B GIO DC V O TO I HC NNG

BO CO TNG KT

TI KHOA HC V CNG NGH CP I HC NNG

NG DNG PHNG PHP SINH HC TNG HP CHITOSAN T PH LIU CH BIN THY SN

M s: 2013-02-53

Ch nhim ti: TS. Bi Xun ng

Nng, 12/2013
1

B GIO DC V O TO I HC NNG

BO CO TNG KT

TI KHOA HC V CNG NGH CP I HC NNG

NG DNG PHNG PHP SINH HC TNG HP CHITOSAN T PH LIU CH BIN THY SN

M s: 2013-02-53

Xc nhn ca c quan ch tr ti (k, h v tn, ng du)

Ch nhim ti (k, h v tn)

Nng, 12/2013
2

NHNG THNH VIN THAM GIA NGHIN CU TI TT 1 H v tn KS. Trng Vn Thin KS. Phm Th Kim Tho KS. Nguyn Xun Hong n v cng tc v lnh vc chuyn mn Khoa Ha - Trng i hc Bch Khoa, i hc Nng Khoa Ha - Trng i hc Bch Khoa, i hc Nng Khoa Ha - Trng i hc Bch Khoa, i hc Nng

N V PHI HP CHNH

Tn n v trong v ngoi nc Cng ty c phn XNK Thy sn Min trung: Cng ty ch bin v xut khu thy sn Th Quang

H v tn ngi i din n v Nguyn Anh Tun

MC LC

MC LC ................................................................................................................................. 4 DANH MC BNG BIU ....................................................................................................... 6 DANH MC HNH V............................................................................................................. 7 CC CH VIT TT ............................................................................................................... 9 THNG TIN KT QU NGHIN CU ............................................................................... 10 INFORMATION ON RESEARCH RESULTS....................................................................... 12 M U ................................................................................................................................. 14 Chng I - TNG QUAN TI LIU ..................................................................................... 15 1.1 Tng quan v cng ngh sn xut chitin/chitosan ......................................................... 15 1.1.1 Gii thiu v chitin ................................................................................................. 15 1.1.2 Gii thiu v chitosan ............................................................................................. 16 1.1.3 Tnh cht vt l v tnh cht ha hc ca chitin/chitosan ....................................... 17 1.1.4 Cc phng php thu nhn chitin ........................................................................... 19 1.1.5 Thu nhn chitosan ................................................................................................... 25 1.2 Tng quan v vi khun Bacsillus subtilis v enzyme protease ...................................... 30 1.2.1 Gii thiu v vi khun Bacillus subtilis. ................................................................ 30 1.2.2 Gii thiu v enzyme protease. ............................................................................... 32 1.2.3 Mt s nghin cu ng dng Bacillus subtilis ca cc tc gi trong nc ............. 37 1.3 Cc lnh vc ng dng ca chitin/chitosan .................................................................... 37 1.3.1 Trong y hc ............................................................................................................. 38 1.3.2 Trong x l nc thi v lm trong nc sinh hot................................................ 39 1.3.3 Trong nng nghip .................................................................................................. 39 1.3.4 Trong m phm ....................................................................................................... 40 1.3.5 Trong thc phm ..................................................................................................... 40 Chng 2 - NGUYN LIU V PHNG PHP NGHIN CU ..................................... 42 2.1. Nguyn liu, ha cht v thit b .................................................................................. 42 2.1.1. Nguyn liu: ........................................................................................................... 42 2.1.2. Ho cht v thit b: .............................................................................................. 43 2.2. Phng php nghin cu .............................................................................................. 43 2.2.1 nh lng nc ..................................................................................................... 43 2.2.2 nh lng mui Calci carbonat ............................................................................. 43 2.2.3 nh lng chitin .................................................................................................... 44 2.2.4 Phng php xc nh deacetyl ca chitosan .................................................... 44 2.2.5 Mt s phng php xc nh khc: ....................................................................... 44 2.3 S cc m hnh th nghim (hnh 2.2) ....................................................................... 44 2.4 X l s liu ................................................................................................................... 46 Chng 3 KT QU V THO LUN ............................................................................. 47 3.1 Kt qu nghin cu kho st thnh phn ha hc ca v tm, nguyn liu ni tng c v phng php bo qun nguyn liu ni tng c. ............................................................. 47 3.1.1 Thnh phn ha hc ca mt s nguyn liu cha chitin, ph bin thnh ph Nng ................................................................................................................................. 47 3.1.2 Kt qu xc nh thnh phn ha hc ca nguyn liu ni tng c ........................ 48 3.1.3 Kt qu nghin cu bo qun nguyn liu ni tng c phc v thu hi ch phm enzyme protease ............................................................................................................... 48 3.2 Chn phng php thu hi ch phm enzyme protease t ni tng c, kho st hot ca ch phm enzyme protease thu c. ............................................................................ 49 4

3.3. nh hng ca nhit v thi gian bo qun ln hot tnh ca ch phm enzyme protease tch chit t ni tng c ......................................................................................... 52 3.4 Cc kt qu nghin cu sn xut chitin/chitosan ng dng enzyme protease tch chit t ni tng c ........................................................................................................................ 54 3.4.1 Kt qu nghin cu phng php x l nguyn liu v tm trc khi loi b protein: ............................................................................................................................. 55 3.4.2 Kt qu nghin cu phn ng thy phn protein trong nguyn liu v tm .......... 56 3.4.3 Kt qu nh gi cht lng chitin c thu nhn bng phng php ng dng enzyme protease t ni tng c ........................................................................................ 59 3.4.4 nh gi cht lng chitosan sn xut t chitin ng dng ch phm enzyme protease t ni tng c ..................................................................................................... 60 3.4.5 Kt qu nghin cu x l cc loi ph thi sinh ra trong qu trnh x l nguyn liu thnh chitin/chitosan ........................................................................................................ 61 3.5 Kt qu nghin cu ng dng vi khun B. subtilis loi b protein trong nguyn liu v tm .................................................................................................................................. 61 3.5.1 Kt qu nghin cu nhm chun b ging vi khun Bacillus subtilis. .................... 61 3.5.2 Kt qu ng dng Bacillus Subtilis loi b protein trong v tm ...................... 65 3.6 So snh cht lng chitosan sn xut theo cc phng php ha hc v ng dng cng ngh sinh hc ....................................................................................................................... 70 3.7 xut s cng ngh sn xut chitin/chitosan ng dng cng ngh sinh hc ......... 71 3.6.1 Dy chuyn cng ngh phc hp sn xut chitin/chitosan ng dng ch phm enzyme protease t ni tng c (hnh 3.18) ..................................................................... 71 3.6.2 Dy chuyn cng ngh sn xut chitin/chitosan ng dng vi khun B.subtilis (hnh 3.19) ................................................................................................................................. 72 KT LUN .............................................................................................................................. 74 TI LIU THAM KHO ....................................................................................................... 75 PH LC................................................................................................................................. 78 Ph lc 1: Phng php pha long thp phn ...................................................................... 78 Ph lc 2: Mi trng hot ho ging (pH=6,5) ................................................................. 78 Ph lc 3: Mi trng kim tra hot lc enzyme ................................................................ 78 Ph lc 4: Thnh phn thuc nhum Amido-Black............................................................. 78 Ph lc 5: Mi trng nhn ging cp 1 ............................................................................. 78 Ph lc 6: Mi trng nhn ging cp 2 ............................................................................. 79 Ph lc 7: Phng php chun phooc-mn .................................................................... 79 Ph lc 8: Phng php chun Kjeldahl ......................................................................... 80 Ph lc 9: Mi trng kim tra hot lc enzyme ................................................................ 81 Ph lc 10: Thu nhn chitin/chitosan bng phng php ha hc (mu i chng)........... 81 Ph lc 11: Thu nhn ch phm enzyme protease t ni tng c ........................................ 82 Ph lc 12: Nghin cu qu trnh thy phn protein trong v u tm bng ch phm enzyme protease, thu nhn t ni tng c ............................................................................ 82 Ph lc 13: Nghin cu qu trnh thy phn protein trong v u tm bng vi sinh vt Bacillus subtillis ................................................................................................................... 83 Ph lc 14: Cc bc tin hnh nghin cu phng php tch chit chitin s dng vi khun B. Subtilis ............................................................................................................................. 83

DANH MC BNG BIU

Bng 3.1 Thnh phn ha hc ca nguyn liu v tm ........................................................... 47 Bng 3.2 - Thnh phn ha hc ca nguyn liu ni tng c .................................................. 48 Bng 3.3 - Mt s tnh cht ca ch phm protease thu nhn t nguyn liu ti v nguyn liu ng lnh ........................................................................................................................... 51 Bng 3.4 Mt s ch tiu cht lng ca chitin sn xut bng phng php ng dng enzyme protease t ni tng c ................................................................................................ 60 Bng 3.5 Mt s c im cht lng chitosan thu nhn t chitin tch chit t v tm ng dng enzyme protease t ni tng c ....................................................................................... 60 Bng 3.6 Mt s c im ca ph thi rn v lng ............................................................. 61 Bng 3.7 - Mt khun lc khi nui cy trn mi trng hot ho vi cc pha long khc nhau .......................................................................................................................................... 62 Bng 3.8 - ng knh vng thu phn ca cc khun lc ..................................................... 63 Bng 3.9 - S gim khi lng v tm qua tng cng on ................................................... 70 Bng 3.10 Mt s ch tiu cht lng ca chitin sn xut bng phng php ng dng vi khun B. subtilis ....................................................................................................................... 70 Bng 3.11 So snh mt s c im cht lng ca chitosan sn xut theo phng php ha hc v ng dng cng ngh sinh hc ....................................................................................... 71

DANH MC HNH V
Hnh 1.1: Cng thc cu to ca chitin.................................................................................... 16 Hnh 1.2 Cng thc cu to ca chitosan ............................................................................. 17 Hnh 1.3 S tng qut thu nhn chitin bng phng php ha hc ................................. 19 Hnh 1.4 - Cc phng n thu nhn chitin ............................................................................... 20 t cc ngun nguyn liu khc nhau ........................................................................................ 20 Hnh 1.5 Kriel ....................................................................................................................... 21 Hnh 1.6 Phn ng tm tt qu trnh iu ch chitosan ........................................................ 26 Hnh 1.7 - My sy, to ht loi FL ......................................................................................... 30 Hnh 1.8 - Thit b nghin ti kch thc siu nh Cage-Paktor .......................................... 30 Hnh 1.9 - Vi khun Bacillus subtilis sau khi nhum gram ..................................................... 31 Hnh 1.10 - Hnh nh a khun lc ca vi khun Bacillus subtilis ......................................... 31 Hnh 2.1 S khc nhau ca mt s nguyn liu tm, v ca chng dng thu nhn chitin/chitosan .......................................................................................................................... 42 Hnh 2.2 S m hnh cc bc thc hin trong nghin cu ............................................ 46 Hnh 3.1 - nh hng ca thi gian bo qun ln ch tiu cht lng ni tng c bo qun nhit -18 -200C ................................................................................................................. 49 Hnh 3.2 - Biu s ph thuc hot cc nhm enzyme protease ..................................... 50 trong cc vng pH khc nhau................................................................................................... 50 Hnh 3.3 - S quy trnh tch chit enzyme protease ........................................................... 51 t ni tng c ............................................................................................................................ 51 Hnh 3.4 - Ch phm enzyme protease tch chit t ni tng c ng lnh ............................ 52 Hnh 3.5 S ph thuc ca hot ca ch phm enzyme protease .................................... 53 vo nhit mi trng ........................................................................................................... 53 Hnh 3.6 - nh hng ca thi gian bo qun ln hot enzyme protease tch chit t ni tng c ...................................................................................................................................... 53 Hnh 3.7 - nh hng ca thi gian ln kh khong v hm lng cht khong cn li trong v tm ............................................................................................................................. 56 Hnh 3.8 S ph thuc kh protein trong nguyn liu v tm vo thi gian thy phn . 57 Hnh 3.9 S ph thuc ca kh protein vo nng ch phm enzyme protease t ni tng c ...................................................................................................................................... 58 Hnh 3.10 - nh hng ca nhit ln phn ng enzyme thy phn protein trong nguyn liu............................................................................................................................................ 59 Hnh 3.11 - a khun lc sau khi nhum bi thuc nhum Amido-Black. ........................... 62 Sau khi nhum chng ti quan st thy chung quanh cc khun lc xut hin cc vng thu phn mu sng, nhng vng mi trng cn li c mu xanh m ca thuc nhum nh hnh 3.11........................................................................................................................................... 63 Hnh 3.12 - Hnh thi vi khun Bacillus subtilis soi di knh hin vi ................................... 64 Hnh 3.13 - Vng thu phn ca vi khun Bacillus subtilis sau khi nhn ging cp 1 ........... 64 Hnh 3.14 - Vng thu phn ca vi khun Bacillus subtilis sau khi nhn ging cp 2 ........... 64 Hnh 3.15 S thay i hm lng m ha tan trong mi trng cha vi khun B. Subtilis v v tm nhit 480C ....................................................................................................... 66 Hnh 3.16 - Hm lng m ha tan thu c khi thy phn protein trong v tm vi cc t l b sung ging khc nhau 480C ............................................................................................. 67 Hnh 3.17 S ph thuc ca qu trnh thy phn protein trong v tm vo nhit ........... 68 Hnh 3.18 S cng ngh phc hp sn xut chitin/chitosan ng dng ch phm enzyme protease t ni tng c ............................................................................................................. 72

Hnh 3.19 S cng ngh phc hp sn xut chitin/chitosan ng dng vi khun B. subtilis .................................................................................................................................................. 73

CC CH VIT TT

Chitin Chitosan KK KP KA EDTA G20X V tm VSV QMAFAM

: : : : : : : : : :

poly (1,4) - 2 acetamido - 2 deoxy - - D glucose poly (1,4) -2-amino-2-deoxy--D-glucan Qu trnh kh khong Qu trnh kh protein Cng on deacetyl Etilendiamin tetraaxetic axit Enzyme proteinase protosubtilina u tm + v tm Vi sinh vt Quantity of Mesophilic Aerobic and Facultative Anaerobic Microorganisms

I HC NNG
TRNG I HC BCH KHOA

THNG TIN KT QU NGHIN CU

1. Thng tin chung: - Tn ti: ng dng phng php sinh hc tng hp chitosan t ph liu ch bin thy sn - M s: 2013-02-53 - Ch nhim: TS. Bi Xun ng - Thnh vin tham gia: KS. Phm Th Kim Tho, KS. Nguyn Xun Hong, KS. Trng Vn Thin. - C quan ch tr: Trng i hc Bch Khoa i hc Nng - Thi gian thc hin: t 10/04 30/12/2013 2. Mc tiu: ng dng phng php sinh hc sn xut chitin/chitosan nhm hn ch s dng ha cht, gy nhim mi trng 3. Tnh mi v sng to: S dng kh nng xc tc phn ng ca enzyme protease trong ni tng c v enzyme protease ca vi khun B. subtilis loi b protein ra khi chitin trong v tm 4. Tm tt kt qu nghin cu: Trn c s nghin cu qu trnh kh protein trong v tm bng bin php ng dng cng ngh sinh hc v thc nghim sn xut chitin/chitosan quy m pilot theo cng ngh mi c nghin cu thu c cc kt qu nh sau: - Sn phm chitin sn xut ng dng ch phm enzyme protease tch chit t ni tng c c cht lng tt vi hm lng protein nh hn 0,5%, v ch tiu vi sinh vt t chun an ton (SanPin 2.3.2.1078-01 LB Nga); chitosan sn xut t loi chitin ny c deacetyl l 79-89% v nht cao n 18 dl/g ph hp vi cc bo co khoa hc trong nc v quc t. - Sn phm chitin sn xut ng dng vi khun B. subtilis c ch tiu cm quan thp, v vi sinh vt tng s tng i cao mc 3,3x105 nhng vn nm trong gii
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hn an ton (SanPin 2.3.2.1078-01 LB Nga); chitosan sn xut t loi chitin ny c nht l 18,1 dl/g c cht lng bo m so vi bo co trong nc v quc t. 5. Tn sn phm: - 2 s dy chuyn cng ngh sn xut chitin/chitosan - cc sn phm chitin, chitosan v cht mu carotenoid 6. Hiu qu, phng thc chuyn giao kt qu nghin cu v kh nng p dng: - Hiu qu v cng tc ging dy: Hng dn thnh cng hai sinh vin lm n tt nghip nghin cu nm hc 2012-2013 l: Trng Quang Thm (08SH) v V Th Ngc Thy (11SHLT). - Chuyn giao kt qu nghin cu: Dy chuyn cng ngh sn xut chitin/chitosan ng dng cng ngh sinh hc c ng ti trn ch trc tuyn: Ch cng ngh v thit b Vit nam. http://www.techmartvietnam.vn

C quan Ch tr (k, h v tn, ng du)

Ngy thng nm Ch nhim ti (k, h v tn)

11

DANANG UNIVERSITY OF TECHNOLOGY UNIVERSITY OF DANANG

INFORMATION ON RESEARCH RESULTS

1. General information: Project title: Produce chitosan from the wastes of aquatic processes by using biotechnique Code number: 2013-02-53 Project Leader: Doctor Bui Xuan Dong Coordinator: Pham Kim Thao, Nguyen Xuan Hoang, Truong Van Thien Implementing institution: Danang University of Technology University of Danang Duration: from April 10, 2013 to December 30, 2013 2. Objective(s): Produce chitin/ chitosan by bio-technique to eliminate the using of chemicals to prevent environmental pollution. 3. Creativeness and innovativeness: Applying the catalization of protease in internal fish organs and protease from Bacillus subtilis to discard protein from chitin in shrimp shells. 4. Research results: Base on the using of bio-technique to demineralise the shrimp shells and the implementation of pilot studies with new technique, we received some given results: - Chitin product produced by using protease extracted from internal fish organs had high quality such as protein content was less than 0.5% and the microorganism quota was obtained the Safety Standard (SanPin 2.3.2.1078-01 Russia); Chitosan produced from these chitin products had deacetylation degree from 79% to 89% and the viscosity was up to 18 dl/g. The product quality can be compared to products reported from inside and outside of Vietnam. Chitin product prepared by Bacillus subtillis fermentation had low perceptible showing and the total microorganism in the sample was quite high, at 3.3x10-5 but still
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under the Safety Standard (SanPin 2.3.2.1078-01 Russia). The viscosity of Chitosan produced from these chitin products was 18.1dl/g. The product quality can be compared to products reported from inside and outside of Vietnam. 5. Products: - Two production lines of Chitin and Chitosan; - Chitin, chitosan and carotenoid pigment products 6. Effects, transfer alternatives of research results and applicability: - Teaching: Conducting two students to do their final research thesis, including Truong Quang Thom (08SH) and Vo Thi Ngoc Thuy (11SHLT). - Hand over the research results: Production lines to produce chitin/ chitsan by bio-technique were placed in the online market: The Technology and Equipment Market of Vietnam (http://www.techmartvietnam.vn). Date:. IMPLEMENTING INSTITUTION PROJECT LEADER

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M U Trong nguyn liu v tm, chitin lin kt rt cht ch vi protein, lipid v cc cht khong (pha rn). Vic thu nhn chitin t ngun nguyn liu ny xy dng trn cn bn l a protein v cc cht khong v trng thi lng (pha lng), v t loi b chng ra khi chitin. sn xut chitin t cc tiu chun cht lng quc t cn loi b hon ton protein v khong cht, cng nh lipid. Hin nay phng php ha hc c dng ph bin thu nhn chitin bng cch s dng cc ha cht m c, tc l x l nguyn liu trong nhng iu kin khc nghit (mi trng axit mnh, baz mnh, nhit cao v thi gian ko di), thng dn ti lm gim cht lng chitin thng phm. Cc h hng ca sn phm chitin thng gp khi thu nhn theo phng php ha hc l: bin dng cu trc phn t, gim nht ca dung dch chitin v chitosan. Bn cnh vic bo qun v s dng cc ha cht c m c cao m i hi s tip xc trc tip ca cng nhn gy nhiu quan ngi trong vn bo m an ton v sc khe cho ngi lao ng. Theo phng php ha hc c th thu nhn c sn phm phn gii protein l hn hp cc axit amin t do ha ln cng cc cht c hi, cc cht c hi sinh ra do protein b thy phn su dn n phn gii c cc axit amin nn vic s dng sn phm dch thy phn gp phi nhiu hn ch, v d khng s dng c trong thc phm hay trong sn xut thc n chn nui. Cc hng s dng chitin v dn xut t chitin trong thc t trong nhng nm gn y khng ngng c m rng dn ti cn tm kim v th nghim cc phng php mi thu nhn cc polymer sinh hc ny. Mt vn c tnh thi s cn tn ti l nghin cu phng php x l nguyn liu v tm to iu kin thu hi chitin v ton b cc hp cht t nhin c hot tnh sinh hc c trong n, bn cnh vic hng ti pht trin bn vng trn c s gim thiu nhim mi trng. Mc tiu ca nghin cu ny l ng dng phng php sinh hc tng hp chitosan t ph liu ch bin thy sn nhm thu c cc sn phm chnh nh: chitin, chitosan v cc sn phm ph nh cht mu carotenoid v ch phm enzyme protease t ni tng c.

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Chng I - TNG QUAN TI LIU 1.1 Tng quan v cng ngh sn xut chitin/chitosan V tm, gh, cua, mai mc v nhiu loi v gip xc khc chim 15 ti 35% tng khi lng khai thc, l ngun nguyn liu qu iu ch cc hp cht polymer t nhin nh aminopolysaccharide (chitin v chitosan), v nhng dn xut ca chng l cc oligosaccharite v glucosamine, nhng sn phm ngy nay tr nn thit yu trong cc lnh vc khc nhau. V d: + s dng lm cc cht ph gia thc phm, hay thnh phn ca thc phm chc nng + s dng lm cc ch phm y dc ( gim cn, iu ha p huyt, gim colesteron; cc ch phm chng ung th v cc loi thuc khng th) + s dng trong m phm bo v da v tc + s dng lm sch nc trong cc m h t nhin v nhn to (keo t v hp ph cc hn hp c hi, cc loi vi khun, cc kim loi nng) + dng trong cc ch phm nng nghip (cc ch phm cha bnh cho ng vt, ch phm chng li bnh cho thc vt) [21, 27]. 1.1.1 Gii thiu v chitin Chitin phn b rng ri trong t nhin, cng vi xellulo l nhng hp cht hu c ph bin nht trn b mt tri t. N l ngun ti nguyn di do, c tnh vo khong 100 nghn t tn/nm. Nhng khc vi xellulo, chitin cn l ngun sinh khi t nhin t c khai thc. Chitin ln u tin c Gio s khoa hc t nhin, gim c vn thc vt thuc Vin hn lm khoa hc ca TP Nensi (Php) Henri Braconnot pht hin nm 1811 khi nghin cu thnh phn ca nm v ng t tn l fungin. Nm 1823 A.Odier iu ch chitin t cnh b cnh cng v t tn l chitin (Ting Hy-lp. citwu- ngha l o). Cn chitosan ln u tin pht hin nm 1859 bi nh khoa hc C. Rouget, cng t thi im ny gii khoa hc bt u nghin cu thc nghim v s dng nhng polymer ny.

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Nhng cng trnh u tin Nga, lin quan ti vic iu ch chitin c th c hin di s ch o ca Vin s P. Sorugin nhng nm 1934-1935. Cc th nghim s dng chitosan c F. Cadov thc hin nm 1941. Hin nay nhm mc ch pht trin cc nghin cu v chitin/chitosan Nga c Hip hi lin bang v chitin (The Russian chitin society: www.chitin.ru), c thnh lp t nm 2000. Chitin l mt poly (1,4) - 2 acetamido - 2 deoxy - - D glucose. Cng thc cu to ca chitin c trnh by trn hnh 1.1

Hnh 1.1: Cng thc cu to ca chitin Lin kt (1,4) glucoside ca mi mt xch cu to nm lch nhau mt gc 1800 to nn mch xon. Lin kt ny km bn, d b ct t bi tc nhn ion H + ca axit. S c mt ca nhm amino trong chitin lm cho chng c nhng c tnh sinh hc chuyn bit v c th tham gia cc phn ng c trng. Chitin l polymer c nhng tnh cht xc nh, bao hm c kh nng b phn hy bi vi sinh vt v c hot tnh sinh hc. Nn chitin cn c quan tm nghin cu nh mt loi vt liu chc nng c bit mi. iu khc bit ca chitin so vi cc polysaccharide khc l phn t chitin tch in dng mnh, n gip cho chitin to ra lin kt vi cc phn t mang in tch m trn b mt. Trong mi nguyn liu khc nhau th chitin c s khc bit v thnh phn cu to v hm lng chitin khc nhau [12, 21, 25]. 1.1.2 Gii thiu v chitosan Chitin c th chuyn ha thnh nhiu dn xut khc nhau, hay hp nht l
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chitosan. Chitosan l poly (1,4) -2-amino-2-deoxy--D-glucan. N c to thnh khi N-deacetyl ca phn t chitin, c cng thc cu to nh trn hnh 1.2. Chitosan c mt s u vit so vi chitin, c th k n nh l tnh tan trong nc v trong dung dch acid. Khi lng phn t chitosan ph thuc vo iu kin x l chitin hay v tm, thng thng t 10 n 1000 kDal. n v phn trm deacety c xc nh bng mc deacetyl (y l t l ca khi lng glucosamine so vi s monomer chung trong phn t polymer DD = m(m+n) = Namin/Ntng), thng t 70-90%.

Hnh 1.2 Cng thc cu to ca chitosan 1.1.3 Tnh cht vt l v tnh cht ha hc ca chitin/chitosan Do c trng v cu to nn chitin v chitosan c tnh cht c trng ca nhm amino (-NH) v nhm hydroxyl (-OH). Chng c kh nng lin kt vi nhiu kim loi nng to nn hp cht gi l chelate. Chitin c c tnh hot ha thp do trong phn t chitin ch c hai nhm hydroxyl trn mi mt xch (monomer) c th tham gia vo phn ng ha hc. Chitosan nh c nhm amino t do nn c nng lng t do ln, to iu kin cho chitosan c th ha tan trong cc dung dch ca cc acid hu c v mt s acid v c. Ngoi ra, cc polymer ny cn c th b thy phn do cha cc lin kt glucoside v lin kt peptide. C th d dng to ra cc nhm a nc bng cch bin tnh chitin/chitosan, nn m ra kh nng m hnh ha tnh cht ca chng. Chitin c 3 trng thi tinh th l: , v chitin. Ph bin nht v bn vng nht l -chitin, trong phn t c cc lin kt i song song, chnh nh cc lin kt hydro. Phn t v chitin km bn vng hn v t gp hn. Chitosan cng c th tn ti dng tinh th, s to ra polymer chitosan ph thuc vo trng thi vt l ca n.
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Chitin/chitosan bn vng di tc dng ca kim, nhng c th b thy phn di tc dng ca cc acid. Khi b thy phn hon ton bi dung dch acid HCl hoc acid sunfuric nhit cao chitin v chitosan b thy phn n monosaccharide-Dglucosamine. iu kin mm hn c th to thnh hn hp cc oligosaccharide. Dung dch HCl thng c dng thy phn tng phn, sn phm to ra trc tip l glucosamine t cc on oligosaccharide vi kh nng polymer ha 2-4. X l bng siu m c th lm phn hy chitin v chitosan. S thy phn lin kt glucoside ca phn t chitin/chitosan di tc dng ca cc yu t khc nhau (oxy ha, x l nhit) lm t mch polymer ca chng, lm gim khi lng phn t, tng kh nng ha tan, tnh cht ny thng c ng dng iu ch dung dch, v sau dng vo cc mc c khc nhau. S thy phn lin kt amid (peptit) ca chitin to ra chitosan. V nguyn tc khng c danh gii r rng gia chitin v chitosan. trng thi t nhin chitin lun c mt s nhm amino t do (5-15%). Thng thng chitosan c cho l sn phm, c th ha tan trong cc dung dch acid ha c long (nh acid acetic), tng ng vi deacetyl khng di 55-60%.S thy phn ca cc lin kt peptide trong dung dch acid v baz hoc di tc dng ca cc enzyme (chitosanase). Mt phng php deacetyl vi hiu xut cao thng thy phn bng baz mnh. Qu trnh deacetyl lm bin i cu trc polymer, dn n lm gim khi lng phn t polymer. Trong t nhin chitin/chitosan b phn gii bi nhiu sinh vt khc nhau, chng c kh nng sinh enzyme chitinase v chitosanase. Phn ln enzyme chitinase trong vi sinh vt thy phn lin kt N-acetyl--(1,4)glucosamin theo c ch khc nhau. Chitinase trong thc vt bc bao khng c chitin nh thnh phn cu to. Enzyme chitosanase thy phn chitosan bng c ch ct endo. Nh vy chitosan c th b phn gii di tc ng ca nhiu ch phm enzyme khc nhau nh glycanase, lipase v protease t nhng ngun khc nhau. Enzyme cellulose T. viride phn gii chitosan n chitooligosacharide. Enzyme papain xc tc thy phn chitosan v lm gim nht ca chitosan. X l chitin bng cc enzyme thy phn m ra kh nng sn xut N-acetyl-Dglucosamin bng cc enzyme chitinase. Nghin cu nh hng ca nhm N -acetyl ln qu trnh thy phn vi lysim chng minh rng, deacetyl ti u bng 0,7, khi c
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th thu c cc on oligosaccharide n tetramer. Oligosaccharide c di polymer t 2-8 ng dng nh ph gia thc phm, thnh phn ch phm y dc c iu ch bng cch phn gii chitosan bng enzyme chitosanase t vi khun Bacillus 7-M bn pH t 5-11. Chitosan ha tan cng c iu ch bng phng php enzyme nh sau: x l chitosan vi deacetyl 60-90% bng enzyme chitosanase trong mi trng acid nhit 30-60 0C. Sn phm thu nhn c, tch ra bng phng php siu m, c kh nng ha tan trong nc [21,25]. 1.1.4 Cc phng php thu nhn chitin Chitin l polymer khng tan nn khng th thu nhn trc tip t v tm, cua. thu nhn chitin cn thc hin tun t vic tch protein v cc cht khong, cu trc nn lp v cng cha chitin, ngha l chuyn protein v khong cht v trng thi ha tan, v loi b chng. T nhng phng php thu nhn chitin bit chng ta c th phn chia chng thnh hai nhm phng php c bn: nhm 1 - phng php ha hc, x l bng acid, kim; nhm 2 - cc phng php cng ngh sinh hc, ng dng cc ch phm enzyme v cc vi sinh vt phn gii protein.

Hnh 1.3 S tng qut thu nhn chitin bng phng php ha hc a) X l ha hc:
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Phn ln cc phng php thu nhn ca nhm ny xy dng trn mt hoc hai bc lm sch chitin khi protein v cc hp cht khong - gi l kh protein v kh khong. Mt s phng php khc c thm bc kh lipid v cc cht khc [13]. Thu nhn chitin t nhng ngun nguyn liu khc nhau c s tng qut nh hnh 1.3 Ph thuc vo ngun nguyn liu v cht lng chitin cn thu nhn, cng nh thu nhn chitosan t chitin m s lng cc bc kh protein v kh khong khc nhau, trt t c th thay i theo s nh hnh 1.4

Hnh 1.4 - Cc phng n thu nhn chitin t cc ngun nguyn liu khc nhau Cc phng n ny th hin vic thu nhn chitin t cc ngun nguyn liu khc nhau, ph thuc vo yu cu v cht lng sn phm chitin. V d x l v tm, b cnh cng (lp v mng) ch cn cng on x l kim (x l 1 ln). X l kp (2 ln x l kim v acid) kh hon ton protein khi chitin. Cc khong cht cn li x l bng acid khng vt qu 1-3%. Vic lp li bc kh khong c th thu nhn chitin khng cha tp cht, bc ny cn thc hin vi cc nguyn liu c lp v dy (nh v cua,) [17]. Vic p dng tun t cc bc KK v KP c mt s li ch v cht lng chitin. Vic p dng tun t ny nh hng ln cht lng chitin thu nhn c v t chitin thu nhn chitosan. V d, khi x l nguyn liu cha chitin theo s KK -KP nht ca ca chitosan thu c cao hn rt nhiu so vi s cng ngh KP-KK. Hiu sut cc qu trnh KK v KP ph thuc vo nghin nh ca v tm, v cua, ngha l tng din tch tip xc ca nguyn liu vi cc cht ha hc, nhng khi
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nghin nh nguyn liu cng lm hiu sut sn phm gim do tht thot trong qu trnh x l [18]. Cc thng s ca cng on KP v KK ca cc nghuyn liu khc nhau cng khc nhau. Theo phng php Hackman [12] v crab c x l bng dung dch HCl 2N trong 5h nhit phng, sau hn hp c nghin nh v kh protein bng dung dch acid NaOH 1000C trong 12h. kh hon ton protein cn thc hin cc cng on trong 4 ln. Phng php Whisler v BeMiller [12] thc hin kh protein ca nguyn liu nghin nh trong dung dch NaOH 10% trong thi gian 72 h, kh mu bng etanol 95%, sau ra bng dung mi hu c, kh protein bng dung dch HCl 37% trong 4h. Kh khong theo phng php Horowits, Rosoman v Blumental [12] c thc hin trc khi kh protein, nguyn liu x l bng dung dch acid fomic 90% trong 18h nhit phng, sau kh protein bng dung dch NaOH 10% trong 2,5h un nng. Cc nh khoa hc u khng nghin cu nh hng ca trt t cc bc kh khong, kh protein ln cht lng chitin/chitosan. Nhng sau ny, ngi ta pht hin trt t KK, KP nh hng n cht lng chitosan thu nhn c. T cc phng php trn, c mt iu r rng l cc cng on u thc hin iu kin khc nghit, nguyn liu cha chitin c x l trong dung dch acid v dung dch kim mt thi gian di v nhit cao kh protein. iu ny dn n bin dng cu trc, v mt phn kh deacetyl (KA) ca chitin.

Hnh 1.5 Kriel Ti LB Nga t nhng nm 1970-1980 ( Vladivostoc, Murmansk, VNIRO) ng dng cng ngh sn xut chitin v chitosan t v nhuyn th (kriel) v v tm
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(hnh 1.5). Chitin c thu nhn nh kh protein bng dung dch NaOH 1N trong 30 pht nhit 70-800C, sau kh khong bng dung dch HCl 1N trong 30 pht nhit phng. tng hiu sut qu trnh kh protein bng dung dch kim nh, cc nh khoa hc ng dng cc cht c hot tnh b mt. Phng php thu nhn chitin t v nhuyn th (kril), c xut bi A.I. Gamzazade cng cng s [22]: v nhuyn th (krel) c kh protein trong dung dch NaOH 2-5% nhit phng trong 6-8h. Trong cng on kh protein dng phi hp cc cht hot ng b mt anion (sodium dodecyl sulfate, mui natri acid aurino), lm gim nng NaOH, bn cnh lm tng hiu sut kh mu v lipid. Phng php sn xut chitin t v nhuyn th (kriel) ng dng Ba lan [23] xy dng trn c s x l s dng nguyn liu ng lnh. Nguyn liu c gi ng, nghin nh, sau ra sch v kh khong bng dung dch acid HCl 3,5% trong 1,5h nhit phng. Sau tin hnh loi b lipid v v cht mu bng hn hp gim v etanol (acetate ester). Kh protein bng dung dch NaOH 3,5% nhit 250C trong 2h. S pht trin cng ngh sn xut chitin cc quc gia ch khc nhau vic chn ha cht phc v qu trnh KK v KP [14]. n trong cng nghip sn xut chitin t ph liu ch bin tm Matapenaeus dobsoni. Kh protein c tin hnh trong dung dch NaOH 0,5% un nng trong 30 pht. Phn dung dch c tch khi chitin v tn dng sn xut protein. Bc kh protein c thc hin ln th hai bng dung dch NaOH 3%. Nguyn liu tip tc c kh mu bng dung dch hypochloride cha 0,3-0,5% clo lng, sau ra bng nc v tin hnh kh khong bng dung dch HCl 1,25N nhit phng trong 3h. Chitin sn xut ra c dng sn xut chitosan. M ngi ta s dng cng ngh sn xut chitin ca cc nh khoa hc Peniston K.P. v Jonson E.L. [25]. Nguyn liu cha chitin c nghin nh dng m t ti kch thc 1,3-1,8 mm v tin hnh KP nhit 65-710C trong thi gian 1-2h trong dung dch NaOH 0,05-0,2N theo c ch chy ngc.Trong dung dch NaOH trc khi dng kh protein cho thm 0,1% Na2SO4, kch hat qu trnh thy phn. Sau tch ra phn dung dch cha protein sn xut protein, phn rn chuyn qua cng on kh khong bng HCl.
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Tt c cc phng php thng k trn y ch khc nhau trnh t thc hin hai cng on KK v KP, v cc thng s ca cng on trn cn bn phng php ha hc. Kh khong (KK) l cng on quan trng trong sn xut chitin. kh khong xc nh bng nht v cc thng s vt l, ha hc khc, kh khong ca chitin cng ging nh ca chitosan, tc l mang cng mt ngha. kh khong (kh CaCO3 v Ca3(PO4)2) t nguyn liu cha chitin ngi ta thng dng acid HCl, acid formic, HNO3 v acid sunfuric. Hn ch y l khi x l chitin bng acid c th dn ti ph hy mt phn cu trc v deacetyl ca chitin, gim nht ca dung dch chitosan. C cc phng php khc thu nhn chitin, khng ging nh cc phng php tiu chun nh trn. l phng php ca Foster v Hackaman [12] thc hin iu kin mm, khng dn ti lm hng cu trc v deacetyl ca chitin. Tt c cng ngh ch bao gm 1 bc x l nguyn liu cha chitin trong dung dch etilendiamin tetraaxetic axit (EDTA). V cua c x l mt thi gian di 2-3 tun trong dung dch EDTA khi pH = 9. Mt s loi nguyn liu mng nh v tm, nhuyn th (kriel) sau khi nghin nh ch mt 15 pht x l. Sau khi x l chitin cha 1% tro v 5% protein. Peniston K.P. v Jonson E.L. [26] cn c phng php: nghin nh nguyn liu cha chitin n kch thc 3-6 mm. Sau hn hp cho vo thit b phn ng cng vi NaOH 0,5-2% trong thi gian 1-4h nhit 50-700C kh protein. Sau tin hnh theo m hnh KK v KP bng dung dch NaOH 30-50% nhit 1201500C. Khi xy ra ng thi qu trnh deacetyl ca chitin chuyn ha CaCO3 v dng Ca(OH)2 theo phng trnh:

nc thi sau cng on ny cha cc thnh phn nh hydroxit natri, acetat natri va cacbonat natri. Dung dch NaOH c th ti s dng. Hin tng ny hay gp l hin tng n mn da. im tn ti chung ca tt c cc phng php trn l x l nguyn liu trong
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iu kin v mi trng khc nghit ca acid v baz, nh vy cn cc thit b bn vng vi kh nng chng n mn, nh vy lm tng gi thnh sn xut v sn phm. Ngoi gia khi thit k nh my cn thit k cc khu vc pha ch ha cht, cng nh bo qun chng, gy nguy hi n an ton sn xut v mi trng. Protein sau khi tch khi acid v baz t dch thy phn cha nhiu mui, lm hn ch khi s dng chng. b) Tng quan cc qu trnh sinh hc sn xut chitin ng dng enzyme to iu kin mm x l nguyn liu cha chitin, c th gp nhiu cng on lm mt nn n gin ha cng ngh. ng thi gim thiu tnh khc nghit ca mi trng phn ng ln thit b v tng tui th ca chng. Phng php n gin nht thu nhn chitin l li dng qu trnh t phn hy di tc dng ca enzyme c trong nguyn liu (qu trnh t gii). Nhng phng php ny c ng dng sn xut chitin t ph phm kriel [27] v v cua [28]. Qu trnh kh protein (KP) c tin hnh bng cch nho trn nguyn con kriel vi nguyn liu cha chitin theo t l 1:2 nhit 500C. Hn hp thu c cn nho trn lin tc trong 5 gi. chuyn dch protein t nguyn liu vo pha lng t 68%. Kt qu thu c chitin cha 34% khong cht v 12% protein. Nh vy phng php ny khng loi b c hon ton protein ra khi chitin, tc l qu trnh KP khng hon ton v cn cng on ph loi b protein. Ngoi ra, trong hn hp phn ng cha enzyme chitinase, tc ng ln chitin, lm gim khi lng phn t ca chitin. T quan im t c hiu sut kh protein cao, mt phng php c tnh ng dng cao hn l s dng vi khun phn gii protein stam Pseudomanas maltophilia [29]. Nguyn liu v cua (crab) c kh khong bng dung dch HCl 2N trong thi gian 2 ngy nhit phng, sau nghin nh n kch thc 0,5 cm. Mi trng nui cy c b sung 0,8 g nguyn liu cha chitin vi so vi 80 ml dung dch K2HPO4 0,2%, tip tc cho vo hn hp dung dch HCl iu chnh pH v pH 7,0. P. maltophilia cy trong ng nghim, lc u n nhit 300C. Cui qu trnh chitin c ra sch bng nc. Hm lng protein trong chitin gim xung cn 1%. Phng php kh protein trong v nhuyn th (kriel) ti bng vi khun Bacillus Subtilis, thy phn protein t 98% sau 36 gi, thu c chitin cha protein di 1% [30].
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Cc nh khoa hc nghin cu cc phng php sn xut chitin t v cua, v gh ng dng cc enzyme khc nhau. Phng php thu nhn chitin [12] bng cch KK bng HCl 1,4N nhit phng trong 24h. Sau kh protein bng enzyme papain, pepsin hoc tripsin. Phng php ca Rogozina v cng s [31] th cng on KK v KP c gp lm mt bng enzyme ngun gc vi sinh vt hot ng trong mi trng acid: protorizinase v protavamorinase. Qu trnh xy ra pH 3,0 nhit 35-400C trong 24h. Ch phm enzyme protease c thu hi t nm Aspergillus niger, A. fietidus, A. orizai. Chitin thu c khng cha tro, cha 5-10% protein. C th loi b hon ton protein cng on cui bng dung dch kim. Ti LB Nga, trong cng nghip ngi ta sn xut chitin t v nhuyn th (kriel) bng phng php s dng enzyme proteinase protosubtilina G20X [32] v pankreatuna [33] hot ng trong mi trng kim. Cc phng php cng ngh sinh hc thu nhn chitin c nhc im chung l khng loi b ht protein ra khi chitin ( tinh sch di 95%), c th nh hng xu n cht lng chitin, v ng dng ca chitin. Ngoi ra, khi la chn ch phm enzyme cn c bit ch trng n kh nng chng c cha enzyme chitinase, v trong qu trnh x l nguyn liu nu hot lc chitinase mnh c th dn n ph hng cu trc ca chitin thnh phm. 1.1.5 Thu nhn chitosan a) Deacetyl ha (DA) chitin Dn xut deacetyl ha u tin khi bin tnh chitin l chitosan, l polymer cao phn t ca glucosamin, ha tan trong dung mi acid hu c v v c long (ngoi tr acid sunfuric). im khc bit gia chitin v chitosan l kh nng ha tan ca chitosan trong dung dch acid, nn c nhiu ng dng trong cng nghip, nng nghip v y dc. thu nhn chitosan cn kh nhm acetyl t N-acetyl-D-glucosamin (chitin) hay cn gi l phn ng deacetyl ha (DA). Phn ng iu ch chitosan thu gn nh hnh 1.6. Phn ng DA i km vi vic ct t lin kt glucosit ca polymer. Nh vy, chitosan l cht trng hp phn tn theo khi lng phn t - polymer D-glucosamin,
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cha 5-15% cc nhm acetal amid v gn 1% cc nhm c lin kt vi acid amin v cc peptid.

Hnh 1.6 Phn ng tm tt qu trnh iu ch chitosan B ngoi chitosan c cu trc hnh vy vi ng knh nh hn 10 mm hoc dng bt vi nghin nh khc nhau, c mu trng n mu kem, hay gp chitosan mu hi ng vng, mu hi nu hoc mu hng, chitosan khng mi. Tnh cht khc ca chitosan dng kh l tnh dn in v c v ng. Theo c th chitosan thuc lp 4 v khng c. Qu trnh DA thng c tin hnh trong dung dch kim mnh nhit cao. Th nghim u tin thu nhn chitosan l lm nng chy chitin vi kim rn 180 0C. Theo phng php ny thu c chitosan vi deacetyl ti 95%, nhng lm bin dng mnh cu trc (n 20 n v) ca chitosan. Ph bin trong k thut ngi ta thc hin qu trnh DA bng dung dch kim 30 50%, v l mi trng mm hn. C nhiu cch khc nhau bin tnh theo phng php ny. Thc hin qu trnh DA trong dung dch kim c th t deacetyl ln n 100% khi thc hin li nhiu ln v t lm bin dng cu trc chitin. Khi thu nhn chitosan trong nhng iu kin nh trn mt mt l qu trnh DA, ng thi xy ra qu trnh song song l lm hng cu trc chitin, ngha l lm t cc lin
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kt glucosit, dn ti gim khi lng phn t v gim nht ca chitosan. Trong sn xut chitosan theo phng php trn, tc l cc qu trnh xy ra trong mi trng kim mnh v nhit cao nn cn s dng cc thit b chuyn dng lm t hp kim niken hoc ph ngoi bng niken. Thng thng thc hin qu trnh DA s dng cc thit b phn ng lm t thp hp kim chu nhit, c b phn nho trn v lp v dn hi. Cng on kh khn, phc tp nht trong cng ngh sn xut chitosan l ti s dng v x l nc thi l dung dch kim vi nng cao. Trong nhiu trng hp qu trnh DA thc hin bng dung dch kim 30-50%, mc d trong qu trnh phn ng nng kim gim, nhng thi ngay nc thi vo h thng l iu khng c cho php. Cho nn khi thit k nh my sn xut chitosan cn c phng n x l nc thi v b phn thu nhn protein t trong nc thi. C hng lot phng php thay th mt phn hay hon ton nc trong qu trnh DA bng cc dung mi khc nhau hoc cc cht mang nhit. V d trong [34] cht mang nhit c s dng l paraphin lng, hexan, ru isopropyl. Khi gim mnh lng nc v dung dch kim trong qu trnh DA, gim s ph hng cu tr c polymer, deacetyl t 92%. Phng php Brossignac [12] khng s dng nc, ngi ta thay th nc hon ton bng etanol vi monoetylenglicol. Khi DA bng KOH nhit 1200C acetyl t 83%. Takeda [34] nghin cu phng php x l chitin trong kh tr hydrocylamin pH 13 v nhit 1000C trong 10-20h. Hn ch ca phng php ny l deacetyl thp (70%), thi gian x l di, v lm hng cu trc ca mch polymer. Phng php deacetyl ca chitin 2-4 ln trong dung dch hydrazine hydrate [25] nhit 120-1500C. Khi hn ch s bin dng cu trc mc thp nht deacetyl t 90-97%. Ngoi ra, qu trnh DA c th thc hin bng enzyme - deacetylase, c ngun gc vi sinh vt. T nhng nghin cu ca cc nh khoa hc nh trn lit k v qu tr nh deacetyl trong kim c th rt ra mt s nhn nh. Trc ht, bn ca chitin trong qu trnh DA c l gii bi s c mt ca lin kt hydro gia nhm carboxyl v nguyn t ni-t ca nhm amid trong cu trc micell. ph v lin kt (lin kt bn
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vng) cn tin hnh nhit cao (100-1600C). Khi tng nhit v gi nng baz mc thp (30%) deacetyl t gi tr 98%, i khi khi lng phn t gim lm cho nht ca chitosan cng gim theo. bo ton khi lng phn t trong qu trnh x l chitin cn gim nhit . Phn ng deacetyl xy ra nhanh nht gi u tin trong qu trnh x l, thng t hiu sut 70% khi nng baz l 50% nhit 1000C. Phn ng chm li gi th 5 khi deacetyl t 80%. Nh vy, ko di qu trnh x l khng lm tng c deacetyl, m cn nh hng n cu trc ca chitosan. Cu trc chitin l cu trc mng tinh th nn ha tan v ht nc ca chitin thp. Kch thc ca chitin sau khi nghin nh hng n ng nht ca sn phm chitosan sau khi deacetyl. Nghin nh chitin lm tng b mt tip xc ca chitin vi ha cht t ni phn t. Khi kch thc ht chitin qu ln qu trnh deacetyl xy ra khng hon ton, lp b mt ca chitin b deacetyl nhiu hn so vi cc lp pha trong. Khi ha tan trong acid axetic nhng lp mt ngoi to thnh dung dch, lp pha trong ca ht chitin khng b deacetyl hon ton ch ht nc. Hin tng deacetyl khng hon ton nh trn ca chitosan lm nh hng n kh nng ng dng ca chng. Trong trng hp chng ta nghin chitin vi kch thc nh v ng u th cc ht chitin c deacetyl nh nhau to iu kin thu c sn phm vi ng nht cao hn. Mi trng thc hin qu trnh deacetyl nh hng ln n bin dng cu trc ca chitin, v nh s c mt ca oxy. Cc nh khoa hc thc hin hng lot cc nghin cu loi b oxy ra khi mi trng phn ng DA. Mt gii php n gin nht l nn cht nguyn liu, ht ht kh ra khi mi trng phn ng v a kh ni -t vo ri mi trng kn. Thc hin qu trnh DA trong mi trng nh trn so vi thc hin ngoi khng kh l lm tng khi lng phn t v nht ca sn phm chitosan, khng lm gim deacetyl. Yu t quan trng trong qu trnh DA l lin tc khuy o hn hp phn ng. Thc t s dng c thit b c b phn khuy o v khng c b phn khuy o vi cc nguyn liu polymer khc nhau. Trong trng hp s dng thit b khuy o cn ch trng n c ca hn hp phn ng, xc nh bng t l gia pha lng v pha rn. T l khi lng ti u gia chitin v dung dch kim l 1:5 - 1:12, t l ny la
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chn ph thuc vo cht lng chitin. Mt hn hp nh vy d dng khuy o, m khng cn s dng lng NaOH d tha. Chitin c cu trc v thnh phn ha hc ging nh cellulose, nn c tnh cht gn ging nhau. Nh bit, cellulose c tnh ht nc (s trng n) trong cc dung dch kim long. Nhng tnh cht tng t cng c pht hin chitin. V nh, khi chng ta thc hin 12 ln ng lnh v gi ng chitin, sau cho chitin vo dung dch kim 15-20%, th xy ra s trng n mnh ca chitin, tan cng tng. S trng n v ha tan ca chitin trong dung dch kim nhit thp l do s gin mch ca cu trc chitin, m ra kh nng lm bin tnh v thu nhn polymer vi cht lng ng nht. Cc nh khoa hc nghin cu phng php thu nhn dung dch chitin kim tnh bng mt chu k ng lnh v gi ng v sau tin hnh cc phn ng DA trong mi trng ng nht [26]. Tm li, tt c cc phng php DA bit c ng dng trong sn xut chitosan quy m cng nghip u thc hin nhit cao v mi trng kim mnh. Ngi ta nghin cu phng php DA lnh [27], cc phn ng xy ra nhit phng (khng thp hn 20-220C) s dng cc thit b lm t thp khng r hoc vt liu polymer. Nng NaOH gim xung 30-40% vi t l chitin: dung dch kim l 1:10 - 1:15. iu kin mm nh trn c th thu c chitosan vi nht v deacetyl cao. b) Sy chitosan. Nghin v bo qun. Sau khi deacetyl v ra n pH trung ha sn phm chitosan c kh nng hydrat ha mnh, chitosan trng n cha hn 70% nc.

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Hnh 1.7 - My sy, to ht loi FL bo qun chitosan cn sy nhit 50-550C. Khi sy nhit cao chitosan b ng tng, ti mu v gim ha tan, gim kh nng ng dng ca chitosan. Ch sy tt nht ca chitosan thc hin trong my sy si phn tng 50 0C. Chitosan vi khi lng phn t thp v tan trong nc th phi sy phun. Chitosan kh c m 8-10% (hnh 1.7). ng dng chitosan trong y dc lm bao bc thuc v lm vin cn nghin nh chitosan n kch thc 100-200 mkM. Chitosan cn gi cu trc tinh th ca chitin nn kh nghin nh, nn thu c chitosan dng bt cn nghin nh, sau ct, mi v p trong my phay hay my nghin bi (hnh 1.8).

Hnh 1.8 - Thit b nghin ti kch thc siu nh Cage-Paktor Kh khn nht trong vic nghin chitosan l sy khng ng quy cch hoc chitosan b sng ha, trong nhng trng hp ny chitosan c bn cao, rt kh nghin nh. Trong qu trnh bo qun chitosan, quan st di nh sng thy mu ca chitosan bin i n mu nu v gim ha tan. Cho nn cn bo qun chitosan trong bao b kn, khng cho nh sng i qua (v d, trong hp, ti, bao) trong cc phng kn, kh ro v nhit thng. 1.2 Tng quan v vi khun Bacsillus subtilis v enzyme protease 1.2.1 Gii thiu v vi khun Bacillus subtilis.
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Vi khun Bacillus subtilis thuc gii Bacteria, ngnh Firmicuter, lp Bacili, b Bacillales, h Bacillaceae, chi Bacillus, loi subtilis.

Hnh 1.9 - Vi khun Bacillus subtilis sau khi nhum gram

Hnh 1.10 - Hnh nh a khun lc ca vi khun Bacillus subtilis Nm 1835, vi khun ny c tm ra v t tn l Vibrio subtilis bi Christian Gottfried Ehrenberg. n nm 1872 c Ferdinand Cohn i thnh Bacillus subtilis. Bacillus subtilis l loi trc khun gram dng, c hnh que ng ring l hoc kt thnh chui hoc thnh si v c kh nng sinh ngoi bo t. Ngoi bo t c hnh thnh khi gp cc iu kin bt li ca mi trng nh thiu ht cc thnh phn dinh dng, nhit qu cao, hoc cc sn phm ph to thnh trong mi trng c ch hot ng ca vi khun, ... Bo t c th tn ti trong mi trng mt thi gian
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kh di v khi gp iu kin thun li tr li, n s ny mm v hnh thnh nn t bo vi khun mi. Vi khun Bacillus subtilis c hot tnh proteaza v amilaza, c kh nng phn hu nhiu dng hp cht hu c c bit l protein v tinh bt. Trc y, ngi ta ngh rng y l loi trc khun hiu kh bt buc, nhng iu khng chnh xc. Trong iu kin ym kh vi khun Bacillus subtilis vn pht trin, mc d c km hn trong iu kin hiu kh. Nhit thch hp cho chng pht trin l 35 450C (mi trng nui cy k 370C) v nhit ti a l 600C. Mi trng c pH di 4,5 chng ngng pht trin [4]. Vi khun ny c tm thy ph bin trong t nhin, nhiu nht trn c kh, v vy cn c gi l trc khun c kh. Trn y l hnh nh ca vi khun Bacillus subtilis c soi di knh hin vi sau khi nhum gram v hnh nh khn lc thu nhn c sau khi nui cy (hnh 1.9). Khun lc ca vi khun c mu trng c, mp vin rng ca, b mt khun lc khng nhn bng m nhn v c nhng np gp nh (hnh 1.10). 1.2.2 Gii thiu v enzyme protease. a) Lch s pht trin Enzyme protease l nhm enzyme xc tc cho cc qu trnh thy phn lin kt peptide trong cc peptide hoc protein. Trong cc protease, cc enzyme ca h tiu ho c pht hin sm hn c. T th k 18, nh bc hc ngi Php Recomur pht hin trong dch d dy ca chim n tht c kh nng tiu ho tht. Nm 1836, Schwam quan st c hot ng phn gii protein ca dch v. Tuy nhin, mi n 30 nm sau mi tch c enzyme ny. Nm 1859, Corvisart tch c tripxin t dch ty, l protease u tin nhn c dng ch phm nhng ch phm ny vn cn cha nhu protein v cc tp cht khc. Nm 1862, Danilevxki dng phng php hp ph trn colodion tch c tripxin vi amylaza tu tng. Phng php hp th la chn ca Danilevxki c ngha ln trong nghin cu tinh ch enzyme cng nh protein. Nm 1861 Brunke cng tch c cc pepxin t dch d dy ch dng tng i tinh khit. Nm 1872, Hommarster tch c ch phm chymozin (renin).
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Cc protease thc vt c pht hin mun hn. Nm 1874, Group Besanez cng b nhn c protease t ht ca mt loi cy h u ... Cc protease ca vi sinh vt c ch nghin cu nhiu t nm 1950 mc d t nm 1918 1919 Waksman pht hin c kh nng phn gii ca x khun. Trong hn mi nm nay, s cng trnh nghin cu v protease vi sinh vt ang tng ln ng k nhiu hn cc cng trnh nghin cu protease v ng vt v thc vt [9]. b) Tnh cht ca enzyme protease Protease l cc enzyme xc tc s thu phn lin kt peptide (CO-NH) trong phn t protein v cc c cht tng t. Nhiu protease cng c kh nng thu phn lin kt este v vn chuyn axit amin. Di tc dng ca protease, protein b thu phn theo s sau: Protein pepton polypeptit peptit axit amin y l qu trnh thu phn tng i phc tp v c s tham gia ca nhiu loi protease khc nhau gm hai loi chnh l exoprotease v endoprotease. c) Phn loi enzyme protease phn loi protease, ta da vo 2 cch sau: Cch 1: Da vo trung tm hot ng: Theo phn loi quc t cc enzyme thuc nhm ny chia thnh 4 phn nhm: Aminopeptidase: xc tc s thu phn lin kt peptit u Nit ca mch polypeptide; Cacboxypeptidase: xc tc s thu phn lin kt peptide u cacbon ca mch polypeptide. C hai phn nhm enzyme trn u l cc exo-peptidase; Dipeptihydrolase: xc tc s thu phn cc lin kt dipeptide; Proteinase: xc tc s thu phn cc lin kt peptide ni mch [2]. Cch 2 : Da vo c ch thu phn, protease c phn thnh hai nhm chnh: Exoprotease: nhng enzyme thu phn cc lin kt u mt ca chui mch polypeptide; Endoprotease: nhng enzyme xc tc thu phn cc lin kt peptide gia mch polypeptide. Ngi ta cng phn loi cc protease theo vng pH hot ng ti thch: protease axit, trung tnh, v kim tnh. Tuy nhin s phn loi ny ch c ngha thc dng khng tht s chnh xc v pH hot ng ti thch ca enzyme cn ph thuc vo bn
33

cht c cht v nhiu yu t khc na [2]. d) Ngun thu enzyme proteaza Protease thu nhn t thc vt: chng ta c th thu mt lng ln protease t thc vt. Tuy nhin vic thu nhn protease t thc vt gp nhiu vn : phi c t trng trt, ph thuc vo iu kin thi tit kh hu, t ai, phi tri qua mt thi gian di... Tuy vy, mt s cc protein thu nhn t thc vt c cht lng rt tt nh Papain, Bromelain, Ficin ... Protein thu nhn t ng vt: Cc protein c ngun gc t ng vt nh tripxin, chymotrypxin, pepxin... c th thu c vi s lng tinh khit. Tuy nhin vic sn xut n phi ph thuc vo vt nui cn git m, cc iu kin nng nghip... Protease c ngun gc t vi sinh vt: Kh nng khng p ng c nhu cu trn th gii ca enzyme ng vt v thc vt dn n s quan tm ngy cng tng ca enzyme vi sinh vt. Vi sinh vt l ngun cung cp enzyme tuyt vi do tnh a dng sinh ho v tnh nhy cm v k thut di truyn ca chng. Protease vi sinh vt chim khong 40% tng lng enzyme bn trn th gii [2], chng c nhiu u im hn so vi protease ca ng vt v thc vt do cc tnh cht ca chng ph hp vi kh nng ng dng cng ngh sinh hc. Cc protease c th c thu nhn t vi khun, nm v x khun. e) Cc yu t nh hng n qu trnh sinh tng hp protease t vi sinh vt Qu trnh tng hp enzyme ni chung cng nh tng hp protease vi sinh vt ni ring cng chu nh hng ca nhiu yu t khc nhau nh: m mi trng, nhit nui, pH mi trng, thng kh, thnh phn mi trng nh hng ca nhit : Nhit nh hng n s sinh trng pht trin, kh nng sinh tng hp enzyme ca vi sinh vt cng nh tnh cht ca enzyme c tng hp. Ty tng vi sinh vt, nhit thch hp c khc nhau: cc loi nm mc pht trin thch hp nhit 22-320C, cn cc vi khun nhit cao hn 36-550C. Ni chung a s vi sinh vt sinh tng hp enzyme khng bn vi nhit v b km hm nhanh chng nhit cao hn nhit thch hp nh hng ca pH mi trng: Khi dng phng php nui cy b mt, pH mi trng t nh hng n qu trnh sinh tng hp enzyme vi sinh vt. Hn na pH mi trng hu nh khng thay i trong qu trnh pht trin ca vi sinh vt. i vi
34

nm mc pH thch hp cho qu trnh sinh tng hp protease vo khong 6 - 6,5. Cc vi khun pht trin tt v to thnh nhiu enzyme pH gn trung tnh 6,6 - 7,4. Khi dng phng php b su, pH mi trng c nh hng rt ln, nhiu khi c vai tr quyt nh i vi s tch lu protease trong mi trng. ng lu l pH mi trng thng c th thay i sau khi kh trng v trong qu trnh pht trin ca vi sinh vt. pH ban u ca mi trng thch hp cho s pht trin ca cc vi sinh vt khc nhau khng ging nhau. i vi cc nm mc, pH ny vo khong 3,8 5,6, cn i vi vi khun l 6,2 7,4 [2]. nh hng ca thng kh ca mi trng: Tu theo tnh cht ca tng loi vi sinh vt khc nhau v sn phm cn thu nhn chng m s nh hng ca yu t ny l khc nhau. Trong mt s trng hp, thiu oxy tuy kim hm s sinh trng ca vi sinh vt nhng li lm tng qu trnh sinh tng hp protease v c th ngc li. thng kh ca mi trng c nh hng ln n qu trnh tng hp protease. Tuy nhin nh hng ny c khc nhau tu theo vi sinh vt. Trong mt s trng hp, thiu oxy tuy c kim hm s sinh trng ca vi sinh vt nhng li lm tng qu trnh tng hp protease. Lng oxy thch hp cho qu trnh tng hp protease cc vi sinh vt khc nhau l khng ging nhau (Aleeva 1973-Aravina v cng s nm 1976). Ngay c i vi mt s vi sinh vt nht nh, s hiu kh cng nh hng khc nhau n qu trnh tng hp cc protease khc nhau (Crivova v tp th nm 1977). Trong mt s trng hp, s hiu kh mnh li km hm tng hp protease. i vi nm mc, ch sc kh thch hp 10-12m3 khng kh v trng (c nhit khng cao qu 400C) trn 1m3 mi trng trong 1h vi thi gian nui 68-73h. Vi thi gian nui ngn hn trong cc thng ln men ging (48h) v trong cc thng ln men sn xut (48-52h) th lng khng kh cn sc vo mi trng nui Asp.oryzae phi l 30m3/m3 mi trng/gi i vi thng nhn ging v m3/m3 mi trng/ gi cho thng sn xut. Khi nui Bacillus subtilis trong cc thng nhn ging c cnh khuy lm vic lin tc v sc khng kh v trng vi lng l 4 0 m3/m3 mi trng/1h. V nui vi khun ny trong cc thng ln men trong sn xut (trong 48h 370C) c cnh khuy lm vic lin tc v sc khng kh v trng vi lng 60 m 3/m3 mi trng/h.
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nh hng ca thnh phn mi trng: Thnh phn mi trng nh hng rt ln n kh nng sinh tng hp enzyme. tng lng enzyme trong mi trng cn la chn ngun C, N, mui khong thch hp, chn t l nng thch hp gia cc thnh phn ny v c bit l cn s dng cc cht cm ng v protease l enzyme cm ng. i vi mt vi sinh vt nht nh, iu kin thch hp cho qu trnh sinh tng hp cc enzyme khc nhau th khng ging nhau. Nm vng cc qui lut nh hng ca cc yu t ny c th iu khin vi sinh vt sinh tng hp nh hng mt loi enzyme xc nh. * nh hng ca ngun cacbon: Ngun cacbon thng dng nui cy vi sinh vt thng dng l cc glucid: monosacarit, disacarit v c polysacarit. Cc cht ny nh hng khc nhau n s sinh tng hp enzyme ni chung cng nh tng hp protease vi sinh vt. c tnh tc dng ca ngun cacbon tu thuc vo bn cht ho hc ca chng v c tnh sinh l ca vi sinh vt. Ngun cacbon v nit t nhin thng dng trong mi trng nui cy l bt m, bt u tng, cm v nc chit ca chng. Trong s monosacarit, glucose nh hng rt khc nhau n qu trnh sinh tng hp enzyme ni chung cng nh sinh tng hp protease. * nh hng ca ngun nit: Cc cht c th dng lm ngun nit trong mi trng nui cy vi sinh vt c th l cc cht hu c (protein v cc sn phm thu phn ca chng) hoc cc mui v c (mui amn, mui nitrat). Nhiu vi sinh vt cn c th s dng c nit ca HNO3, HNO2. Nng dng nit trong mi trng c nh hng r rt v c quy lut n qu trnh tng hp protease ngoi bo vi sinh vt. Ngun nit hu c thng c nh hng tt n qu trnh sinh tng hp protease v chng c vai tr nh cht cm ng ca qu trnh ny. Cc ngun nit hu c thng dng l: cc loi bt khc nhau (c gluten), nc chit ca cm, nm men t phn gii, pepton, protein Ngoi vic chn ngun v lng cacbon, nit thch hp cn cn phi chi chn t l thch hp gia C : N. Bn cnh photpho v lu hunh cng c nh hng quan trng n qu trnh sinh tng hp protease. Ngoi cc yu t k trn cc nguyn t vi lng (Steiner 1961), NaCl (Kovaleva v tp th 1977), vitamin (Emxva 1975) v mt s yu t khc (Aravina v tp th
36

1977) cng c nh hng n lng protease trong mi trng nui cy vi sinh vt. 1.2.3 Mt s nghin cu ng dng Bacillus subtilis ca cc tc gi trong nc Nm 1997, TS. L c Mnh tin hnh nghin cu: Nghin cu thu nhn v ng dng -amylase trong cng nghip sn xut bia dng Bacillus subtilis CNTP-B08 c kh nng sinh tng hp -amylase cao, hot ca n t mi trng nui cy b mt l 2610SKB/g thay th mt malt i mch bng cc loi nguyn liu sn c trong nc nh: ng 30%, go 30%, i mch 40%, do gi thnh sn phm h. Nm 2004, tc gi V Ngc Bi c cng trnh Nghin cu qu trnh thu phn protein t Bacillus subtilis S5 trong iu kin nhit l 50oC, pH t nhin, t l ph phm 0,3% v nng mui thch hp l 3%. Nm 1998, tc gi L c Mnh c nghin cu Nghin cu thu nhn v bo qun protease t ch phm ln men b mt ca vi khun Bacillus subtilis. Protease ca vi khun hot ng mnh nht nhit 42 48oC, nu nhit cao hn hoc thp hn th protease s b mt hot ng k ch cn li khong 40%, pH thch hp cho protease hot ng l 6,4 7. Protease ca vi khun kh bn vi nhit , nhit 50oC, hot ca protease khng gim sau 6 gi. nhit cao hn th hot protease gim dn theo thi gian v c bit nhit 70oC hot ca protease gim rt nhanh. Nm 2004, Th.S Th Bch Thu v TS. Trn Th X thc hin ti Nghin cu mt s nh hng ln kh nng sinh protease ca Bacillus subtilis. Kt qu ca nghin cu cho thy ngun cacbon v nit c tc dng kch thch cao nht l tinh bt tan v casein. Thnh phn nui cy chng Bacillus subtillis trong phng th nghim bao gm: Pepton 1%, cao tht 0,3%, mui 0,5%, tinh bt tan 1,75%, cao nm 0,1%, ph ban u l 6,5 v nhit nui cy thch hp nht ng vi mi trng ti u Bacillus subtillis sinh protease mnh nht l 35oC. 1.3 Cc lnh vc ng dng ca chitin/chitosan Chitin v chitosan c th d dng b vi khun phn hy, tc l chng cng thn thin vi mi trng hn cc loi polymer tng hp. Hin nay chitosan c ng

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dng trong rt nhiu lnh vc nh: y hc, thc phm, nng nghip, mi trng 1.3.1 Trong y hc Chitin v chitosan l nguyn liu sn xut thuc iu tr bng, vim lot d dy, h cholesterol, tr bo ph, gim au, chng ng t mu, tng sc khng, cha xng khp v chng c c bnh ung th. Tc nhn h cholesterol: Chitosan c chc nng h cholesterol trong rut ng vt. Ngi ta tin hnh th nghim vi th v thy khi c cho n thc n giu cholesterol 0.9% trong 39 ngy, lng cholesterol huyt thanh tng t 79 ln 650 mg/ trn 1kg th trng. Trong trng hp vi khu phn n nh trn nhng c b sung 2% chitosan, lng cholesterol huyt thanh ch tng khong 300 mg/1kg th trng, trong lng cholesterol c ch (HDL cholesterol) gim khng ng k. V t lu, mt s chuyn gia Trung tm Huyt hc thuc Vin Hn lm Y hc Nga cng pht hin chitosan c th ngn chn s pht trin ca chng nhi mu c tim v bnh t qu iu tr bo ph: Khi vo ng tiu ha, chitosan c kh nng bao cc ht cu bo v ko chng thi ra ngoi theo (ng vt khng tiu ha chitosan) nh n c ng dng lm thuc gim bo. Nc ta hin nay c thuc Chitozan gim bo. Cc vt liu y sinh hc v dc phm: chitin v cc dn xut c s dng nh nhng vt liu y sinh hc hay vt liu bao gi cc loi thuc tan chm. Film chitosan bao thuc cng c cng dng nh cc dng con nhng thng phm v c s dng nh vt liu dng cy gii phng chm cc loi thuc chng ung th. Chitosan cng c a vo cng thc cc loi thuc ung lm gia tng s hp thu ca thuc vo mu. Vt liu v vt thng: Cc vt thng m ng, thc vt c th c bao bng mt tm mng hay mt ming xp chitin v chitosan dng bng hoc dng bt mn. Cc vt thng cng c th c tr liu bng cc dung dch hay kem chitin v chitosan. Kt qu l s pht trin ca cc t bo vng m b thng c kch thch, chitinase v lysozyme c tng cng dn n mau lnh vt thng v hn ch nhim trng. Ti cuc chin Iraq va qua, M cng s dng loi bng cu thng
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kiu mi, k thut cao, c thnh phn cu to bi cht chitosan. So vi cc loi bng thng, tc cm mu, tnh st khun v thi gian lnh m khi s dng loi bng ny c hiu qu hn gp nhiu ln. Cha bnh khp: in hnh trn th trng dc hin nay l loi thuc cha khp lm t v tm c tn Glucosamin ang c thnh hnh trn ton th gii. So vi sn phm cng loi th Glucosamin c u th hn, do sn xut t ngun v tm t nhin nn sn phm t gy phn ng ph, khng c hi v khng b ri lon tiu ho cho ngi bnh (iu ny c ngha rt quan trng). Nc M tiu th c hn 1 t vin nang Glucosamin. Nhng nm gn y, loi thuc cha khp ny cn c ph cp rng nhiu nc trong c Vit Nam. Nc ta cng c thuc Glusivac c tr thoi ha khp [19]. 1.3.2 Trong x l nc thi v lm trong nc sinh hot Dng chitosan hp th cc ion kim loi nng gy nhim mi trng sinh thi nh Cu, Hg, Cr, Pb, Ni, Cd. Chitosan cationit to cc phc hp a in ly vi nhng polymer polyanionic v to phc hp chelate vi cc ion kim loi to kt ta. Cc phn ng ny c ng dng lm trong nc thi nhim. Vo nm 1975, mui chitosan acetate ln u tin c mt cng ty Nht gii thiu nh mt tc nhn cationit t nhin ng t v loi cc cht thi trong nc cng. Hin nay h thng ny vn cn c s dng x l nc thi sinh hot, ti s dng nc thi (v d nc h bi), thu hi cc protein v khong t nc thi cng nghip, bao bc cc ht bo, phn lp cc cht c hot tnh sinh hc trong nc tiu v tch cc cht c ni bo t dung dch long. Chitosan cng c s dng nh mt cht hp ph tch cc ng v phng x nguy him t nc nhim v thu hi uranium t nc bin v nc ngt [27]. 1.3.3 Trong nng nghip Lm phn bn rau sch: Chitin v chitosan ch yu c bn dng bt, dng ming hay dng dung dch vo t nng nghip hay mi trng nui cy lng. Ngoi ra dung dch chitosan c th c phun ln l cy.

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X l ht ging: ht ging c th c nhng vo dung dch chitosan long, bao ph b mt bng mt mng chitin hay chitosan mng hay bt mn. Khi c bao ph nh vy, hot ng ca chitinase ca ht c tng cng trong giai on ny mm. S tng cng chitinase trong ht s lm gia tng kh nng t v sinh hc ca ht qua vic nhim VSV, kt qu l lm cho cy trng pht trin tt. 1.3.4 Trong m phm Chitosan l nguyn liu sn xut kem dng da, kem chng tia t ngoi Cc mui hu c ca chitosan phn t thp ha tan trong ethanol long v c s dng nh mt thnh phn ca keo xt tc. CM-chitin v HP-chitosan cationic ha tan trong nc v bn trong mt khong pH rng, chng c s dng nh mt thnh phn ca m phm chm sc da. Chitosan, CM-chitosan, HP-chitosan c chc nng to m cho da, ngn cn s hy hoi c hc tc. c tnh gi m tng ng vi dung dch propylenglycol 20% v dung dch hyaluronic long. Nhng dn xut trn ca chitosan c th ngn chn s nhim khun trn da v hot ha t bo da dn n s ngn chn s lo ha ca da [19, 27]. 1.3.5 Trong thc phm Hin nay chitosan c s dng rt ph bin trong sn xut thc phm. C th s dng thu ht nc v bo (qu trnh nh ha), kt hp vi thuc nhum (qu trnh ng ha), qu trnh ng c. Chitosan cng c chng minh l c kh nng to dng mng mng s dng nh l nhng lp mng mng hoc nhng lp bao khng c hi (c th n c). Mng bao Chitosan c th ci thin kh nng bo qun cc loi thc phm d b thi ra bng cch gim lng khng kh bn trong bao gi cng nh gim qu trnh thot hi nc. C th nhng trc tip thc phm vo dung dch chitosan pha sn ri kh, to thnh mt lp mng mng t nhin trn b mt sn phm: trng, tht c, rau qu, gi , bnh go, nhng ci bp trc khi lm kim chi Hoc cng c th to thnh mng trc ri mi cho sn phm vo: bnh m, xc

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xch Hoc cho chitosan trc tip vo sn phm dng lng: x l nc qu, lm trong gim, bo qun tu h, ng ha sa, kem v mayonaise, bo qun m[12, 19, 24].

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Chng 2 - NGUYN LIU V PHNG PHP NGHIN CU 2.1. Nguyn liu, ha cht v thit b 2.1.1. Nguyn liu: Nguyn liu c s dng bao gm: a) Chng vi khun Bacillus subtilis c phn lp v gi ging ti phng th nghim CNSH - Khoa Ho - trng i hc Bch Khoa - i hc Nng; b) V u tm c thu nhn t cng ty c phn XNK Thy sn Min trung: Cng ty ch bin v xut khu thy sn Th Quang; Ph phm sau khi thu nhn t nh my ch bin thy sn c sy cho kh rn nhit 400C ri nghin nh v bo qun lm ngun nguyn liu thu nhn chitin, v sau t chitin thu nhn chitosan. Trong nghin cu ny nguyn liu v tm c thu nhn t hai loi tm ph bin trong ch bin trn a bn TP Nng hin nay l tm s (tiger shrimp Penaeus monodon Fabricius) v tn th chn trng (White Shrimp - Penaeus vannamei). Cc th nghim c tin hnh trn hn hp 2 loi u v tm ny.

a thu nhn chitin/chitosan a) Tm s; b) tm th chn trng

Hnh 2.1 S khc nhau ca mt s nguyn liu tm, v ca chng dng

42

c) Ni tng c thu nhn ch phm enzyme protease c thu gom t cng ty c phn XNK Thy sn Min trung: Cng ty ch bin v xut khu thy sn Th Quang. Ni tng c sau khi c thu nhn t nh my v c ra sch, x l bng nc mui 5%, sau c bo qun ng lnh nhit -220C phc v thu nhn ch phm enzyme protease. 2.1.2. Ho cht v thit b: Ho cht chnh: - Cc ho cht tinh khit nh : HCl m c, Foocmol 40%, cn tuyt i, KOH 0,01N, H2SO4 - Cc ch th mu : thimolphtalein, phenolphtalein - Pepton, casein, cao tht, cao nm - V cc ha cht phc v cc th nghim phn tch khc. Thit b: - T n nhit, my lc, thit b tit trng, t lnh ng, t cy v trng. - Thit b li tm - Thit b c c chn khng - Thit b phn tch m Kjeldahl. - V cc thit b thng dng trong phng th nghim CNSH khc. 2.2. Phng php nghin cu 2.2.1 nh lng nc V tm c sy 1200C trong 3 gi, n khi lng khng i. ngui, sau cn xc nh lng nc theo cng thc: 2.2.2 nh lng mui Calci carbonat Mui calci carbonat trong v tm c nh lng bng cch ngm v tm c xay nh v sy kh trong dung dch HCl 10% trong 6 gi, nhit phng. Sau lc, ly nc ci dng acid H2SO4 30% kt ta Ca2+ trong dung dch. T trng lng CaSO4 kt ta v sy kh suy ra hm lng calci carbonat
43

2.2.3 nh lng chitin Sauk hi loi b calci carbonat v protein, chitin c un nng vi dung dch K2CO3 m c 1600C chitin khng tan. Lc, ra sch, sy 1000C, thu c chitin. 2.2.4 Phng php xc nh deacetyl ca chitosan n v phn trm deacety trong sn phm chitosan c xc nh bng mc deacetyl (y l t l ca khi lng glucosamine so vi s monomer chung trong phn t polymer DD = m(m+n) = Namin/Ntng), thng t 70-90%. 2.2.5 Mt s phng php xc nh khc: -Hm lng lipid (%), protein (%), tro (%) v hm lng nc (%) c xc nh theo GOST 7636-85 (LB Nga) -Hm lng khong c phn tch theo chun ca AOAC (1990) - Phng php xc nh N-formon theo Sorensen - Hot enzyme, v ch phm enzyme protease xc nh theo phng php Anson ci tin - Phng php xc nh QMAFAM (CFU/g) theo GOST 10444.15 94 (LB Nga) - kh khong trong v tm c xc nh theo cng thc KK=[(mK1mK2)/mK1]x100%; mk1, mk2-hm lng cht khong ban u v sau khi kh khong trong v tm . - kh protein trong v tm c xc nh theo cng thc KK=[(mP1mP2)/mP1]x100%; mP1, mP2-hm lng protein ban u v sau khi kh protein trong v tm. - deacety trong sn phm chitosan c xc nh theo t l ca khi lng glucosamine so vi s monomer chung trong phn t polymer DD = m(m+n) = Namin/Ntng), thng t 70-90%; nht ca chitosan c xc nh trn nht k Roto Brookfield 2.3 S cc m hnh th nghim (hnh 2.2)
44

Tng quan cc nghin cu trong v ngoi nc v chitin/chitosan

Tnh cht vt l v ha hc ca chitin/chitosan

Cc phng php thu nhn chitin: ha hc, sinh hc

ng dng vi khun B. subtilis trong sn xut chitin

Phng php thu nhn chitosan

Cc lnh vc ng dng chitin/chitosa n

Xy dng cc m hnh th nghim gii quyt cc vn cn nghin cu ca ti

Nghin cu thnh phn ha hc ca v u tm

Nghin cu thnh phn ha hc v hot enzyme ca ni tng c Tch chit enzyme protease t ni tng c

Nghin cu mi trng nui cy v kh nng sinh protease ca B.subtilis

Kh mu ca v u tm

Kh khong ca v u tm

Nghin cu kh protein ca u v tm

Kh protein bng ch phm enzyme protease, tch chit t ni tng c

Kh protein bng ch phm enzyme protease

Kh khong ln 2

nh gi cht lng chitin

Chitin

Chitosan

nh gi cht lng chitosan

45

Hnh 2.2 S m hnh cc bc thc hin trong nghin cu 2.4 X l s liu S liu trong nghin cu ny l trung bnh ca ba ln phn tch. Kt qu c phn tch thng k bng phn mm Minitab Professional v16.1.0.0. . Gi tr ca p < 0,05 c xem l c ngha v mt thng k

46

Chng 3 KT QU V THO LUN 3.1 Kt qu nghin cu kho st thnh phn ha hc ca v tm, nguyn liu ni tng c v phng php bo qun nguyn liu ni tng c. 3.1.1 Thnh phn ha hc ca mt s nguyn liu cha chitin, ph bin thnh ph Nng Phn tch thnh phn ha hc ca v tm sau khi c sy kh nhit 400C nhn thy hm lng chitin tng i cao t 33-35% tng khi lng v tm kh. Bn cnh hm lng protein l 12-14%, iu ny i hi cn c bin php ph hp loi b lng protein tng i ln trong nguyn liu ny. ng thi hm lng cht khong rt cao 37-40% tng khi lng nguyn liu, nn cn s dng phng php loi khong pH ph hp t hiu sut tt nht trong thu nhn chitin/chitosan. Bng 3.1 Thnh phn ha hc ca nguyn liu v tm STT Loi nguyn liu V tm s V tm th chn trng Hn hp v tm s v v tm th chn trng Thnh phn ha hc, % Protein 141,3 121,2 Lipid 1,50,2 0,40,1 Cht khong 371,9 402,1 Chitin 331,2 352,5 Nc 14,51,9 12,61,7

1 2 3

131,6

0,950,5

38,52,4

39,51,6

8,051,5

Ngoi ra, hm lng lipid trong hn hp v tm s v v tm th chn trng g n 1%, cc lipid ny cha cht mu c trng ca v tm l carotenoid, carotenoid c th gy nh hng n cht lng chitin v tnh cm quan, nhng chng ta c th tn dng cht mu ny ng dng trong cng nghip thc phm, nn cn c bin php ph hp thu hi ngun cht m t nhin qu gi ny.

47

3.1.2 Kt qu xc nh thnh phn ha hc ca nguyn liu ni tng c Cc kt qu th nghim xc nh thnh phn ha hc ca nguyn liu dng tch chit ch phm enzyme protease c trnh by trong bng 3.2. T bng 3.2 ta thy sau qu trnh bo qun lnh ng hm lng nc trong nguyn liu gim xung. Nguyn nhn l do kh nng gi nc ca nguyn liu gim. Hm lng protein b gim t 12% xung 11%, nguyn nhn c th l do protein b phn gii di tc dng ca vi sinh vt hoc b tht that khi chng ta tin hnh gi ng nguyn liu. Bng 3.2 - Thnh phn ha hc ca nguyn liu ni tng c Nguyn liu Nguyn liu ti Nguyn liu ng lnh Protein 121,0 111,2 Hm lng, % Lipid Nc 16,361,3 702,0 17,41,6 671,4

Tro 1,640,2 2,60,4

iu ng quan tm khi phn tch c im cng ngh ca nguyn liu l hm lng lipid tng i cao (16-18%), y s l tr ngi v mt cng ngh khi chng ta tin hnh tch lng lipid ny ra khi dch enzyme. 3.1.3 Kt qu nghin cu bo qun nguyn liu ni tng c phc v thu hi ch phm enzyme protease Ni tng c s dng thu nhn ch phm enzyme protease c thu gom trong qu trnh m v tch fi-l. Nguyn liu l hn hp ni tng ca hai loi c: c nc s (Decapterus maruadsi) v c nc di (Decapterus lajang Bleeker) thc n ch yu ca chng l cc loi ng vt: chn cho (Copepoda), c v (Ostracoda), lng tc (Amphipoda) [Nguyn Nht Thi, 1991]. Ni tng c sau khi ra sch cn c bo qun trong mi trng lnh (4 50C) hoc iu kin lnh ng su (-18 -200C) ph thuc vo vic thu hi ch phm enzyme protease. Kt qu kho st, chng minh rng, phng php bo qun mi trng 4
48

50C m bo khng lm gim hot enzyme protease ni ti trong nguyn liu vi thi gian bo qun khng c vt qu 12 gi. Phng php ti u hn l bo qun ni tng c trong iu kin lnh ng su nhit -18 -200C. xc nh thi gian ti u bo qun nguyn liu chng ti da trn quan im xc nh lng ni-t formol (Nf) bng phng php chun formol Sorensen v lng ni-t tng s (Nt) ca Kjeldahl. Nhiu ti liu chng minh rng nguyn liu thy sn cha h hng khi t l Nf/Nt thp hn 10% [Chernogorsev, 1960]. Nh vy t th hnh 3.1 chng ti xc nh c thi gian bo qun ti u ni tng c nhit -18 -200C l 2 thng, sau khi thu gom t nh my.

Hnh 3.1 - nh hng ca thi gian bo qun ln ch tiu cht lng ni tng c bo qun nhit -18 -200C 3.2 Chn phng php thu hi ch phm enzyme protease t ni tng c, kho st hot ca ch phm enzyme protease thu c.

49

Hnh 3.2 - Biu s ph thuc hot cc nhm enzyme protease trong cc vng pH khc nhau Cc kt qu xc nh hot enzyme trong nguyn liu ni tng c ti v ng lnh sau thi gian bo qun 1 thng cc pH = 1, 2, 3, 4, 5, 6, 7, 8, 9 c biu din trn hai ng cong hnh 3.2 Theo hnh 3.2 ta thy c 2 vng pH ti u cho hot ng ca enzyme trong ni tng c l vng pH kim (7 8) v vng pH acid (2 2,5) . Tuy nhin theo mt s ti liu tham kho th vng pH kim khng thch hp tch chit enzyme protease v dch enzyme rt d b nhim khun, cn vng pH acid thch hp hn cho vic tch chit enzyme protease vi hot tng i cao (2,5 Kat), mt khc pH ny enzyme protease kh nhim khun hn. V vy trong cc phn tip theo s tin hnh tch chit enzyme protease trong iu kin pH acid. Da vo nhng tnh cht ca enzyme cn thu nhn v cc cht kh c trong thnh phn ni tng c chng ti thc hin theo quy trnh hnh 3.3 v thu c 2 ch phm enzyme protease dng lng t nguyn liu ti v nguyn liu ng lnh, ch phm enzyme protease thu c c bo qun 0 50C.

50

Hnh 3.3 - S quy trnh tch chit enzyme protease t ni tng c Bng 3.3 - Mt s tnh cht ca ch phm protease thu nhn t nguyn liu ti v nguyn liu ng lnh Ch tiu nh gi ng nht ca dung dch Mu sc pH Mi v Ch phm enzyme protease t nguyn liu ti ng nht cao t nguyn liu ng lnh ng nht cao

Mu vng nht Mu vng ti nu 2 2,5 2 2,5 Mi c trng ca cc sn Mi c trng ca cc sn phm t c phm t c Khi lng ring, g/ml 1,1 1,15 Hot , kat 2,31 2,1 Mt s c im ca ch phm enzyme protese thu nhn t nguyn liu ni tng c ti v ng lnh c trnh by trong bng 3.3
51

Theo bng 3.3 ta thy tnh cht ca ch phm enzyme thu c t ngun nguyn liu ti v ngun nguyn liu ng lnh khng c c s khc bit ln. ch phm enzyme thu c t nguyn liu ng lnh c mu sm hn, nguyn nhn ca s khc bit ny l c th do qu trnh bo qun (1 thng) mt phn protein trong nguyn liu b bin tnh, v mt s phn ng oxy ha lipid. Trong hnh 3.4 l hnh nh ch phm enzyme thu c dng lng

Hnh 3.4 - Ch phm enzyme protease tch chit t ni tng c ng lnh T kt qu xc nh hot ch phm enzyme protease theo phng php Anson ci tin trong bng 3.3 ta thy rng hot ch phm enzyme protease gim so vi trong nguyn liu ti trong vng pH acid tng ng (2,5). Nguyn nhn ca s gim ny l do trong qu trnh tch chit mt phn protein b bin tnh v hng. Ch phm enzyme protease c bo qun nhit 4-50C phc v nghin cu qu trnh loi b protein bng phn ng thy phn trong v tm cc nghin cu tip theo. Vic thu gom v tch chit ch phm protease t ni tng c ti c nhiu li th, nhng trong cc th nghim tip theo chng ti ch dng ch phm enzyme protease tch chit t ni tng c c bo qun ng lnh. 3.3. nh hng ca nhit v thi gian bo qun ln hot tnh ca ch phm enzyme protease tch chit t ni tng c
52

Kt qu xc nh nh hng ca nhit ln hot ca ch phm enzyme protease tch chit t ni tng c ng lnh c trnh by trn hnh 3.5.

Hnh 3.5 S ph thuc ca hot ca ch phm enzyme protease vo nhit mi trng

Hnh 3.6 - nh hng ca thi gian bo qun ln hot enzyme protease tch chit t ni tng c T ng cong biu th nh hng ca nhit ln hot ca ch phm enzyme
53

protease r rng rng, ch phm enzyme protease c hot lc cao nht vng nhit 40-450C xc nh thi gian ti u bo qun ch phm enzyme protease nhit 450C, chng ti tin hnh xc nh hot sau mi 15 ngy bo qun. Kt qu xc nh ng th hin trn hnh 3.6 Kt qu nghin cu chng minh rng c th bo qun ch phm enzyme protease tch chit t ni tng c n 6 thng m khng nh hng n cht lng. iu ny c l gii bi vai tr ca HCl nh l mt cht bo qun, bn cnh pH thp cng hn ch ti a nh hng ca cc vi khun gy hng ch phm enzyme. 3.4 Cc kt qu nghin cu sn xut chitin/chitosan ng dng enzyme protease tch chit t ni tng c Trong nguyn liu v tm, chitin lin kt rt cht ch vi protein, lipid v cc cht khong (pha rn). Vic thu nhn chitin t ngun nguyn liu ny xy dng trn cn bn l a protein v cc cht khong v trng thi lng (pha lng), v t loi b chng ra khi chitin. sn xut chitin t cc tiu chun cht lng quc t cn loi b hon ton protein v khong cht, cng nh lipid. Hin nay phng php ha hc c dng ph bin thu nhn chitin bng cch s dng cc ha cht m c, tc l x l nguyn liu trong nhng iu kin khc nghit (mi trng axit mnh, baz mnh, nhit cao v thi gian ko di), thng dn ti lm gim cht lng chitin thng phm. Cc h hng ca sn phm chitin thng gp khi thu nhn theo phng php ha hc l: bin dng cu trc phn t, gim nht ca dung dch chitin v chitosan. Bn cnh vic bo qun v s dng cc ha cht c m c cao m i hi s tip xc trc tip ca cng nhn gy nhiu quan ngi trong vn bo m an ton v sc khe cho ngi lao ng. Theo phng php ha hc c th thu nhn c sn phm phn gii protein l hn hp cc axit amin t do ha ln cng cc cht c hi, cc cht c hi sinh ra do protein b thy phn su, thm tr phn gii c cc axit amin nn vic s dng sn phm dch thy phn gp phi nhiu hn ch, v d khng s dng c trong thc phm hay trong sn xut thc n chn nui. Cc hng s dng chitin v dn xut t chitin trong thc t trong nhng nm gn
54

y khng ngng c m rng dn ti cn tm kim v th nghim cc phng php mi thu nhn cc polymer sinh hc ny. Mt vn c tnh thi s cn tn ti l nghin cu phng php x l nguyn liu v tm to iu kin thu hi chitin v ton b cc hp cht t nhin c hot tnh sinh hc c trong n, bn cnh vic hng ti pht trin bn vng trn c s gim thiu nhim mi trng. 3.4.1 Kt qu nghin cu phng php x l nguyn liu v tm trc khi loi b protein: Phn tch cc kt qu xc nh thnh phn ha hc ca v tm nhn thy rng, hm lng cht khong (38,5%) ln hn hm lng protein (13%) nn v nguyn tc khi xy dng cng ngh sn xut chitin cn tin hnh qu trnh kh khong trc khi loi b protein. Bn cnh cc cht khong v protein trong v tm cn cha mt lng lipid. V gc ha sinh trong lipid cha hp cht carotenoid. Carotenoid l cht mu, lm cho v tm c mu hng n khi gia nhit, cht mu ny c th nh hng ti gi tr cm quan ca chitin thnh phm. Hin nay nhiu nghin cu chng minh carotenoid ca v tm l cht mu c th dng trong cng nghip ch bin thc phm [Lebskaya, 2000]. Nhm mc ch thu hi cht mu t nhin qu gi ny chng ti thc hin cc th nghim tch chit chng cng vi lipit. thc hin tin hnh tch cht mu bng phng php phi chn hn hp v tm nghin nh vi cn 96 nhit 60-700C vi t l 1:2 v 1:3 (w nguyn liu: w cn), sau hn hp c t trong mi trng chn khng 1 2 gi tin hnh phn ng tch chit hn hp lipit-carotenoid ra khi nguyn liu. loi b nc v thu c cht mu carotenoid tan trong lipit cn phi trn hn hp lipit-carotenoid vi hexan. Sau , loi b hexan tin hnh trong ni chn khng. Hiu sut tch chit cht mu carotenoid trong lipit l 0,3-0,32% khi lng v tm ban u. V tm sau khi ty mu c sy kh loi b cn c bo qun lm nguyn liu thu nhn chitin. thc hin qu trnh kh khong v tm sau khi ty mu c nho trn vi nc vi t l 1:8 1:10 v axit HCl m c, hm lng axit HCl theo tnh ton l
55

4% th tch phn ng. Qu trnh kh khong c tin hnh nhit phng v lin tc khuy o hn hp phn ng. Axit HCl c cho dn dn vo bng tng lng nh trnh s to thnh nhiu bt lm gim tc phn ng. Kt qu qu trnh kh khong trong 140 pht c trnh by trn th hnh 3.7

Hnh 3.7 - nh hng ca thi gian ln kh khong v hm lng cht khong cn li trong v tm T th 3.7 v bng phng php thng k chng ti xc nh c thi gian ti u cho qu trnh kh khong l 1,8 2 gi Hm lng cht khong cn li trong nguyn liu sau qu trnh kh khong l 2,0 n 3,2% (theo khi lng cht kh), t tiu chun cht lng chitin. Quan st thy, mt phn cc cht khong vn cn st li trong nguyn liu v tm, do chng nm nhng v tr khng c kh nng tip xc vi ha cht. loi b hon ton cn tin hnh lp li bc kh khong ny. 3.4.2 Kt qu nghin cu phn ng thy phn protein trong nguyn liu v tm Nhm gim thiu s khc nghit ca cc phn ng ha hc ng dng trong thu nhn chitin theo phng php ha hc, chng ti tin hnh cc th nghim ng
56

dng ch phm enzyme protease, tch chit t ni tng c thc hin phn ng thy phn protein trong nguyn liu v tm sau loi b khong. Sau qu trnh kh khong chng ti thc hin iu chnh pH hn hp phn ng v pH = 2-2,5 bng cch cho thm nc vo hn hp, nhm to iu kin cho ch phm enzyme protease t ni tng c hot ng. Sau cho vo hn hp phn ng mt lng ch phm enzyme pha long tng ng vi t l 1:4 v th tch. Qu trnh thy phn protein c tin hnh nhit 400C trong 72 gi. Kt qu xc nh kh protein c trnh by trn th hnh 3.8

Hnh 3.8 S ph thuc kh protein trong nguyn liu v tm vo thi gian thy phn Phn tch kt qu ca qu trnh thy phn trn hnh 3.8 cho thy, ch phm enzyme protease hot ng tt mi trng phn ng c pH 2-2,5. kh protein sau 48 gi xy ra phn ng thy phn t 74-75%, khi hm lng protein cn li trong nguyn liu l 4-6%. Bn cnh qu trnh phn ng enzyme trong mi trng pH 2 2,5 c kh nng lm gim hm lng cht khong trong chitin xung 0,3-0,4%, nh vy khng cn tin hnh kh khong ln hai nh cng ngh ng dng phng php ha hc, gim
57

c mt lng ha cht HCl ng k. Nng ch phm enzyme protease t ni tng ti u b sung vo hn hp phn ng c tnh l 1 (100%), sau khi ha long vi nc. Nh vy 100 ml ch phm enzyme long s thy phn c 400 ml hn hp phn ng (t l 1:4). Kt qu th nghim cho php ta xc nh thi gian ca phn ng enzyme loi b protein l 72 gi nhit 400C. Trn hnh 3.9 l kt qu kho st nh hng ca nng ch phm enzyme protease ln kh protein trong nguyn liu v tm sau khi ty mu v kh khong trong 72 gi nhit 400C

Hnh 3.9 S ph thuc ca kh protein vo nng ch phm enzyme protease t ni tng c T th xc nh c nng ch phm enzyme ti u thc hin qu trnh thy phn l 0,45 0,5 % tnh theo khi lng hn hp phn ng. Phn ng enzyme ph thuc vo nhit nn chng ti tin hnh kho st nh hng ca yu t nhit ln kh protein trong nguyn liu, kt qu c th hin trn hnh 3.10

58

Hnh 3.10 - nh hng ca nhit ln phn ng enzyme thy phn protein trong nguyn liu Phn tch cc d liu t th nhn thy rng, khi tng nhit qu trnh kh protein trong v tm t 30 ln 500C th tc phn ng enzyme tng thi im gi th 30-36, tng ng vi kh protein t 70 -72%. Trong qu trnh tin hnh phn ng enzyme kh protein nhit 600C thy phn protein sau 54 gi ch t trn 50%, s gim kh protein c th gii thch bng qu trnh bin tnh mt phn protein ca ch phm enzyme protease t ni tng c. Nh vy nhit ti u tin hnh qu trnh kh protein c th xc nh l 400C. Theo nghin cu ca Hamzazade A.I. enzyme protease khng c kh nng loi b protein dng cu trc nm gia cc lp chitin [Hamzazade, 2002], nn loi b hon ton nhm protein ny cn thc hin bc ra chitin bng kim sau khi thc hin phn ng kh protein. 3.4.3 Kt qu nh gi cht lng chitin c thu nhn bng phng php ng dng enzyme protease t ni tng c nh gi cht lng chitin chng ti dng phng php nh gi cm quan v xc nh thnh phn ha hc ca chitin thnh phm. Kt qu xc nh c trnh by
59

trong bng 3.4 Bng 3.4 Mt s ch tiu cht lng ca chitin sn xut bng phng php ng dng enzyme protease t ni tng c Kt qu Mu sc Mu trng Hm lng nc, % 7,8-8,0 Hm lng protein, 0,4-0,6 Hm lng cht khong, % 0,3-0,4 QMAFAM , CFU/g 3,3102 Chitin thu c c mu trng t nhin, hm lng nc 7,8-8,0%, hm lng protein 0,4-0,6% v hm lng cht khong chim 0,3-0,4%. nh gi an ton thng qua ch tiu vi sinh vt tng s (QMAFAM, CFU/g) l ph hp vi yu cu cht lng tiu chun chitin quc t SanPin 2.3.2.1078-01 (ca LB Nga). 3.4.4 nh gi cht lng chitosan sn xut t chitin ng dng ch phm enzyme protease t ni tng c Trn c s ngun chitin thu nhn c c hm lng protein di 1%, v hm lng khong di 0,5% chng ti thc hin qu trnh deacetyl theo phng php hin hnh hin ang ng dng ti Vit Nam v mt s nc trn th gii. Qu trnh deacetyl c tin hnh trong dung dch NaOH 50% nhit 90-1350C trong 1 gi, t l chitin : NaOH l 1:3 1:5. Chitosan thu c sau qu trnh deacetyl c ra bng nc lc v sy kh nhit 55-600C. Trong bng 3.5 trnh by kt qu nh gi cht lng chitosan Bng 3.5 Mt s c im cht lng chitosan thu nhn t chitin tch chit t v tm ng dng enzyme protease t ni tng c Ch tiu cht lng deacetyl, % nht [], dl/g Hm lng, % Nc Cht khong Cc cht khng ha tan
60

Ch tiu

c im sn phm chitosan 79-80 14,9 - 18 8,7 0,2 0,3

Hiu sut thu hi chitosan t chitin theo phng php nghin cu t 84-86% so vi khi lng chitin kh. Cht lng chitosan ph hp vi tiu chun quc t. 3.4.5 Kt qu nghin cu x l cc loi ph thi sinh ra trong qu trnh x l nguyn liu thnh chitin/chitosan Trong qu trnh sn xut chitin to thnh cc sn phm ph pha rn v pha lng. Pha rn l phn protein cha thy phn ht to thnh khi tch chit enzyme protease. Pha lng bao gm: dung dch axit to thnh sau qu trnh kh khong; dch thy phn sau qu trnh kh protein; v dung dch baz trong qu trnh deacetyl. c im v thnh phn ha hc c trnh by trong bng 3.6 Bng 3.6 Mt s c im ca ph thi rn v lng Loi ph liu pH nc 80-84 Hm lng, % tro protein 0,6-0,7 13,1- 13,8

Phn cha b thy 5,5-6 phn Dung dch axit 3-3,5 97-97,5 2,5-2,9 0,5-0,6 0,03 Dch thy phn 2,5-3 89-90 0,3 -0,35 8-8,5 0,5-0,6 Dung dch baz 14 97-98 0,5-0,6 0,8-0,8 0.05 Phn protein cha b thy phn sau qu trnh tch chit ch phm enzyme protease c th tn dng sn xut thc n gia sc.Phn dung dch cha cc cht dinh dng t hn hp dch thy phn chng ti s nghin cu bin php thu hi trong cc nghin cu tip theo, nhm mc ch s dng trong nui cy vi sinh vt. 3.5 Kt qu nghin cu ng dng vi khun B. subtilis loi b protein trong nguyn liu v tm 3.5.1 Kt qu nghin cu nhm chun b ging vi khun Bacillus subtilis. s dng c ging vi khun B. Subtilis trong x l v tm theo quy tc trong cng ngh sinh hc cn tri qua mt s bc c nh: hot ha ging hot ha ging chng ti tm ra pha long ti u l 10-5, sau 24 gi nui cy trn mi trng hot ha c cha cao tht, cao nm, pepton v NaCl (ph lc 2) nhn thy mt khun lc nhiu v mc ring r, hon ton ph hp cho yu cu th nghim tip
61

lipid 0.8-1

theo (bng 3.7). Sau khi chn c pha long ph hp hot ha vi khun tin hnh kim tra hot lc sinh tng hp enzyme protease ca vi khun Bacillus subtilis bng cch s dng phng php o vng thy phn da trn kh nng bt mu ca thuc th Amido-black vi cc protein m khng bt mu vi cc acid amin (ph lc 3 v 4) Bng 3.7 - Mt khun lc khi nui cy trn mi trng hot ho vi cc pha long khc nhau STT 1 2 3 4 5 6 7 8 pha long 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 S khun lc m c Khun lc mc nhiu, trn a thch, cc khun lc mc dnh nhau. Cc khun lc mc nhiu, a s cc khun lc mc dnh lin nhau, ch c mt s mc ring r. 127 khun lc mc ring r, 8 cm khun lc mc lin nhau 80 khun lc mc ring r, 3 cm khun lc mc dnh lin vo nhau. 17 khun lc mc ring r. 1 khun lc mc ring r. Khng c khun lc no mc. Khng c khun lc no mc.

Hnh 3.11 - a khun lc sau khi nhum bi thuc nhum Amido-Black.

62

Sau khi nhum chng ti quan st thy chung quanh cc khun lc xut hin cc vng thu phn mu sng, nhng vng mi trng cn li c mu xanh m ca thuc nhum nh hnh 3.11 chn c vi khun c hot tnh mnh, chng ti tin hnh o ng knh vng thu phn ca su khun lc trn. ng knh vng thu phn c tnh bng hiu s ng knh ngoi ca vng thy phn (D) v ng knh ca khun lc (d). Kt qu o c nh trong bng 3.8 Kt qu cho thy vng thu phn ca khun lc M1, M2, M5 l ln nht, cn vng thu phn ca khun lc M4 l nh nht. Chng ti gi ging c ba chng M1, M2, M5. Ring chng M1 c ng knh vng thu phn ln nht c chn nhn ging cho cc th nghim tip theo. Bng 3.8 - ng knh vng thu phn ca cc khun lc Mu khun lc D - d (mm) M1 2,8 M2 2,0 M3 1,4 M4 0,8 M5 1,8 M6 1,2

Dng vi khun Bacillus subtilis c hot tnh mnh sau khi chn la c tin hnh nhn ging cp1. Da theo thnh phn mi trng c kho st ca TS. Th Bch Thu (Lun n tin s nm 2001), chng ti s dng mi trng c b sung cao nm, cao men, pepton v cc mui (ph lc 5). Thnh phn mi trng ny c cha nhiu cht dinh dng thch hp cho vi khun B. subtilis sinh trng v pht trin. Chng M1 vo c cy vo mi trng v nui 35oC trong 24 gi trn my lc th thu c canh trng ging cp 1. kim tra thun khit ca chng Bacillus subtilis bng cch quan st di knh hin vi ng thi kim tra hot lc sinh tng hp enzyme ca ging cp 1 (hnh 3.12 v 3.13). T kt qu thu c khi quan st di knh hin vi chng ti thy cc vi khun ng nht, khng c vi khun l xut hin chng t ging cp 1 khng b nhim v vng thu phn thu c c kch thc ng knh ln t yu cu cho th nghim tip theo.

63

Hnh 3.12 - Hnh thi vi khun Bacillus subtilis soi di knh hin vi

Hnh 3.13 - Vng thu phn ca vi khun Bacillus subtilis sau khi nhn ging cp 1

Hnh 3.14 - Vng thu phn ca vi khun Bacillus subtilis sau khi nhn ging cp 2 Chng ti tin hnh nhn ging cp 2 t ngun ging cp 1 thu nhn c bng cch trong mi trng nhn ging cp 2 gim t l thnh phn cc cht giu dinh
64

dng nh pepton, cao tht, cao nm nhng b sung thm tinh bt v v tm kh nghin nh cho vi khun lm quen dn nhm mc ch x l ph liu v tm trong qu trnh tch chit chitin. Mi trng sau khi kh trng v ngui, chng ti b sung thm 10% th tch ging cp 1, nui trn my lc 35oC trong 24 gi. Ging cp 2 thu c cng c kim tra kh nng sinh tng hp enzyme protease, kt qu thu c nh trn hnh 3.14 Kt qu thu c cho thy vng thu phn ca ging cp 2 ln hn so vi vng thu phn ca ging cp 1. iu ny c th gii thch l do cc cht dinh dng trong mi trng nhn ging cp 1 dng n gin c khi lng phn t thp v ph hp cho vi khun B. subtilis hp th. Cn mi trng nhn ging cp 2, cc cht dinh dng n gin t hn v dng cao phn t nn khi vi khun B. subtilis hp th ht chng buc phi tit ra thm nhiu enzyme phn hu tinh bt v v tm vi mc ch b sung thm cht dinh dng cho nhu cu sng ca chng. Vi nhng dn liu khoa hc thc nghim v ln ng knh vng thu phn c th i n kt lun rng ging cp 2 thu c hon ton p ng c vic ng dng phn gii protein 3.5.2 Kt qu ng dng Bacillus Subtilis loi b protein trong v tm Cc nghin cu trong v ngoi nc chng minh rng, vi khun Bacillus subtilis l loi vi khun hiu kh tu tin v a m. Nhm mc ch tm iu kin ti u cho vi khun Bacillus subtilis sinh trng pht trin tt nht sinh ra nhiu enzyme protease c hot lc mnh phc v cho mc ch loi protein v tm, chng ti tin hnh xy dng 12 m hnh th nghim kho st mc thy phn protein trong v tm c phi kh v nghin nh vi cc t l ging b sung khc nhau ln lt l 10% ,20%, 30% vi cc mc nhit l 35oC, 42oC, 48oC, v 54oC, trong 35 gi. m bo cho v tm c tip xc trc tip v ton din trong dch cha vi khun ng thi c oxy m bo cho vi khun h hp, pht trin cho v tm nghin nh vo trong cc bnh tam gic 250 ml ngp dch cha t l ging tng ng v sc kh. Theo cc m hnh th nghim thm d nhm loi b protein trong v tm theo cch trn, chng ti tin hnh cc th nghim phn tch thu phn protein nhit l 48oC vi hm lng ging vi khun B. subtilis l 20% da trn s thay i v
65

mu sc dung dch, cng nh c. Hm lng m ho tan c xc nh sau mi 3 gi thu phn v kt qu c trnh trn hnh 3.15

Hnh 3.15 S thay i hm lng m ha tan trong mi trng cha vi khun B. Subtilis v v tm nhit 480C T kt qu nghin cu chng minh rng chng Bacillus subtilis sinh trng v pht trin mnh sinh ra nhiu enzyme protease xc tc qu trnh thy phn protein trong v tm phi kh nghin nh. Trong qu trnh thy phn quan st thy trong hn hp phn ng c to ra bt kh vi s lng nh, iu ny c th l gii l do khi protein b thy phn s to ra cc axit amin t do c th lm gim pH mi trng phn ng, mi trng pH thp to iu kin cho qu trnh kh khong c trong v tm to ra bt kh. Th nghim cng chng minh nhit 480C l iu kin tt cho s sinh trng v pht trin ca vi khun B. subtilis. Tuy nhin khng nh iu ny cn c cc th nghim kho cu nh hng ca nhit ln qu trnh thy phn. 3.5.2.1 Kt qu nghin cu nh hng ca t l ging ln thy phn kho st nh hng ca t l ging ln qu trnh thy phn chng ti tin hnh thy phn protein trong v tm vi 3 t l ging b sung ln lt l 10%, 20% v 30% theo th tch, cc th nghim c tin hnh nhit c chn trc l 48 0C. Sau 8 gi nui cy vi khun B. subtilis, nh k c 3 gi ly mu ra v chun bng phng php phooc-mn, thu c kt qu nh trn hnh 3.16

66

Hnh 3.16 - Hm lng m ha tan thu c khi thy phn protein trong v tm vi cc t l b sung ging khc nhau 480C Theo th, nhn chung lng m ho tan thu c c ba mu th nghim vi cc t l ging b sung l 10%, 20%, 30% theo th tch sau cc khong thi gian khc nhau u c xu hng tng. mu th nghim b sung 10% ging, trong giai on u hm lng m ho tan chun tng mnh, c lc vt qua c mu cha lng ging l 20%, 30%. Tuy nhin, bt u gi th 24 27, lng m ho tan thu c li tng chm dn v n cui th nghim th lng m thu c thp hn so vi cc mu cn li khong 0,4 mg/ml. Vi cc mu th nghim b sung 20% v 30% ging, mc thu phn tng kh ng u. Mc du giai on cui lng m thu c mu cha 30% ging cao hn so vi mu cha 20% ging nhng t l ny ln hn khng ng k v nu xt c v hiu qu phn hu protein cng nh vn kinh t th chng ti chn t l ging cp cho qu trnh thu phn l 20%. 3.5.2.2 Kt qu nghin cu nh hng ca nhit v thi gian ln thy phn protein trong v tm Vi t l ging vi khun B. subtilis thch hp l 20% chng ti tin hnh kho st nh hng ca nhit ln qu trnh thy phn protein. Qu trnh thy phn c tin
67

hnh cc mc nhit : 350C, 420C, 480C v 540C. Sau 8 gi nui, nh k c 3 gi ly mu ra v chun bng phng php phooc-mn, thu c kt qu nh th trn hnh 3.17

Hnh 3.17 S ph thuc ca qu trnh thy phn protein trong v tm vo nhit Phn tch s liu thng qua cc ng cong trn th 3.8 c th rt ra mt s nhn xt nh sau: nhit 35oC lng m thu phn khong thi gian 17 gi u tng, t l m ho tan ny v cao hn so vi hm lng m ho tan thu c 54oC nhng li khng bng vi t l thu c mc phn hu ti nhit 42 oC v 48oC. Thm vo , chng ti nhn thy ti nhit ny mi sinh ra trong qu trnh phn hu mnh v ti gi th 12 sau khi nui cy c gii xut hin. Thi gian ko cng di, lng gii sinh ra cng nhiu c bit l phn v u tm nguyn khng hp. Vi l do nn mc nhit ny chng ti ch nghin cu trong khong thi gian 17 gi nui cy; mc nhit 42 oC, giai on u, hm lng m ho tan thu c c tng ln nhng sau gi th 21 th hm lng m ho tan khng tng na v c xu hng gim xung. iu ny c th do nhit ny cng thch hp cho nhiu vi sinh nhim tp khc nn sau mt thi gian nui cy, chng B. subtilis b c ch v km hm hot ng bi cc vi sinh vt khc; nhit 48 oC, trong 14 gi u, hm lng m ho tan thu c c thp hn so vi mu nui

68

42oC. Tuy nhin sau hm lng m ho tan tng ln r rt vt qua cc mu cn li. Nh chng ti trnh by trn, chng B. subtilis l loi a m. Mc nhit 48oC c th c ch c nhiu loi vi sinh vt nhim tp khc nhng li l nhit ph hp cho chng B. subtilis sinh trng, pht trin v sinh tng hp enzyme. Mc khc, theo nhiu ti liu nghin cu nhit 48oC cng nm trong khong nhit ti thch cho enzyme protease hot ng. Do vy lng protein b thu phn mc nhit ny l rt cao; nhit 54oC hm lng m ho tan thu c khng ng k v yu hn ba mc nhit cn li rt nhiu. iu c th do mc nhit ny qu cao nn c ch s sinh trng, pht trin cng nh s sinh tng hp enzyme protease ca vi khun Bacillus subtilis. T kt qu thu c trn c th chng minh mc nhit 48oC l nhit ti u cho qu trnh phn gii protein v u tm bng enzyme protease ca chng vi khun Bacillus subtilis. Bn cnh , t th hnh 3.6; 3.7; 3.8 chng ti nhn thy thi gian ko di th lng m b thu phn sinh ra tng dn, tuy nhin qu trnh thy phn protein c xu hng chm li sau gi ln men l 32 gi. Nu tip tc ko di thi gian ca phn ng th hiu qu thy phn protein tng khng ng k v iu ny c th ko di thi gian sn xut nn y chng ti chn thi gian thu phn ti u l 32 gi. 3.5.2.3 Kt qu x l v tm sau qu trnh thy phn bng vi khun B. subtilis Phn v tm sau khi loi protein c chng ti ra sch bng nc, phi kh, cn trng lng. Sn phm v u tm c loi protein m chng ti thu c c mu hng nht, c mm nht nh, tuy nhin phn mt tm vn cn mu en v vn cn mt lng nh protein trong v tm vn cha c thu phn sch trit . Sn phm ny chng ti tin hnh loi khong bng dung dch acid HCl 1M trong 2 gi. Sn phm sau c ra sch, phi kh v cn trng lng. So snh s gim khi lng cc mu x l sau cc cng on loi protein v loi khong bng phng php sinh hc v bng phng php ho hc (bng 3.9) Da vo kt qu thu c trong bng 3.9 chng ti nhn thy hiu qu phn hu protein bng enzyme protease ca Bacillus subtillis trong v u tm ban u thp hn so vi x l bng NaOH, cng nh mu sc ca sn phm sau khi phi kh khng sng v p bng.
69

Bng 3.9 - S gim khi lng v tm qua tng cng on Khi lng Khi lng Khi lng cn v tm ban cn li sau khi li sau khi loi u loi protein khong Phng php sinh hc 100 (g) 69 (g) 48 (g) Phng php ho hc 100 (g) 53 (g) 36 (g) nh gi cht lng chitin chng ti dng phng php nh gi cm quan v xc nh thnh phn ha hc ca chitin thnh phm. Kt qu xc nh c trnh by trong bng 3.10 Bng 3.10 Mt s ch tiu cht lng ca chitin sn xut bng phng php ng dng vi khun B. subtilis Ch tiu Mu sc Hm lng nc, % Hm lng protein, Hm lng cht khong, % QMAFAM , CFU/g Kt qu Mu hng nht 7,8-8,0 0,4-0,6 0,3-0,4 3,3105 Cch x l

Qu trnh deacetyl c tin hnh trong dung dch NaOH 50% nhit 90 1350C trong 1 gi, t l chitin : NaOH l 1:3 1:5. Chitosan thu c sau qu trnh deacetyl c ra bng nc lc v sy kh nhit 55-600C. Trong qu trnh sn xut chitin/chitosan cn x l dch thy phn cha enzyme sinh ra hai cng on ln men v deacetyl c c im khc nhau v pH. Chng ti tin hnh thu gom hn hp dch thy phn, sau khuy u v gia nhit 100 C trong 5 pht thu hi ngn protein b bin tnh to thnh kt ta nhit cao. Ngun protein c c c v hng ti s dng lm thc n chn nui. 3.6 So snh cht lng chitosan sn xut theo cc phng php ha hc v ng dng cng ngh sinh hc T kt qu xc nh mt s ch tiu cht lng ca chitosan trong bng nhn thy rng chitosan sn xut theo cng ngh ng dng ch phm enzyme protease tch chit t ni tng c v ng dng vi khun B. subtilis c deacetyl v nht cao
70

hn so vi chitosan sn xut theo phng php ha hc. Tuy nhin trong qu trnh bo qun chitin sn xut theo phng php ng dng vi khun B. subtilis khi phn tch ch tiu vi sinh vt, nhn thy vi sinh vt tng s (QMAFAM) kh ln cc thng bo qun th 2 v 3 trn mc 3,3105 CFU/g, iu ny nh hng n cht lng chitin. So snh mu sc cho thy chitin/chitosan sn xut theo phng php ha hc v ng dng ch phm enzyme protease tch chit t ni tng c c u im hn so vi phng php ng dng vi khun B. subtilis. Bng 3.11 So snh mt s c im cht lng ca chitosan sn xut theo phng php ha hc v ng dng cng ngh sinh hc
c im sn phm chitosan sn xut theo cc phng php ha hc ng dng protease ng dng vi khun t ni tng c B. subtilis 79 79-80 82 10,9 14,9 - 18 18,1 8,8 0,1 0,3 8,7 0,2 0,3 8,7 0,2 0,4

Ch tiu cht lng deacetyl, % nht [], dl/g Hm lng, %: Nc Cht khong Cc cht khng ha tan

Vn x l cht mu trong chitin v bin php kh khong trong cng ngh sn xut chitin bng phng php ng dng vi khun B. subtilis chng ti s tip tc nghin cu trong cc nghin cu tip theo. 3.7 xut s cng ngh sn xut chitin/chitosan ng dng cng ngh sinh hc T cc kt qu nghin cu nh trn, chng ti xut 2 dy chuyn cng ngh x l v tm v thu hi cc hp cht t nhin c hot tnh sinh hc: cht mu carotenoid, chitin, chitosan trn cn bn ng dng cc gii php cng ngh sinh hc nh sau: 3.6.1 Dy chuyn cng ngh phc hp sn xut chitin/chitosan ng dng ch phm enzyme protease t ni tng c (hnh 3.18)

71

V tm Ra Sy (t=400C; = 48h)

Nghin (d = 1-3mm) Tch lipid (bng cn 96 ; t=600C, = 2h) Sy (t=600C; = 5h) KK (HCl, t=250C, = 2h)

Tch pha nc

Dch chit

X l vi hexan

Loi b hexan

Carotenoid iu chnh pH =2-2,5

KP bng CP enzyme (t=400C; =72h) Ra (pH = 7)

Dch thy phn

Thc n gia sc

Sy (t=600C; = 5h)

Chitin Deacetyl vi NaOH 50% (t=900; = 1h; w/w = 1:3) Sy (t=600C; = 5h)

Chitosan

Hnh 3.18 S cng ngh phc hp sn xut chitin/chitosan ng dng ch phm enzyme protease t ni tng c

3.6.2 Dy chuyn cng ngh sn xut chitin/chitosan ng dng vi khun B.subtilis (hnh 3.19)
72

V tm Ging B. subtilis Ra Hot ha ging Sy (t=40 C; = 48h) Nhn ging cp 1 Nghin (d = 1-3mm) Cy ging vi khun (w/w = 20:100) Ln men KP (t=400C; =32h; sc kh) Ra (pH = 7) KK (HCl, t=250C, = 2h) Nhn ging cp 2
0

Thu dch thy phn

Gia nhit ( t=1000C, = 5 pht)

Thu protein kt ta

Chitin C c

Deacetyl vi NaOH 50% (t=900; = 1h; w/w = 1:3)

Thu dch thy phn

Sy (t=600C; = 5h)

Thc n gia sc

Chitosan

Hnh 3.19 S cng ngh phc hp sn xut chitin/chitosan ng dng vi khun B. subtilis

73

KT LUN Da trn kt qu nghin cu v sn xut th nghim chitin/chitosan ng dng bin php cng ngh sinh hc c chng ti rt ra mt s kt lun nh sau: - Sn phm chitin sn xut ng dng ch phm enzyme protease tch chit t ni tng c c cht lng tt vi hm lng protein nh hn 0,5%, v ch tiu vi sinh vt t chun an ton (SanPin 2.3.2.1078-01 LB Nga); chitosan sn xut t loi chitin ny c deacetyl l 79-89% v nht cao n 18 dl/g ph hp vi cc bo co khoa hc trong nc v quc t. - Sn phm chitin sn xut ng dng vi khun B. subtilis c ch tiu cm quan thp, v vi sinh vt tng s tng i cao mc 3,3105 nhng vn nm trong gii hn an ton (SanPin 2.3.2.1078-01 LB Nga); chitosan sn xut t loi chitin ny c nht l 18,1 dl/g c cht lng bo m so vi bo co trong nc v quc t. - Cng ngh sn xut chitin/chitosan ng dng cng ngh sinh hc trn cn bn s dng ch phm enzyme t ni tng c v vi khun B. subtilis gp phn gim ng k lng ha cht dng trong cng on kh protein - Bn cnh sn phm chnh cn thu c sn phm ph l cht mu carotenoid l cht mu t nhin c th s dng trong cng nghip thc phm. Tuy nhin, khi so snh v mc phc tp ca cng ngh p dng trong thc t th cng ngh ng dng enzyme protease t ni tng c c nhiu u vit hn.

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TI LIU THAM KHO Ting Vit 1) Th Bch Thu, Trn Th Luyn, Nghin cu nui cy trc tip vi khun Bacillus subtillis loi protein ra khi phn v ca ph liu tm (PLT), Tp ch Khoa hc Cng ngh Thu sn s 2/2006. 2) L Ngc T, Ho sinh cng nghip, NXB khoa hc- k thut, H Ni. 3) L Xun Phng, Vi sinh vt cng nghip, NXB xy dng H Ni. 4) Lng c Phm, 2003, Cng ngh x l nc thi bng bin php sinh hc, NXB Gio dc. 5) Nguyn c Lng, Cao Cng, 2003, Th nghim cng ngh sinh hc - NXB i hc quc gia TP H Ch Minh. 6) Nguyn Vn Mi (2001), Thc hnh ho sinh hc, NXB i hc Quc Gia H Ni. 7) Nguyn Nht Thi, 1991 C bin Vit nam: C xng vnh Bc B. NXB Khoa hc v k thut, H Ni 8) Nguyn Vn n, Ng Ngc Khuyn. Hp cht thin nhin dng lm thuc, Nxb Y hc, H Ni, 47 - 48. 9) Phan Th Cm Vn, Nghin cu thu nhn v ng dng ch phm enzyme protease th dng bt t Bacillus subtilis. 10) Trn Th Luyn, Bi Vn T, Nghin cu s dng Lactobacillus plantarum ln men u tm s (Penaeus monodon) thu hi chitin, Tp ch Khoa hc Cng ngh Thu sn s 03 04/2006 11) V Ngc Bi, Nghin cu qu trnh thu phn protein c bng enzyme protease t Bacillus subtilis, lun n tin s hc, H Quc Gia TP H Ch Minh. Ting nc ngoi 12) Muzzarelli R.A.A. Chitin, Oxford: Pergamon Press, 1977, 309 tr. 13) . . . .: , 1994, trang 125-127. 14) .., .., .. , 1995.
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.64, 1, trang 26-30. 15) .. . , 2001. 5, trang 50-51. 16) .. . , 2000. 5, trang 51-57. 17) .. , IV . . .,1995, trang 3-5. 18) .. III . . .: , 1992, trang 7-15. 19) Hood M.A. Absract. First. Inter. Conf. Chitin/Chitosan. Boston: 1997. P.4445 20) Girand-Guille M.M., Bouligand Y. Chitin in Nature and Technology. Ed. By Muzzarelli R.A.A., C. Jenniaux, Gooday G.W.N. Y. and L.: Plenum Press, 1986. P.29-35 21) .., .. .. . .. . ., 1974, .11. trang 3-7 22) .. 665683 . 08 37.8. 23) .. II . . . , , 1982. . . -57232 : , 1983. 15 trang. 24) .., .. I . . . . . : , 1983, trang 19-22. 25) 4199496 USA . . .. -49589. : , 1984. 20 trang 26) 3862122 USA. .. -49584. : , 1984. 25 trang 27) Chitin and Chitosan: production, properties and usage/ Ed. By Academician of the Russian Academy of Agricultural sciences K.G. Skriabin, G.A. Vikhoreva Dr. Sc. (Chem), V.P. Varlanmov Dr. Sc. (Chem). Moscow nouka 2002, 360 trang. 28) Ricardo A.A, Muzzarelli. Fabio Tanfani. Carbohydrate Resea rch, No. 107 (1982) 189 - 214.
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29) Shigehiro Hirano. Chitin biotechnology applications, Elsevier Science, 2 (1996) 237 - 257. 30) Shigemasa. Y, Morimoto. M, Saimoto. H, Okamoto. Y and Miami. S, Applications of chitin and chitosan for biomaterials, Tottori, Japan, 680-855. 31) Wenlung Chen and Robin Y - Y Robin. A modified chemical procedure for rapid determination of glucosamine and its application for estimation of mold growth in Peanut Kernels and Koji, Agri Food Chemistry, 47 (1999) 32) Timothy J. Maher. Glucosamine continuing Education Module, New Hope Institute of Retailing (5/2000) 2 - 6. 33) Noirot P (2007). "Replication of the Bacillus subtilis chromosome", Bacillus: Cellular and Molecular Biology (Graumann P, ed.), Caister Academic Press. ISBN 978-1-904455-12-7 34) Theruvathil K. Sini, Sethumadhavan Santhosh* and Paruthapara T. Mathew (2007), Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation, Science Direct.

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PH LC Ph lc 1: Phng php pha long thp phn Cch tin hnh: ht 1 ml dung dch t ng ging cho vo ng nghim th nht cha sn 9 ml dung dch nc mui sinh l c tit trng, o trn u. Sau , ta ht 1 ml dung dch t ng nghim th nht cho vo ng nghim th hai cha sn 9 ml dung dch nc mui sinh l c tit trng qu trnh tip tc n ng nghim th 8. Tng ng vi mi pha long, ht 30 l cho vo a petri cha sn mi trng hot ho ging c tit trng (ph lc 2), dng que trang phn b u dch trn mt a. Cui cng cho vo t m nui 35oC, trong 24 gi. Ph lc 2: Mi trng hot ho ging (pH=6,5) Cao tht NaCl Pepton Aga Cao nm :0,3% :0,5% :1% :2% :1%

Ph lc 3: Mi trng kim tra hot lc enzyme Aga Casein :2% :2%

Ph lc 4: Thnh phn thuc nhum Amido-Black Amido-Black 0,1% pha trong dung dch axit acetic 7% Ph lc 5: Mi trng nhn ging cp 1 Pepton Cao tht NaCl Cao nm :1% :0,3% :0,5% :1%
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Tinh bt tan

:1,75%

Ph lc 6: Mi trng nhn ging cp 2 Cao tht Pepton Cao nm NaCl Bt sn th PLT :0,05% :0,5% :0,1% :0,5% :5,2% :1,1%

Ph lc 7: Phng php chun phooc-mn Nguyn tc: Nhm carboxyl ca cc amino acid lng tnh, cc peptid ln v protein c th c xc nh bng k thut chun trong dung dch nc-etanol khng c formalin. Mi trng nc-etanol gy cn tr s phn ly nhm amin, kt qu l n tr trong mi trng nc, amino acid tr thnh acid, nhm carboxyl ca amino acid c th c chun li bng kim. Vi c tnh c bn ny, c th nh lng amino acid bng phng php micro. Ho cht: 1.Dung dch KOH 0,01N trong ethanol 90% 2.Dung dch CuCl2 amoniac 0,0025M 3.Cn tuyt i. 4.Dung dch tymolphtalein 0,1% trong ethanol 90% Cch tin hnh: Ly 2ml dung dch cha amino acid, cho thm 2 git dung dch tymolphtalein 0,1% trong ethanol 90% v chun bng KOH 0,01 N trong ethanol 90% n khi xut hin mu xanh. Sau cho thm 10 ml th tch cn tuyt i vo 1ml th tch dung dch cn chun . Mu dung dch bin mt, chun li cho n khi xut hin mu xanh. Mu so snh vi mu chun c th s dng dung dch amoniac CuCl2 0,0025M.
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 Tnh kt qu: 1 ml dung dch KOH 0,01N tng ng vi 1,4 mg Nit. Vy s mg nit trong 1ml dung dch l: (x*1,4)/ 2 Ph lc 8: Phng php chun Kjeldahl Nguyn tc: V c ho thc phm bng H2SO4 m c v cht xc tc. Dng kim mnh NaOH y NH3 t mui. nh lng NH3 bng mt dung dch axit c nng xc nh. Ho cht: H2SO4 m c ( D= 1,84), NaOH 30%, H2SO4 0,1N, NaOH 0,1 N Cht xc tc: hn hp ca CuSO4.5H2O: K2SO4= 3,15: 0,35. Thuc th : - phenolphtalein trong cn tuyt i. - Thuc th hn hp : bromtymol xanh 0,1% trong nc ct, crezola 0,1% trong nc ct v t l ca hai dung dch l 1:1 Cch tin hnh : Giai on v c ho: cn 1 g thc phm cho vo bnh Kjeldahl vi 10ml H2SO4 m c v 3,5 xc tc. Lc u un nhit thp sau 30 pht tng dn nhit ln cho dung dch si u. Trong qu trnh un dch chuyn t mu nu sm sang mu cnh gin, n vng nht v cui cng l dung dch trng trong, kt thc giai on v c ho. Pha long dung dch v c: ngui bnh Kjeldahl sau chuyn ton b mu v c ho vo bnh nh mc 100ml v nh mc. Giai on ct m: ly 10 ml dung dch mu v c ho cho vo bnh cu thm 3 git thuc th phenolphtalein v 30 ml dung dch NaOH 30% n khi dung dch chuyn sang mu hng l c. t bnh cu ln h thng ct m. Tin hnh ct mu bng cch un si dung dch trong bnh cu khong 30 pht ri dng giy pH th xem NH3 ht cha. Kt qu: Hm lng nit ton phn X1 theo %: (mg/ml).

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X1

(V 1 V 2) * 0,0014 *1000 G (V 1 V 2) * 0,0014 *1000 G

Hm lng nit ton phn tnh X2 trong 1000 ml:

X2

V1 V2

: s ml NaOH 0,1 N tiu tn cho mu trng : s ml NaOH 0,1 N tiu tn cho mu th nghim.

G : s gam thc phm cn nh lng V : s ml thc phm cn nh lng. 0,0014 1000 : s gam nit tng ng vi 1 ml NaOH 0,1 N : h s quy i ra lt.

Ph lc 9: Mi trng kim tra hot lc enzyme Aga Gelatin :2% :2%

Ph lc 10: Thu nhn chitin/chitosan bng phng php ha hc (mu i chng) thu nhn chitin/chitosan bng phng php ha hc tin hnh cc bc sau: kh protein, kh khong, ty mu v lipid. Sau bng phng php deacetyl chitin thu c chitosan. Trong nghin cu ny s dng phng php thu nhn chitin/chitosan c cng ty TNHH Phng Duy (Suntze chemical Co.,LTD) c tr s ti TP Cn Th lm mu i chng. -Nghin v tm: trong nghin cu ny ph phm c nghin ti kch thc 1 -3 mm, iu ny c hai mt li, th nht l m bo c vn tc phn ng xy ra, mt khc gim tn tht cc ht nh khi loi b dung dch sau phn ng -Kh protein: V tm c kh protein pH=7 bng dung dch NaOH 0,5-1M nhit 800C trong 2 gi - Kh khong: Sau khi kh protein, tin hnh kh khong bng dung dch HCl 11,5M nhit 20-250C trong 2 gi Theo quan im ha hc, qu trnh kh khong c th dng li khi khng cn kh
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CO2 thot ra t thit b phn ng. Phng trnh phn ng ca qu trnh kh khong.

- Kh mu: dng dung dch 0,25% NaOCl trong 5 pht - Deacetyl chitin thu nhn chitosan: qu trnh c tin hnh trong mi trng kim m c nhit cao kh hon ton cc nhm acetyl ca mch polymer, t thu nhn chitosan. Qu trnh deacetyl c tin hnh trong dung dch NaOH 50% nhit 90-1350C trong 1 gi. Ph lc 11: Thu nhn ch phm enzyme protease t ni tng c Ni tng c sau khi gi ng nhit phng n nhit -1 00C c xay nh bng my xay tht vi kch thc 1-3 mm. Hn hp ni tng c sau khi xay nh c phi trn vi nc ct theo t l 1:0,5 (w ni tng: w nc). Cho dung dch HCl m c vo hn hp iu chnh pH v 1,5 2, l pH ti u thu nhn enzyme hot ng trong mi trng axit. Hn hp c t trong t m nhit 40-500C trong 40-60 pht thc hin qu trnh ln men, trong qu trnh ln men cn thc hin khuy o sau mi 5 pht. Sau khi ln men, hn hp c ly tm trong my ly tm vi vn tc 5000 vng/pht trong 15 pht. Sau tch phn cn, lc thu c dch enzyme. Dch enzyme th c xc nh hot bng phng php Anson ci tin. Sau bo qun trong t lnh s dng thy phn loi b protein trong v tm. Ph lc 12: Nghin cu qu trnh thy phn protein trong v u tm bng ch phm enzyme protease, thu nhn t ni tng c nghin cu qu trnh kh protein bng ch phm enzyme protease thu nhn t ni tng c, chng ti tin hnh xc nh hot ca ch phm enzme protease, v xc nh s ph thuc ca hot enzyme protease vo nhit mi trng cc mc nhit 20, 30, 40, 50 ,60, 70 v 800C. T xc nh nhit ti u tin hnh thy phn protein. Sau , tin hnh kho st s ph thuc ca thy phn protein trong v tm vo nng ch phm enzyme protease. T xc nh nng protease ti u kh protein trong v u tm.
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Hiu qu ca qu trnh thy phn protein c xc nh bng vic xc nh hm lng protein trong v tm sau khi thc hin qu trnh thy phn. Ph lc 13: Nghin cu qu trnh thy phn protein trong v u tm bng vi sinh vt Bacillus subtillis nghin cu kh nng phn gii protein ca chng Bacillus subtillis ln v tm nhm tm iu kin ti u cho qu trnh ny xy ra hiu qu nht, chng ti tin hnh cc th nghim loi protein trong v tm bng cch s dng dch khun Bacillus subtilis cc iu kin th nghim khc nhau chn ra kin ti u. Sau so snh sn phm ca phng php ny vi phng php ha hc. Ph lc 14: Cc bc tin hnh nghin cu phng php tch chit chitin s dng vi khun B. Subtilis Bc 1: Hot ho ging bng phng php pha long thp phn Ging sau khi ly t t lnh -20oC, chng ti tin hnh hot ho ging bng phng php pha long thp phn (ph lc 1), ri cy tri cc pha long tng ng trn mi trng hot ho (ph lc 2). T vic pha long ny, chng ti s chn c cc khun lc ring bit phc v cho cc bc tip theo ca th nghim. Bc 2: Xc nh kh nng tng hp protease ca Bacillus subtilis bng phng php o ng knh vng thu phn Sau khi thu c cc khun lc ring bit t phng php pha long, chng ti tin hnh xc nh hot lc ca vi khun nhm chn ra cc dng c hot lc sinh tng hp mnh nht. S dng mi trng nui cy c b sung c cht cm ng casein (ph lc 3) v thuc nhum Amido black (ph lc 4), chng ti o ng knh vng thu phn ca tng dng t so snh v chn ra dng c hot lc mnh nht. Bc 3: Nhn ging cp 1 Sau khi chn c dng vi khun c hot lc mnh nht, tin hnh nhn ging cp 1 trn mi trng lng c cha cao nm, cao tht, pepton v cc mui (ph lc 5). Nui trn my lc trong 24 gi 35oC. Vi mi trng ny, vi khun s c cung cp y cc cht dinh dng m bo s sinh trng v pht trin tt nht.

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Ging cp 1 thu c s c em i kim tra hot tnh trn mi trng c cha gelatin (ph lc 9) v nhum bng dung dch Amido black. Bc 4: Nhn ging cp 2 T ging cp 1, chng ti tin hnh nui ging cp 2 trn mi trng c b sung bt ph liu tm, bt sn th (ph lc 6). iu kin nui cy tng t nh khi nui ging cp 1. Mi trng ny s gip vi sinh vt lm quen dn vi mi trng c cha ph liu v u tm sau ny. Ging sau khi em nhn ging cp 2 s c kim tra hot lc mt ln na. Bc 5: Kho st nh hng ca t l ging b sung vo v u tm ln qu trnh thu phn protein V u tm c b sung ging cp 2 vi t l kho st ln lt l 10%, 20%, 30% nhit nui cy l 48oC trong 35 gi c sc kh. Tin hnh xc nh lng m ho tan thu c khi thu phn v u tm vi c ba t l ging b sung bng phng php chun phooc mol nh trn. T so snh chn ra t l ging b sung thch hp nht. Bc 6: Kho st nh hng ca nhit ln qu trnh thu phn protein V u tm khi chn c cch x l v b sung lng ging thch hp, chng ti tip tc kho st nh hng ca nhit ln qu trnh thu phn. Bn mc nhit c chn nghin cu ln lt l 35oC, 42oC, 48oC, 52oC. Cc bc xc nh m ho tan c tin hnh nh trn. T kt qu thu c, chng ti chn ra mc nhit m ti lng protein trong v u tm c thu phn l cao nht. Bc 7: Kho st nh hng ca thi gian ln qu trnh thu phn protein Thi gian thu phn l yu t khng nhng nh hng ln hiu qu ca qu trnh thu phn m cn nh hng n vn kinh t ca qu trnh sn xut. y chng ti kho st qu trnh thy phn bt u t gi th 8 ca qu trnh nui cy, c 3 gi mt ln ly mu dch kim tra hm lng m ho tan to thnh cho n gi th 35 k t khi cho ging cp 2 vo. Da trn nhng kt qu thu c, chng ti s chn ra thi gian ti v tm c thu phn sch protein v t hiu qu kinh t nht. Bc 8: Phng php nh hng nghin cu x l dch sau khi thu phn v u tm Sau khi thu phn chng ti tin hnh thu nhn phn v v phn dch. Phn dch
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c em i c c nhit 80oC trong 4 gi, sau em i xc nh hm lng m ton phn bng phng php Kjeldahl (ph lc 8) v nng cht kh c trong mu. Bc 9: Phng php nghin cu thu nhn chitin t v u tm sau khi thu phn Phn v tm thu c sau khi thu phn c chng ti ra sch bng nc sau em i phi kh. Chng ti th loi khong sn phm thu c bng dung dch HCl 1N trong thi gian 2 gi 350C thu nhn chitin. Lc v ra sch sn phm di nc my, sy kh nhit 600C cho n khi sn phm kh hon ton.

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