v nhit lnh, khng phi l vn cho s tn ti Bifidobacteria ngay c sau 4 tun. Trong th nghim ny, trn 95% kh nng tn ti ca Bifidobacteria c gi li sau 14 ngy lu tr 40C. Tuy nhin, s gim mt Bifidobacteria c quan st trong 4 tun sau , c bit l gia 28 v 35 ngy. Khng c s khc bit ng k gia mu b sung v mu thng thng v mt ny. Tuy nhin, trong ton b thi gian lu tr, s lng Bifidobacteria tn ti trong sa ln men ABT b sung Spirulina vn cao hn so vi mu thng thng. Tm quan trng ca vic lu tr nhit thp c lm sng t v thc t rng s tn ti ca Bifidobacteria sau 28 ngy lu tr 40C l tng t kt qu thu c sau 3 ngy 150C. Khng c s tng trng ca nm men, nm mc, coliforms, E.coli pht hin trong mu bt k, iu cho thy sinh khi S.platensis c tim nng v thuc tnh khng sinh. Bng 1.22 cho thy s thay i chua v pH ca sa ln men ABT trong sut thi gian lu tr lnh 40C.
178
Bng 1.22: pH v chua ca sn phm sa ln men b sung Spirulina v ln men thng thng trong thi gian lu tr 40C. Thi gian lu pH chua tr (ngy) B sung Bnh thng B sung Bnh thng Spirulina Spirulina 0.96 0.01 0.98 0.02 4.28 0.01 4.33 0.01 0 1.11 0.05 1.13 0.03 4.28 0.02 4.30 0.01 7 1.14 0.01 1.17 0.01 4.26 0.01 4.27 0.01 14 1.13 0.01 1.19 0.01 4.24 0.01 4.23 0.01 21 1.17 0.01 1.19 0.01 4.25 0.01 4.24 0.02 28 1.18 0.01 1.20 0.01 4.26 0.01 4.25 0.01 35 1.19 0.01 1.21 0.01 4.23 0.01 4.24 0.01 42 [55] Khng c s acid ha xy ra nhit ny. C s st gim pH t hn 0,1 n v trong sn phm c b sung Spirulina sut 42 ngy lu tr, cn pH ca mu thng thng gim vi mc t hn. Bt k vic tng nhit bo qun dng nh c tm quan trng thit yu bi v Medina v Jordano (1995) quan st thy s suy gim pH ti 0,3 n 0,4 n v trong trng hp sn phm sa ln men ABT lu tr 36 ngy 70C. Trong th nghim ny, s lng acid lactic v acid acetic sinh ra sau 42 ngy bo qun 40C c th so snh vi kt qu o sau 3 ngy 150C. Kt lun: Kt qu ca nghin cu ny chng minh rng sinh khi S.platensis c nh hng tch cc n s tn ti ca vi khun mi ABT bt k nhit lu tr. S hin din ca L. acidophilus dao ng trong phm vi 107CFU/ml trong mi ln ly mu, v tng s S. thermophilus cng vt qu gii hn, t gi tr cao hn 109 CFU/ml trong hu ht cc trng hp. Mt iu ng quan tm l Bifidobacteria rt d b tn thng bi cc acid c hi. S lng ca chng gim nhanh hn so vi Lactobacilli v Streptococci. Tuy nhin, vic b sung sinh khi S.platensis to ra tc ng c li ln s tn ti ca chng.
179
Khng c vi sinh vt gy h hng pht hin trong bt k ln ly mu no, iu cho thy kh nng st khun cao trong sut qu trnh ch bin v ng gi sn phm. Nhit bo qun 150C dn n mt s trng hp xy ra s acid ha, trong khi , pH n nh trong qu trnh bo qun lnh 40C. Ngoi nhng li ch trn, sinh khi vi khun lam gip tng amino acid thit yu, vitamin ca sa b v n cng ci thin thnh phn acid bo. S phong ph cc thnh phn hot tnh trong S.platensis c tm quan trng rt ln nh mt ngun dinh dng, v l do , sinh khi vi khun lam m ra mt c hi mi cho vic sn xut cc sn phm sa chc nng. 1.4. Gii thiu mt s sn phm Spirulina hin nay Spir @ Cid: Cng dng:

H tr iu tr v phng bnh ung th. Hn ch cc tc dng ph do x tr v ha tr trong Nng cao sc khng cho ngi nhim HIV.
iu tr ung th.
Spir @ HA: Cng dng:

iu ho huyt p. Lm chc thnh mch, gim m mu. Chng tai bin mch mu no. Gim cng thng tr no khi hc, lm vic; ci Bi b sc khe, gip tng cng sc khng.
thin tr nh, tng kh nng t duy, tng th lc.
180
Spir @ B: Cng dng:

Chng suy dinh dng. Bi b sc khe. Gip tng cng sc khng. Gip lm chm qu trnh lo ha ca c th. Kim sot trng lng c th. Lm gim m trong mu.
Dia Spir @: Cng dng:
H tr dinh dng v phng bnh tiu ng. Gm Hn ch cc tc dng ph do mt s thuc tr bnh Trng hp mi mc bnh, nu dng dia Spir@ lu
ng huyt khi dng km vi thuc tr tiu ng.
tiu ng gy ra.
di kt hp vi ch sinh hot v n ung hp l th ng huyt c th nhanh chng tr li bnh thng. Spir @ C: Cng dng:

Chng v gii c cho gan thn do bia, ru. Phng chng cc bnh do cc gc t do trong mi Lm chm qu trnh lo ho c th. Gim Cholesterol v chng x va ng mch. Tng sc khng cho c th, bi b sc kho
181
trng sng gy ra.

Bt to Spi 1: Cng dng:
Bt to Spi-1 b sung vitamin, cht dinh dng gip Hn ch qu trnh lo ha. Ngn nga cc bnh cc bnh Lm p da, gip ph n c ln da hng ho hn
tng cng sc khng cho c th.
thi i nh bo ph, tim mch ,tiu ng huyt p cao
Ngoi ra, Spirulina cn c b sung vo cng on ch bin cc sn phm thc phm thng thng nhm tng hm lng dinh dng: Sa chua, kem, m n lin, nc ung th thao, bnh biscuit
1.5. Kt lun To xon Spirulina l mt loi vi to, c gi tr dinh dng cao. Loi to ny c cha trn 60g protein/ 100g to kh, bao gm 18 loi acid amin cn thit khng th thiu cho c th, nht l i vi tr em nh: isoleucin, lysine, methionin, phenylalanine, threonin, tryptophan v valine. Ngoi ra, protein ca Spirulina c c tnh l mng t bo d b ph v nn hiu sut hp thu protein trong loi to ny rt cao, trn 95%. Cht bo trong to chim khong 6%, nhng c tnh cht u vit l mt ngun dinh dng rt giu acid bo omega-3 ( DHA) v acid bo Gamma Linolenic (GLA), l cc loi acid bo c bit qu gi c tc dng trong hon thin t bo thn kinh trung ng cho tr em, gip tng cng tr nh cho tr em, v ngn nga suy gim tr nh ngi cao tui. To Spirulina cn l ngun cung cp nhiu vitamin v khong cht cn thit cho c th. Giu cc vitamin B1, B2, B6, B12, vitamin E, c bit l B12 rt cao, gp 3 ln gan ng vt, vt xa cc ngun thc n khc, c tc dng quan trng trong vic to thnh cc
182
t bo mu. -caroten c hm lng gp 10 ln trong c rt, c tc dng chng oxy ha, ngn nga x va ng mch. Cc cht khong v vi khong c st, ng, mangan, canxi, magnegium...Vi nhng u im k trn to Spirulina l ngun thc n tim nng, c gi tr dinh dng rt tt gip ngn nga, phc hi suy dinh dng v thiu vi cht dinh dng cho mi la tui. Do c nhiu cht dinh dng v cng dng nh vy, to xon Spirulina tht s l loi thc phm qu gi cho sc khe con ngi. Tuy nhin, ngun nguyn liu ny hin nay vn cha c ph bin rng ri nc ta do quy m nui trng cn hn hp. V vy, vi vic m rng quy m nui trng, ti u cc phng php x l to sau thu hoch nhm tng gi tr cm quan v tn dng trit nhng hp cht qu gi trong to, t s to ra mt c hi mi cho th trng to Vit Nam.
183
Chng 2: Fructooligosaccharide
CHNG 2 FRUCTOOLIGOSACCHARIDE (FOS)
2.1. Tng quan v FOS 2.1.1. Khi nim FOS Gn y trong dinh dng, ngi ta ch n carbohydrate trong khu phn n, m trc tin l cht x. Tuy c th khng ln men tiu ha c, nhng cht x ng vai tr quan trng trong cuc chin chng li nhng bnh tt v tim mch v mt s trng hp ung th ng rut [40]. Nhiu loi carbohydrate c nghin cu ng vai tr nh mt prebiotic, vi cc c tnh nh: Khng b hp thu tuyn tiu ha trn. Ln men bi vi sinh vt trong rut. Kch thch chn lc s pht trin v hot tnh ca mt s vi khun c kh nng ci thin sc khe. ng chc nng l mt b phn quan trng trong nhm thc phm chc nng c tp trung nghin cu nhiu trong nhng nm gn y v c tim nng tr thnh loi cht ngt mi thay th cho sucrose, loi ng truyn thng. C nhiu loi ng chc nng: palatinose, maltitol, sorbitol, lactitol, fructooligosaccharide (FOS), galactooligosaccharide (GOS), isomaltooligosaccharide (IMO) trong , ng FOS ni bt t nhng nm 1980 v c nghin cu nhiu hn c, khng ch v cng ngh sn xut n gin, m n cn c nhiu c tnh sinh hc c li cho sc khe con ngi: kch thch tiu ha, gim cholesterol, phospholipid, triglyceride, chng bo ph, an ton cho ngi b bnh tiu ng, ngn nga su rng c bit,
SVTH: V Th Ngc An
184
FOS cn gip tng kh nng hp thu Ca, Mg, Fe, ngn nga bnh thiu mu, thiu st, cn bng ion Mg2+, Ca2+ trong c th, chng long xng [39]. FOS l cht to ngt nng lng thp, khng c hp thu tuyn tiu ha trn, tuy nhin c s dng chn lc bi h vi khun ng rut Bifidobacteria, nn c c tnh prebiotic. M, inulin v FOS l nhng sn phm dn u th trng, tip sau l GOS. Nht, ngoi nhng loi ng trn cn c IMO, oligosaccharide u nnh, xylooligosaccharide, gentiooligosaccharide v lactosucrose. Cng ty Meiji Seika Nht l cng ty u tin sn xut thnh cng sm phm FOS thng mi bng enzyme chuyn ha t Asp. niger v bn trn th trng vi tn thng mi l Neosugar (1984). Gn y hn, cng ty Cheil Food & Chemicals Hn Quc thnh cng trong vic c nh t bo nm si Aureobasidium pullulans sn xut FOS quy m cng nghip. FOS ang dn thay th cc loi ng truyn thng nh ng knh v syrup giu fructose, c s dng rng ri trong thc phm v thc n gia sc [58]. 2.1.2. Ngun gc FOS Cu to FOS c nhiu trong t nhin, tn ti trong cc loi rau qu nh chui, mn, o, qut, atiso, c chua, hnh, ti Hm lng FOS mt s loi rau qu c th tham kho bng 2.1 [18]. FOS l nhng oligosaccharide m trong phn t ca chng gm mt phn t ng sucrose lin kt vi 1, 2 hay 3 gc fructose thng qua mi lin kt -2,1-glucoside. Cng thc tng qut ca ng FOS l GFn, trong n l s nhm n = 2, 3, 4 (G l gc ng glucose, F l gc ng fructose), tng ng l cc ng 1-kestose (GF2), nystose (GF3) v fructofuranosylnystose (GF4) vi cng thc cu to nh hnh 2.1 [58]. Tuy nhin, nhiu nh nghin cu khc cng gp c cc loi ng khc nh fructan, glucofructosan v inulin vo nhm ng FOS [58].
SVTH: V Th Ngc An
185
Bng 2.1: Hm lng FOS ca mt s loi cy qu (mg/g cht kh) Loi cy qu Chui Qut o L Mn Da hu Mng Rau dn Rau cn ty Hnh ta Da Nht Bn C chua Ti Cy atiso Hnh ty 1-Kestose (GF2) 5.9 1.7 3.5 0.8 1.8 2.8 0.3 0.1 0.2 0.4 0.5 0.2 8.7 93.9 15.5 Nystose (GF3) 0.1 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 0.3 0.0 0.0 1.2 94.3 6.7 Fructofuranosylnystose (GF4) 0.0 1.11 0.0 0.0 0.0 0.1 0.0 0.0 0.4 0.4 0.0 0.4 0.4 98.1 4.2 Tng FOS (GFn) 6.0 2.8 3.5 0.8 2.0 3.0 0.3 0.1 0.6 1.1 0.5 0.6 10.3 286.2 26.4 [18]
SVTH: V Th Ngc An
186
Hnh 2.1: Cng thc cu to ca FOS [58]
Trong t nhin, FOS c hnh thnh di tc dng ca enzyme chuyn ha fructose. Qu trnh trch ly FOS t nhng loi thc vt ny quy m cng nghip khng c tnh kinh t do nng rt thp, lng enzyme li b gii hn bi iu kin thi tit, v vy, FOS thng mi c sn xut bng phng php tng hp hoc thy phn. Bng 2.2 nu mt s loi thc vt c enzyme tng hp FOS [58].
SVTH: V Th Ngc An
187
Bng 2.2: Ngun enzyme tng hp FOS t thc vt Ngun Agave americana (Cy tha) Agave vera cruze (Cy tha) Asparagus officinalis (mng ty) Allium cepa (c hnh) Cichorium intybus (rau dip xon) Crinum longifolium L c ci ng Helianthus tuberosus (atis Jerusalem) Lactuca sativa L. (rau dip) Lycoris radiata (ht cy n t dip) Taraxacum officinale (cy b cng anh) Tc gi Bhatia v cs. (1979); Nandra v Bhatia (1980) Bhatia v cs. (1954, 1955); Satyanarayana (1976) Shiomi v cs. (1976, 1979a, 1979b, 1980, 1981, 1982) Darbyshire v cs. (1978); Henry v Darbyshire (1980) Shingh v Bhatia (1971); Chandorkar v Collins (1972) Bhatia v cs. (1959) Allen v Bacon (1956) Edelman v Dickerson (1966); Scott v cs. (1966); Praznik v cs. (1990) Chandorkar v Collins (1972) Nagamatsu v cs. (1990) Chandorkar v Collins (1972) [58] FOS c th c sn xut quy m cng nghip t ngun nguyn liu sucrose, vi xc tc l enzyme thu nhn t cc loi nm mc nh Aureobasidiums sp. v Aspergillus niger [58]. Bng 2.3 cho bit cc ngun thu enzyme chuyn ha FOS t vi sinh vt [58].
SVTH: V Th Ngc An
188
Bng 2.3: Vi sinh vt sn xut FOS Ngun Aureobasidium pullulans Tc gi Jung v cs. (1987, 1989); Yun v cs. (1990, 1992, 1993a, b, c, d); Smith v cs. (1980) Hayashi v cs. (1989, 1990, 1991a, b) Fujita v cs. (1990) Duan v cs. (1994) Hidaka v cs. (1988, 1989); Bealing v Bacon (1953) Pazur (1952); Kida v cs. (1988); Bealing v Bacon (1953) Balken v cs. (1991) Muramatsu v cs. (1988); Dickerson (1972); Arcamone v cs. (1970) Gupta v cs. (1982); Maruyama v cs. (1979); Patel v cs. (1994) Usami v cs. (1991) Bealing v Bacon (1953) Hankin v Meintype (1977) Takeda v cs. (1994) Straathof v cs. (1986) [58] 2.1.3. Tnh cht ca FOS: Theo Gross, kestose l loi ng kt tinh mu trng c gc quay cc []D20 l +28.50 v nhit nng chy l 199 2000C. ngt ca kestose, nystose v fructofuranosylnystose so vi sucrose ln lt l 31%, 32% v 16%. ngt tng ca ba loi ng ny (FOS) ch bng 30% ngt ca ng sucrose [58]. V ngt ca FOS tng t sucrose. ng FOS ht m mnh, nn kh bo qun trng thi tinh th trong thi gian di. cng mt nng , FOS c nht cao hn
Aureobasidium sp. Arthrobacter sp. Aspergillus japonicus Aspergillus niger Aspergillus oryzae Aspergillus phoenicis Aspergillus sydowi Claviceps purpurea Fusarium oxysporum Penicillium frequentans Penicillium spinulosum Phytophthora parasitica Scopulariopsis brevicaulis Saccharomyces cerevisiae
SVTH: V Th Ngc An
189
nht ca sucrose. bn nhit ca FOS cng cao hn sucrose. ng FOS bn trong di pH 4.0- 7.0 v nhit cao ti 1400C [58]. dng lng, FOS mch ngn l cht lng trong, khng mu hoc mu vng nh syrup, c mi tri cy v v ngt. dng bt, FOS c mu trng n vng nht, mi tri cy v v ngt. FOS ha tan trong nc [11]. Mt s tc gi khi nghin cu v tnh cht ho l ca FOS u a ra mt kt lun chung l c nhiu tnh cht ha l tng t sucrose nh ha tan, ng c, nhit si v cc thng s v qu trnh kt tinh. Khc vi ng sucrose FOS mang nhiu c tnh sinh hc u vit hn. y chnh l c im khin cc nh thc phm ch v tp trung khai thc loi ng ny [58]. Bng 2.4: c tnh ca FOS so vi mt s loi ng khc n nh nht Tnh ht ngt (Sucrose = 1) Sorbitol Xylitol Mannitol Erythritol Maltitol Isomalt Lactitol Maltidex FOS 0.7 0.9 0.5 0.65 0.75 0.60 0.40 0.75 0.30 Nhit < 1600C < 1600C < 1600C < 1600C < 1600C < 1600C < 1600C < 1600C < 1600C pH 2 10 2 10 2 10 2 10 2 10 2 10 2 10 2 10 2 10 Thp Rt thp Thp Rt thp Cao Cao Rt thp Cao = sucrose m Cao Cao Thp Thp Trung bnh Thp Trung bnh Trung bnh Trung bnh [18]
ng
SVTH: V Th Ngc An
190
2.1.4. nh hng ca FOS ln c th v sc khe con ngi 2.1.4.1. nh hng ca FOS n s chuyn ha carbohydrate FOS khng hoc rt t b thu phn bi h enzyme ng rut, nn khi n lng ng trong mu khng b bin ng.Th nghim v s thay i hm lng ng v insulin trong mu theo thi gian sau khi n FOS cho kt qu nh hnh 2.2; 2.3 v 2.4 [18]. Kt qu cho thy tri vi sucrose, khi n FOS, trong cng mt khong thi gian, lng ng trong mu khng h thay i.
Hnh 2.2: S thay i nng insulin trong mu [18]
Hnh 2.3: S thay i nng glucose trong mu [18]

SVTH: V Th Ngc An 191
Hnh 2.4: S thay i nng fructose trong mu [18] Liu lng FOS cn cho ngi trng thnh l 8g/ngy [51]. T kt qu ca mt s nghin cu khc cho thy, i vi nhng ngi b mc bnh tiu ng nu mi ngy n 8g FOS, lng ng trong mu s gim nhanh trong vng 14 ngy. Nhng bin i c tnh tch cc pht sinh khi chuyn ho glucose v cht bo trong qu trnh trao i cht vi s hin din ca FOS. Nguyn nhn ca cc hiu ng ny l do FOS khng b tiu ho rut non bi tc dng ca h enzyme ng rut, m b ln men trong rut gi di tc dng ca vi khun Bifidobacterium (l mt chi trong h Lactobacteraceae, t bo hnh que, phn nhnh, thng hoc cong, bt ng, gram +, sng k kh, a m, khng sinh bo t, thng gp trong rut gi). Kt qu ca qu trnh l sinh ra cc acid bo on mch (SCFA) nh acid acetic, acid propionic v acid butyric. Cc cht ny sau c thm vo thnh rut gi hoc chuyn ln gan, c ch s gia tng ca glucose v cht bo trong mu. i vi bnh nhn mc bnh tiu ng (c hoc khng ri lon tit insulin) ngoi s bt bnh thng v hm lng ng trong mu, cht bo trong mu cng lun b ri lon. V th vic hn ch s gia tng hm lng cht bo trong mu cng l mt bin php cha bnh tiu ng. Vi tng trn, Agheli v cc cng s gn y nghin cu
v ch ra rng nhm chut mc bnh tiu ng c khng tit isulin khi n thc n c cha 10% FOS gim ng k hm lng phospholipid v triglyceride trong mu. Kt qu ny khng nh li bo co trc ca Delzenne, ngi ch ra rng FOS c kh nng lm gim lng cht bo trong mu ca chut. Cng theo Agheli, FOS khng ch lm thay i hm lng cht bo trong mu m cn c th iu chnh s to ra cc enzyme tng hp acid bo. Trong gan, hot lc ca enzyme trn tng ln nu ch n sucrose nhng s c bnh thng ho bi s cung ng FOS. Nh vy FOS c vai tr kh tch cc trong vic phng v cha bnh tiu ng xt gc bnh l lin quan n s gia tng ca lipoprotein trong mu. V th FOS hin nay c dng nhiu nh l mt cht ngt thp nng lng c bit dnh cho cc i tng mc bnh tiu ng [18]. 2.1.4.2. nh hng ca FOS n h vi sinh vt ng rut Tc ng ca FOS n h vi sinh vt ng rut c nhiu tc gi nghin cu. Bng th nghim ngoi th Gross. D khi nghin cu h vi sinh vt trc trng cho thy cc ni Bifidobacterium spp. v Bacteroides spp c th pht trin mnh trong mi trng dinh dng c cha FOS, ngc li Escherichia coli v Clostridium perfringens li b tiu dit trong mi trng ny. Cn kt qu th nghim trn c th cho thy c hai i tng ngi v ng vt cc la tui khc nhau sau khi n FOS cho thy s lng vi sinh vt Bifidobacterium v Lactobacilli trong i trng tng ln, trong khi s lng vi sinh vt Clostridium perfringens li gim xung. Th nghim cn ch r rng do FOS c cu to mch thng ngn nn tc ng n kh nng ln men ca cc vi sinh vt ni trn. Cn cc loi fructoligosaccharides mch nhnh hoc inulin mch thng nhng di th nh hng trn rt hn ch. Hin nay Bifidobacterium c c bit quan tm trong lnh vc sinh hc bi n mang nhiu li ch cho c th sng. Vi khun Bifidobacterium tn ti v pht huy tc dng ngay trong c th ch, iu ny rt c ngha v n c th phng chng cc bnh tt v ng rut, do Bifidobacterium c kh nng sn sinh cc acid mch ngn nh acid acetic, acid lactic trong qu trnh ln men ng. S gia tng hm lng acid s c tc
SVTH: V Th Ngc An
193
dng gim pH trong ng rut, gi gn hot ng trao i cht ca cc vi sinh vt ng rut khc n nh, ng quy lut, hn ch hoc ngn nga s pht trin ca cc vi sinh vt gy bnh v cc vi sinh vt gy thi ra. mc cao hn nghin cu cn ch ra rng qu trnh gim pH trong ng rut c tc dng trc tip phng v tr bnh ung th rut thng qua vic ngn tr s pht trin ca bacterium hoi sinh v c ch hot lc ca cc enzyme nh nitroreductase, -glucoronidase v decarboxylase. y l nhng enzyme tham gia qu trnh chuyn ho nit to cc sn phm trung gian c kh nng gy ung th (nhng dn xut phenol ca tyrosin v tryprophan). Ngoi ra vi khun Bifidobacterium cn c kh nng to vitamin, nht l vitamin nhm B, gim cholesterol trong mu v ti sinh h vi sinh vt ng rut cho cc bnh nhn sau khi dng nhiu khng sinh iu tr bnh [18]. 2.1.4.3. nh hng ca FOS n bnh su rng Bnh su rng ch yu l do vi khun Streptococcus mutans v cc lin cu khun gy nn. Cc vi sinh vt trn c rt nhiu trong khoang ming ca ngi v ng vt. Khi thc n a vo, chng s la chn cc thnh phn dinh dng ph hp ln men, pht trin v gy bnh. Streptococcus mutans s dng ngun ng to thnh cc acid v glucans khng tan l nguyn nhn chnh gy su rng.V vy nu trong thnh phn thc n ca ta khng cha hoc cha t cht thch hp cho qu trnh dinh dng ca loi vi sinh vt trn s c th ngn nga c bnh. xt nh hng ca FOS n bnh su rng ngi ta th nghim trn c th chut. Kt qu cho thy nhm chut th nghim (n FOS) b su rng t hn nhiu so vi nhm chut i chng (n sucrose). Cc th nghim nui cy vi sinh vt phn lp t khoang ming ln mi trng FOS chng t c ch v kh nng phng bnh su rng ca n. l do FOS khng phi l mi trng thch hp cho cc vi sinh vt gy bnh trn pht trin. Ngoi ra Ikeda. T cn nghin cu cho thy FOS khng nhng ch c kh nng phng m cn c kh nng cha bnh bnh su rng. V th ngy nay nhiu ni trn th gii ngi ta dng FOS thay th cho ng knh trong thnh phn n hoc trong ch bin bnh ko, c bit l bnh ko cho tr em phng bnh su rng [18].
SVTH: V Th Ngc An
194
2.1.4.4. Vai tr thc y qu trnh hp th calcium ca FOS: Nhiu nghin cu cho thy, s dng FOS c th tng cng s hp th calcium ca t bo. Nh c tnh ny, FOS c th gip cho con ngi phng v chng cc bnh v chuyn ho v bnh long xng. Qu trnh thc y hp th calcium ca FOS xy ra trong rut gi. C ch thc y trn cha c xc nh mt cch r rng nhng c mt kt lun chung l do ba yu t sau: ng FOS trong rut gi b cc vi sinh vt sinh acid ln men, lm gim pH ca mi trng, dn n s ti ho tan ca cc mui calcium. Trong rut gi vi khun Bifidobacterium ln men mnh khi mi trng c cha FOS sinh ra cc acid mch ngn, cc acid ny khuych tn vo t bo biu b thnh rut, t thc y s hp th calcium. S dch chuyn FOS trong rut gi s ko theo s dch chuyn ca hp cht calcium-protein, nh calcium c tip xc nhiu hn vi cc t bo thnh rut, to iu kin tt cho s hp th vo mu. Ngoi calcium, FOS ng thi cn thc y s hp th c magi. iu ny thc y qu trnh pht trin xng, tng cng hm lng calcium trong xng [18]. 2.1.5. Tnh an ton v ng dng ca FOS khng nh tnh an ton ca FOS i vi c th sng, c nhiu nghin cu v t bo v gen ca ng vt n FOS. Cc nghin cu ny ch yu tp trung tr li cc cu hi l khi ng vt s dng FOS c nh hng n kh nng khng khun ca t bo hay khng? C gy ra t bin v gen dn n s tng hp ADN bt qui tc hay khng? Tt c cc nghin cu i n kt lun l FOS khng gy c hi n t bo ca ch th. Tc gi Clevenger M.A. cn lm th nghim trn chut kim tra v c t cp tnh v mn tnh cng nh kh nng gy ung th ca FOS. Kt qu cho thy FOS khng c biu hin ca cc c t khi cho chut n hn 2,17mg/kg/ngy [18]. Ph thuc vo tinh khit, FOS thng phm ch yu c hai loi l FOS ph thng ( tinh khit t 45% n 60%) v FOS cao ( tinh khit cao hn 75%). ng FOS
ph thng thng c dng trc tip nh mt loi thc phm hoc nh mt cht ngt b sung trong ch bin cc loi thc phm khc. Cn FOS cao thng chuyn dng cho cc i tng mc bnh, n king. Ngoi ra cn c loi tinh khit 100 % dng cho cng vic phn tch ho hc. Loi c tinh khit thp c th dng lm cht b sung trong mi trng nui cy vi khun Bifidobacterium. Theo ti liu cng b th c th ngi mt ngy ch c th hp th 2-12g FOS, nn vic dng FOS b sung vo thc phm khc nh mt cht ph gia tng cng hot tnh sinh hc ca thc phm l ng dng u tin v quan trng ca FOS trong lnh vc ch bin thc phm. Do FOS c c tnh l c v ngt, thp nng lng v c hot tnh sinh hc nn thng c b sung vo cc loi bnh, ko, bnh quy v cc sn phm ca sa hoc kt hp trn ln vo trong cc cht ngt khc. Nht Bn FOS c dng b sung vo hn 500 loi thc phm. Nm 1984 nc ny c mt th trng rng ln cho FOS. n nm 1990 lng FOS tiu th trong c nc ln n 4000 tn. ng FOS bn trn th trng Nht Bn rt a dng, t loi c hm lng rt thp n loi c tinh khit cao n 98%, phc v cho nhiu mc ch khc nhau, loi hm lng thp dng lm cht kch thch trong mi trng ln men cho vi khun Bifidobacterium, cht c hm lng trung bnh dng cho ngi n trc tip hoc b sung vo thc phm cn loi tinh khit dng trong k thut phn tch. M v Nht Bn ngi ta cn s dng FOS lm cht b sung trong thc n cho gia sc [58]. Nh c tnh ca FOS l c th tng cng hot ng ca h tiu ho nn gip cho gia sc n nhiu, tng cn nhanh . Ti Trung Quc n tn nm 1992 cng nghip sn xut FOS mi bt u, nhng trong vng mi nm ngnh cng nghip ny c bc pht trin vt bc. Nu nh nm 1999 ton Trung Quc mi ch c 2 nh my sn xut FOS ti Vn Nam v Giang T vi cng sut 2000 tn/nm th n u nm 2002 s nh my sn xut FOS trong ton quc ln n mi c s vi cng sut t 2000 tn n 4000 tn/nm. Sn phm FOS nc ny c dng nhiu cho cc i tng gi, tr em v nhng ngi bnh trong thi
SVTH: V Th Ngc An
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k phc hi sc kho tng cng hot ng tiu ho, gip cho cc i tng trn nhun trng, n tt. M v Chu u, nhiu cng ty v ang xin chng ch GRAS (Generally Recognized As Safe) cho vic s dng sn phm FOS ca mnh, v nh th FOS c cng nhn chnh thc bng vn bn v an ton thc phm. Nh s lng ngi tiu dng FOS ngy cng tng cao [18]. 2.1.6. Tnh hnh nghin cu v sn xut FOS trn th gii Fructooligosaccharide c th thu nhn c bng cch chit xut chng t cc loi cy qu, hoc bng cc phng php ho hc, ho l, ho sinh... Nhng cho ti nay phng php c coi l hiu qu v c kh nng cng nghip ho nht l phng php cng ngh sinh hc. Bt u bng s pht hin mt s chng nm men v nm mc c kh nng sinh tng hp enzyme xc tc qu trnh chuyn ho sucrose thnh FOS, cho ti nay c rt nhiu nghin cu v lnh vc sn xut ng ny theo cng ngh sinh ha. Cc nghin cu ch yu tp trung vo vic phn lp tuyn chn ging sinh tng hp enzyme, ng dng enzyme trong sn xut FOS v nghin cu tinh ch FOS ... 2.1.6.1. Nghin cu sinh tng hp enzyme cho sn xut FOS V enzyme chuyn ho fructose trong thc vt c hot tnh thp v thng l hn hp ca mt vi loi v li b hn ch khai thc do c tnh thi v, nn vic chit tch enzyme t thc vt phc v cho cng nghip sn xut FOS khng c tnh kh thi. Cng vi s pht trin ca cng ngh sinh hc, vic ng dng vi sinh vt trong sn xut enzyme ngy cng nhiu. Cng nh cc loi enzyme khc, ngun cung cp enzyme chuyn ho fructose cho cng nghip ch bin FOS ch yu l t vi sinh vt. Hidaka v cc cng s l nhng ngi u tin nghin cu kh nng cng nghip ho sn xut FOS t ng knh. ng cng l ngi a ra phng php xc nh hot lc fructosyltransferase (FTS) v i lng biu th mi lin quan gia hot lc FTS v hiu sut chuyn ho sucrose thnh FOS (t l hot tnh chuyn ho vi hot tnh thu
SVTH: V Th Ngc An
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phn UT/UH) m cho ti nay vn c coi nh l phng php c s cho hu ht cc nghin cu lin quan ti FOS. Bng nghin cu ca mnh Hidaka tm ra nhiu chng vi sinh vt thuc cc loi nh Aspegillus awamori, Saccharomices cerivisiae, Aspegillus niger c kh nng tng hp FTS. Thng qua qu trnh phn lp, tuyn chn theo hot lc chuyn ho v t l hot tnh chuyn ho vi hot tnh thu phn (UT/UH), tc gi khng nh nm mc Asp. niger l ngun vi sinh vt tt nht cho ln men tng hp FTS. Kh nng sinh tng hp FTS ca Aureobasidium pullulans cng c Jung K.H v cc cng s nghin cu. Tc gi ny kt lun rng sucrose l ngun carbon l tng cho ln men sinh tng hp FTS v hiu sut tng hp FTS ca chng vi sinh vt trn tng t l thun vi nng sucrose trong mi trng ln men. Khi nghin cu thu nhn FTS t F.oxysporum, tc gi ngi Anh Patel V. v cc cng s cn i n kt lun rng qu trnh ln men ca vi sinh vt trong mi trng cha sucrose c s tng hp FTS mang hot tnh invertase v hot tnh chuyn ho. Nm 1992 Bo Jiang phn lp c mt chng Asp. niger AS0023 c kh nng tng hp FTS vi hot lc cao. Ngoi ra cn rt nhiu tc gi khc nh Hayashi nghin cu chn ra chng Aureobasidium sp. dng sinh tng hp FTS cho sn xut FOS. Jung v Smith cng c cc cng b v kh nng sinh FTS ca loi Aureobasidium pullulans. Van Balken nghin cu thnh cng qu trnh tng hp FTS t Aspegillus phoenicis v ng dng sn xut FOS t tinh khit 60%. Fujita pht hin ging A. bacterium cng c kh nng tng hp FTS. Gn y Takeda cn tm ra loi mc Scopulariosis brevicaulis c kh nng tng hp FTS ch xc tc to ng 1kestose. Ngoi ra cc vi sinh vt khc nh A.japonicus, Penicillium rigolosum, Acetobacter diazotrokicus SRT4 cng c kh nng tng hp FTS, cc vi sinh vt ny c nghin cu v ng dng trong cng nghip sn xut FOS. Mt c im chung cho tt c cc kt qu nghin cu trn cho thy, hu ht FTS trong cc vi sinh vt tm c ch yu l enzyme ni bo v lun cha ng thi c hai hot tnh l thu phn v chuyn ho. T l hot lc ca hai hot tnh trn thay i ph thuc vo chng loi ging v iu kin ln men, iu kin phn ng [18].
SVTH: V Th Ngc An
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FTS c sn xut bng cch ln men chm hiu kh vi mt s chng nm. Cc thng s ca qu trnh ln men nh thong kh, khuy o, pH v nhit cn c xc nh cho tng loi vi sinh vt. iu kin chung cho tng hp FTS thng qua nui cy vi sinh vt trong mi trng c nghin cu k. V d nh sucrose l ngun carbon tt nht cho s pht trin sinh khi v hot ng ca enzyme. pH cn duy tr ln hn 5.5 v nhit ti u cho t bo sinh trng l khong 300C. Aureobasidium sp., enzyme ni bo sau c tit ra ngoi mi trng. Hot ng ca enzyme ni bo c kch thch mnh khi tng nng ion Mg2+. Khi hm lng sucrose trong mi trng b gii hn, FOS s c s dng lm ngun cung cp carbon. Sau 6h nui cy vi 20% sucrose, khong 55% (w/w) FOS c to thnh. T bo vi sinh vt sau c loi ra khi canh trng bng cch ly tm v enzyme c chit tch nh lysozyme hoc c th c nh s t bo vi sinh vt cha enzyme v a vo sn xut FOS [58]. 2.1.6.2. Nghin cu v sn xut FOS 50 (FOS ph thng) Hin nay Nht, Trung Quc, Hn Quc, M v cc nc khc, FOS c sn xut qui m cng nghip v hng nm cho ra th trng hng ngn tn sn phm. Thng thng FOS ph thng c sn xut theo phng thc lin tc v khng lin tc. Trong phng thc sn xut lin tc cng ngh c nh enzyme hoc c nh t bo c s dng, cn i vi phng thc sn xut khng lin tc th s dng enzyme chit tch. Gii php c nh enzyme v c nh t bo trong sn xut FOS cho hiu qu cao hn v sn xut c lin tc, tiu hao nng lng t, nh xng nh nhng tnh n nh km v cn my mc thit b hin i, nh xng tiu chun. Mc d vy, y li l phng php c kh nng cng nghip ho cao v c tp trung nghin cu nhiu cc nc c nn cng nghip pht trin nh Nht Bn, Trung Quc, Hn Quc Phng thc khng lin tc trong sn xut FOS i hi phi trang b thm mt cng on trch ly enzyme v lm sch tp cht sau qu trnh chuyn ho. B li k thut sn xut li n gin v thit b cng r tin hn. Phng php ny c s dng nhiu cc qui m nh, u t thp. Nghin cu cng ngh ny c Yun, J. W., theo phng php ny
SVTH: V Th Ngc An
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tc gi kt hp FTS vi cc enzyme khc sn xut ra FOS c tinh khit cao ti 98%. Ngoi hai gii php trn hin nay ngi ta cn dng phng php n gin, vi phng php ny t bo vi sinh vt c s dng nh mt ngun enzyme. Phng php n gin ny ang c dng trong mt s nh my Trung Quc. Cc cng b kt qu nghin cu v c nh enzyme sn xut FOS rt nhiu. in hnh c Hayashi S., tc gi ny c nh FTS tng hp t A. pullulans sp sn xut FOS. Ngoi ra Yun J.W., mt tc gi ngi Hn Quc cng nghin cu thnh cng cng ngh sn xut FOS lin tc bng phng php c nh FTS trn cc ht nha trao i ion. i vi cng ngh c nh t bo, Jiang Bo tm ra cht mang thch hp cho c nh t bo pht trin Asp. niger AS0023 sn xut FOS l calcium alginate. Tc gi sau cng xc nh c cc thng s k thut thch ng cho qu trnh chuyn ho FOS t sucrose trn ct phn ng [b,i]. Phng php c nh enzyme thot u c v c nhiu u im hn l c nh t bo nh phng php c nh n gin v nng sut th tch cao hn do qu trnh tng hp din ra nhanh hn. Tuy nhin, t bo c nh c u im l n nh hn trong qu trnh hot ng. Chn la phng php c nh enzyme hay t bo cng cn ang l vn tranh ci do c nng sut v n nh u l yu t cn thit trong sn xut cng nghip [58]. Quy trnh chung cho sn xut FOS nh FTS tng hp t vi sinh vt qua cc bc nh hnh 2.5 [50]
SVTH: V Th Ngc An
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Hnh 2.5: Quy trnh sn xut FOS t FTS vi sinh vt [50]
SVTH: V Th Ngc An
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Quy trnh sn xut FOS thng mi theo phng php lin tc v khng lin tc c trnh by trn s 2.1 Canh trng nui cy t bo
Tch chit enzyme
C nh t bo
Phn ng enzyme Dung dch sucrose
Thit b phn ng ct
Tinh sch
C c
Thanh trng
Sn phm
Hnh 2.6: S quy trnh sn xut FOS lin tc v khng lin tc [58]
SVTH: V Th Ngc An
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2.1.6.3. Nghin cu v sn xut FOS cao Qu trnh chuyn ha sucrose thnh FOS ph thng di tc dng ca FTS c th biu din mt cch tng qut nh sau: GF Sucrose + GF Sucrose G Glucose + GF Sucrose + GF2 + GF3 + GF4 FOS
Qua phng trnh phn ng ta thy, ngoi FOS ra, sn phm thu c cn cha mt lng glucose nht nh. ng glucose xut hin trong phn ng khng nhng lm gim tinh khit ca FOS m cn c ch hot tnh ca enzyme. V cht c ch ny m ta ch thu c dch ng ph thng c nng FOS khng qu 55% cn li hn 45% l sucrose v glucose. V th mun thu c FOS c tinh khit cao (FOS cao ) hoc FOS tinh khit, cn thit phi tch b hoc chuyn ho lng glucose v sucrose cn st li. Cho ti nay cc phng php tinh sch c p dng ch yu bao gm: Lc gel: Nht gn y phng php ny c a vo p dng qui m cng nghip. Da trn nguyn l ca s khc nhau v kch c phn t ca cc cu t phn tn trong dch m ngi ta phn li c ring r tng loi ng, nh c th tch, loi b glucose ra. Phng php ny cn c h thng thit b tinh lc ng b t tin do hiu qu kinh t khng cao. Lc Nano: y l mt phng php mi, s dng k thut thm thu ngc, c nhiu tnh u vit rt ph hp vi qui m cng nghip [11]. Phng php trao i ion: trong phng php ny cc ng thnh phn c phn li khi i qua ct c cha cc ht nha trao i ion. Nh ta c th tch glucose v sucrose ra khi dch FOS. Dng cng ngh ln men: Phng php ny c thc hin thng qua vic s dng vi sinh vt ln men glucose c trong dung dch, loi vi sinh vt hay c dng l nm men. Phng php ny c s dng rt nhiu bi n cho hiu qu kinh t cao, tuy th n cng cn tn ti l sn phm FOS b nhim bn v gy mi v l.
SVTH: V Th Ngc An
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Dng cng ngh enzyme: C hai loi enzyme c s dng phn gii glucose l glucoseisomerase (GI) v glucooxidase (GOD). S dng enzyme GI: Trong phng php ny enzyme GI c vai tr trong chuyn ho glucose c trong dch thnh fructose. V l thuyt fructose mi sinh ra li gn kt vi sucrose to thnh FOS. Nhng trong thc t th nghim cho thy fructose mi to ra khng c kh nng gn kt vi sucrose, kestose hoc nystose. V th trong phng php ny ch c th lm gim glucose trong dch thy phn ch khng nng cao tinh khit ca dch FOS c. S dng enzyme GOD: Bng cch ny enzyme GOD c nhim v phn gii glucose thnh acid gluconic. y l loi enzyme c coi l c hiu qu nht trong vic chuyn ho glucose trong sn xut FOS cao (c th n 98%). Cng nh trong sn xut FOS ph thng, cng ngh sn xut FOS tinh khit c th thc hin bng hai phng php l c nh v khng c nh enzyme (t bo). i a s cc nghin cu chn h enzyme FTS, GOD v catalase (CAT), c nh trn cht mang nh calcium alginate v ht trao i ion ri c nhi vo ct phn ng. Vi cng ngh ny Yun, J. W. lin tc sn xut c FOS c tinh khit cao hn 90%. Jiang Bo cn kt hp c nh t bo nm mc AS0023 vi GOD v GI sn xut FOS t tinh khit 72%. Cn Lamia bng phng php khng lin tc cng dng h enzyme GOD v CAT sn xut FOS cao . Tc gi tm c ra iu kin ti u cho hot ng ca h enzyme trn v nng cao c tinh khit ca FOS ph thng t 50% ln 82% [18, 57, 58]. 2.1.7. Gii thiu cc enzyme trong sn xut FOS 2.1.7.1. Enzyme fructosyltransferase (FTS) FTS l tn gi chung cho cc enzyme c hot tnh xc tc qu trnh chuyn dch gc fructose. Qu trnh trn c th xy ra theo hai cch l ct khng gn v ct ri gn. Kh nng xc tc ct khng gn ca enzyme l hot tnh thu phn (vit tt l UH) , cn kh nng ct ri gn l hot tnh chuyn ho (vit tt l UT).
Nhiu nh nghin cu vn gi tn loi enzyme dng sn xut FOS l -Dfructofuranosidase, mt loi enzyme thuc nhm thy phn k hiu EC.3.2.1.26. Nguyn nhn c th l do hot tnh chuyn gc fructose ban u c pht hin enzyme invertase khi tin hnh th nghim vi mu c nng sucrose cao. Trong khi s khc li gi enzyme ny l fructosyltransferase, k hiu EC.2.4.1.9, nhm phn bit vi cc enzyme thuc nhm enzyme thy phn khc [58]. Ph thuc vo v tr gn cc gc fructose ln cht nhn, enzyme ny c phn ra nhiu loi vi cc tn gi khc nhau nh: 6G-fructosyltransferase (6G-FT), 6Ffructosyltransferase (6F-FT) v 1F-fructosyltransferase (1F-FT). Cc enzyme trn xc tc ln phn t sucrose to ra cc loi FOS c phn t lng nh nhau nhng khng ging nhau v cu trc phn t. Ba enzyme 6G-FT , 6F-FT v 1F-FT xc tc gn vo phn t ng cc v tr khc nhau to ra cc ng tng ng. Cc FTS c ngun gc t thc vt ch c hot tnh UT, nhng mang tnh c hiu c cht tng i (gn gc fructose vo phn t ng sucrose khc nhiu v tr khc nhau). Khc vi enzyme tng hp t vi sinh vt, FTS trong thc vt c phn t lng nh hn v trong phn t khng cha nhm glucoside. Nhit thch hp cho hot ng ca chng l 300C 450C [18]. Trong khi FTS c ngun gc t vi sinh vt li c c hai loi hot tnh UT v UH nhng hot tnh gn li c tnh c hiu c cht gn nh tuyt i. Enzyme FTS c ngun gc khc nhau cng khc nhau v cu to ho hc. FTS tng hp t vi sinh vt l nhng enzyme a cu t, c phn t lng cao (>10.000 Da), trong phn t c cha cc nhm glucoside. Nhit hot ng thch hp ca enzyme ny l 500C 600C, rng hn so vi enzyme t thc vt, pH thch hp l 5 - 6.5 v nng sucrose t 700-850g/l [58]. Mt s cht c ch v xc tc hot ng chuyn ha ca FTS cng c nghin cu. loi cy Agave americana, FTS c xc tc bi cc ion Ca2+, Mg2+, Co v Li+ v b c ch bi Ag+, Pb, Hg, Al3+ v Sn. Trong khi , enzyme sn xut bi loi nm si Aureobasidium sp. b c ch bi Hg, Cu v Pb [58].
SVTH: V Th Ngc An
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FTS l loi enzyme c hiu, n ch c th tc dng xc tc phn ct v gn kt gc fructose trong phn t sucrose hoc cc dn xut ca sucrose to thnh FOS. Cn trong dung dch ch c fructose v glucose th enzyme ny khng c kh nng xc tc to FOS. Mt n v hot tnh ca FTS c tnh l lng enzyme chuyn ha 1 mol fructose trong 1 pht. C ch chung cho phn ng chuyn ha gc fructose nh sau: GF + fructosyltransferase F fructosyltransferase + G F-fructosyltransferase + GF GF2 + fructosyltransferase Hnh 2.6 cho thy c ch xc tc phn ng to cc FOS t sucrose ca
fructosyltransferase thu t A.pullulans.
Hnh 2.7: C ch chuyn ha to FOS t sucrose ca FTS A.pullulans [58] G, GF, GF2, GF3, GF4 ln lt l glucose, sucrose, 1-kestose, nystose, 1Ffructofuranosylnystose.
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2.1.7.2. Gii thiu v enzyme glucooxidase (GOD) Cc nghin cu v cu to v c tnh ca enzyme GOD c A. Crueger v W.Crueger thc hin nm 1990. Cc tc gi kt lun enzyme GOD l hp cht glucoprotein, c phn t lng khong 155.103 DA. GOD cha 2 phn t FAD v 16% khi lng phn t l carbohydrate (mannose, galactose v glucosamine di dng Nacetyl). Mch carbohydrate trong phn t GOD khng tham gia xc tc v cu to ca n cho ti nay cng cha c nghin cu r rng. Enzyme GOD sinh tng hp t A. niger c cu to bi 583 acid amin. im ng in ca enzyme ny l pH 4.2. Enzyme GOD c tnh c hiu rt cao, ch cn mt thay i nh trong cu trc th hot lc oxy ha ca enzyme khc hn nhau, v d gc glucose trong phn t enzyme dng - D glucose c hot tnh oxy ha mnh hn - D glucose 157 ln. Enzyme GOD cn c mt c tnh quan trng l rt an ton v mt thc phm. Phn ng oxy ho ca glucose di xc tc ca enzyme GOD c thc hin theo cc phng trnh biu din hnh 2.7. Theo phng trnh phn ng ta thy, u tin glucose di tc dng ca enzyme GOD b oxy ho to thnh -D-glucolacton, sau t phn thnh acid gluconic. S to thnh acid gluconic s lm gim pH ca mi trng, nn trong qu trnh phn ng phi ng thi b sung kim iu chnh pH (phng php potentionmetric). Theo c ch hot ng ca enzyme GOD th qu trnh oxy ho glucose lun c s hnh thnh H2O2., chnh sn phm ph ny li c ch hot ng ca enzyme GOD.
SVTH: V Th Ngc An
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Hnh 2.8: Qu trnh oxy ha glucose di tc dng ca enzyme GOD [18] V nh hng ca H2O2 trong phn ng oxy ho glucose c nhiu tc gi nghin cu nh Kleppe, Bourdillon, Tse v Googh. Cc kt qu u cho thy hot lc ca GOD gim t l vi s tng ca nng H2O2. V v th trong sn xut FOS nu ch dng mt enzyme GOD th ta ch c th chuyn ha c mt phn nht nh lng glucose sinh ra v tinh khit ca FOS cng s b hn ch mc thp. Kt qu nghin cu ca Lamia cho thy mc d dng nhiu phng php ci thin iu kin phn ng oxy ha glucose c enzyme GOD xc tc nhng hm lng FOS trong sn phm khng th vt qu 61.26 % v lng glucose b chuyn ha ch hn ch trong khong 16%. Hot lc ca enzyme GOD lun biu hin gim mnh sau 5 gi phn ng. gii quyt tn ti trn, cn thit phi loi b nhn t c ch enzyme GOD l H2O2. iu ny c th tin hnh bng cch b sung thm mt loi enzyme khc c kh nng chuyn ha H2O2 l catalase (CAT). Qu trnh oxy ha glucose di tc dng ca h enzyme GOD- CAT c th hin theo phng trnh phn ng hnh 2.8. [18, 57]
SVTH: V Th Ngc An
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Hnh 2.9: Phn ng oxy ha glucose di tc dng ca h 2 enzyme GOD v CAT [18]
2.1.8. Tim nng cho sn xut FOS ti Vit Nam Vit Nam l mt nc c ngnh cng nghip ma ng pht trin t rt sm. Thc hin ch trng v chnh sch ca chnh ph, n nm 2000 c 47 nh my trrn ton quc i vo hot ng v sn lng ng t ti mt triu tn. Cy ma c th trng tt c cc a danh ton quc. Cc vng tp trung trng ma nhiu l cc tnh thuc Bc Trung B, Nam Trung B, ng Nam B v ng Bng Sng Cu Long. Sn lng ma v ng ma hng nm khng ngng c nng cao. Qu trnh tng trng ca ngnh cng nghip ma ng t nm 1998 n nm 2001 c trnh by trn bng 2.5 S liu thng k trn bng 2.5 cho thy Vit Nam c sn lng ng ma tng i cao, l ngun nguyn liu di do cho ngnh sn xut FOS, mt mt hng cha c pht trin trong th trng ni a.
Bng 2.5: Din bin tng trng ca ngnh cng nghip ma ng trn ton quc Nm 1998 1999 2000 2001 Sn lng ma (nghn tn) Sn lng ng (nghn tn) Din tch trng ma (nghn hecta) [18] Cng vi ngun nguyn liu di do, Vit Nam cn c ngun nhn lc ln, c trnh khoa hc lm ch cng ngh sinh hc ni chung v ch bin thc phm ni ring. V vy vic m ra mt ngnh sn xut mi s dng cng ngh hin i to nn sn phm FOS vi nhiu c tnh u vit, c gi tr cao hn ng knh v c tnh chc nng, cung cp cho th trng trong nc v xut khu, lm nguyn liu cho cc sn phm chc nng nh bnh ko, thc phm cho ngi mc bnh tiu ng, sa v bt dinh dng cho tr em l cn thit v hon ton kh thi. 283.0 344.2 302.3 291.0 736.0 947.3 1208.7 1957.8 13843.5 17760.3 15044.3 14325.4
SVTH: V Th Ngc An
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2.2. Thu nhn v tinh sch enzyme -fructofuranosidase t chng nm mc Aspergillus niger IMI303386 [43] 2.2.1. Tm tt nghin cu Enzyme ni bo -fructofuranosidase (EC 3.2.1.26) thu nhn t Aspergillus niger IMI303386 c tinh sch t n ng nht bng phng php in di SDSPAGE vi cht to ta (NH4)2SO4, sc k trao i ion DEAE Sepharose v lc gel Ultrogel AcA44. Quy trnh ny em li tinh sch gp 50 ln v hiu sut thu hi enzyme l 42%. Khi lng phn t -fructofuranosidase thu c nm trong khong 120 130 kDa. Phn tch ti im ng in nhn thy enzyme ny gm 2 loi protein. pI ca 2 loi protein ny l 5.4 v 4.4. Ba2+, Mg2+, Ca2+ v sodium-EDTA ng vai tr l cc cht hot ha trong khi Hg2+, Ag+ v Ni2+ l cc cht c ch mnh hot ng ca enzyme -fructofuranosidase. Enzyme -fructofuranosidase hot ng n nh trong khong pH t 4 8 v t ti u ti pH 5.5. Nhit ti u ca enzyme l 500C v c th hot ng n nh ti 550C. Trn 90% hot tnh ca enzyme vn c gi li sau 5h ln men. fructofuranosidase thu t Aspergillus niger IMI 303386 l mt glycoprotein vi 17% hm lng carbohydrate. 2.2.2. Nguyn liu v phng php Nguyn liu: Ging A. niger IMI 303386 c mua t Vin nm quc t (International Mycological Institute), Surrey, UK v c lu gi trn mi trng agar dextrose khoai ty. Ct trao i ion resin DEAE-Sepharose v b kit xc nh pI t Pharmacia (Uppsala, Thy in). Thang chun cho in di SDS-PAGE t Bio-Rad Laboratories (CA, USA). Ultrogel AcA44 t Biosep (CA, USA). Tt c cc ha cht s dng khc u c phn tch v mua t ngun thng mi c sn.
Nui cy A. niger v thu nhn -fructofuranosidase: Cho 5ml nc ct v trng vo ng thch nghing cha ging A. niger IMI 303386 trn mi trng agar dextrose khoai ty chun b dch treo bo t. 5ml dch bo t sau c cy truyn vo bnh 250ml cha 100ml mi trng nui cy gm cc cht t l (w/v): 1% glucose, 1% dch chit nm men, 2% NaNO3, 0.05% MgSO4. 7H2O, 0.5% K2HPO4. pH mi trng c iu chnh n 7.4 trc khi em hp. Canh trng c trong 24h 280C vi ch lc 200 vng/pht. Tip sau , cho 15ml dch cha h si nm vo 5 bnh 1lt cha sn 300ml mi trng vi t l (w/v) cc cht nh sau: 2% sucrose, 0.4% L-asparagine, 0.1% K2HPO4, 0.05% MgSO4. 7H2O, 0.01% CaCl2. Cc bnh sau c ln men trong 48h 280C, ch lc 200 vng/pht. Thu nhn enzyme: Sau 48h ln men, h si nm c thu nhn bng cch lc qua giy lc Whatman No.1 v ra nhiu ln vi m phosphate 0.1M, pH 6.5. Sau si nm c nghin vi lng ct gp i v lng ti thiu dd m. Sau khi si nm c nghin ng u th em ly tm ch 15000 g trong 20 pht v thu nhn dch enzyme th l phn ni. Phn tch hot tnh enzyme: Hot tnh thy phn v chuyn ha: 3ml hn hp phn ng cha 0.75ml m phosphate 0.2M pH 6.5, 1.5ml sucrose 5% (w/v) v 0.65ml nc ct c 500C trong 10 pht. Sau thm vo 0.1ml enzyme pha long vi nng thch hp v tip tc thm 15 pht na. Phn ng kt thc khi t cc ng nghim vo ni polyethylene glycol 400 un si trong 10 pht. Sau khi lm ngui, ly 2ml v 1ml mu phn ng i xc nh hm lng ng kh v glucose ln lt theo phng php Somogyi/ Nelson v phng php glucose oxidase-peroxidase (GOD/POD). 2 mu sau c em i so mu bc sng 520nm, t xc nh hm lng ng kh v glucose thng qua ng chun. Hot tnh thy phn c xc nh bng cch o lng ng kh to thnh t sucrose (R). Hot tnh chuyn ha xc nh
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bng cch o c lng ng kh v glucose (G) to thnh t sucrose. Hm lng fructose t do (F) v fructose chuyn ha (F) trong hn hp phn ng c xc nh bng cng thc: F=RG F = G F = 2G R Mt n v hot tnh thy phn c xc nh l lng enzyme cn thit gii phng 1mol ng kh trong 1 pht. Mt n v hot tnh chuyn ha c xem l s mol fructose c chuyn nhm trong 1 pht. Xc nh hm lng protein: Hm lng protein c c lng bng cch o hp thu 280nm hoc s dng b kit phn tch protein ca Pierce (USA). Tinh sch enzyme: Tt c cc bc tinh sch c tin hnh 40C s dng h thng FPLC ca hng Pharmacia, Uppsala, Thy in. Cc phn on sau c em i kim tra hm lng protein 280nm v xc nh hot tnh enzyme. Phn tch in di: Quy trnh c m t bi Laemmli. Protein c nhum vi 0.25% Coomassie brilliant blue G-250. Cc cht sau c s dng lm du chun (Bio-Rad): myosin (200 kDa), -galactosidase (116.25 kDa), phosphorylase B (97 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), cht c ch trypsin (21.5 kDa), lysozyme (14.4 kDa) v aprotinin (6.5 kDa). Gel agarose 1% c s dng xc nh im ng in pI ca -fructofuranosidase 40C trong khong pH 2.5 10.
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Siu lc: Qu trnh siu lc c tin hnh trong b nc nhm c c v thm tch dung dch enzyme, s dng h thng siu lc Amicon (USA) vi mng lc PM-10. nh hng ca pH v nhit ln hot tnh enzyme: Tin hnh kho st trong khong pH t 2.0 11 xc nh nh hng ca pH. Xc nh nh hng ca nhit trong khong 25 750C pH 5.5. nh hng ca ion kim loi v cc hp cht khc ln hot tnh ca enzyme: Ha tan 1mM cc mui kim loi (HgCl2, ZnSO4, CuSO4, AgNO3, CoCl2, MnSO4, FeSO4, BaCl2, CaCl2, MgSO4, NaN3, NiSO4), 10mM dd disodium-EDTA v 10mM urea trong 50mM dd m phosphate pH 5.5. Sau em phn tch hot tnh enzyme nh trn. nh hng ca pH v nhit ln tnh n nh ca enzyme: Xc nh nh hng ca pH: dd enzyme 8.5 U c em 400C. Cc mu th nghim c nhng khong thi gian khc nhau v em xc nh hot tnh. Xc nh nh hng ca nhit : dd enzyme c t trong b n nhit nhit khc nhau trong khong 25 750C ti pH 5.5. Cc mu th nghim sau c t ngay vo trong nc trc khi em xc nh hot tnh. Xc nh hm lng carbohydrate ca enzyme: Phng php dng phenol-sulfuric c m t bi Dubois v cng s. 10L dd enzyme cha 50g protein (nh lng bi phng php BCA) c pha long vo 1mL nc ct. Thm vo 1ml thuc th phenol v 5ml H2SO4. Khuy u hn hp v lm ngui xung nhit phng trc khi o hp thu 490nm. Hm lng carbohydrate c xc nh thng qua ng chun xc nh sn bi glucose. Th nghim tin hnh vi 3 mu ging nhau.
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2.2.3. Kt qu v bn lun Tinh sch -fructofuranosidase Bng 2.6: Kt qu tinh sch -fructofuranosidase t A. niger IMI 303386 Phng php Protein tng tinh sch Enzyme th Kt ta DEAESepharose Ultrogel AcA44 [43] 40ml enzyme sau khi chit tch cha 93mg protein v hot tnh chuyn ha ca enzyme l 96.5U c ta bng (NH4)2SO4 bo ha 80% trong bn nc . Hn hp c gi qua m 40C to ta. Lng protein kt ta c thu nhn sau khi ly tm 15000 g, 40C trong 20 pht v ha tan vi mt lng ti thiu m phosphate 0.02M (pH 6.5). Khong 90% hot tnh tng ca -fructofuranosidase c gi li sau qu trnh ny vi tinh sch l 2 ln. Ton b s mu enzyme sau khi ha tan vo m trn tip tc c np qua ct sc k trao i ion DEAE Sepharose dng chy nhanh (25cm x 2.5cm) vi h thng FPLC (Fast Protein Liquid Chromatography) v m Natri acetate 50mM pH 5.5. Protein b gi li trn ct s c tch ra khi ct bng m Natri acetate cha 0.2% Natri azide (NaN3) vi gradien nng 0 0.5mL NaCl trong 500ml th tch dd m. 0.78 40.30 51.67 49.8 41.8 (mg) 93.00 43.60 1.76 Hot tnh tng (U) 96.50 86.40 73.04 Hot tnh ring (U/mg) 1.04 1.98 41.50 tinh sch 1 1.9 40 Hiu sut (%) 100.0 89.5 75.7
SVTH: V Th Ngc An
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Hnh 2.10: Sc k trao i ion DEAE-Sepharose [43]
Theo hnh 2.9 th mt lng ln protein tp c tch loi v khong 75% hot tnh thy phn ca -fructofuranosidase c thu hi vi tinh sch l 5.7. Cc phn on cha peak c gp li v c c nh phng php siu lc bng mng chng thm 10kDa, sau thm tch vi m phosphate 0.02M pH 6.5. Mu sau khi c c c cho vo ct Ultogel AcA44 (90cm x 1.6cm) dng m phosphate 50mM pH 6.5 cha 0.2% NaN3. Protein c ra gii bng m ging nh vy vi tc dng chy 16mL/h. o hp thu bc sng 280nm xc nh c 2 protein thnh phn v hot tnh enzyme tng ng. Cc phn on cha enzyme c gp chung v c c bng phng php lc mng (Amicon UV 10). tinh sch enzyme khong 50 ln v hiu sut thu hi hot tnh enzyme tng ln 42%.
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Sau khi tin hnh qua 3 bc tinh sch (ta bng amoni sulphate, sc k trao i ion v lc gel) th enzyme -fructofuranosidase t tinh sch l 50 ln v hiu sut thu hi hot tnh l 42%.
Hnh 2.11: Sc k lc gel Ultrogel AcA44 ca -fructofuranosidase [43]
Cc tnh cht ha l ca -fructofuranosidase thu nhn t A. niger: Kt qu in di SDS-PAGE cho thy ch c mt loi protein trong dd enzyme em phn tch. Enzyme sau c tinh sch t n mt ng nht biu kin. Khi lng phn t ca enzyme nm trong khong 120 130 kDa. Kt qu nghin cu ca LHocine v cng s cho thy A. niger AS0023 to ra 2 dng enzyme: fructosyltransferase vi khi lng phn t t 81 168 kDa khi phn tch SDS-PAGE v invertase khi lng phn t t 71 111 kDa. Hirayama v cng s cng
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phn tch -fructofuranosidase t A. niger ATCC 20611 v cho kt qu khi lng phn t da trn SDS-PAGE l 100 kDa, lc gel l 340 kDa. Nh vy c th kt lun rng -fructofuranosidase thu nhn t A. niger gm t nht 2 thnh phn c khi lng phn t nh nhau. Khi lng phn t ca mt thnh phn nm trong khong t 40 200 kDa. Da trn kt qu phn tch im ng in, fructofuranosidase t A. niger cho ra mt vch m pH 5.4 v vch m hn pH 4.4. Hin nay vn c rt t thng tin v pI ca invertase t A. niger.
Hnh 2.12: Kt qu in di SDS-PAGE ca -fructofuranosidase [43] M: Du chun ; P: enzyme tinh sch
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nh hng ca pH ln hot tnh v tnh n nh ca enzyme fructofuranosidase: -fructofuranosidase thu nhn t A. niger IMI 303386 c hot tnh mnh trong khong 5.0 6.5 v ti u pH 5.5. Kt qu ny gn vi gi tr pH ti u ca frutctosyltransferase (pH 5.8) nhng cao hn invertase (pH 4.4) (Theo kt qu ca LHocine v cng s).
Hnh 2.13: nh hng ca pH ln hot tnh v n nh ca -fructofuranosidase [43]
Da vo th trn hnh 2.12, khong pH hot ng n nh ca fructofuranosidase t A. niger l 4.0 8.0. Khi enzyme 400C cc gi tr pH ny th t nht 90% hot tnh ca enzyme vn c gi li sau 5h. Enzyme b mt hot tnh nhanh chng cc gi tr pH thp hn 4.0 hoc cao hn 8.0.
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nh hng ca nhit n hot tnh v n nh ca enzyme fructofuranosidase: Trn hnh 2.13 cho thy enzyme hot ng mnh nht 500C v mt hot tnh nhanh chng khi tng nhit cao hn 600C. Sau 5h pH 5.5, cho thy -fructofuranosidase hot ng n nh nhit thp hn 600C. Hn 90% hot tnh ca enzyme c gi li. Kt qu ny cho thy cc phn ng chuyn ha nn thc hin trong khong nhit t 50 550C.
Hnh 2.14: nh hng ca nhit ln hot tnh v n nh ca -fructofuranosidase [43]
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nh hng ca ion kim loi v cc hp cht khc ln hot ng ca fructofuranosidase: Kt qu bng 2.7 ch ra khi thm ln lt 1mM Ba2+, Mg2+, Ca2+ v 10mM NatriEDTA, hot tnh ca enzyme mnh hn. Kt qu r nht khi thm 1mM MgSO4 (tng 15%) v 10mM Natri-EDTA (tng 17%). Hg2+, Zn2+, Ag+ v Ni2+ l cc cht c ch mnh. Khi thm 1mM cc ion ny th -fructofuranosidase gn nh mt ht hot tnh. Khi b sung 1mM Co2+ v Cu2+, enzyme mt 15 20% hot tnh. Bng 2.7: nh hng ca cc ion kim loi v hp cht ln hot tnh enzyme fructofuranosidase ca A. niger Hp cht (1mM) Khng b sung HgCl2 ZnSO4 CuSO4 AgNO3 FeSO4 CoCl2 MnSO4 BaCl2 MgSO4 CaCl2 Natri-EDTA (10mM) NaN3 NiSO4 Urea (10mM) Hot tnh cn li (%) 100 2 0 83 0 92 78 91 108 115 112 117 98 15 103 [43]
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Hm lng carbohydrate ca -fructofuranosidase t A. niger: -fructofuranosidase t A. niger IMI 303386 l mt glycoprotein vi hm lng carbohydrate l 17% xc nh theo phng php phenol-H2SO4. Kt qu ny ph hp vi gi thuyt cho rng enzyme t nm l glycoprotein vi hm lng carbohydrate t 2 20%.
2.3. Ln men ti s dng nhiu chu k chng Aspergillus oryzae CFR 202 thu nhn enzyme fructosyltransferase v ng dng sn xut ng fructooligosaccharide [49] 2.3.1. Tm tt nghin cu Tin hnh ln men chm Aspergillus oryzae CFR 202 nhm thu nhn fructosyltransferase ng dng sn xut FOS. Dng pellet (cu trc bi si do h si nm cun xon li trong qu trnh ln men) ca nm mc A. oryzae thu nhn sau 48h ln men c b sung mi trng sau mi 24h v thu enzyme fructosyltransferase (FTS). Enzyme thu c em s dng cho qu trnh sn xut FOS vi c cht l sucrose 60% nhit 550C, pH 5.15. Hot tnh enzyme FTS uc duy tr trong khong 15 2U/ml/pht ln n 6 chu k nui cy. Hiu sut to FOS l 53% chu k th 6. Pellet khng c ti s dng trn 6 chu k do sau qu trnh t phn xy ra. H thng ny mang nhiu li ch v c tnh kinh t cao do khng cn b sung thm cht dinh dng v khng i hi phi nhn thm ging mi trc khi ln men. Mi trng dng nhn ging c ti u trc . Vi iu kin pht trin ti u, nm si A. oryzae to thnh dng pellet chc, trn, n nh trong sut 90h ln men. Dng pellet c nhiu thun li nh nh gn, h si nm khng b mc vo thit b Enzyme FTS to ra bi A. oryzae l enzyme ngoi bo. Sau khi kt thc 1 chu k ln men thu enzyme trong 24h th thu nhn dch ln men, sinh khi nm c thu nhn li bng lc chun b cho chu k ln men k tip.
2.3.2. Nguyn liu v phng php Ha cht: Tt c ha cht s dng u c phn tch. Cc loi ng FOS chun 1-kestose, 1-nystose, 1-fructofuranosyl nystose c cung cp bi Wako Pure Chemical Industries, Osaka, Nht bn. Chng vi sinh vt v iu kin nui cy: Aspergillus oryzae CFR 202 c ly t b su tp CFTRI, Mysore. Ging c lu gi trn mi trng agar dextrose khoai ty 40C. Cy ging: Bo t cy 5 ngy trn ng thch nghing c cy truyn vo 100ml mi trng cha 1% sucrose v 0.2% dch chit nm men (pH 5.5) trong cc bnh 500ml, em hp 1210C trong 15 pht. Sau cc bnh ging c 30 10C trn my lc vng ch 250 vng/pht trong 24h. Sn xut enzyme fructosyltransferase bng ln men ti s dng t bo: Cc bnh ging sau khi 24h th c chuyn vo 100ml mi trng ln men cha 10% sucrose, 0.8% dch chit nm men, 1% NaNO3, 0.03% MgSO4. 7H2O, 0.4% K2HPO4, 0.9% KH2PO4, 1% NH4Cl v 0.6% NaCl vi pH ban u l 5 trong bnh 500ml. Cc bnh ny sau li em 30 10C trn my lc vng ch 250 vng/pht. Qu trnh ln men kt thc sau 48h. Dch ln men c gn ra trong iu kin v khun. Mi trng mi li c b sung vo bnh cha pellet. Sau mi 24h, canh trng li c gn ra v b sung mi trng mi. Qu trnh ln men c th tip tc trong 144h tip theo. Canh trng gn ra c kim tra pH, hot tnh enzyme, v a vo sn xut FOS. Phn tch fructosyltransferase: Hn hp phn ng cha 1.5ml sucrose 60% (w/v) pha trong m citrate 0.1M v 0.5ml dch enzyme th. Th nghim tin hnh 55 10C trong 1h trong b nc. Kt thc
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phn ng bng cch ngm hn hp trong nc si trong vng 15pht. Glucose gii phng trong phn ng c xc nh bng b kit glucose s dng phng php GOD/POD (n ). Mt n v hot tnh fructosyltransferase c xc nh l lng enzyme cn gii phng 1mol glucose trong mi ml trong 1 pht di iu kin phn ng cp trn. Sn xut FOS: Hn hp phn ng (1.5ml sucrose 60% trong m citrate 0.1M pH 5.5 v 0.5ml dch enzyme th) c vi iu kin phn ng nu trn trong 18h. Sn phm sau c phn tch bng h thng HPLC ca Nht. Pha ng l dung dch acetonitrile : nc (t l 3:1) vi tc chy l 1.0ml/pht. Cc mu sau c pha long nng thch hp v lc qua mng lc Millipore c kch thc l 0.45m. 2.3.3. Kt qu v bn lun Sau 48h ln men th pellet c ti s dng sau mi 24h sn xut FTS trong mi trng mi. Trong qu trnh , pH ca dch ln men tng t 5.0 6.03 sau 48h ln men. Sau tip tc tng ln 6.18 sau 96h ln men v gi nguyn cho n khi kt thc ln men trong 7 ngy (Hnh 2.14) Hot tnh enzyme l 7.8U/ml/pht sau 48h ln men. Hot tnh t gi tr cao nht l 16.5U/ml/pht sau 2 chu k lin tip v gim cn 11.9U/ml/pht cui chu k 6 (hnh 2.15).
SVTH: V Th Ngc An
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Hnh 2.15: S thay i pH v hot tnh FTS trong qu trnh nui cy nhiu chu k A. oryzae CFR 202 [49]
Hnh 2.16: Hm lng FOS sinh ra bi FTS qua cc chu k nui cy A. oryzae [49]
Sau 6 chu k ln men lin tip pellet A. oryzae khng th ti s dng c na do n mt cu trc bi si chc, trn v bt u phn r. Hot ng sn xut FOS khng b suy gim trong sut 6 chu k ln men. Hiu sut thu nhn FOS gi mc 53% (w/w) trong 6 chu k ti s dng pellet.
2.4. C nh t bo nm mc Aspergillus japonicus vo cht mang gluten v ng dng sn xut ng fructooligosaccharide [23] 2.4.1. Tm tt nghin cu Nghin cu nhm mc ch c nh Aspergillus japonicus bng cch by vo mng gluten ng dng sn xut FOS t sucrose nh enzyme -fructofuranosidase. Khi em 1g h si nm c nh trong cha 20% (w/w) t bo vi 100ml dd sucrose nng ban u l 400g/l th tng lng FOS thu c l 61% (w/w) lng ng tng sau khi tin hnh 1 m phn ng trong 5h. Tc phn ng tng theo lng t bo by trong mng gluten v t gi tr cao nht khi lng t bo l 20% (w/w). Cc si gluten c nh si nm c nhi vo trong ct phn ng sn xut FOS lin tc. Sn lng thu c l 173g/h trong 1 lt th tch ct phn ng vi tc dng chy l 0.8ml/pht. Khi lng cc phn on FOS tng t 0.2 0.54 w/w khi dng chy gim t 1 cn 0.1ml/pht, cng vi thi gian lu tng t 0.35 ln 3.5h. H si nm c by trong mng gluten v t bo c nh trong n nh trong thi gian di. Gluten c nhn thy l vt liu thch hp c nh si nm cng vi enzyme sn xut FOS. Gluten l mt dng polymer t nhin, v l ph phm ca cng nghip sn xut bt m. S dng gluten c cc u im nh n l cht c th phn gii c, r tin, khng c v l ngun nguyn liu c sn.
SVTH: V Th Ngc An
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2.4.2. Nguyn liu v phng php Chng v t bo nui cy: Aspergillus japonicus TIT-KJ1 c cung cp bi tin s K-J Duan b mn cng ngh sinh hc, trng i hc Tatung, Taipei, i Loan. Mi trng lng cha 20% ng sucrose, 1% dch trch ng maltose, v 1% dch chit nm men c dng. Cc t bo c nui ch lc (200v/p) 300C trong 36 gi. thu c nhng si nm dng cho c nh, t bo s c thu nhn, v trn u vi cht lm ng nht polytron trong dung dch m cha Triton X-114 40C trong 24 gi, v lm ng kh -850C. C nh t bo: Bt gluten (10g, Sigma) c ha tan trong 200ml dung dch NaOH 0.05N. Sau khi ly tm loi nhng phn khng tan, phn dd gluten cn li c iu chnh vi HCl 0.1N. Khi pH gim cn 11, tip tc thm 5ml dung dch tinh bt b oxy ha vo (22%, w/v), v sau pH c gim tip xung 6. Sau khi lm lng gluten dng keo v loi b nhng phn ni ln trn b mt, phn keo lng c trn u vi si nm nghin nh ca A.japonicus. Phn gel ny sau c lm kh dng nhng si t bo c nh vi ng knh 0.25cm. Nhng gel kh sau cng c ct thnh nhng mnh nh c kch thc 0.25cm x 0.4 cm v tr 40C trc khi em s dng. Sn xut fructooligosacchride: B sung 1g (hoc 1 lng c th no ) mnh t bo c nh vo 100 ml dung dch ng sucrose (40%, w/v) sn xut FOS 370C di ch lc 125v/p t trong b nc. Qu trnh sn xut FOSs c kim sot bng cch o hm lng FOS to thnh trong dung dch. FOS c sn xut lin tc bng cch nhi 8g cc mnh t bo c nh vo ct kch thc 18cm x 1.55cm. Dung dch sucrose (40%, w/v) c cho i qua ct vi vn tc dng chy xc nh. Nhit ca ct c duy tr 370C. Sn phm trong hn hp phn ng c phn tch bng HPLC s dng ct Econosphere NH2 (5
SVTH: V Th Ngc An
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cartridge, 250 mm x 4.6 mm). Sc k tin hnh trong iu kin nh sau: nhit ct 300C, tc dng chy 1.5 ml/pht; pha di ng l acetonitrile H2O (75:25); my d Water 410 RI. Tng FOS to thnh c gi nh l tng ca 1-kestose v nystose. Lng FOS cao hn thng ch chim 3% v c b qua. Trong tt c cc php phn tch, lng nh fructose c xem nh l glucose. Vn tc phn ng gi u tin: xc nh tc phn ng c xc tc bi -D-fructofuranosidase, 55 ml dung dch sucrose (40%, w/v) c vi 0.2g t bo c nh 370C, trong 1 gi. Hm lng cc loi ng trong hn hp c xc nh bng HPLC. Vn tc phn ng c tnh nh l lng sucrose b chuyn ha trn mt n v thi gian vi hot ng xc tc ca enzyme km theo si nm c nh. 2.4.3. Kt qu Sn xut FOS theo m vi t bo c nh: Hnh 2.16 biu din ng hc ca phn ng transfructosyl sucrose, s dng cc mnh gluten nh by nhng si nm ca Aspergillus japonicus. Khi 100 ml dung dch sucrose 40% (w/v) vi 1g t bo c nh c mt t bo l 20% (w/w), sucrose nhanh chng bin i thnh glucose v 1-kestose (GF2) theo tin trnh phn ng. Sau 4 gi hm lng 1-kestose to ra t gi tr cao nht. 1-kestose sau c chuyn ha thnh nystose (GF3). Tng lng FOS, m ch yu l 1-kestose v nystose, t n gi tr cc i l khong 61% lng ng trong hn hp phn ng sau 5h. Phn khi lng ca FOS tng to thnh gn nh khng i cho n khi hm lng nystose vt qua 1-kestose ti thi im phn ng din ra c 8 gi. Mc d vy, lng FOS tng h xung dn sau .
SVTH: V Th Ngc An
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Hnh 2.17: Nng v phn khi lng ca cc loi ng theo thi gian [23] Khi dng mt lng nh t bo c nh, v d nh 0.2g, th thi gian t ti hm lng FOS cc i l 15h, v hm lng 1-kestose v nystose bng nhau l 18 gi. Nh biu din hnh 2.17, phn khi lng FOS tng ti nhng thi im ny th ging nh khi s dng 1g t bo c nh. Khi mt lng tng ng si nm kh (0.04g khi lng t bo kh) c dng trc tip sn xut FOS t ng sucrose th tc phn ng din ra nhanh hn v thi gian thu lng FOS ln nht cng c rt ngn so vi khi s dng 0.2 g t bo c nh. Thi gian hm lng FOS t cc i v lng 1-kestose v nystose bng nhau ln lt l 12 v 15 gi khi xc tc phn ng bng t bo nguyn vn.
SVTH: V Th Ngc An
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Hnh 2.18: Sn xut FOS s dng t bo c nh v t bo khng c nh [23]
nh hng ca mt t bo trong gluten: Hnh 2.18 biu din nh hng ca hm lng t bo c trong gluten ln hot tnh enzyme sn xut FOS. xc nh vn tc phn ng, 0.2 g t bo c nh c trn vi 50 ml dung dch sucrose (40% w/v) v phn ng xy ra trong 1 gi. Tc phn ng gi u tin (g/h) c tnh da trn lng sucrose bin i. Ngoi tr trng hp t l t bo l 2.5% hoc t hn, nu nng c cht vt qu 5% c th lm tc phn ng khng t nh thi k u. Tc phn ng gi u tin c th gn vi vn tc ban u. hnh 2.18, tc phn ng tng n gi tr cc i l 5.2 g/h cng vi s tng hm lng t bo, trong khi vn tc ring trn 1 n v khi lng t bo t n gi tr ti a (190g/hg trng lng kh t bo) t l t bo 5% w/w, v sau gim xung mc d hm lng t bo tng do s cn tr khng gian v s khuch tn vt cht qua thnh t bo gp kh khn.
Hnh 2.19: nh hng ca lng t bo c nh trong mng gluten [23]
Sn xut FOS lin tc: Sucrose vi nng 400 g/l c bm lin tc vo ct phn ng nhi cc mnh gluten cha si nm c nh. Hnh 2.19 bin din nh hng ca lu lng dng chy ln nng sut to FOS. Phn khi lng ca FOS tng ln cng vi s gim i ca tc dng chy ng thi thi gian lu tng ln. Phn khi lng FOS tng t 0.2 n 0.54 w/w khi tc dng chy gim t 1 n 0.1 ml/pht, tng ng vi thi gian tng t 0.35 n 3.5 gi. Hnh 2.19 cng cho thy nng sut theo tc dng chy v phn khi lng FOS tng, tng theo tc dng chy, v kh nng to FOS cao nht khi s dng tc chy 0.8 ml/pht. Khi th tch hn hp phn ng trong ct cn 34 ml, nng sut cao nht t c khi tc dng chy l 0.8 ml/pht c tnh l 173 g FOS sn xut trong 1 gi trn 1 lt th tch phn ng. Ct phn ng c hot ng trong thi gian di vi ng dn sucrose c nng 400g/l. Khi lu lng dng chy l 0.1 ml/pht, tng ng vi thi gian lu trung bnh l
3.5 gi, phn khi lng ca FOS tng ch thot ca ct gn bng 0.53 w/w sau 4 gi khi ng. Phn khi lng ca FOS duy tr hm lng t 0.52-0.54 w/w trong khong 4 ngy u. Nhng si nm sau khi c by trong mng gluten v nhng t bo lin kt c nh sn sinh ra enzyme c hot tnh duy tr mc cao. Mt khc, phn khi lng FOS gim dn khong 0.4 w/w khi tip tc hot ng trong 18 ngy sau . Hot ng sn xut FOS gim khong 50% gi tr ban u sau khi ct phn ng c dng lin tc 34 ngy.
Hnh 2.20: Phn khi lng FOS v nng sut to FOS theo tc dng chy [23]
SVTH: V Th Ngc An
232
2.5. Sn xut Fructooligosaccharide cao t sucrose s dng h enzyme fructofuranosidase v glucose oxidase [57] 2.5.1. Tm tt nghin cu FOS cao c nghin cu sn xut t sucrose bng phng php ln men khng lin tc s dng h enzyme -fructofuranosidase v glucose oxidase. H enzyme ny c tip vo thit b phn ng dng khuy trn cha 0.7l sucrose, phn ng xy ra trong 25h. Nghin cu tp trung vo vic ti u ha cc iu kin phn ng cho th nghim nhm sn xut hng lot FOS cao vi h 2 enzyme -fructofuranosidase v glucose oxidase. Cc iu kin hot ng ti u cho h 2 enzyme nh sau: pH 5.5, nhit 400C, hm lng sucrose 400 g/l, tc khuy trn 550 vng/pht, lu lng oxy 0.7 l/pht., t l enzyme phi trn: 10 n v -fructofuranosidase kt hp vi 15 n v glucose oxidase trong 1g sucrose. Phn ng din ra trong iu kin ti u, h 2 enzyme phn ng ht vi sucrose v glucose, FOS to thnh vi hm lng ln n 98%. So vi phng php sn xut FOS ch s dng enzyme -fructofuranosidase, FOS cao c sn xut bi h enzyme -fructofuranosidase v glucose oxidase c im khc bit c bn l hm lng nystose tch ly cao hn, v ch c mt lng nh fructofuranosyl nystose to thnh. 2.5.2. Nguyn liu v phng php Ha cht: ng sucrose s dng trong th nghim l ng thc phm, cc ha cht khc dng tinh ch. Oxy 99.5% c s dng cho phn ng enzyme glucose oxidase. Enzyme: -fructofuranosidase thu nhn t vic nui cy chng Aureobasidium pullulans KFCC 10524 (Hn Quc) c m t bi Jung v cng s. Dung dch enzyme c chun b nh sau: cc t bo log phase c thu nhn bng ly tm v ha tan li vo
Chng 2: Fructooligosaccharide nc kh ht ion. Dch treo t bo c x l vi 2% (w/v) kitalase trong 2h 450C sau lc s dng cht tr lc iatomit. Nc lc cha nhiu protein khc nhau c s dng lm enzyme th m khng cn tinh sch. Glucose oxidase (t Aspergillus niger, hot tnh ring 25000 U/g) mua t Sigma (USA), s dng ngay khng cn tinh sch thm. Phn tch enzyme: Hot tnh ca -fructofuranosidase c xc nh bng cch o lng glucose sinh ra trong iu kin sau: hn hp phn ng gm 7.5ml sucrose 800g/l, 2.3ml m Natri citrate 0.1M pH 5.5, v 0.2ml dung dch enzyme; pH 5.5; nhit 550C; thi gian phn ng 1h. Mt n v hot tnh -fructofuranosidase c xc nh bng lng enzyme cn thit gii phng 1mol glucose trong mt pht iu kin th nghim. Hot tnh ca glucose oxidase c tnh theo lng glucose bin mt trong bnh phn ng khuy trn 2l theo cc iu kin kho nghim sau: c cht 0.7l -D-glucose 400g/l, lu lng oxy 0.7 l/pht, tc khuy trn 550 vng/pht, 1ml dd glucose oxidase ha trong m citrate 0.1M, nhit 400C, pH 5.1. Mt n v hot tnh ca glucose oxidase l lng enzyme cn thit oxy ha 1mol glucose trong iu kin phn ng m t. Lng glucose sinh ra c xc nh theo phng php glucose oxidase-peroxidase (dng b kit chn on Sigma). Phng php phn tch: Tt c sn phm phn ng c phn tch bng phng php HPLC s dng ct HPX 42C (0.78 x 30cm, Bio-Rad) v my d khc x RI-4. Nhit ct c gi 850C. Nc c s dng lm pha ng vi tc chy 1.0ml/pht. FOS tng s c cho l tng ca 1-kestose (GF2) v nystose (GF3). FOS mch di hn thng nh hn 3% nn b qua.
SVTH: V Th Ngc An
234
Xc nh pH v nhit ti u cho -fructofuranosidase v glucose oxidase: nh hng ca nhit v pH ln hot ng ca enzyme glucose oxidase c nghin cu trong thit b phn ng 2l ca BIOLAB. Th tch phn ng l 0.7l, lu lng oxy 0,7l/pht, v lng enzyme s dng l 10 n v cho mi gram sucrose. Tuy nhin, c cht c thay th bi -D-glucose 400 g/l v thi gian phn ng gim xung cn 30 pht. Nhit tin hnh 400C. nh hng ca nhit c nghin cu pH 5.5. ti u ha pH v nhit cho -fructofuranosidase, phn ng c tin hnh trong 30 pht vi 10 n v enzyme cho mi gram sucrose , s dng hn hp phn ng tng t nh cho th nghim phn tch enzyme. nh hng ca nhit c nghin cu pH 5.5 v nh hng ca pH th tin hnh nhit 55C. Cc kt qu c trnh by l hot tnh tng i (% ln nht) sau khi phn tch cc sn phm phn ng bng HPLC. 2.5.3. Kt qu v bn lun Nhit v pH ti u cho glucose oxidase v -fructofuranosidase: Nhng tc ng ca nhit v pH ln hot tnh enzym ca -fructofuranosidase v glucose oxidase c kim tra ring bit (Hnh 2.20). Nhit ti u ca 2 enzyme c s khc bit ng k. Cc nhit ti u cho glucose oxidase v -fructofuranosidase tng ng l 35C v 55C. i vi glucose oxidase, nhit tng c ngha l nng oxy gim. V c bn tnh ha tan ca oxy gim khi nhit tng ln. Mt khc, pH ti u cho glucose oxidase gn nh tng t vi fructofuranosidase, trong khong 5 6. Kobayashi v cs. bo co mt kt qu tng t rng iu kin phn ng ti u cho glucose oxidase ngun gc t Penicillium amagasakiense l nhit 40C v pH 5 6. Cn c vo kt qu ny, tt c cc th nghim phn ng c thc hin trong phm vi nhit 35 - 45C v pH c nh l 5.5 ti u ha h 2 enzyme.
SVTH: V Th Ngc An
235
Hnh 2.21: nh hng ca nhit (A) v pH (B) ln hn hp enzyme [57]
nh hng ca nhit ln hn hp enzyme: nh hng ca nhit ln qu trnh sn xut FOS cao c tin hnh nghin cu vi 600g/l sucrose ba nhit 35, 40 v 45C (Hnh 2.21). c ba nhit , ban u FOS c to ra rt nhanh, v gim dn theo thi gian phn ng. Ngoi ra, tc to FOS ban u nhanh hn khi nhit phn ng tng, mc d hiu sut chuyn ha FOS t gi tr tng ng 83% sau 25h. iu ny c th c gii thch do mc kch hot enzyme khi nhit tng khng b p cho lng oxy ha tan gim trong h thng. V vy, 40C c chn lm nhit phn ng cho sn xut FOS cao .
SVTH: V Th Ngc An
236
Hnh 2.22: nh hng ca nhit n t l FOS to thnh [57]
nh hng ca tc khuy trn: nh hng s truyn khi n hot tnh chung ca hn hp enzyme c kho nghim vi 600 g/l sucrose (vi cc tc khuy trn khc nhau), vi kt qu a ra l t l sn xut FOS. B phn truyn ng l mt turbin 6 cnh khuy (ng knh 5.8cm, lm bng thp khng g) c cch y thit b phn ng 5cm. Tsukamoto v cs. nghin cu vai tr quan trng ca tc khuy trn n t l phn ng ca glucose oxidase c nh trn cht mang v thy rng t l ban u l hu nh khng i vi tc khuy trn 1.300 vng/pht. Tuy nhin, trong h thng ny, khng c s khc bit ng k trong t l sn xut FOS nu tc khuy trn 550 vng/pht (Hnh 2.22). iu ny cho thy vai tr ca s truyn khi, khng ging nh
SVTH: V Th Ngc An
237
qu trnh phn ng bng enzyme c nh, l khng quan trng khi tc khuy cao hn 550 vng/pht. Sau y, tt c cc th nghim tip theo c thc hin vi ch khuy o 550 vng/pht.
Hnh 2.23: nh hng ca tc khuy trn n t l FOS to thnh [57]
nh hng ca lu lng oxy: Hnh 2.23 cho thy nh hng ca tc dng chy kh oxy ln sn xut FOS. Khi khng cung cp oxy, c t l sn xut v chuyn i c rt thp, nng sut gn nh ging nhau khi ch s dng mt loi enzyme -fructofuranosidase. Lu lng oxy cn thit t c lng FOS mong mun c xc nh l phi ln hn 0.7 l/pht. S hn ch khuch tn ca oxy vo dd ng nng cao (nng sucrose ban u 600 g/l) lm hm lng oxy ha tan trong hn hp phn ng khng t
SVTH: V Th Ngc An
238
bo ha. Kt qu l, 0,7 l/pht c chn l lu lng oxy cho tt c cc th nghim tip theo.
Hnh 2.24: nh hng ca lu lng oxy n sn xut FOS [57]
nh hng ca nng sucrose: Tin hnh kho nghim nng sucrose trong khong 400 700 g/l xc nh gi tr ti u. Hnh 2.24 cho thy hn hp enzyme c hot ng tng t trn mt di nng sucrose v sau 15h phn ng th hu nh khng i. nng ng cao th oxy ha tan gim nn t l to FOS cng gim. Do chn nng sucrose cho cc th nghim v sau l 400 g/l.
SVTH: V Th Ngc An
239
Hnh 2.25: nh hng ca nng sucrose n t l to FOS [57]
nh hng ca nng glucose oxidase: Theo bo co trc ca nhm tc gi th hm lng -fructofuranosidase ti u cho sn xut FOS l 5 n v cho mi gram sucrose 55C. Trong nghin cu ny th h hn hp 2 enzyme cn mt lng -fructofuranosidase cao hn bi v nhit ti u ca hn hp enzyme (40C) l thp hn nhiu so vi 55C. Vi lng c nh 10 n v fructofuranosidase cho mi gram sucrose th lng glucose oxidase dao ng trong khong 5-15 n v cho mi gram sucrose (400 g/l). Khi thm 5 hoc 10 n v glucose oxidase th lng glucose cn li sau 25h phn ng tng ng l 18% v 5%.
SVTH: V Th Ngc An
240
Tuy nhin, hot tnh ban u ca glucose oxidase khng c duy tr mc cao trong sut thi gian phn ng 25h v enzyme b v hiu ho bi H2O2, mt trong nhng sn phm phn ng. Hnh 2.25 v 2.26 cng cho thy khi s dng 15 n v glucose oxidase, sucrose v glucose phn ng ht v lng FOS to thnh t gn 98%. C th kt lun 15 n v glucose oxidase sn xut FOS cao . Lng catalase ln trong glucose oxidase (di 1%) cng gp phn phn hy H2O2 (Lu rng ngay c mt du vt nh ca catalase cng l mt s ln n v).
Hnh 2.26: nh hng ca nng glucose oxidase ln t l to FOS [57]
SVTH: V Th Ngc An
241
Hnh 2.27: T l cc loi ng trong hn hp phn ng iu kin ti u [57]
Hnh 2.28: Sc k HPLC ca FOS v FOS cao [57] Thi gian lu 5.2, 5.9, 6.9, 8.2 pht tng ng nystose, 1-kestose, sucrose, glucose
Bng 2.8 cho thy thnh phn ng trong sn phm FOS cao thu c di cc iu kin phn ng ti u m t v mt s khc bit ch yu khi so snh vi sn xut FOS ch s dng -fructofuranosidase. Bng 2.8: Thnh phn ng so snh gia FOS v FOS cao a ng Glucose Sucrose 1-kestose Nystose FOS tng FOSb 27.73 14.01 39.78 18.48 58.26 FOS cao c 0 1.68 43.63 54.69 98.32d [57] a: % theo hm lng cht kh; b: iu kin phn ng: 10 n v -fructofuranosidase cho 1 gram sucrose, 400C, pH 5.5, 25h c: iu kin phn ng: 10 n v -fructofuranosidase v 15 n v glucose oxidase cho 1 gram sucrose trong iu kin ti u m t. d: acid gluconic c loi ra bng ct resin trao i cation trc khi phn tch HPLC. Cc dng ng FOS cao hn nystose ch thy dng vt, mc d hm lng cao ca nystose c duy tr trong sut thi gian phn ng. Theo c ch phn ng ca fructofuranosidase th dng ng FOS cao hn, chng hn nh fructofuranosylnystose (GF4), s c to ra bng cch s dng nystose nh c cht nn. Kt qu ny cho thy s khc bit ng k trong ng hc ca -fructofuranosidase gia mt h thng ch s dng duy nht -Fructofuranosidase v h thng hn hp 2 enzyme ny. C th kt lun y l mt phng php y ha hn cho sn xut FOS cao ln n 98% bng cch s dng mt h hn hp enzyme -fructofuranosidase v glucose oxidase. H hn hp enzyme r rng ng mt vai tr quan trng trong vic nng cao tinh khit ca FOS, khng ch bi vic loi b glucose, m sucrose cng c s dng ti a.
SVTH: V Th Ngc An
243
2.6. ng
dng
cng
ngh
enzyme
thu
nhn
ng
chc
nng
fructooligosaccharide t dch ma Vit Nam [10] 2.6.1. Tm tt nghin cu Nc ta l nc c ngnh cng nghip ma ng kh pht trin, cc vng nguyn liu ma phn b kh rng ri (5 vng sinh thi) tri rng t Bc vo Nam. Do , chng ta sn xut ng FOS t nguyn liu ban u l ma kh thun li. Mc ch ca nghin cu l tm hiu cc c tnh ca enzyme -fructofuranosidase trong ch phm enzyme thng mi Pectinex Ultra SP-L (Novozymes) nhm ng dng thu nhn ng chc nng FOS t dch ma. Ch phm Pectinex Ultra SP-L cha -fructofuranosidase vi hot lc 58.1 U/ml. Cc iu kin ti u c chn: nhit 400C, pH 5.6, 240ml v t l enzyme : dch ma l 2 : 100 (v/v). Sir FOS thu c sau khi s dng ch phm Pectinex Ultra SP-L cha cc thnh phn ng nh sau: 50.4% FOS, 10.4% saccharose, 3.9% fructose v 35.3% glucose. 2.6.2. Vt liu v phng php nghin cu Vt liu: Dch ma ca ging ma chn trung bnh F156 trng trong v 2005 - 2006 c s dng nghin cu. ng saccharose l loi ng trng tinh luyn c hm lng 99,62%, c m 0,05% v ng kh: < 0,05%. Ch phm enzyme pectinex Ultra SP-L (hng Novozymes, an Mch) l ch phm dng lng, mu nu en, c snh, c mi thm c trng, trong c cha enzyme -Dfructofuranosidase c sn xut t chng aspergillus niger. Phng php: Tin hnh 4 t ly mu ma 4 giai on (thi im) khc nhau kim tra thnh phn ng trong dch ma. Mi t tin hnh kim tra 3 mu ly kt qu trung bnh.
SVTH: V Th Ngc An
244
Hot tnh ca enzyme b-D Fructofuranosidase c xc nh theo phng php do Seikagaku xut (tham kho ti liu ca Whitaker &cs, 2004), hm lng ng kh theo phng php Lane - Eynone (tham kho ti liu ca ng Th Thu & cs, 1997), hm lng ng theo phng php c Nguyn Vn Mi m t (2001) v thnh phn v hm lng ng trong sn phm FOS thu c bng phng php sc k lng hiu nng cao (HPLC) trn h thng sc k HP 1100 (hng Aligence, M) ti Phng phn tch v kim nh cht lng thc phm, Vin Cng Nghip Thc Phm. Cc loi ng trong mu chun (Trung Quc) v mu nghin cu c phn chia trn ct sc k supelco Ca 30cm x 7,8mm ID (hng Supelco, M), tc dng chy 1ml/min, Detector l Detector o ch s khc x - RI. 2.6.3. Kt qu nghin cu v tho lun: Xc nh hot tnh ca enzyme -D-fructofuranosidase trong ch phm Pectinex Ultra SP-L v hm lng ng saccharose trong dch ma: c nh hng s dng ch phm enzyme pectinex-Ultra SP-L, hot tnh ca enzyme -D fructofuranosidase c xc nh. S n v hot tnh trong 1ml dung dch ch phm enzyme l 58,1 U/ml. Do ch phm Pectinex Ultra SP-L cha enzyme -D fructofuranosidase, do c th s dng ch phm ny thu nhn cc fructooligosaccharide (FOS) t ng saccharose. Hm lng 3 loi ng chnh trong dch ma thu c trnh by bng 2.9. Bng 2.9: Thnh phn ng trong dch ma t kim tra Saccharose Glucose Fructose (% w/w) (% w/w) (% w/w) 1 90.6 6.2 3.2 2 91.6 6.3 2.1 3 90.6 6.2 3.2 4 90.6 6.2 3.2 Trung bnh 90.9 6.2 2.9 [10]
S liu ca bng 2.9 cho thy thnh phn ng trong nc ma tng i ng u trn 4 mu nc ma phn tch. Trong s 3 loi ng xc nh, dch ma cha hm lng ng saccharose l ch yu (~ 90%). Ngoi ra dch ma cn cha ng glucose (~6%) v ng fructose (~3%). Xc nh iu kin ti u cho enzyme hot ng: nh hng ca nhit , pH v thi gian phn ng n hot tnh ca enzyme v kh nng vn chuyn ng fructose to thnh ng FOS c nghin cu. Hnh 2.28 cho thy hm lng fructose c vn chuyn to FOS tng dn t nhit 25- 400C v t cc i ti gi tr nhit 400C vi hm lng l 6,9%. Sau hm lng fructose c vn chuyn gim dn t nhit 45- 500C. iu chng t kh nng phn ng th hin hot tnh transferase ca enzyme t cc i ti gi tr nhit 400C.
Hnh 2.29: nh hng ca nhit n kh nng vn chuyn Fructose [10] Hnh 2.29 cho thy khi gi tr pH tng t 4,5-5,5, hm lng fructose c vn chuyn to FOS cng tng ln v t cc i ti gi tr pH 5,5 vi hm lng fructose chuyn ha t 6,9%. Sau , khi tip tc tng t pH 5,5-7, hm lng fructose c vn chuyn li gim xung. Ti gi tr pH = 7 hm lng fructose c vn chuyn ch bng 09%. Ti gi tr pH 5,5 hm lng fructose c vn chuyn to FOS t gi tr cao nht.
Hnh 2.30: nh hng ca pH n kh nng vn chuyn Fructose [10] Hnh 2.30 cho ta thy thi gian phn ng cng tng th hm lng fructose c vn chuyn to FOS cng tng. Thi gian phn ng tng t 30 n 240 pht th hm lng fructose c vn chuyn to FOS tng cao trn 1%. Hm lng fructose c vn chuyn mc thp nht l 0.3% 30 pht. Vi thi gian t 240 pht n 300 pht th hm lng fructose c vn chuyn tng ln khng ng k. V vy, thi gian phn ng ca ch phm enzyme c chn l 240 pht. Lc ny, hm lng fructose c vn chuyn to FOS t 7,0%. Trn c s cc kt qu thu c nhit 40oC, pH 5,5 v thi gian phn ng 240 pht c chn cho phn ng thu nhn ng FOS t dch ma.
Hnh 2.31: nh hng ca thi gian n kh nng vn chuyn Fructose [10]

nh hng ca t l enzyme/c cht ti s bin i thnh phn ng: Lng ch phm enzyme b sung l mt yu t rt quan trng nhm tng kh nng thy phn v vn chuyn, cht lng v gi thnh sn phm. Nu b sung vi lng enzyme thp th kh nng thy phn thp, thi gian thy phn ko di lm cho sn phm bin mu, cht lng v thnh phn ng bin i khng nh mong mun. Ngc li nu lng ch phm enzyme b sung cng cao th hiu qu thy phn cng cng cao (t n im cc i). Tuy nhin b sung enzyme trong qu trnh sn xut cn phi quan tm n gi thnh ca enzyme. V vy m bo c yu t kinh t v yu t k thut phi chn ra mt t l thch hp gia cc yu t . Khi b sung enzyme vi cc lng khc nhau t 0,005 n 0,04 v tin hnh phn ng iu kin: nhit : 400C, pH 5,5 v thi gian phn ng 240 pht, lng enzyme tng th hm lng fructose vn chuyn to FOS cng tng theo (bng 2.10). iu chng t hot tnh transferase ca enzyme cng tng khi nng enzyme tng. Khi lng enzyme tng t 0,005 - 0,02 th hm lng fructose c vn chuyn to FOS tng t 0,6% n 6,9%. Nhng khi lng enzyme tng t 0,02 n 0,04 th hm lng fructose c vn chuyn ch tng t 6,9% n 7,9% (=1%). Nh vy l hm lng fructose c vn chuyn tng rt chm t nng ezyme trong khong ny. Bn cnh hm lng saccharose+FOS gim dn theo chiu tng ca lng enzyme, cn ngc li hm lng glucose tng dn theo chiu tng ca lng enzyme. S liu cng cho thy kh nng thy phn ca enzyme tng dn khi tng nng enzyme. Kt qu thu c bng 2.10 cho php chn t l enzyme/c cht: 2ml ch phm enzyme cho 100ml dch ma trong quy trnh sn xut l hp l hn p ng yu cu v gi thnh cho sn phm.
SVTH: V Th Ngc An
248
Bng 2.10: nh hng ca t l Enzyme/c cht ti s bin i thnh phn ng Saccharose + FOS Glucose Fructose Fructose T l (%w/w) (%w/w) (%w/w) vn chuyn Enzyme/nc ma (%w/w) (v/v) 0 90.6 6.2 3.2 0 0.005 85.7 9.0 5.3 0.6 0.01 82.2 11.7 6.1 2.6 0.015 80.1 13.9 6.0 4.8 0.02 73.5 18.2 8.2 6.9 0.025 73.4 18.4 8.1 7.2 0.03 71.7 19.5 8.8 7.6 0.035 70.6 20.1 9.3 7.7 0.04 69.7 20.6 9.7 7.9 [10] Thnh phn ng FOS c trong sn phm: Thnh phn v hm lng cc loi ng c trong ch phm FOS c xc nh v nh lng (hnh 2.31, 2.32 v bng 2.11). Siro FOS thu nhn c theo quy trnh sn xut trnh by s 1 c phn tch thnh phn v hm lng cc loi ng trn my sc k lng hiu nng cao (HPLC). Mu i chng l ng FOS bn trn th trng Trung Quc. S liu bng 2.11 v sc k (hnh 2.31 v 2.32) cho thy hm lng FOS t gi tr cao nht, chim 50,4%, hm lng fructose chim t l thp nht 3,9%. Bn cnh hm lng saccharose cn li vi t l thp (10,4%) v lng glucose chim t l kh cao (35,3%). iu ny chng t hiu qu thy phn v kh nng vn chuyn fructose ca ch phm enzyme pectinex Ultra SP-L t t l cao. Vi hm lng fructose chim 3,9% cho thy hiu sut chuyn ha rt cao. Vi vic s dng ch phm enzyme Pectinex Ultra SP-L cho siro FOS c cht lng tng ng nh ng FOS ca Trung Quc bn trn th trng.
SVTH: V Th Ngc An
249
Hnh 2.32: Sc k ca ng FOS v cc loi ng trong mu nghin cu [10]
Hnh 2.33: Sc k ca ng FOS chun [10]
Bng 2.11: Thnh phn v hm lng ng c trong sn phm FOS Loi ng FOS Saccharose Glucose Fructose Hm lng (%) 50.4 10.4 35.3 3.9 [10]
Quy trnh sn xut ngh:
Hnh 2.34: S quy trnh sn xut siro FOS t nc ma s dng Pectinex Ultra SPL [10] 2.6.4. Kt lun Hot tnh ca enzyme -D-fructofuranosidase c trong ch phm Pectinex Ultra SPL l 58.1U/ml. Cc iu kin cho vic x l enzyme Pectinex Ultra SP-L c la chn gm: Nhit thch hp l 400C, pH thch hp l 5.5, thi gian phn ng l 240 pht, t l enzyme/nc ma (200Bx) l 2ml/100ml dch ma.
SVTH: V Th Ngc An
251
T nghin cu xut mt quy trnh sn xut siro FOS t nc ma c s dng ch phm Pectinex Ultra SP-L vi Siro FOS thnh phm c thnh phn v hm lng cc loi ng l: ng FOS: 50,4%, saccharose: 10,4%, fructose: 3,9% v glucose: 35,3%. 2.7. Nghin cu s dng ng FOS trong sn xut mt s thc phm chc nng Vit Nam [18] 2.7.1. Sn xut bt dinh dng tr em Nghin cu sn xut dt dinh dng tr em c thc hin ti Vin Cng nghip thc phm t nhng nm 90 ca th k trc. Kt qu nghin cu c chuyn giao n nhiu c s sn xut v ngay ti Vin Cng nghip thc phm chng ti cng c mt dy chuyn sn xut theo cng ngh nu p n nghin cu. t c mc ch l a sn phm ng FOS vo bt dinh dng tr em nhm nng cao gi tr dinh dng cng nh gi tr sinh hc, chng ti tn dng dy chuyn thit b c sn, ci tin cng ngh c s trong nghin cu s dng ng FOS vo sn xut bt dinh dng tr em. i tng nghin cu ca chng ti l bt dinh dng loi ngt.
SVTH: V Th Ngc An
252
Hnh 2.35: S quy trnh sn xut bt dinh dng tr em c b sung FOS [18] Bt dinh dng c b sung ng chc nng FOS sau c em i phn tch thnh phn dinh dng v nh gi cm quan. Kt qu c trnh by bng 2.12 v 2.13. Bng 2.12: Thnh phn ca bt dinh dng tr em Cc ch tiu m m Bo Glucid tng ng FOS n v tnh % % % % % Bt dinh dng thng 6.0 12.5 4.5 75.5 0 Bt dinh dng FOS 6.0 12.0 4.0 76.5 5 [18]
Bng 2.13: Kt qu nh gi cm quan bt dinh dng tr em (thang im 10) Cc ch tiu Mu Mi V Hng Trng thi Tng th Bt thng 9 9 8 9 9 8.8 Bt c b sung FOS 9.5 9 9 9 8.5 9 [18] Kt qu nghin cu cho thy vic b sung ng FOS thay th ng knh trong sn xut bt dinh dng tr em l hon ton d dng v kh thi trong sn xut ln v cng ngh sn xut mt hng mi ny khng cn s thay i ln v thit b cng nh cng ngh m sn phm thu c li c hng v thm ngon hn so vi sn phm thng thng li mang c tnh chc nng gip cho tr d tiu ho, n ngon ming v cn phng chng mt s bnh khc na nh c tnh ca ng FOS gii thiu. V mt gi thnh sn phm ny s t hn so vi mu i chng l 2334 /kg. S tng gi ny qu nh so vi gi thnh bt dinh dng bn trn th trng. 2.7.2. Sn xut bnh bch quy Bnh bch quy l sn phm c sn xut t hai nguyn liu chnh l bt m v ng cng vi cc cht b dng nh b, trng, sa... thng qua qu trnh nng, di tc dng ca nhit , sn phm thu c l mt loi thc phm thm ngon d tiu ho, giu hydratcabon, protein v lipit. Vi c tnh l sn phm kh, hm lng nc thp nn bnh quy rt d bo qun v thun tin cho vic chuyn ch cng nh tiu dng. V th bnh quy t bao i nay lun l sn phm c ngi tiu dng a thch. Cc i tng s dng bnh quy rt a dng, t tr em n ngi gi, t ngi kho n ngi bnh. Phm vi s dng cng rt rng t thnh ph n cc min xa xi ho lnh. V ng FOS c cc tnh cht v hng v gn ging nh ng knh, nn hng b sung ng FOS vo bnh bch quy nh mt cht thay th ng knh. Cng nh trong
SVTH: V Th Ngc An
254
sn xut bt dinh dng, mc tiu ca sn phm l phi cha t 5% 10 % ng FOS. i tng s dng s l FOS 50 gim gi thnh. Sn phm bnh bch quy c b sung ng FOS sau c em i phn tch thnh phn dinh dng v nh gi cht lng cm quan, kt qu c th hin trn bng 2.14 v 2.15. T cc kt qu trn ta thy vic s dng ng FOS vo sn xut bnh bch quy cng nghip l hon ton kh thi. Sn phm mi ny ngoi gi tr ci thin cht lng ca bnh quy ra n cn c tc dng ci thin v phng chng bnh tt ca ng FOS na.
Hnh 2.36: S quy trnh sn xut bnh bch quy s dng ng FOS [18]
SVTH: V Th Ngc An
255
Bng 2.14: Thnh phn ca bnh bch quy Cc ch tiu m m Bo Glucid tng ng FOS n v tnh % % % % % Bnh quy thng 6.20 6.80 3.90 69.3 0 Bnh quy FOS 6.2 6.8 3.92 70.0 7 [18]
SVTH: V Th Ngc An
256
Bng 2.15: Kt qu nh gi cm quan bnh bch quy (thang im 10) Cc ch tiu Mu Mi V Hng Trng thi Tng th 2.7.3. Sn xut ko Ko l mt trong nhng sn phm chnh, chim khi lng bc nht trong cng nghip ch bin ng. Ph thuc vo c tnh nh trng thi, thnh phn, mu m v hng v, ko c chia ra lm nhiu chng loi nh ko cng, ko mm, ko dai ... Do c tnh d bo qun, thun tin s dng v hng v thm ngon nn ko c sn xut v tiu dng rng ri t bao i nay trn khp th gii, cho mi i tng. tng cng gi tr ca ko chng ti tin hnh nghin cu s dng ng chc nng FOS vi mc ch gip cho ng o ngi tiu dng c tip nhn sn phm ng chc nng FOS ci thin v tng cng kh nng phng chng bnh tt. Trong khun kh thi gian v kinh ph c hn nn ch chn loi ko cng khng nhn lm i tng nghin cu. Bnh quy thng 9 9 8 9 9 9 Bnh quy FOS 9.5 9 9 9 9 9.1 [18]
SVTH: V Th Ngc An
257
Hnh 2.37: S quy trnh sn xut ng cng s dng ng FOS [18] Vic b sung ng FOS vo sn xut ko thc t cho kt qu rt tt, ngoi nhng c tnh c c nh hm lng ng FOS c cha trong n, gi tr cm quan ca ko cn c nh gi rt cao bi hng thm v ngt du mt v xp do. Trong s cc chuyn gia ca hi ng cm quan cn cho bit, v ngt mt ca ko khi thay th ng FOS c c l do khng nhng t bn thn ng FOS mang n m hnh nh ng FOS cn c kh nng ci thin v ngt ca ng knh v cc loi ng khc lm cho chng du hn, mt hn.
SVTH: V Th Ngc An
258
Bng 2.16 v 2.17 di y l kt qu phn tch so snh thnh dinh dng v im cm quan ca sn phm ko cng b sung 200 g/kg ng FOS vi mu ko i chng. Bng 2.16: Thnh phn ca hai loi ko Cc ch tiu m m Bo Glucid tng ng FOS n v tnh % % % % % Ko cng thng 6.2 2.2 3.1 80 0 Ko cng FOS 6.2 2.15 3.0 82 7.5 [18]
Bng 2.17: Kt qu nh gi cm quan ca hai loi ko Cc ch tiu Mu Mi V Hng Trng thi Tng th Ko cng thng 9 8.5 7.5 9 9 8.6 Ko cng FOS 9.5 9 9 9 9 9.1 [18] 2.8. Kt lun chung v sn xut FOS v ng dng cho n nay FOS c sn xut trn quy m cng nghip s dng h enzyme fructosyltransferase t vi sinh vt. Cho n nay quy trnh sn xut c nhiu bc tin vt bc cng vi s pht trin ca cng ngh sinh hc trn ton cu. Cc phng php c nh enzyme, c nh t bo gip FOS c sn xut hng lot vi quy m ln hn. FOS cao c sn xut vi cc phng php s dng h a enzyme, lc nano, ln men m bo tinh khit ca FOS khi b sung vo thc phm. Phng php phn tch thnh phn FOS c s dng ph bin hin nay vi chnh xc cao l phng php sc k lng hiu nng cao HPLC.
Mt bc tin mi trong k thut di truyn m ra mt k nguyn mi cho FOS. Cc nh khoa hc nghin cu to dng gene sn xut fructosyltransferase vo E.coli, gip vic sn xut FOS trn quy m cng nghip d dng hn vi nng sut cao hn gp bi. Nh vy, vi cc c tnh chc nng quan trng, FOS ngy cng c bit n l mt trong nhng thc phm chc nng hng u hin nay, to nn bc tin mi trong ngnh cng nghip thc phm. nc ta, FOS cha c sn xut hng lot m mi ch th nghim trn quy m nh. Trong tng lai khng xa, vi ngun nguyn liu ti ch di do l ma, tim nng cho sn xut FOS ca Vit Nam l rt ln v cn c u t ng mc.
SVTH: V Th Ngc An
260
KT LUN CHUNG TI
n Cng ngh sinh hc ng dng trong sn xut v ch bin thc phm chc nng trnh by mt cch tng qut nhng vn lin quan n thc phm chc nng hin nay v i su vo 2 hng ln: ng dng cng ngh di truyn lm giu cc cht dinh dng chc nng vi nhm sn phm b sung folate v ng dng cng ngh vi sinh trong sn xut v ch bin thc phm chc nng vi 2 sn phm c quan tm hin nay l to Spirulina v ng chc nng Fructooligosaccharide. Bc u cc hng nghin cu ca ti khi qut tnh hnh pht trin v sn xut cc sn phm nu, tng hp li mt s nghin cu c khoa hc cng nhn v ra hng pht trin trong thc trng Vit Nam hin nay./.
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TI LIU THAM KHO
Ti liu tham kho trong nc: [1]. Clive James (2007). Bo co tm tt : Hin trng cy trng CNSH/ cy trng chuyn i gen trn ton cu nm 2007, ISAAA. [2]. Cng ngh gen trong nng nghip. Gio trnh i hc Hu. [3]. Dng Thanh Lim (2009), Thc phm chc nng v sc khe bn vng, NXB H Nng Lm, Tp. H Ch Minh. [4]. Glick, B. R., Pasternak, J. J. (2007). Cng ngh sinh hc phn t Nguyn l v ng dng ca DNA ti t hp. Nh xut bn Khoa hc v K thut. [5]. Hunh Lm Minh Thy (2008), ng dng Spirulina vo sn xut sa chua, Lun vn thc s, H Bch Khoa Tp. H Ch Minh. [6] L Tun Ngha, Trn Duy Qu (2005). Nghin cu v ng dng cng ngh sinh hc phc v sn xut nng nghip Vit Nam trong 20 nm qua (1985-2005). Tp ch Sinh hc, 28, 1-9. [7]. L Vn Lng (1999), Spirulina, NXB Y Hc. [8]. Lng nh Qut (2007), Nghin cu cc phng php x l sinh khi vi khun lam S.platensis sn xut mt s loi nc ung giu dinh dng, Lun vn thc s, H Bch Khoa Tp. H Ch Minh. [9]. Mai Ngc Tho (2008), ng dng Spirulina vo sn xut bnh m ngt v bnh m lt, Lun vn thc s, H Bch Khoa Tp. H Ch Minh. [10]. Ng Xun Mnh v cng s (2006), ng dng cng ngh Enzyme thu nhn ng chc nng Fructooligosaccharide (FOS) t dch ma, Tp ch Khoa hc k thut Nng nghip, tp IV, s 6, 105 111.
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[11]. Nguyn nh Th Nh Nguyn (2009), Nghin cu qu trnh tinh sch Fructooligosaccharide bng phng php lc nano, Lun vn cao hc cng ngh thc phm, i hc Bch Khoa TP. H Ch Minh. [12] Nguyn c Lng v cng s (2002). Cng ngh gen. Nh xut bn i hc Quc gia Tp.HCM. [13]. Nguyn c Lng (2006), Cng ngh vi sinh tp 2 - Vi sinh vt hc cng nghip, NXB H Quc gia Tp. H Ch Minh. [14]. Nguyn Ti Lng (2005). Thc phm chc nng-Thc n ca con ngi th k 21. ng dng cng ngh sinh hc to cc ch phm thc phm chc nng phc v sc khe ca cng ng. Tp ch sinh hc, 27, 1-5. [15]. Nguyn Th Nh Ngc (2003), Nghin cu ch bin mt s thc phm c b sung to Spirulina, Lun vn thc s, H Bch Khoa Tp. H Ch Minh. [16]. Nguyn Tin Thng, Nguyn nh Huyn (1998). Gio trnh sinh ha hin i. Nh xut bn Gio Dc. [17]. Trn Hng H (2004). An ton sinh hc- nh gi v qun l ri ro cc sinh vt bin i gen, Nh xut bn B ti nguyn v mi trng. [18]. Trnh Th Kim Vn v cng s (2004), Nghin cu sn xut ng fructooligosaccharide bng cng ngh a enzyme v ng dng trong sn xut thc n tr em v bnh ko chc nng, Bo co tng kt Khoa hc v k thut, B Khoa hc v cng ngh - Vin cng nghip thc phm. Ti liu tham kho nc ngoi: [19]. Ahsan, M., Habib, B., Parvin, M. (2008), A review on culture, production and use of spirulina as food for humans and feeds for domestic animals and fish, Department of Aquaculture, Bangladesh Agricultural University, Mymensingh, Bangladesh.
- 263 -
[20]. Bagley, P. J. & Selhub, J. (2000). Analysis of Folate Form Distribution by Affinity Followed by Reversed-Phase Chromatography with Electrochemical Detection. Clin. Chem. 46, 404411. [21]. Basset, G., Quinlivan, E. P., Ziemak, M. J., Daz de la Garza, R., Fischer, M., Schiffmann, S., Bacher, A., Gregory, J. F., III, & Hanson, A. D. (2002). Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I. Proc.Natl. Acad. Sci. USA, 99, 1248912494. [22]. Bekaert, S. et al. (2008). Folate biofortification in food plants. Trends in Plant Science. 13, 28-35. [23]. Chien, C.S., Lee, W.C, Lin, T.J. (2001), Immobilization of Aspergillus japonicus by entrapping cells in gluten for production of fructooligosaccharides, Enzyme and Microbial Technology 29, 252 257. [24]. Daniell. H; Singh, N. D; Mason, H; Streatfield, S. J (2009) Plant-made vaccine antigens and biopharmaceuticals. Trends in Plant Science, 14, 669-679. [25]. Datta, S. and Bouis, H.E. (2000) Application of biotechnology to improving the nutritional quality of rice. Food Nutr. Bull. 21, 451456. [26]. Deikman, J., Kline, R. & Fischer, R. L. (1992) Organization of Ripening and Ethylene Regulatory Regions in a Fruit-Specific Promoter from Tomato (Lycopersicon esculentum). Plant Physiol. 100, 20132017. [27]. Daz de la Garza, R. et al. (2004) Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis. Proc. Natl. Acad. Sci. U. S. A. 101, 1372013725. [28]. Daz de la Garza, R.I. et al. (2007) Folate biofortification of tomato fruit. Proc. Natl. Acad. Sci. U. S. A. 104, 42184222. [29]. Fell, D. A. (1998) Increasing the flux in metabolic pathways: A metabolic control analysis perspective.Biotechnol. Bioeng. 58, 121124.
- 264 -
[30]. Fukushima, T. & Nixon, J. C. (1980) Anal. Biochem. 102, 176188. [31]. Hanson, A.D. and Roje, S. (2001) One-carbon metabolism in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 119137. [32]. Henrichkson, R. (2009), Earth Food Spirulina, Ronore Enterprises, Inc., Hana, Maui, Hawaii. [33]. Hossain, T., Rosenberg, I., Selhub, J., Kishore, G., Beachy, R. & Schubert, K.(2004). Enhancement of folates in plants through metabolic engineering. Proc. Natl. Acad. Sci. USA 101, 51585163. [34]. Klee, H. J. & Kishore, G. M. (1996). U.S. Patent 5,512,466. [35]. Kohashi, M., Tomita, K. & Iwai, K. (1980). Analysis of Conjugated Pterins in Food Resources and Human Urine. Agric. Biol. Chem. 44, 20892094. [36]. Konings, E. J., Roomans, H. H., Dorant, E., Goldbohm, R. A., Saris, W. H. & van den Brandt, P. A. (2001). Folate intake of the Dutch population according to newly established liquid chromatography data for foods. Am. J. Clin. Nutr, 73, 765776. [37]. Koziel, M. G., Carozzi, N. B. & Desai, N. (1996) Optimizing expression of transgenes with an emphasis on post-transcriptional events. Plant Mol. Biol. 32, 393405. [38]. Lin, T.J., Lee, Y.C. (2008), High content fructooligosaccharides production using two immobilized microorganisms in an internal-loop air [39]. Lobo, A.R., Colli, C., Tullia, M.C, Filisetti, C. (2006), Fructooligosaccharide improve bone mass and biomechanical properties in rats, Nutrition Research 26, 413 420. [40]. Mesa, M.D., Silvn, J.M., Olza, J., Gil, A., Castillo, M.D. (2008), Antioxidant properties of soy proteinfructooligosaccharide glycation systems and its hydrolyzates, Food Research International 41, 606 615.
- 265 -
[41]. Murr, C. et al. (2002) Neopterin as a marker for immune system activation. Curr. Drug Metab. 3, 175187. [42]. Nestel, P. et al. (2006) Biofortification of staple food crops. J. Nutr. 136, 1064 1067. [43]. Nguyen, Q.D., Szab., J.M.R., Bhat, M.K., Hoschke, A. (2005), Purification and some properties of -fructofuranosidase from Aspergillus niger IMI303386, Process Biochemistry 40, 2461 2466. [44]. Patarca, R. J. (2003). The Role of Pteridines in Physiology and Pathology Environ. Pathol. Toxicol. Oncol. 22, 117127. [45]. Pfeiffer, C. M. & Gregory, J. F., III (1996). Enzymatic deconjugation of erythrocyte polyglutamyl folates during preparation for folate assay: investigation with reversedphase liquid chromatography .Clin. Chem. 42,18471854. [46]. Quinlivan, E. P., Roje, S., Basset, G., Shachar-Hill, Y., Gregory, J. F., III, & Hanson, A. D. (2003). The Folate Precursor p-Aminobenzoate Is Reversibly Converted to Its Glucose Ester in the Plant Cytosol. J. Biol. Chem. 278, 2073120737. [47]. Jimnez, C., Cosso, B.L, Labella, D., Niell, F.X. (2007), Screening of functional compounds in supercritical fluid extracts from Spirulina platensis, Food Chemistry, 102, 1357-1367. [48]. Sakakibara, M., Fukuda, Y. (2006), Process for treating Spiruilna, United States Patent. [49]. Sangeetha, P.T., Ramesh, M.N., Prapulla, S.G. (2005), Fructooligosaccharide production using fructosyl transferase obtained from recycling culture of Aspergillus oryzae CFR 202, Process Biochemistry 40, 1085 1088.
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[50]. Sangeetha, P.T., Ramesh, M.N., Prapulla, S.G. (2005), Recent trends in the microbial production, analysis and application of Fructooligosaccharides, Trends in Food Science & Technology 16, 442 457. [51]. Santos, A.M.P., Maugeri, F. (2007), Synthesis of Fructooligosaccharides from sucrose using inulinase from Kluyveromyces marxianus, Food Technol. Biotechnol. 45 (2) 181 186. [52]. Stein, A.J. et al. (2006) Potential impact and cost-effectiveness of golden rice. Nat. Biotechnol. 24, 12001201. [53]. Storozhenko, S. et al. (2007) Folate fortification of rice by metabolic engineering. Nat. Biotechnol. 25, 12771279. [54]. Stover, P.J. (2004) Physiology of folate and vitamin B-12 in health and disease. Nutr. Rev. 62, S3S12. [55]. Varga, L., Szigeti, J., Kovcs, R., Fldes, T., Buti, S. (2002), Influence of a Spirulina platensis Biomass on the Microflora of Fermented ABT Milks During Storage, Journal of Dairy Science, 85, 1031-1038. [56]. Wang, L., Pan, B., Sheng, J., Xu, J., Hu, Q. (2007), Antioxidant activity of Spirulina platensis extracts by supercritical carbon dioxide extraction, Food Chemistry, 105, 36-41. [57]. Yun, J.W., Lee, M.G., Song, S.K. (1994), Batch production of High-content Fructooligosaccharids from sucrose by the mixed-enzyme system of -fructofuranosidase and glucose oxidase, Journal of Fermentation and Bioengineering Vol.77, No.2, 159 163. [58]. Yun, J.W. (1996), Fructooligosaccharide occurrence, preparation and application, Enzyme and Microbial Technology 19, 107 117.
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Web tham kho: [59].http://www.khoe24.vn/home/Kien-thuc-san-pham/Thong-tin-san-pham/tao-spirulinathuc-an-ky-dieu. [60]. http://www.thucphamsuckhoe.vn/NewsDetails.aspx?NewsID=30462d3d-c65e-40038603-f8c9285051b6. [61]. http://vi.wikipedia.org/wiki/Vitamin. [62].http://www.medinet.hcmcity.gov.vn/ttyh/bshkhkt/vtchatbeodoivoitreem04-112003.htm. [63]. http://tpcnhocvienquany.com/?tab=detail_news&catid=MQ==&id=NTY. [64]. http://www.vihuco.com/modules.php?name=News&file=article&sid=20. [65]. http://images.google.com/ [66]. http://www.genco.com/ [67]. http://www.ext.vt.edu/pubs/biotech/443-003/443-003.html#L3 [68]. www.healthlinkbc.ca/healthfiles/index.stm [69]. http://www.agbiotech.com/vn [70]. http://ods.od.nih.gov/factsheets/folate.asp [71]. http://www.cfsan.fda. Gov/dms~/OPA-appa.html [72]. http://www.nal.usda.gov/fnic/foodcomp/search/ [73].http://thucphamvadoisong.vn/
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I HC QUC GIA TP.

H CH MINH TRNG I HC BCH KHOA KHOA K THUT HA HC B MN CNG NGH SINH HC

TP. H Ch Minh, thng 06/2010

Phn 1: Tng quan v thc phm chc nng

PHN 1 TNG QUAN V THC PHM CHC NNG

Chng 1: Khi nim thc phm chc nng

CHNG 1 KHI NIM THC PHM CHC NNG

Chng 1: Khi nim thc phm chc nng

Chng 1: Khi nim thc phm chc nng

thng c th hin qua s sau :

Chng 1: Khi nim thc phm chc nng

Thc phm thuc Thc phm chc nng

Thc phm thng thng

Chng 1: Khi nim thc phm chc nng

Lch s nghin cu thc phm chc nng trong nc v trn th gii

Chng 1: Khi nim thc phm chc nng

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Thc phm ci thin ng tiu ho

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Hnh 2.1: Cng thc cu to Soyasaponin I IV [3]

Hnh 2.2: Cng thc cu to isoflavone v dn xut [3] - 17 -

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Hnh 2.3: Cng thc ha hc ca lycopene [65] - 18 -

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Chng 2: Phn loi thc phm chc nng Vai tr sinh hc

Hnh 2.6: Gii thiu hnh nh mt s loi nm [65]

Chng 3: Phng php lm giu cc cht dinh dng chc nng

i vi ging rau ci: V d gia cc ging ci bp v ci b ngi ta nhn thy loi

i vi cc loi c qu th loi c mu vng, mu nh khoai lang ngh, b , c

i vi cc loi ht cc th loi c mu vng c cha nhiu carotenoid hn so vi

Chng 3: Phng php lm giu cc cht dinh dng chc nng

Chng 3: Phng php lm giu cc cht dinh dng chc nng

Chng 4: Nhng quy nh chung v qun l thc phm chc nng

Chng 4: Nhng quy nh chung v qun l thc phm chc nng

Chng 4: Nhng quy nh chung v qun l thc phm chc nng

SVTH: Nguyn Kiu Oanh

Chng 1: Thnh tu pht trin ca thc phm chuyn gen

Chng 1: Thnh tu pht trin ca thc phm chuyn gen

SVTH: Nguyn Kiu Oanh

Chng 1: Thnh tu pht trin ca thc phm chuyn gen

SVTH: Nguyn Kiu Oanh

Chng 1: Thnh tu pht trin ca thc phm chuyn gen

SVTH: Nguyn Kiu Oanh

Chng 1: Thnh tu pht trin ca thc phm chuyn gen