AI HOC QUC GIA HA NI TRNG AI HOC KHOA HOC T NHIN KHOA SINH HOC ____________

LP K12 CNKHTN SINH HOC BI TIU LUN MN VI SINH VT CNG NGHIP

SN XUT PROTEIN N BO (SCP)

Ha Ni, 1/2012

 AI HOC QUC GIA HA NI TRNG AI HOC KHOA HOC T NHIN KHOA SINH HOC ____________

LP K12 CNKHTN SINH HOC BI TIU LUN MN VI SINH VT CNG NGHIP

SN XUT PROTEIN N BO (SCP)

Ging vin: PGS.TS. Ng T Thnh Sinh vin thc hi n: Trn V Qunh Giao Nguyn C m Linh L Hi Vn

Ha N i, 1/2012

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Sn xut Protein n bo

Mc lc
PHN I. TNG QUAN ............................................................................................ 3 1. Cc khi nim chung . ....................................................................................... 3 1.1. Protein n bo ......................................................................................... 3 1.2. Cng ngh sn xut protein n bo ......................................................... 3 2. Lch s nghin cu v sn xut protein n bo............................................... 4 3. Cc nhm vi sinh vt c s dng sn xut protein n bo..................... 6 3.1. Yu cu i vi cc chng vi sinh vt s dng trong sn xut. ................. 6 3.2. Cc nhm vi sinh vt s dng trong sn xut protein n bo. ................ 6 4. Cc ngun nguyn liu dng sn xut protein n bo ............................... 9 4.1. Cc sn phm thi trong nng nghip ....................................................... 9 4.2. Cc sn phm ph ca cc qu trnh ln men cng nghip. ..................... 9 4.3. Cc sn phm, dch chit v dch thy phn t nguyn liu thc vt...... 10 4.4. Cc ngun nhin liu c gi tr thng mi cao nh kh t, methan, methanol v cc n-alkan.................................................................................. 10 5. Quy trnh sn xut protein n bo ................................................................. 11 6. Ch bin protein n bo thnh thc phm .................................................... 11 6.1. Ph hy thnh t bo: .............................................................................. 12 6.2. Loi b axit nucleic .................................................................................. 12 7. u nhc im ca protein n bo. .............................................................. 13 7.1. u im: ................................................................................................... 13 7.2. Nhc im: ............................................................................................. 13 1

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Sn xut Protein n bo

PHN II. MT S QUY TRNH SN XUT PROTEIN N BO ................ 14 1. Sn xut protein n bo s dng chng Cellulomonas t bin [3] ............. 14 2. Mt s quy trnh sn xut protein n bo t nm men: ................................ 18 2.1. Mt s ngun nguyn liu thng s dng sn xut protein n bo.20 2.2. Quy trnh sn xut protein n bo t nm men ..................................... 26 3. Mt s quy trnh sn xut protein n bo t nm mc: ................................ 29 3.1. Sn xut protein n bo t cht thi cng nghip: ................................ 30 3.2. Sn xut protein n bo t rc thi nng nghip t phn b i ca qu ................................................................................................................... 30 4. Mt s quy trnh sn xut protein n bo t nhm vi sinh vt quang hp ... 31 4.1. Nhm vi sinh vt quang hp- ngun cung cp protein. ........................... 31 4.2. ng dng trong sn xut SCP.................................................................. 34 PHN III. KT LUN ........................................................................................... 54 TI LIU THAM KHO: ...................................................................................... 55

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Sn xut Protein n bo

PHN I. TNG QUAN

1. Cc khi nim chung . 1.1. Protein n bo Protein n bo (Single-cell protein SCP) l thut ng thng dng ch phn protein thu c trong sinh khi kh ca cc t bo hoc tng lng protein tch chit c t mi trng nui cy vi sinh vt, c s dng lm ngun thc n cho con ngi hay ngun thc n chn nui. Thut ng protein n bo c GS. C.L.Wilson a ra vo nm 1966, c dng thch hp hn i vi hu ht cc vi sinh vt n bo hoc c th dng si. Cc protein n bo c thnh phn protein cao (60-80% khi lng kh ca t bo), cht bo, carbohydrate, axit nucleic, vitamin v cht khong. Chng cng cha nhiu cc axit amin thit yu nh Lysin v Methionine. 1.2. Cng ngh sn xut protein n bo Cng ngh sn xut protein n bo l cng ngh nui cy v thu sinh khi cc vi sinh vt. N ra i c coi l mt phng php ha hn c th gii quyt c vn thiu protein trn ton th gii. Cng ngh sn xut protein n bo bao gm c cc qu trnh chuyn v sinh hc, bin i cc sn phm ph t gi tr v chi ph thp, thng l cc cht thi, tr thnh sn phm vi gi tr dinh dng v gi tr th trng cao hn. Sn xut protein n bo i hi phi vn dng cc kin thc v ln men, cng ngh sn xut sinh khi, nui cy vi sinh vt. u th k 20, s p dng rng ri cc chng trnh pht trin nng nghip lm cho cc ngun thc n thc vt, nh u nnh, ng, la m v go lun trong tnh trng sn c. Ngoi ra, s pht trin v chnh tr v kinh t thay i trt t th gii t mt h thng cc khi nc tr nn ton cu ha, to iu kin thun li cho vic m ca giao dch cc sn phm nng nghip. Cc sn phm nng nghip ny vt tri so vi protein n bo trn c s gi c sn xut thp hn. Tuy nhin, s kt hp gia vic sn xut phc tp vi cc cng ngh ch bin thc phm mang li mt th h sn phm protein n bo mi, c th s dng thay th cc loi th hay dng lm cht tng cng hng v. Cc ng dng trong tng lai ca vic biu hin protein d hp th c th pht trin hn na tim nng ca dng thc phm ny, to ra cc sn

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Sn xut Protein n bo

phm ph hp vi yu cu ca ch n ung hay to thnh cc dng sn phm c gi tr cao. Sn xut protein n bo quy m cng nghp c mt s c im nh sau: S dng a dng cc phng php, loi nguyn liu th v loi vi sinh vt khc nhau. C hiu qu bin i c cht ban u cao Nng sut cao nh vo tc tng trng nhanh ca cc vi sinh vt. Khng ph thuc vo cc yu t ma hay thi tit. 2. Lch s nghin cu v sn xut protein n bo Ngi tin phong nghin cu v xy dng cng ngh ny l nh khoa hc ngi c Max Delbruck v cc cng s ca ng cch y gn 1 th k. H l ngi u tin ch n gi tr ca cc nm men ru d tha trong vic to ngun thc n cho vt nui. Trong chin tranh Th gii th nht, ngi c thay th phn na ngun protein quan trng bng nm men. V cc nm men ru t cng nghip sn xut bia khng s lng p ng nhu cu protein thc n, mt t l rt ln sinh khi nm men c sn xut trong iu kin hiu kh trong mi trng bn tng hp cha ngun nit l cc mui ammonium. Phng php ny hiu qu hn so vi ln men, nhng vn c hin tng ln men mt s cht trong ngun cacbon, ng thi nng sut sinh khi thu c l khng ti u. Vo nm 1919, Sak (an Mch) v Hayduck (c) pht minh ra mt quy trnh mi, a dung dch ng vo dch huyn ph cha nm men thay v a thm nm men vo mt dung dch ng pha long. Cng ngh ny n ngy nay vn c s dng. Sau khi Chin tranh Th gii th nht kt thc, s quan tm ca ngi c n nm men gim xung, nhng n 1936, ngi ta tip tc s dng nm men ru v mt s loi nm men khc, c bit l cc loi c nui cy vi s lng ln lm thc n b sung cho con ngi v ng vt. K t , u im ca vic nui cy nm men trong mi trng giu dinh dng v iu kin hiu kh c nhn ra mt cch y v nhanh chng tr thnh mt phng tin sn xut thc n trn quy m cng nghip.

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Sn xut Protein n bo

Vo thi im bt u Chin tranh th gii th II, nm men c s dng trc ht lm thc n trong qun i, sau p dng cho tt c cc cng dn. Ngi ta hi vng c th sn xut c hn 100,000 tn mi nm, tuy nhin trn thc t, sn lng cha bao gi vt qu 15,000 tn, do vic sn xut lin tc b gin on bi chin tranh. Vic s dng nm men lm thc n trong giai on ny bt u t c, sau c p dng rng ri nhiu ni trn th gii. Phng th nghim cc sn phm rng ca S nng nghip Hoa K tin hnh hng lot th nghim nui cy nm men Candida utilis trn mi trng cha sulfit l cht thi trong qu trnh sn xut giy. Sau chin tranh, vn sn xut protein n bo ngy cng c ch trng gii quyt cc vn ca nhn loi trn quy m ton cu. u nhng nm 60, mt s cng ti a quc gia quyt nh nghin cu sn xut sinh khi vi sinh vt lm ngun protein thc n. Cc c ch c bn quyt nh s tng trng ca cc sinh vt nh nm men v nm si c lm sng t, tuy nhin vn cn nhiu kh khn trong ng dng cc c ch trn trong cng nghip, do ngi ta tip tc y mnh nghin cu. Ngi ta tip tc tm kim cc loi c cht vi gi thnh thp. Cc sn phm ph trong cng nghip nh sa phomat, r ng hay cc c cht gi thnh r nh tinh bt, ethanol v methanol, sulfit t qu trnh sn xut giy c chn s dng trong qu trnh sn xut protein n bo. Li ch ca vic sn xut protein n bo khng ch dng li vic cung cp thm ngun thc n cho con ngi na m cn hn ch s lng ph cc sn phm ph trong cc qu trnh sn xut khc v bo v mi trng. Vo gia thp nin 60, ngi ta sn xut c hng triu tn nm men thc phm cc vng khc nhau trn th gii. Lin bang Soviet ln k hoch sn xut thng nin 900,000 tn k t nm 1970 b p li s thiu ht ca protein nng nghip. n nm 1980, qu trnh sn xut protein n bo tip tc c m rng cc nc pht trin v c d kin tin hnh cc quc gia ang pht trin. T n nay, quy trnh sn xut protein n bo c khng ngng nghin cu pht trin hon thin, tm kim cc c cht v cc loi vi sinh vt phong ph hn, phc v nhu cu ca con ngi khp cc quc gia trn th gii.

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Sn xut Protein n bo

3. Cc nhm vi sinh vt c s dng sn xut protein n bo 3.1. Yu cu i vi cc chng vi sinh vt s dng trong sn xut. Thi gian sinh trng ngn. C kh nng to thnh lng protein ln, t 40-70% sinh khi kh ca t bo. C kh nng tn dng c ti a cc cht dinh dng trong mi trng nui cy. Khng c c t, khng c kh nng gy bnh C sc bn cao, t b nhim trong qu trnh nui cy, d tch khi dch nui cy. Ngoi ra, phn trm axit nucleic ca t bo vi sinh vt cng l mt yu t quan trng cn xem xt. Qu trnh hp th axit nucleic qu mc c th dn n lng ng axit uric, gy ra mt s bnh nh gout hay hnh thnh si thn, nn i vi ngi th thnh phn axit nucleic ny phi gim xung di 2%. Do , ngi ta p dng mt vi k thut gim thnh phn axit nucleic trong t bo vi sinh vt, bao gm c quy trnh v ha hc v enzyme. 3.2. Cc nhm vi sinh vt s dng trong sn xut protein n bo. Ngi ta s dng nhiu nhm vi sinh vt khc nhau sn xut protein n bo, bao gm: Cc nhm vi khun: Cellulomonas, Alcaligenes, Nm men: Candida, Saccharomyces, Nm si: Trichoderma, Fusarium, Rhizopus, Cc nhm to: Spirulina, Chlorella, Gi tr dinh dng ca chng c trnh by trong Bng 1 [1]: Thnh phn Protein Cht bo Cc cht v c Axit nucleic Nm 30-45 2-8 9-14 7-10 To 40-60 7-20 8-10 3-8 Nm men 45-55 2-6 5-10 6-12 Vi khun 50-65 1-3 3-7 8-12

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Sn xut Protein n bo

Bng 1: Trung bnh cc thnh phn trong t bo cc nhm vi sinh vt chnh (% khi lng kh) theo Miller and Litsky (1976) Ngi ta c tnh rng, 100 pound nm men c th sn xut ra 250 tn protein trong vng 24h. i vi to nui cy trong ao th nng sut protein thu c l 20 tn sinh khi kh/acre/nm. Vi khun th cho lng protein cao, c th ln n 80% v c tc sinh trng nhanh hn, tuy nhin li c nhiu nhc im. Nm men l loi c nhiu u im, v d nh kch thc ln, d thu sinh khi, hm lng axit nucleic trong t bo thp, hm lng lysine cap, c kh nng sinh trng pH axit. Tuy nhin, c im quan trng nht l n rt ph bin v c chp nhn rng ri v c s dng t rt lu trong cng ngh ln men truyn thng. Nhc im ca nm men bao gm tc sinh sn chm, hm lng protein cng nh methionine thp hn so vi vi khun. Nm si cng c nhng u im tng t, d thu hoch, tuy nhin hn ch v tc sinh trng, hm lng protein v t c chp nhn hn so vi nm men. Mt nhm vi sinh vt khc l to th li c nhc im l thnh t bo cu to t cellulose loi phn t m con ngi khng tiu ha c, bn cnh n cn cha nhiu kim loi nng. Ngoi ra, cn nhn mnh rn, v l do k thut v kinh t th ngi ta thng thu c sinh khi to ch khng tch ring protein ca n, do vy i lc thut ng protein n bo y cha hon ton chnh xc. Protein ca to thng c cht lng cao, c th snh c vi protein t thc vt. Sn xut protein n bo t cc vi sinh vt khc nhau, c th l t nm v vi khun nhn c s quan tm ng k, ngc li nghin cu sn xut v s dng thnh cng protein trn to hin nay cn cha nhiu, do gi thnh sn xut cao hn v kh khn hn v mt k thut. Mt vi loi to, nm si, nm men v vi khun c s dng lm protein n bo v hin ang c sn xut thng mi, cng vi ngun carbon ca n c th hin trong Bng 2: Vi sinh vt Vi khun Aeromos hydrophylla Acromobacter delvacvate Acinetobacter calcoacenticus Lactose n-alkan Ethanol C cht

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Bacillus megaterium Bacillus subtilis, Cellulomonas sp., Flavobacterium sp., Thermomonospora fusca Lactobacillus sp. Methylomonas methylotrophus, M. clara Pseudomonas fluorescens

Sn xut Protein n bo
Cc hp cht nit (ngoi protein) Cellulose, hemicellulose Glucose, amylose, maltose Methanol Axit uric v cc hp cht cha nit ngoi protein khc. Glucose

Rhodopseudomonas capsulata Nm si Aspergillus fumigatus Aspergillus niger, A. oryzae, Cephalosporium eichhorniae, Chaetomium cellulolyticum Penicillium cyclopium Rhizopus chinensis Scytalidium aciduphlium, Thricoderma alba Nm men Amoco torula Candida tropicalis Candida utilis Candida novellas Candida intermedia Saccharomyces cereviciae To Chlorella pyrenoidosa, Chlorella sorokiana, Thricoderma viridae,

Maltose, glucose Cellulose, hemicellulose Glucose, lactose, galactose Glucose, maltose Cellulose, pentose

Ethanol Maltose, glucose Glucose n-alkan. Lactose Lactose, pentose, maltose

CO2 dng cho quang hp

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Chondrus crispus, Scenedesmus sp, Spirulina sp., Porphyrium sp.

Sn xut Protein n bo

Bng 2: Mt s loi vi sinh vt s dng trong sn xut protein n bo v ngun carbon ca chng theo Bhalla v cng s (2007) 4. Cc ngun nguyn liu dng sn xut protein n bo Cc ngun nguyn liu hin ang c s dng sn xut protein n bo rt phong ph v a dng, c th lit k mt s loi chnh nh sau: 4.1. Cc sn phm thi trong nng nghip Cellulose t ngun nng nghip v lm nghip l ngun nhin liu ti to nhiu nht trn hnh tinh ny, ng thi l ngun nguyn liu tim nng cho sn xut protein n bo. Trong t nhin, cellulose thng dng phc hp vi lignin, hemicellulose, tinh bt v c cu to phc tp, do vy, s dng di dng c cht n phi c x l ha hc (phn gii bng axit hay kim) hoc bng cc enzyme (cellulase) phn gii cellulose thnh cc ng m t bo vi sinh vt s dng c. Lignocellulose l ngun nguyn liu t g, i hi phi x l trc khi s dng lm c cht cho sn xut protein n bo. C nhiu cch x l khc nhau, v d nh phn gii bng axit hay kim, bng hi nc hay thm ch dng phng x tia X [2]. Ngi ta c th nu g trong dung dch cha Canxi sulfit. n ngy nay, tn dng cc ngun lignocellulose l sn phm thi ngi ta dng cc nhm nm, trong c bit r nht l Agaricus bisporus v mt s cc nhm khc cha enzyme phn gii lignocellulose. 4.2. Cc sn phm ph ca cc qu trnh ln men cng nghip. Dch kim sulfit sn phm ph ca qu trnh sn xut giy c s dng lm c cht ln men k t nawmg 1909 Thy in v sau nhiu ni trn th gii. Dng vi sinh vt u tin c s dng trong qu trnh ny l Saccharomyces cerevisiae, mc d loi vi sinh vt ny khng c kh nng chuyn ho pentose loi hp cht c mt vi lng ln trong sn phm thi ny. Sau , ngi ta tm ra cc loi vi sinh vt thch hp hn nh Candida tropicalis hay Candida utilis. R ng l mt sn phm ph ca qu trnh ln men ng. Dch ng nng cao thu c t qu trnh xay xt ma hay c ci ng c lm lnh ng kt tinh. Khi ng khng kt tinh na th ngi ta thu c phn dung dch cn li, l r ng. i vi 100kg thc vt c th thu c 3.5 n 4.5 kg r ng. Bn cnh nng ng cao, r ng cn cha cc cht khong, cc hp cht hu c v

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Sn xut Protein n bo

vitamin c gi tr cao trong cng nghip ln men. Tuy nhin, sn xut sinh khi vi sinh vt t r ng i hi phi b sung thm ngun nit v photpho ph hp. Ngun nit truyn thng l cc mui amonium, ngun photpho thm vo thng dng mui. 4.3. Cc sn phm, dch chit v dch thy phn t nguyn liu thc vt. Tinh bt thu c t cc loi thc vt c c cc nc nhit i v n i, t go, ng v ng cc l ngun nguyn liu sn xut protein n bo. cc nc nhit i, ngi ta cn s dng sn nh mt ngun nguyn liu sn xut protein n bo. Quy trnh sn xut ny s dng mt s loi vi sinh vt c enzyme amylase nh Endomycosis fibuligira, sau a dch nui cy sau khi phn hy tinh bt qua ni ln men c cha mt loi vi sinh vt c tc sinh trng nhanh nh Candida utilis. Dch chit ca mt s loi qu nh , da, chui cng l ngun c cht tng hp protein n bo. Dch chit u c hm lng cht dinh dng cao: saccharride chim 9.6%, protein, protein chim 0.2%, ng ha tan chim khong 7%; ng ha tan trong u dng glucose, fructose v sucrose, ng thi y l loi thc vt c nng sut cao, sinh trng tng i nhanh. Mt loi qu khc l chui cng l nguyn liu sn xut protein n bo. iu ny c rt c ch trong vic tn dng ngun sn phm d tha do khng cht lng xut khu cc quc gia c trng nhiu loi cy n qu ny. 4.4. Cc ngun nhin liu c gi tr thng mi cao nh kh t, methan, methanol v cc n-alkan. Cc vi sinh vt lin quan n qu trnh sn xut protein n bo t ngun c cht trn ch yu l cc vi khun v nm men. Nhiu qu trnh hin nay ang trong giai on nghin cu. Vic s dng ngun hp cht nhin liu trn sn xut thc n t lu c nhiu nh khoa hc t ra. Cng ti du m ca Anh s dng hai loi nm men Candida lipolytica v C. tropicalis vi ngun c cht l cc alkan c mch Carbon t 12 n 20 nguyn t c trong phn do ca kh gas. Mt vi loi du th c cha phn ny chim khong 15%. Sn phm thu c t qu trnh ny cn gi l TOPRINA, c kim tra c tnh v kh nng gy ung th trong vng 12 nm, sau c s dng lm ngun thc n thay th c hm lng protein cao cho c hoc dng lm bt sa khng bo. Tuy nhin, do gi du m ngy cng cao, nhiu nc ngng s dng ngun nguyn liu ny sn xut protein n bo. Thay vo , ngi ta tng cng nghin cu s dng methane lm ngun c cht. Vi sinh vt ph hp vi ngun c cht ny m c nghin cu k l Methylomonas methanica, nui cy trong mi trng c cha mui nitrat hay mui ammonium lm ngun nit.

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Sn xut Protein n bo

Mt khc, methanol cng l mt ngun cht c quan tm. Ln men trn quy m ln thu sinh khi ca Methylophilus methtlotrophus t ngun c cht l methanol l phng php c xy dng sn xut protein n bo t ngun ny, v sn phm ca quy trnh ny c s dng lm thc n cho vt nui. Methanol vi vai tr l ngun carbon sn xut protein n bo c nhiu u im vt tri hn so vi n-paraffin, kh metan hay thm ch mt s hp cht carbohydrate. 5. Quy trnh sn xut protein n bo Qu trnh ln men to sinh khi yu cu la chn loi vi sinh vt c c giai on sinh l ph hp, kh trng mi trng nui cy v cc thit b dng gi mi trng nui trng thi sn sng, tch t bo, thu cc t bo phn dch ni, tinh ch v x l, b n nhit iu ha nhit , dng c xc nh pH v cc thit b iu khin khc iu ha cc yu t cn thit cho s sinh trng mc n nh. Sau khi chun b, nui cy vi sinh vt iu kin thch hp trong mi trng chun b. Qu trnh ny i hi phi kim sot nng carbon mc thch hp cng nh cung cp oxy duy tr iu kin hiu kh cn cho s sinh trng ca vi sinh vt. V y l mt qu trnh ph thuc vo thi gian, nn vic bin i lin tc cc yu t ca mi trng s nh hng n sinh l ca t bo, trong nng c cht c nh hng ln nht. V th, ngi ta s dng phng php nui cy lin tc c th kim sot ngun Carbon a vo mi trng, ng thi ly ra mt lng vi sinh vt nht nh m bo cung cp oxy cho lng vi sinh vt trong thng nui cy. Cc phng php thng c p dng bao gm: ln men ni, ln men chm v ln men trn pha rn. Sau khi nui cy, cn thu sinh khi vi sinh vt. Hin nay c nhiu phng php c c dch nui cy nh lc, lng, li tm v dng cc mng bn thm. Ngi ta thng thu sinh khi nm men bng cch li tm, cn i vi nm si thng thu bng cch lc. Tuy nhin, cc thit b cn thit tng t v khng ph hp vi sn xut quy m nh cng nh m rng sn xut. Vic loi b lng nc l rt cn thit gi cc cht n nh lu tr nhng li khng d thc hin. Protein n bo cn phi c sy n m 10% hoc phi lm c c li v bin tnh phng chng b hng. 6. Ch bin protein n bo thnh thc phm s dng hiu qu cc protein vi sinh vt lm thc n cho ngi, cn cc iu kin sau: Gii phng protein t bo bng cch ph hy hay phn hy thnh t bo.

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Tiu lun mn Vi sinh vt hc Cng nghip

Lm gim hm lng axit nucleic trong t bo. 6.1. Ph hy thnh t bo:

Sn xut Protein n bo

C th ph hy thnh t bo vi sinh vt bng nhiu cch: p, b vn, nghin, dng p lc hay ph hy bng sng m. Ngoi ra ngi ta cn s dng cc enzyme hay hn hp enzyme phn hy tng phn hoc hon ton thnh t bo. Thy phn thnh t bo nh enzyme c hiu qu cao hn do tc ng c hiu. Phng php enzyme c th dng thay th ph hy c hc, c bit l i vi cc nguyn liu c th b bt hot do yu t c hc, ngi ta s dng c cc enzyme ni bo v ngoi bo do cc vi sinh vt khc tit ra. Tuy nhin, qu trnh ny din ra chm hn so vi dng phng php c hc hoc ha hc, do vy, ngi ta thng s dng phi hp nhiu phng php c hiu qu cao hn. Phng php phi-c hc X l ha hc: dng axit, baz, cc cht ha tan, cht ty ra Phn gii bng enzyme: enzyme thy phn, dng phage, t phn hy Dng cc phng php vt l: lm ng lnh- tan, sc thm thu, dng nhit v sy kh Phng php c hc Dng p lc cao Nghin t, s dng cc ht nghin Dng sng m ph hy t bo Nn t bo Dng p sut cao nhit thp

Bng 3: Mt s phng php thng s dng ph hy thnh t bo vi sinh vt 6.2. Loi b axit nucleic Cc phng php dng loi b axit nucleic trong t bo bao gm phng php x l ha hc v x l bng enzyme. Mi phng php u c nhc im l gi thnh t v c th nh hng n thnh phn dinh dng trong t bo. Mt s phng php: Tch axit nucleic bng cc ru, mui, acid v kim. Tch axit nucleic khi sinh khi vi sinh vt bng kim nhit cao, tuy nhin phng php ny c th to ra cc cht c, v d nh lysinoalanine. X l bng anhydrid bin i cu trc nucleoprotein nm men. S dng cc enzyme nuclease phn gii axit nucleic.

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Tiu lun mn Vi sinh vt hc Cng nghip

7. u nhc im ca protein n bo. 7.1. u im:

Sn xut Protein n bo

Vi sinh vt c tc nhn i v tng trng nhanh, thu sinh khi trong thi gian ngn. Vi sinh vt c hm lng protein tng i cao. Cc vi sinh vt c kh nng s dng mt s lng ngun carbon phong ph to thnh nng lng, trong c mt s nguyn liu c ti s dng t ngun cht thi nng nghip hay cng nghip. Cc chng vi sinh vt vi nng sut cao cng nh thnh phn cht dinh dng ph hp c th c chn lc v nui cy vi s lng ln trong iu kin phng th nghim, ng thi cng c tim nng p dng quy m cng nghip. Sinh khi vi sinh vt dng thu protein n bo khng ph thuc vo ma cng nh bin i kh hu. 7.2. Nhc im: Bn cnh cc u im trn, vic s dng cc ngun vi sinh vt hay sinh khi vi sinh vt lm thc n hay sn xut protein n bo cng c nhiu nhc im cn phi khc phc, bao gm: Nhiu loi vi sinh vt c th to ra cc cht gy c cho c th ngi v c th ng vt, V vy, khi chn la mt loi vi sinh vt tin hnh sn xut phi m bo n khng cha bt k cht c no. i khi s dng sinh khi vi sinh vt lm ngun thc n b sung c th dn n kh tiu hoc khng tiu ha c, thm ch gy phn ng d ng cho ngi. Hm lng axit nucleic cao trong sinh khi kh ca nhiu loi vi sinh vt cng l mt yu t gy nh hng khng mong mun cho con ngi. i khi hm lng axit nucleic cao ny c th dn n s hnh thnh si thn hay bnh gout. Kh nng cha cc hp cht gy c hay gy ung th cho con ngi v ng vt. Sn xut protein n bo l mt qu trnh t tin, v n cn c cc cht iu khin c tit trng cao khi nghin cu trong phng th nghim.

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

PHN II. MT S QUY TRNH SN XUT PROTEIN N BO

1. Sn xut protein n bo s dng chng Cellulomonas t bin [3] Cellulase l mt h enzyme c hot ng theo c ch kim ch bng sn phm cui cng, tc l s c mt tha ca glucose (sn phm ca qu trnh phn gii cellulose bi enzyme cellulase) s lm gim s tng hp ra enzyme ny, t hn ch s sinh trng ca t bo. Trong nghin cu ny, ngi ta gii quyt vn trn bng cch s dng chng Cellulomonas t bin lm gim hm lng cellulase trong t bo. Quy trnh: Mi trng nui cy c bn bao gm cc cht sau (tnh theo s gam trn 100ml nc my pH=7.0) Cht ha hc NaNO3 K2HPO4 KCl MgSO4.7H2O Dch chit nm men Glucose Bo qun mi trng -20 oC trn thch Bng 4: Thnh phn ca mi trng c bn dng trong th nghim nui cy chng Cellulomonas t bin Ngun nit v cellulose: Ngi ta nghin cu 5 s dng 5 ngun nit khc nhau bao gm: ammoni bisulfat, ammoni bicacbonat, ammoni nitrat, ure v natri nitrat thu c tc tng trng ca t bo tng t nhau, tuy nhin ure cn c th ng vai tr l cht iu khin pH nn s tng trng ca t bo ngun nit ny cao hn. S gam trn 100 ml nc 0.1 0.1 0.05 0.05 0.05 0.1

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

Cellulose c s dng di dng bt cellolose kt tinh (Avicel, FMC Corp.) Nui cy vi sinh vt trong ni ln men dung tch 250l, cha dung dch c bn nh trn nhng khng c glucose, b sung Avicel 0.5 % v ure.

Hnh 1: Nui cy lin tc . iu kin nui cy: Tc dn kh vo: 0.09 m3/pht Tc lc: 100 vng/pht. Tc ph bt kh: 1800 vng/pht p lc 2 lb. Nui cy trong vng 72- 96h trc khi thu t bo. Thu t bo v sy kh sinh khi vi sinh vt. Xc nh hm lng cc cht dinh dng v phn tch thnh phn cc amino acid c mt trong protein ca vi sinh vt. Sau , ngi ta th nghim trn chut bng cch nui chut bng ngun thc n c cha sinh khi Cellulomonas v so snh vi cc ngun thc n khc. Cc l th nghim c th hin trong bng 5: Cc thnh phn Ch t protein Lng (g/kg) Ch Casein Ch Cellulomonas 573

Tinh bt ng

633

578

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Tiu lun mn Vi sinh vt hc Cng nghip

Dextrose Hn hp vitamin Hn hp mui Avicel Du ng Bt trng Casein Cellulomonas Choline DL-Methionine Bt trng c cho vo lm ngun b sung protein 0.75 0.75 100 10 40 122 50 45a 100 100 10 40 122 50

Sn xut Protein n bo
100 10 40

50

222 0.75 4.5

Bng 5: Thnh phn ca cc ch n th nghim Kt qu thu c nh sau: Thnh phn Protein (AA) Protein (N) Carbohydrate Cht v c RNA DNA Nng lng Nc % khi lng kh ca t bo 67.6 74 12 9.2 26 2 4.03 kilocalo/g 76.9 % tng khi lng ca t bo Bng 6: Thnh phn cht dinh dng c trong Cellulomonas

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Tiu lun mn Vi sinh vt hc Cng nghip

Axit amin Theo tiu chun ca FAO _ _ 4.2 4.8 4.2 2.2 2.8 2.8 2.8 4.2
a b

Sn xut Protein n bo
Cn cho chut ang ln 6.0 3.0 5.0 5.0 7.0 6.0a 8.0b Cellulomonas

Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Tyrosine Threonine Valine

10.2 3.6 3.3 3.3 6.5 1.5 3.4 2.6

5.0 6.0

4.7 5.8

n c th c thay th bi cystein n c th c thay th bi tyrosine

Bng 7: So snh thnh phn axit amin cn cho chut v thnh phn axit amin trong Cellulomonas Ch n Thay i v trng lng c th - 8.7 29.0 23.6 Lng cho n Lng N a vo NPU (biu th mc s dng protein) (%)

t protein Casein Cellulomonas

126.4 153.1 163.4

0.61 2.45 2.61 58.1 0.5 50.4 0.4

Bng 8: Thay i cn nng, lng thc n v gi tr NPU cc ch n th nghim T cc kt qu trn, c th thy rng chng Cellulomonas t bin ny c kh nng phn gii cellulose tinh th to thnh mt sn phm c cht lng dinh dng

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

tt, nh ch ra th nghim nui chut. ng thi, c th thy rng Cellulomonas l mt sinh vt c th s dng an ton v c nhiu tim nng sn xut protein n bo t cellulose. 2. Mt s quy trnh sn xut protein n bo t nm men: Trong cc ngun protein sn xut bng con ng vi sinh vt, nm men c nghin cu sm nht v c p dng rng ri trn th gii. Con ngi s dng nm men hoc cc sn phm hot ng sng ca chng t hng nghn nm nay. Nm men l tn chung ch nhm nm c cu to n bo, sinh sn bng cch ny chi. Nm men khng c dip lc v khng th s dng nng lng mt tri. V vy chng dinh dng bng cc hydratcacbon, cc hydrocacbua, trc ht l ng. Trong t bo nm men c cha hu ht cc cht cn thit cho s sng (protein, gluxit, lipit, cc enzim, cc vitamin, cc axit nucleic, cc cht khong). Khng mt sn phm thc vt hoc ng vt no c trong thnh phn ca mnh mt lng cc cht c tc dng c hiu nh trong nm men. Tuy nhin thnh phn cc cht c hiu ca nm men khng ph hp hon ton vi nhng nhu cu sinh l ca ng vt. Nm men c ch nhiu v khng nhng trong t bo ca chng c nhiu cht dinh dng c gi tr, m chng li c kh nng tng sinh khi v cc c im sinh l ph hp vi iu kin sn xut cng nghip. V c im lch s: Men gia sc c sn xut u tin c vo khong nm 1880. Lc ngi ta dng men bia (Saccharomyces cerevisiae). Trong th chin th I, men gia sc v men thc phm c sn xut ch yu c l ging Torula utilis. M, t nm 1946 mi t chc sn xut sinh khi nm men. Lc u, ngi ta nui cy nm men trn sacaroza thu hi sinh khi lm thc n cho ngi. Sau v l do kinh t, dn dn ngi ta thay sacaroza bng dch thy phn t tinh bt v xenluza, ph liu cng nghip ng, bia, ru Nm 1968, Lin X l nc u tin xy dng nh my sn xut nm men t paraphin du m, sau Anh, Php , Nht vv.. tin hnh rt nhanh trong lnh vc s dng ngun nguyn liu di do v r tin ny vo mc ch thu protein ca nm men v a sn lng nm men trn th gii ngy cng tng. V gi tr dinh dng: Nm men rt giu protein v vitamin, c bit l cc vitamin nhm B.

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

Sinh khi nm men cha khong 75-80% nc, 20-25% cht kh trong : cacbon 45-50%, nit 7-10% (tng ng vi 40-60% protein, hydro 5-7%, oxy 25-30%, cc nguyn t v c 5-10% (photpho v kali chim ti 95-97%) tng lng ro, s cn li l canxi, magi, nhm, lu hunh, clo, st, silic. Ngoi ra cn c mt lng rt nh cc nguyn t mangan, km, molipden, bo, cacbon ..). Trong thnh phn qu nht l protein. Hm lng protein tu thuc vo tng loi ging, vo thnh phn mi trng v iu kin nui cy. Dao ng trong khong 40-60%. V tnh cht protein ca nm men gn ging protein ngun gc ng vt. Protein ca nm men cha khong 20 axit amin khng thay th. Thnh phn cc axit amin ca nm men cn i hn so vi la m v cc ht ng cc khc, km cht t so vi sa, bt c, bt xng tht v cc sn phm ng vt ni chung. S thay i thnh phn cc axit amin trong thi gian nui cy c nghin cu cho thy thnh phn ca cc axit amin thay i mt giai on pht trin: giai on tim pht. Sau 3 gi pht trin, tng hm lng cc axit amin trong protein tng ln 17% so vi thi im ban u. Sau tng hp axit amin gim xung v gi mc trn 40%. n cui, t bo gi, cc cht d tr, trc ht l glucogen tiu hao nhiu nn gim trng lng, do t l gia cc axit amin so vi trng lng chung ca cc t bo tng ln gn 50% (tng khng thc cht). Cc ging nm men dng lm thc phm cho ngi v thc n gia sc l: Endomyces vernalis, Hansenula anomala, Hansenula suaveolens, Saccharomyces cerevisiae, Candida arbores, Candida tropicalis, Mycotorula lipolytica, Mycotorula japonica, Torulopis utilis, Torulopis utilis var. major, Torulopsis utilis var thermophilis, Monilia candia, Oidium lactic. Cc tiu chun la chn ging nm men sn xut protein t cc ngun hydrocacon: C kh nng ng ho nhiu ngun cacbon khc nhau, nht l cc loi pentoza (xiloza, arabinoza) v cc axit hu c. C th pht trin tt trn mi trng c nng cht kh cao. C kh nng pht trin nhanh, c sc khng cao i vi nng CO2. Sn lng cao, sinh khi cha nhiu cht dinh dng c gi tr (hm lng protein cao, c nhiu axit amin khng thay th, vitamin ..) Kch thc t bo tng i ln d tch bng li tm.

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

Chu ng c nhit tng i cao, t lm bin i pH mi trng. Trong sn xut nm men thng dng cc chng thuc ba ging Saccharmyces, Candida v Torulopsis. Kh nng chuyn ho ca ba ging ny rt cao v a dng, qui trnh cng ngh tng i n gin. 2.1. Mt s ngun nguyn liu thng s dng sn xut protein n bo. 2.1.1. Sn xut protein n bo t cc sn phm cha saccarose : Sn phm t ngnh cng nghip ch bin ng: (r ng ma, r ng c ci, b ma, cn r ng, nc ra th Dch chit cc loi tri cy c hm lng ng cao ( nh u ) R ng: Thnh phn chnh ca r ng gm: ng, cht phi ng v nc. Cc cht phi ng bao gm cc cht hu c v v c. Cc cht hu c c cha nit ca r ng ma ch yu l cc acid amin cng vi mt lng nh protein v sn phm phn gii ca n. Cc cht phi ng v c l cc loi mui tm thy trong thnh phn tro ca r ng. R ng ma cn cha cc nguyn t khc vi hm lng nh nh: Zn, Mn, Cu, B, Co, Mo. R ng rt giu cc cht sinh trng nh: acid pentotenic, nicotinic, folic, B1, B2 v c bit l biotin. Cc nguyn liu cha sacaroza (r ng..) l dng nguyn liu l tng nht n sn xut protein n bo, v cc nguyn liu ny cha nhiu yu t kch thch sinh trng, kh, biotin v sn phm protein thu c hu nh sch, khng c. R ng c dng lm cc c cht cho nhiu qu trnh ln men v: Gi thnh r hn cc nguyn liu cha ng khc. Ngoi ng sacaroza, r ng cn cha mt s cht v c, hu c v vitamin c gi tr. i vi nguyn liu l r ng, dung dch ng, nm men thng dng l Saccharomysces cerevisiae, Candidas tropicalis, Candidas utilis. R c ci ng: R ng c ci cha nhiu ng sacaroza hn r ng ma v trong r ng c ci hu nh khng c mt loi ng chuyn ho no (c khi ch c khong 1%) trong khi r ng ma c th cha ti 15-25% hidrat cacbon ca n di dng ng chuyn ho. R ng c ci cha nit hu c nm ln cao hn r ng ma, nhng mt na l betain, mt thnh phn khng c Saccharomyces ng ho, trong khi betain khng c mt trong r ng ma.

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

R ng c ci cha axit pantothenic gp 2-4 ln so vi r ng ma. Quy trnh x l r ng R ng cn c x l cht t trc khi nui cy. Thng thng n c axit ho bng axit sunfuric ti pH = 4 v un nng ti 120-1500C trong 1 pht kt ta mt s cht v c v cht l lng. Cn phi loi b mt phn cc cht sinh trng, ng thi b sung cc mui khong cn thit (nh ur 0,15%, KH2PO4 0,35%, Mg, Ca) v c th phi thm hn hp cc axit amin dng protein thy phn (dch nm men t phn, dch thi trong sn xut nc chm, dch b ru giai on nhn ging). Khi chun b phi trn, r ng c ci v r ng ma phi c x l tch bit trong cc khu pha long, iu chnh pH, un nng, lm trong, kh trng ri mi c phi trn. Thng pha long n nng ng khong 5-6%. Sau khi chun b xong mi trng dinh dng, tin hnh thanh trng nhit 1200C. Ngoi ra, h keo trong r ng c nh hng xu n qu trnh ln men. H keo trong r ng hnh thnh bi protein v pectin. H keo ny to ra nht cao lm gim kh nng ho tan ca oxy, lm cn tr qu trnh trao i cht ca t bo nm men. Nu h keo khng c ph s gy thoi ho t bo, dn n hiu sut thu nhn sinh khi nm men thp. 2.1.2. Sn xut protein n bo t dch chit u [4]: u l loi cy trng cho hm lng ng rt cao, trng ph bin nhiu nc nhit i, tng trng nhanh v nng sut cao. Dch chit u c dng lm c cht sn xut protein s dng Saccharomyces cerevisiae l loi nm men c trong cc qu u thi. Theo nghin cu ca C. Maragatham and A. Panneerselvam ta c c mi trng nui cy ti u ca nm men trong mi trng dch chit u . B1: S ch u c ra sch vi nc kh trng v gt v, b rut v ht, lm sch vi H2SO4 2% ct thnh khi v ra li vi nc kh trng, v cho vo my xay, dch lc qua vi lc cho chy vo thng cha v trng cung cp ngun ng v protein th. Sau b sung thm nito. B2. Nui cy to sinh khi Theo nghin cu trn, mi trng nui cy c b sung thm (NH4)2HPO4 v th nghim vi cc nng pha long mi trng khc nhau thu c ngun sinh khi ln nht. lng pha long 200ml nc, lng t bo nm men thu c l nhiu nht. Qua kt qu phn tch sinh khi thu c 34.0% protein, 40.0%, sacchride, 0.003% lipids, 9.54% m v 0.14% tro.

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Tiu lun mn Vi sinh vt hc Cng nghip

Mu khng pha long 9.20x108 8.60x108 7.51x108 4.63x108 4.14x108

Sn xut Protein n bo
Mu pha long

Ngy nui 1 2 3 4 5

200ml 1.22x108 1.28x108 9.31x107 8.25x107 8.27x106

400ml 7.23x105 4.36x105 5.28x104 3.55x103 4.16x103

600ml 7.73x104 3.27x104 4.30x103 8.48x102 5.22x102

Bng 9: Sinh khi Saccharomyces cerevisiae thu c cc mi trng pha long nng v th tch khc nhau 2.1.3. Sn xut protein n bo t dch kim sulfit Dch kim sufit: Nc thi cc nh my giy xenluloza theo phng php sunfit gi l dch kim sunfit (SWL-Sunfit Waste Liquors) cng l ngun nguyn liu tt sn xut nm men. Thnh phn hydrocacbon ca n ch yu l ng pentoza, mt loi ng ch c nm men mi chuyn ho tt. Ngoi ra cn c linhin, phi xenluloza, mt s axit hu c Khi s dng dch kim sunfit cn phi c lm nng v thng kh trc khi nui nm men loi bo cc yu t kim hm (SO2 v furfurol). B sung cht dinh dng vo dch thi trn (nh NH4+ v PO4--), iu chnh pH v khong 5 s c mi trng nui cy nm men kh tt v lng sinh khi nm men sinh ra sau qu trnh ln men c cht lng ng k vi cc thnh phn nh sau: protein (46% cht kh), lipit (7-8%), photpho (1,8%), axit nucleic (10%) Ngi ta tnh rng khong 5 tn bt xenluloza sn xut giy s thi ra mt lng dch kim sunfit cha ti 180 kg ng. Dch ny hp ph nhiu O2 nn khi nui cy nm men c th gim mc cung cp oxi ti 60% so vi bnh thng. Nguyn liu l kim sunfit, chng nm men s dng l Cryptococus diffluens, Candidas tropicalis, Candidas utilis. 2.1.4. Sn xut protein n bo t nguyn liu sng ng vt [5] Sng thu c t l m Erzurum c thy phn bng phng php vt l v ha hc. Thnh phn gm c protein, nit, tro, mt s khong cht, ng tng s v axit amin c xc nh v thy rng n c nguyn liu hu c v v c cho php s dng nh l mt ngun c cht trong sn xut protein n bo. Dung dch

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

thy phn sng th c lm giu bng cch b sung chit xut nm men, glucose v KH2PO4. Cc nh hng ca cc nng khc nhau (1, 2, 3, 4, 5, 6, 7, 8, 9 v 10%) trn tng trng ca C. utilis c iu tra v 4% ca CHH (Horn Broth = HB) c tm thy l ti u. Nng sut sinh khi ca C. utilis v hm lng protein ca n l 6,8 g l-1 v 49,8%. Mt khc, sinh khi c cha cht bo 5,4%, 5,94% RNA, DNA 1,53% v tro 9,7%. Sinh khi c cha tt c cc axit amin thit yu. Kt qu chng minh rng sng c th c s dng nh l mt ngun c cht trong vic sn xut protein n bo. To mi trng: 35gr bt sng c ngm tm vi 50 ml dung dch HCl 6 N. Hn hp 80oC trong 24 gi. Vo cui ca giai on ny, hn hp c 130oC trong 1 gi v thm vo 100 ml nc kh ion. Sau c lm lnh v iu chnh pH n 7 bng 10 N NaOH. c lc hai ln thng qua giy lc Whatman No.1. Khi lng tng n 400 ml bng nc kh Ion. Cc dch lc c gi l sng thy phn (CHH) v c lu tr ti 4oC. CHH c pha long gim nng ca cht c ch. Cc nng khc nhau (1-9 v 10%) ca CHH c lm giu bng cch thm 0.1% dch chit xut nm men (Difco, M), ng 1% (Oxoid, Anh) v 0,1% KH2PO4. 3H2O (Difco, M). Dch pha long c gi l dch sng (HB). pH ca mi trng c iu chnh n 5 vi 1 N HCl, c tit trng v s dng. Thi gian nui (h) 24 48 72 Sinh khi (g l-1) 3.4 5.6 6.8

Bng 10: Sinh khi thu c sau cc khong thi gian khc nhau Thnh phn Protein tng s Lipit tng s Ash RNA DNA g g-100 49.8 5.4 9.7 5.94 1.53

Bng 11: T l phn trm thnh phn sinh khi 2.1.5. Sn xut protein n bo t nguyn liu du m v kh t:

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

Vn la chn cc chng vi sinh vt c hot lc sinh tng hp cao dng trong sn xut c mt ngha quan trng. Trong cng nghip sn xut protein t du m v kh t, phi chn cc chng p ng c cc yu cu sau: C kh nng s dng tt ngun nguyn liu hydrocacbua dng trong sn xut. Sinh trng nhanh chng, cho sn lng cao trong thi gian ngn, khng i hi cc yu t sinh trng b sung trong sn xut ln. C c im ho hc v nui cy n nh, c hm lng protein cao, cha y cc axit amin cn thit, khng c c t v phi c ng vt ng ho tt. Phn ln cc chng nm men c sn lng cao trn c cht hydrocacbua c phn lp t nhng mu t v bn nhng ni c m du hoc chung quanh cc nh my ch bin du m. Ch nhng phn du m nht nh mi c vi sinh vt ng ho nh: Cc alkan (paraphin) vi chiu di chui C10 - C20 Cc alkin, anken, hydrocacbon thm. Cc parafin chui ngn cn li trong phn du m c nhit nng chy thp. S dng n-parafin tinh khit c tch t m da trn cc nguyn tc sang phn t lm c cht c u im l ngun C b tiu th hon ton v khng li nhng cacbua hidro c. C ch ca s hp th ankal cho n nay cng cha c lm sng t y . So vi cc t bo sinh trng trn glucoza th nm men nui trn cacbua hidro c mng t bo dy hn v c np nhn.. Tuy nhin cc t bo ny khng gp kh khn g trong vic hp th nhng c cht khng tan trong nc c b sung vo mi trng vi nng 2 - 4%. i vi cc kh t, v d nh metan, khi s dng lm ngun carbon trong nui cy d to thnh hn hp d chy n, v vy khc phc nhng nhc im , c th s dng methanol thu c t metan nh s oxy ho ho hc. Methanol c nhng u im sau: Methanol d tan trong nc nn c th dng nng cao hn (2-3%). Nhu cu oxy ca s ng ho methanol l thp hn. C th dng nm men ng ho methanol. M nm men c kch thc t bo ln hn vi khun nn nng lng cn thit cho qu trnh li tm tch sinh khi t hn so vi vi khun s dng ng ho metan. Tnh kinh t cao hn.

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Tiu lun mn Vi sinh vt hc Cng nghip

Tuy nhin dng methanol c nhc im sau:

Sn xut Protein n bo

Methanol t hn nhiu so vi metan hoc kh thin nhin. Thu hoch c lng sinh khi t bo t methanol thp hn t metan. Cc chng nm men thng c s dng l Candida, Cytomyces, Debaryomyces. Endomyces, Hansemula, Monolia, Scopuloriopsis.

2.1.6. Sn xut protein n bo t mt s ngun nguyn liu khc Cc ngun xenluloza thc vt (g, rm, r b ma, li ng..) c ch nhiu trong sn xut nm men. Trc ht cn phi thu phn xenluloza bng axit hoc bng enzim. Nu dng g th thng phi thu phn bng axit sunfuric. Nc thi ca nh my ch bin sa, cn gi l nh thanh (lactoserum): trong qu trnh ln men lactic ch bin phomat, sau khi kt ta cazein ra khi sa, phn cn li gi l nh thanh c cha lactoza, protein, axit lactic, axit bo, mt s vitamin v mui khong. Ngi ta chn chng nm men thch hp c th thu phn c lin kt -galactozidaza v thu c sinh khi nm men dng kh c thnh phn protein th khong 32%, lipit 4-5%, lacto khong 23%. Chng nm men C.utilis v C.pseudotropical rt thch hp trong mi trng trn y. Nu s dng lactoserum (nh thanh sa) th chng nm men c chng l Torula cremoris, T. lactosa. Bt ng cc: l ngun sn xut sinh khi nm men rt tt. Bt hoc tinh bt dng vo mc ch ny trc tin phi tin hnh thu phn bng axit hoc bng enzim ca mm m hoc enzim ca vi sinh vt bin cc polysacarit thnh cc dng ng m nm men c th ng ho c. i vi nguyn liu tinh bt hay nc thi tinh bt, dng chng nm men tng ng l Endomycopis fibuligera hoc phi hp gia Endomycopis vi Candidas tropicalis. Trong trng hp dng nm men Saccharomysces cerevisiae th c th kt hp chng ct thu ly cn t dch thi sau khi tch sinh khi. Nh vy trong dy chuyn cng ngh cn phi trang b thm b phn chng ct. Dch ly tm c a vo h li tm tch (separator) v dch thi sau khi c tch ra c chuyn n khu chng ct.

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Tiu lun mn Vi sinh vt hc Cng nghip

2.2. Quy trnh sn xut protein n bo t nm men 2.2.1. Quy trnh chung

Sn xut Protein n bo

Mi trng nui cy chit xut t cc thnh phn khc nhau c s dng nui nm men thu sinh khi. Thnh phm c tch ra khi dch nui cy bng phng php ly tm v sy kh. 2.2.2. Quy trnh p dng cho tng loi nguyn liu nh sau: 2.2.1.1. Nguyn liu t r ng: R ng sau khi c x l nh trn s pha long vi nc kh trng, dung dch c em i thanh trng loi b vi sinh vt gy hi v b sung mui v c to thnh mi trng dinh dng. Nm men c nhn ging s c nui trong mi trng trn thu sinh khi. Tch ly sinh khi bng phng php ly tm. Ly phn kt ta di v sy kh, phn dch thi b. 2.2.1.2. Nguyn liu tinh bt T nguyn liu tinh bt hoc c cha xenlulose c thy phn bng enzyme xenluloza hoc amilaza to mi trng dinh dng nui thu sinh khi. Ly tm tch ly sn phm. 2.2.1.3. Nguyn liu t du m. Qui trnh cng ngh sn xut sinh khi nm men c th t du m th v parafin tinh khit cng tng t nhau. Tuy nhin dng du m th th i hi qui trnh cng ngh phc tp hn, mc d gi thnh tng i r hn. Dng parafin th khu tch nm men c th b bt khu ty ra bng dung mi hu c v thc t paraffin c nm men s dng hon ton. 2.2.2. Quy trnh sn xut protein n bo t nm men trong cng nghip y, ly v d quy trnh sn xut protein n bo trong cng nghip t nguyn liu l xenluloz X l nguyn liu v chun b mi trng i vi cc nguyn liu ban u dng sn xut protein n bo t nm men cn phi c x l s b. Sau tin hnh pha ch mi trng. Tu tng loi nguyn liu v chng vi sinh vt nui cy, c cc thnh phn mi trng thch hp. Ni chung, ngoi ngun c cht c bn l ngun cacbon ra, cn a vo mi trng ngun nit, photpho, kali, magi, cc nguyn t khong khc na. Ngun nit thng l cc mui sunfat, ngun photpho l supephotphat, KKCl, Mg MgSO4. C th dng amoniac

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Sn xut Protein n bo

gi pH xc nh. Trong qu trnh ln men cn cn ngun cht sinh trng nh cao ng, cao nm men, hoc cc dch thu phn khc Cc thnh phn mi trng c ho tan, lc b cn, iu chnh pH n 4,85,2 bng axit sunfuric hoc axit clohydric (i vi mi trng r ng th pH l 4,2 4,5). Thc vt c thy phn bng H2SO4 nng 0.5-0.6%, nhit 179-190oC trong cc b cha ln. Sau , c trung ha bng sa vi v lm trong. cc b lm trong thng c cc cch khuy v ng thng kh, nh cc cht c ch dng bay hi nh fucfurol, SO s c loi b. Sau khi c trung ho v lm trong, dch lng cn nng s c lm ngui n nhit 30 32 oC, ri pha long n mt nng ng thch hp cho nm men v ty theo yu cu m b sung cc mui v c. Tch sinh khi thu c nh qu trnh ln men trn, dch thi s c tinh ch, chng ct thnh cn ethanol. Thu hi sinh khi: Bt v sinh khi trn ra ngoi trong qu trnh ln men c tch trc tin theo phng php to thnh bt cng vi sinh khi tro ra ngoi ri a i li tm tch. Bt v sinh khi trn ra ngoi c thu gom li i x l bng phng php tuyn ni (flotation) ri a i li tm qua cc my li tm tch (Seprator), c c chn khng. Sinh khi c a vo sy my sy 2 trc hoc sy phun. Trong t bo nm men k c vi khun, c nhiu vitamin nhm B (tr vitamin B12): tiamin, riboflavin, axit niconitic, axit folic, c bit rt giu tin vitamin D2 (ergosterin). Di nh sng tia t ngoi (tia cc tm) ergosterin s chuyn thnh vitamin D2. V vy trc khi ng gi sn phm sinh khi nm men c chiu tia t ngoi vitamin ho sn phm.

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Sn xut Protein n bo

Hnh 2: M t quy trnh sn xut protein t nm men trong cng nghip

Ch thch: 1. Ni ln men. 2. Thng tuyn ni. 3. Bm. 4. Bnh tch kh. 5. v 8. Li tm tch I v II. 6. Thng cha men. 7. Bm nc. 9. Thng cha men c. 10. Bnh iu chnh nhit lin tc. 11. Thng tp trung men trc khi c c. 12. Thit b c c chn khng. 13. Thit b to chn khng. 14. Thng cha men trc khi sy. 15. Sy phun. 16, 17. Ct xiclon. 18. Thng tn tr V c bn i vi cc loi nguyn liu khc sn xut protein t nm men cng da trn cc bc nh trn. Ch khc nhau v bc tng hp mi trng nui cy ban u. Ring quy trnh sn xut t kh t c nhiu khc bit, c th nh sau: Qu trnh nui cy: Parafin nng (50-600C) c lin tc cho vo thng ln men, nng paraffin trong mi trng ban u l 1,5 -2 % th tch. S tch t sinh khi nm men trong thi gian nui cy c th thc hin trong hai ni ln men: Ln men chnh: ni th nht; c thi kh mnh.

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Ln men ph: c thi kh nhng yu hn.

Sn xut Protein n bo

Nu so snh qu trnh ln men sinh khi trn mi trng cha parafin vi mi trng hydratcacbon, chng ta thy c nhng im ging v khc nhau sau y: Nui cy nm men trn mi trng cha paraphin thng phi thi kh mnh gp 2,6 - 2,8 ln so vi kh nui cy nm men trn mi trng hydrat cacbon. S sinh trng ca vi sinh vt trn hidrocacbua ph thuc vo pH cng ging nh khi nui trn mi trng sacaroza (pH = 5-6). Tuy nhin, c th tr s pH thp hn trnh tp nhim. Khi sinh trng trn hydrocacbua, nm men to nhit hn v yu cu v thanh trng khng cht ch nh khi nui trn mi trng sacaroza. 3. Mt s quy trnh sn xut protein n bo t nm mc: Nm mc l nhng c th a bo, d dng. Hoi sinh trn mi trng giu cht hu c. C cha vitamin nhm B, cha chng 30-60% protein. Hm lng metionin v tryptophan thp, cn c cc axit amin khc tng t nh protein tiu chun ca FAO. Cc ging nm mc c hm lng protein cao l Fusarium, Rhizopus, Penicillium, Aspergillus. Nh ni, nm mc t c dng trong sn xut protein. Hin nay ch c mt s c s sn xut nh United Parer rills Phn Lan, cng sut 10.000tn/nm, nguyn liu chnh l nc sunfit, RHM Foods ( 10.000tn/nm ) v Tate anotty1 (4.000tn/nm) u Anh. Cc l do s dng nm mc sn xut protein: Sn phm to thnh c dng si nhng d dng chuyn thnh cc dng kt cu khc Nm mc c thi gian tn ti kh lu trong h thng tiu ha Protein thu c rt ln, chim n 50% thnh phn. Chi ph sn xut thp. Tuy vy, n vn c nhc im l: Tc tng trng chm hn so vi cc vi sinh vt khc C nguy c gy nhim Mt s loi c cha cht c nn cn c sng lc k.

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Tiu lun mn Vi sinh vt hc Cng nghip

3.1. Sn xut protein n bo t cht thi cng nghip:

Sn xut Protein n bo

B.Volesky v H.Zajic [6], phn lp c t nc t chng mc thuc chi Graphium, chng ny c cha ti 52% protein, trong c 16 axit amin, methionine chim 1% so vi protein th, lysine chim n 7,7%, cc axit amin khng thay th khc u c hm lng tng ng vi protein tiu chun, tr isoleucine. Chng mc ny c kh nng ng ho etan, metan v c nui trong mi trng cha hn hp hai nguyn liu ny thu sinh khi, s dng amoni sunphat lm ngun nito. Ngi ta vn thng thu h si nm mc, trong qu trnh sn xut cc cht khng sinh, cc enzim, axit xitric di dng sn phm ph ca nh my, nhm s dng protein, vitamin, enzim c trong vo nhng mc ch khc nhau. Nhc im ca sinh khi nm mc thu theo phng php ny l nhanh b h hng, v vy phi ch khu sy ngy sau khi tch sinh khi ra khi dy chuyn cng ngh. Trong cng nghip khng sinh, ngi ta c th thu c sinh khi h si gn 17% cc cht cha nit, trong s cc cht cha nit ng ho khong 14%, gn 10% protein tiu ho, 2% cht bo, 2,5% cht x sinh khi ny c th s dng trong chn nui. 3.2. Sn xut protein n bo t rc thi nng nghip t phn b i ca qu Phn rc thi trong nng nghip, c th l nhng phn khng s dng lm thc phm ca qu, thn, r s l ngun gy nhim mi trng nu khng c x l tt. S dng cc loi ny c th dng lm mi trng to protein t nm mc s em li ngun dinh dng rt ln v c th gp phn bo v mi trng. Theo M. Khan v cng s [7], Rhizopus oligosporus c kh nng pht trin trn cc phn b i ca qu nh: phn tha t u , v da chut, v qu lu v ci da hu. Cc phn tha ny c ra sch v em i kh trng 121OC v p sut 15 Psi trong 15 pht. Sau khi c lm mt, nguyn liu c cho vo cc a petri kh trng v cy Rhizopus oligosporus. Cc tm sau c 28 1 C trong 5-7 ngy. Sau khi tng trng, si nm c lc trn giy lc (Whatmann s 1) v ra sch bng nc ct loi b cc ht nu c. Cc giy t lc c cha cc si nm c sy kh 90 2 C trong 24 gi loi b m. Kt qu thu c l h nm pht trin trn dch u l thu c sinh khi ln nht. C cht Phn tha ca u Sn phm sinh khi (mg/100g ca c cht) 59.5

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V da chut V da Ht lu Ci da hu

Sn xut Protein n bo
57.3 48.0 51.6 43.2

Bng 12: M t sinh khi thu c t cc mi trng khc nhau ca Rhizopus oligosporus 4. Mt s quy trnh sn xut protein n bo t nhm vi sinh vt quang hp 4.1. Nhm vi sinh vt quang hp- ngun cung cp protein. Trong nhng thp nin gn y, c nhiu nghin cu vic tm ra ngun protein thay th s dng nh l ngun cung cp thc n trc tnh trng khng ngun protein cung cp trong tng lai. Khi nim protein n bo (SCP) c dng ch vic sn xut protein t sinh vt lng hay sinh khi, bt u t cc ngun vi sinh vt khc nhau, chng hn nh cc nhm di dng nh nm, hay to v mt s vi khun quang hp. Tuy nhin, trong ba n hng ngy ca chng ta, gi tr dinh dng l mt danh sch di cc cht cn thit, khng ch c protein m cn c cc cht dinh dng khc nh cht bo, carbohydrate, cht khong, vitamin, cc cht vi lng...Ngun nguyn liu t cc vi sinh vt quang t dng khng ch c protein m cn c hng lot cc cht dinh dng cn thit khc, v th, cho n nay, cc cng ngh tin tin trn ton th gii c s dng trong sn xut i tr cc tvi sinh vt quang t dng. 4.1.1. Phn tch thnh phn ha hc. Nhiu phn tch v cc thnh phn ha hc c bn nh lipid, protein, carbohydrate t cc loi vi sinh vt quang hp khc nhau c cng b (Bng 13). Nhn chung, thnh phn protein chim lng ng k (c th ni l nhiu nht) trn c trng lng t bo, y c l l nguyn nhn khin chng c coi l ngun protein thay th v cng phong ph.

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Sn xut Protein n bo

Bng13: Thnh phn ha hc trong cc VSV quang hp Tuy nhin, ngoi nhng thnh phn k trn, ch mt vi loi chnh c chn sn xut trn quy m ln, v d nh to lc Chlorella sp. V Scenedesmus obliquus v cc khun thuc h Cyanophyta nh Spirulina sp. v Anthrospira sp. Chlorella l to n bo hnh cu, nhn thc v c ng knh t 5-10m. To xanh Scenedesmus cng c kch thc tng t nhng gm cm 4 t bo vi nhau. Spirulina v Anthrospira l nhng sinh vt a bo, dng si, xon c, quang hp v chiu di c th t ti 0.5m, chng trc y c phn loi vo to xanh, tuy nhin ngy nay chng c xp vo nhm vi khun do cu trc nhn s. 4.1.2. c tnh ca protein sn xut t cc vi sinh vt quang hp. Trc y ngi ta nh gi hm lng protein t to thng qua vic thy phn sinh khi to v nh lng hm lng nito tng s. Chnh v vy m hm lng protein c nh gi qu co trong khi thc t ngun nito cn c trong cc acid nucleic, cc amin, glucosamine v cc vt liu cu to thnh t bo Theo nh thng k th hm lng nito khng cha trong protein ca Scenedesmus obliquus l 12%, Spirulina 11.5% v Dunaliella 6%. Cht lng ca protein c nh gi qua thnh phn, t l v s c mt ca cc acid amin c trong n. Bng 14 th hin thnh phn acid amin c mt trong mt s loi to v vi khun quang hp vi mt s mu thc phm giu protein hng ngy v hm lng hp l theo thiu chun ca WHO/FAO. C 3 ch tiu nh gi v protein trong to v vi khun quang hp l nh lng gi tr sinh hc (BV), h s tiu th (DC) v NPU (the net protein utilization= BVxDC). Tuy nhin, cc thnh phn ca t bo nh cellulose chim ti 10% trng lng kh, gy ra nhng vn trong vic tiu dng hay s dng sinh khi t to, v y l nhng phn con ngi v cc ng vt khng nhai li khng th tiu ha c. Do , cc

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Sn xut Protein n bo

phng php hiu qu lm ph v t bo thu ly protein cng nh thnh phn khc l cn thit cho cc enzyme tiu ha.

Bng 14: Cc thnh phn aminoacid trong to v mt s thc phm.

Bng 15: So snh mt s ch s v hm lng protein trong mt s loi to. C nhiu nghin cu v nh hng ca vic x l sau thu hoch trn kh nng tiu th ca nhiu loi to bng PER ( protein efficiency ratio) trong sinh khi x l, nu ra nhng vai tr quan trng trong qu trnh ch bin sinh khi ca cc vi sinh vt quang hp (Bng 15). 4.1.3. ng dng v vai tr ca protein t to v vi khun quang hp. Cc mt hng thc phm mun c coi l an ton cho ngi tiu th phi tri qua cc bc th c tnh, iu ny c bit p dng vi ngun protein t c ngun gc t cc vi sinh vt quang hp. Cc th nghim chng minh khng c bt k tc hi no ca ngun protein ny vi sc khe con ngi, khng nhng th, y thc s l nhng thc phm y ha hn vi nhng protein mi. Nhn chung l cht lng cc loi protein t nhm ny l nh nhau, v thm ch nhiu loi cn cao cp hn so vi protein thc vt thng thng. Mc d cha lm lng protein cao, nhng dng to kh li khng t c tm quan trng ng k nh cc thc phm khc. Vn chnh l do cc loi ny bt, mu sc xanh v v tanh ca n, lm hn ch s kt hp ca to vi cc thc phm

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Sn xut Protein n bo

thng thng. Nhiu th nghim c tin hnh nhm ci bin hay t hp li cc vt liu t to hay cc vi khun quang hp vi cc mt hng thc phm ph bin bng cc cch nh nng, trn..tuy nhin th cng rt kh khn. Chng hn nh ch c th c mt lng nh to cho vo bnh, nho trn u bt v mi tr nn kh chu hay khi trn vo m khin n c mu nu khng bt mtTuy nhin, ngy nay trn thi trng c rt nhiu thc phm chc nng c thnh phn l cc vi to, c ng gi bn nh l thuc cha chng li mi chng bnh, tr cc bnh do suy dinh dng protein. Vic s dng to lm thc n cho ng vt ngy cng ph bin. Mt lng ln cc gi tr dinh dng v c hi ch ra s thch hp ca cc sinh khi to nh l ngun b sung dinh dng thay th ngun protein thng thng nh tht, c, u tng Hin nay, vic p dng ngun dinh dng ny em li cho nh chn nui ngun li kinh t ln, chng hn nh trong chn nui gia cm khi trn to vo khu phn n hay l vic s dng cc vi to trong nui trng thy sn. 4.2. ng dng trong sn xut SCP Chnh v nhng c im v vai tr ca cc vi sinh vt quang hp k trn m chng c p dng vi cc quy m ln trong vic sn xut ngun protein hay th. Mc d y l nhng sinh vt quang t dng c th pht trin mt cch t nhin, nhng em li hiu sut ln, cn c nhng u t trong cng nghip vi cc quy trnh tin tin nhm nng cao hn na nng sut, m thc cht l thu c cng nhiu sinh khi ca chng ng ngha vi vic to iu kin ti u cho chng pht trin. mt s quc gia, to c nui cy trn quy m ln trong cc ao nc thi giu oxy trong iu kin nh sang mt tri hay h thng chiu sng nhn to m rng khng gian sn xut. Cc yu t nh hng n sn xut sinh khi. Thi gian chiu sng Cng nh sng Ngun cung cp CO2 Ngun nito Cc t bo ang pht trin duy tr cc t bo trong trng thi l lng

S nui ly sinh khi to bt u t nhiu nc nh Nht Bn, c, Mexico, n n , vin nghin cu cng ngh mi trng quc gia (NEERI) Nagpur pht trin k ngh nui cy to trong h thng ao thi giu oxy (Hnh 3)

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Sn xut Protein n bo

Hnh 3 :S dng nui cy to trong cc b nc thi c s oxy ha v kh nng ng dng ca sinh khi to Vic thu hoch sinh khi to c nhiu vn bi v s lng xung ca cc t bo y v vic pha trn mi trng nui cy. Cc t bo c thu hi bng cch ly tm (phn tch), loi nc v lm kh. i khi 1 s cht kt ta nh nhm sunfat, canxi hydroxide v cc polymer c tnh cation c thm vo mi trng nhng chng khng stheer tch ra khi cc t bo thu c. V th phng php ny m bo vic ng dng cc sn phm SCP trong thc phm v chn nui. Cc phng php phn tch v ly tm cng cho php ly tm, kt ta v ly tm cng vi s kt ta v ly tm i km vi kt ta nhng khng kh thi v mt kinh t. 4.2.1. Sn xut SCP t vi khun quang hp thuc chi Spirulina. Vic thu hoch cc Cyanobacteria, nhm vi khun quang hp, v d nh Spirulina sp. t kh khn hn v chng sng tri ni trn b mt nc vi t bo chng cha cc khng bo cha y kh, kt qu to nn cc thm to ni. Chng c kh nng na l c nh c ngun nito t kh quyn. Trong qu trnh x l chng s c lm kh vi kh nng to ra dng bt mn. Nhng li ch t SCP Spirulina: Vic nui to sinh khi Spirulina am li nhiu li th hn so vi Chloralla v Scenedemus bi nhng l do sau:

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

- L dng si nn Spirulina c th c thu hoch bng cc phng php n gin v t tn km hn nh dung mng lc nylon hay cotton - Cc si Spirulina ni ln trn mt nc do c cc khng bo kh, v th vn thu hoch n gin hn. - C rt t kh nng b nhim trong pha sinh trng ca Spirula khi m n sinh trng trn mi trng pH kim, pH 8-11 - Ch cn lm kh trong nhit l c hiu qu v vch t bo mng, trong kh lm kh bng cch phun sy l yu cu bt buc vi Chlorella v Scenedesmus, rt tn km. - Khng gy tc dng ph cho con ngi cng nh ng vt khi s dng. - Spirulina c tnh tiu hy cao do thnh mng v hm lng acid acetic thp (4%). N cha t l protein d tiu ha cao (62-72%), vitamin, cc aminoacid v cc cht dinh dng khc (bng 16)

Bng 16: Thnh phn cc cht dinh dng c trong Spirulina sp. 4.2.1.1. S nui cy v sn xut ca Spirulina. a. Sn xut t nhin Hu ht cc h thng sn xut thng mi u da trn cc h thng knh nng ni m mi trng nui c mix u bi cc bnh cho. Tuy nhin, vn cn mt vi trng hp thu sinh khi vi mc ch kinh t t cc khu t nhin. V d nh Mexico nhng nm 1967 vn cn thu vt sinh khi ca Spirulina maxima t khu vc h Texcoco, cao 2200m so vi mt nc bin, trong mi trng cn nhit ni m nhit trung bnh hng nm khong 18oC. y c th coi l nh my cho sinh khi to ln nht, dng mi trng nui cy bn t nhin bao gm vic thu sinh khi xuyn sut ngy m v sinh khi to tng gp i ch trong 3 n 4 ngy. Lng thu c s c em lc, phi kh, lm ng nht v kh trng.

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Sn xut Protein n bo

Mc d cc h ny l iu kin l tng cho s pht trin ca to, nhng vn c nhng yu t t nhin khng kim sot c d ti hm lng dinh dng thp. Tuy vy, vn phi khng nh rng SCP nhng h ny hon ton tt cho dinh dng ca c hay 1 s ng vt. b. Nui cy quy m phng th nghim. C tm yu t mi trng nh hng n cht lng ca Spirulina: cng nh sng, nhit (30oC). kch c , tc khuy, cc cht rn ha tan (10-60g/lt) , pH 8.5-10.5 . cht lng nc, cc cht a lng, vi lng (C, N, P, K, S, Mg, Na, Cl, Ca and Fe, Zn, Cu, Ni, Co, Se) Sau y l mt s mi trng ti u cho s pht trin ca chng nhm sinh khi ln nht. S dng nc thi nh my ru c phn gii k kh (ADE) sn xut Spirulina platensis (ARM 730) ( Rajeev Kaushik, 2006) Tin hnh: Chun b chng S.platensis (AMR 730) c gi ging v nui trc trong mi trng Zarrouk. Chun b cc mi trng pha long s dng ADE v cc iu kin nui cy. Thnh phn thu hi ca mi trng ADE v Zarrouk c ch ra trong Bng 17. Mi trng ADE c s dng vi cc pha long khc nhau: 5, 10, 20, 30, 50 v 70%). Mi trng ADE l mi trng c hm lng N, P, K v 1 s cht dinh dng khc. y, mi trng Zarrouk c s dng nh l mi trng i chng.

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Sn xut Protein n bo

Bng 17: Thnh phn ha hc ca mi trng ADE v Zarrouk Pha long 3 ln mi loi ADE v chnh pH n 9 vi NaOH 1N v ng trong cc bnh lc, mi bnh lc ny c vi chng c ly t mi trng ban u vi OD t 0.8- 1 560nm v 0.23g chlorophyll/ml v trong 2 tun nhit khong 30oC , mt nh sng 2500 lux c ly t cc tube hunh quang trng lnh, chu trnh 12/12 gi sng ti. Mi trng c lc 2 ln 1 ngy cho n khi thu hi sinh khi. Phng php phn tch: Dch nui cy c dng nht bng my lm ng nht trc kho mu c nh gi cc thng s khc nhau. nh lng khi lng kh c thc hin bi vic lc 25ml mu chng ng nht qua mng lc Whatman (ng knh 4cm). Mng lc c lm kh trong khng kh v chuyn sang nhit 80oC trong 4-6 gi. Mng lc kh cng vi snh khi sinh vt c lm lnh trong my dessicator v cn. Chlorophyll tng s c nh lng mu sc bng 10ml dch ng nht vi 95% methanol. nh lng protein, 0.5ml NaOH 1N c cho vo dch mu, un cch thy 5 pht v protein c nh lng theo phng php Lowry

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

Hm lng lipid c nh lng vi cc thit b Soxhlet, s dng hn hp chloroform v phenol (2:1) v c nh lng 628nm bng phn ng vi sulfophosphovanilin. Tng lng C c nh lng bng thang chun glucose. BOD,TOC, v tng lng nito trong mi trng ADE u tin v d tha sau khi nui cy Spirulina c nh lng qua s dng SPSS cho Window version 10.0.1. Kt qu: Cc thng s thu c trong bng III.2.3 Sinh khi thu c ADE 50% cao hn so vi mi trng i chng, nng c ao hn ca ADE c l khng thch hp cho s pht trin ca chng, v th m thu c t sinh khi.

Bng 18: Cc thng s sinh trng v thnh phn ha hc sau khi nui cy 14 ngy chng S.platensis trong mi trng c nng ADE khc nhau v mi trng i chng. Hm lng TOC v sinh khi tng dn trong cc dch pha long khc nhau (550%). Gii hn v lng Nito trong mi trng ADE phn nh s thp ca nng protein. Ngoi tr nng 50% ADE, cc nng khc u c hm lng protein thp hn mu i chng. Hm lng C tng s trong mu 5,10, 20 % c ch ra mc trung bnh nh mi trng i chng, nng 75% ch ra t nht v cao nht vn thuc loi 50%.

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

T l C/P khi nui S. platensis cao hn so vi mi trng Zarrouk v khi nng ADE gim, t l C/P cng gim (Hnh 4) Khng c s khc bit v lng chlorophyll thu c. V nh sng khng i nn hm lng lipid to ra ph thuc nng ADE. So vi mu i chng, ngoi tr nng ADE 50%, cc nng khc u thu hi lng lipid thp hn. (Bng 18) S gim BOD, TOC v tng lng N c nhn thy trong mi trng cn li sau nui cy chng. (Hnh 5) y, ngi ta lp mi quan h gia khi lng sinh khi v s gim cc ch s da trn vic nui cy trn nhng nng ADE khc nhau thu c. c bit ch nng ADE 50% ng vi 1.23 mg/ml, hm lng N trong nc c s gim ng k, iu ny c ngha trong vic x l nc thi b nhim N.

Hnh 4:S bin ng t l C/P cho thy nh hng ca nng ADE

Hnh 5: S gim phn trm N, C v BOD trong mi trng ADE nui cy S. plantensis

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Sn xut Protein n bo

Sn xut sinh khi v cc hp cht thc phm chc nng bi Spitulina platensis di iu kin nhit v nito khc nhau ( Luciane Maria 2005) Tin hnh: Chun b mi trng Zarouk nh trn, trong thay i v nng Nito khc nhau: 0.0625, 1.250, 1.875 v 2500g/l. Chun b chng S. platensis LEB 52 v nui cy trong mi trng trn 2 mc nhit khc nhau: 30 v 35 C. Chng S. platensis LEB 52 c nui cy trong b ln men nh sng 20l vi lng ban u l 14l v sinh khi l 0.15g/l. 2 tube knh c ua qua im dng ca b, mt trong 2 mu c thu hi v 1 mu khc tip tc c sc kh CO2 vo. Mi trng c mix u v thng kh s dng khng kh c lc theo lung 170l/gi cung cp bi bm. S dng nh sng hunh quang 40W cung cp 31.35mol photon m-2s-1 . Vic nui cy c duy tr trong nh knh di nh sng 12h sng/ 12 h ti 2 iu kin nhit nu trn. xem xt s thay i ca sinh khi, c 24h li ly mu ra bng k thut v trng. Cho n ngy cui nui cy, mi trng nhn ln cho mi th nghim phn on, lc, ra vi nc ct loi b mui tan, ly tm 15000rpm, lm ng kh v gi -20oC cho nh lng protein, lipid v cc hp cht phenol. Phng php phn tch: Nng sinh khi c inh lng OD 670nm to 1 thang chun lin quan dn khi lng kh S. platensis vi s o OD, sau s c dng nh lng mu nhanh. Hm lng c tnh bng phng trnh P= (Xi- Xo)/ti vi P= sn lng( mg/l/ ngy) Xo l lng ban u (mg/l) v Xi l lng thu c ti thi im I v ti l thi gian gia Xo v Xi. Protein c nh lng bng phng php Kjeldahl. Lipid c nh lng nh s dng phng php Folch bng su dng hn hp 2 chloroform: 1 methanol. Cc hp cht phenol c nh lng bng methanol , theo sau bi s chia cc hexane v kt tu cc cht khng phi phenol vi Ba(OH)2 v ZnSO4. Tng lng phenol c nh lng bng my o OD vi phng php Folin s dng tyrosine nh l thang chun. Kt qu: S sinh trng 30 v 37 c ch ra trong hnh III.2.4. Nhn chung th 30 thu c sinh khi ln hn 37 .

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Sn xut Protein n bo

Cc gi tr: tc sinh trng ti a (max) khong thi gian ca phase cp s nhn (t) , sn lng 450h (P450) v hm lng protein, lip, hp cht phenol c ch ra trong Bng 19. Nhn chung, sinh khi v max cao hn 35 . Gi tr p ch ra nng ca sodium nitrate trong mi trng Zarrouk v c nh hng n s sn xut protein , lipid v cc phenolic, trong khi nhit c nh hng ln tt c cc bin s.

Hnh 6: Mt sinh khi ca S. Platensis trong mi trng c nng sodium nitrate khc nhau (a) 30 C (b) 35 C. Nng sodium nitrate 0.625 , 1.250 , 1.875 and 2.500 g /l

Bng 19: Phn tch cc gi tr khc nhau khi iu chnh tng yu t v tch hp c 2 yu t nhit v nng nito Hm lng protein thu c cao nht nng 1.875g/l c 2 loi mi trng, tuy nhin cng nng , nhit 35 li cho hm lng protein cao

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Sn xut Protein n bo

hn (hnh 7.) Nng lipd nhn chung 35 cao hn 30 v 2 nng cui gn nh tng t nhau. Hm lng phenol 30 c s dao ng ln xung qua cc nng nhng cao nht nng cui trong khi , 35 hm lng ny nhn thy rt thp 2 nng u tin v tng vt ln 2 nng cui vi gi tr tng ng.

Hnh 7: Hm lng cc cht trong mi trng nui cyy c nng nito khc nhau di nh hng nhit khc nhau.(a) protein (b) lipid (c) cc hp cht phenol. Nh vy, trong th nghim ny, chng S. platensis pht trin v cho cc sn phm hm lng cao 35 C vi nng sodium nitate 1.875g/l hay 2.5g/l.

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Sn xut Protein n bo

Vic sn xut Spirulina c nui cy trn cc quy m ln hn, c th xy dng ln cc trang tri tn km hay cng c th nui trn cc b x l nc thi (Hnh 8). 4. 2.1.2. ng dng ca SCP Spirulina Nh ngun thc phm b sung protein. Cc sn phm chc nng (v d nh thc phm dn cho ngi b bo ph hay l cc sn phm cung cp vitamin di dng bt, vin) Cc loi thuc iu tr t nhin: s dng nhiu trong phng nga cc bnh, nh gim t l cholesterol trong c th, gim lng ng trong mu ca bnh nhn tiu ng do c axit gamma linoleic l ngun cung cp vitamin A gip sng mt v c cha cc carotene nh l cht chng ung th

Hnh 8 Mt s m hnh nui trng Spirulina Trong m phm: do sn phm Spirulina c cha hm lng protein v vitamin A v B cao, nn c th duy tr mi tc khe mnh hay sc t phycocyanin gip to

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Tiu lun mn Vi sinh vt hc Cng nghip

Sn xut Protein n bo

cc son mi v mt n dc tho ca Nht chng c th thay th cc cht trong thuc nhum m gy ung th. 4.2.2. Sn xut SCP t to Chlorella To Chlorella l mt chi ca to xanh n bo, thuc v ngnh Chlorophyta. Chlorella c dng hnh cu, ng knh khong 2-10 m v khng c tin mao.Chlorella c mu xanh l cy nh sc t quang hp chlorophyll -a v b trong lc lp.Thng qua quang hp n pht trin nhanh chng ch cn lng kh carbon dioxide, nc, nh sng mt tri, v mt lng nh cc khong cht ti sn xut. Thnh phn ha hc: - To c cha 65-68 % protein, 17% ng (glucan), 6% cht bo(Axit bo) - Vitamin A gp 5,8 ln c rt - B1 gp 1,3 ln men v c - B2 gp 35 ln sa - St gp 13 ln gan ln, 45 ln nho - Cht x gp 1,5 ln khoai lang - Canxi Gp 1,6 ln sa y l loi to cng c sn xut ph bin rng ri, sau Spirulina. Mc d vic sn xut ca Chlorella nhn y ha hn v c lin quan n cng ngh sng to, n s khng chng minh c hiu qu kinh t trn th trng. Trong cuc cnh tranh vi Spirulina v cc sn phm nh u nnh, ng cc, n km thnh cng hn trn th trng thc phm phc v cho sc khe. Trong thc t, Chlorella khng c thu hoch d dng vi gi r nh d on ca 40 nm trc . thc t ho, ton b l nui cy ca to ang pht trin s phi c lp trong nh sng nhn to hoc trong bng rm sn xut vi hiu qu quang hp ti a. Ngoi ra i vi Chlorella, c nh sn xut nh trn th gii s yu cu, n s phi c trng trong nc c gas, chi ph sn xut s rt ln. Mt quy trnh phc tp v nhiu chi ph, yu cu thu hoch theo v ma v s cho ra Chlorella mt ngun thc phm hu hiu c nghin thnh bt. Ngoi vic hiu qu sn xut km, Chlorella cn khng tn dng c cc li ch ca nh sng mt tri nh d on. Sau mt thp k th nghim, nghin cu cho thy rng sau khi tip xc vi nh sng mt tri, Chlorella ch chim 2,5% - khng tt hn nhiu so vi cy trng thng thng. Cc nh khoa hc trong nhng nm 1960 pht hin rng con ngi v ng vt khng th tiu ha Chlorella trng thi t nhin do n c cc thnh t bo, gy kh khn trong vic ly cc cht dinh dng. T ,

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Sn xut Protein n bo

nhiu phng php c pht trin ph v thnh t bo ny ch bin thnh cc cht dinh dng c sn cho tiu ha. 4. 2.2.1. S nui cy v sn xut to Chlorella c nhiu nghin cu trong vic to c sinh khi li to ny cao nht v t tn km nht. Nghin cu nui cy sinh khi ca Chloralla Vulgaris v hiu qu thu hi sinh khi bng phng php gi thnh thp.( N. Mohan, 2009) Tin hnh: Chng c nui cy trn mi trng Bold Basal (BBM) v nui cy trong 15 ngy 24 trong phng iu nhit v n khng hunh quang lnh (Phillips 40W, cool daylight 6500K0 v mt 2000lux theo 12/12 gi sng ti. Cc b nui cy ngoi tri c thit k vi thnh dy 0.25m, kch c di 3.65m v rng 2.3, su 0.44m. To c nui trong mi trng CFTRI vi nhit khong 30 n 40 v vng quay ngy m t nhin (20000-75000 lux). Mt kilo mi trng c chun b bng kha lc nc va ch sau ca mi trng khng qu 15cm. Cc cht dng to c thm vo v sinh trng vi s kch ng 3 ln trong ngy. Thu hi sinh khi sau 30 ngy. S mt nc do bay hi c kim sot thng xuyn. S sinh trng c nh gi qua vic m cc t bo nh my huyt tc k ( Haemocytometer). Cc phng php phn tch: Chlorophyl c nh lng bng phng pho tiu hy ca Jejjery v Humphrey (1975). Tch chit v nh lng carotene theo Shaish (1992), tng lng C c xc nh theo phng php anthrone v tng lng protein c xc nh theo Lowry, tng lipid c tnh ton theo Bligh v Dyer (1959). o pH bng my o pH. Kt qu: Cc mu c ly 5 ngy/ 1 ln v s lng t bo c nh lng trn my.

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Sn xut Protein n bo
ngy u tin, c 85*104 t bo/ml v tng dn ln 221*104 t bo/ml ngy th 5, vo pha log lng t bo ln ti 1224*104 t bo/ml ngy th 25. T bo pha tnh 2-3 ngy trc khi gim xung 495*104 t bo/ml ngy th 30.( hnh 9)

Hnh 9 S pht trin ca Chloralla Vulgaris

Nng pH tng trong sut thi gian nui cy (hnh 10) Hm lng cc sc t cng c ch ra trong hnh 11 v 12, vi hm lng chlorophyll a gim dn, trong khi o hm lng carotene li cao nht 25 ngy. Hnh 10 Trong khi hm lng protein v carbohydrate tng ln trong sut qu trnh nui cy, c bit l hm lng C tng mnh t ngy th 25 n ngy 30 th mt s gim ng k hm lng lipid c nhn thy (Hnh 13, 14, 15) nh lng s lng vi khun c trong mi trng nui cy, nhn thy khi s lng to tng th s lng vi khun gim v ngc li (hnh 16) Nh vy, trong nghin cu ny ch ra s pht trin ca to mnh nht l thi gian nui cy 25 ngy, thi im m thu c lng protein v carbohydrate nhiu nht.

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Sn xut Protein n bo

Hnh 11 Hm lng Chlorophyll a

Hnh 12 Hm lng Catotenoid

Hnh 13 Hm lng protein tng s

Hnh 14 Hm lng carbohydrate

Hnh 15 Hm lng lipid

Hnh 16: S pht trin ca vi khun trong Chloralla Vulgaris

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Sn xut Protein n bo

Nui cy hn hp Euglena gracilis v Chlorella sorokiniana: phng php sn xut sinh khi to trn din rng (Emmanuel T. Friday, 2010) Tin hnh: Chun b 2 chng to ni trn. Chun b mi trng ln men lng bao gm: 20% Nitrogen, 20% Phosphorous, 20% potassium, 0.1% Magnesium, 0.15% Iron EDTA, 0.0755% Manganese EDTA, 0.0755% Copper EDTA, 0.0755% Zinc EDTA; 0.0315% Boron, 0.0012% C0balt EDTA v 0.0012% Molybdenum. Mi trng I: 0.5g glucose c ha tan trong 100ml nc ct v 0.25ml mi trng ln men lng, kh trng 121 /15 pht v lm lnh 25 . Mi trng II: 0.25ml mi trng ln men lng ha tan trong 100ml nc ct, kh trng 121 /15 pht v lm lnh 25 . Subculture v seed cuture: 0.3ml mi trng ln men lng ha tan trong 100ml nc ct, kh trng 121 /15 pht v lm lnh 25 . 0.5ml mi seed cuture c vi mi bnh trn di nh n hunh quang 4 gc phng trong 2 tun. Nui cy t dng: 0.1ml mi subculture c thm bo 100ml mi trng II v lc nhit phng 25 bn ngoi hp c chiu sng vi 4 n hunh quang trong 480 gi. Nui cy d dng: 0.1ml mi subculture c thm bo 100ml mi trng I v lc nhit phng 25 trong bng ti trong 480 gi. Nui cy hn hp: Nui cy t dng: 0.1ml mi subculture c thm bo 100ml mi trng I v lc nhit phng 25 bn ngoi hp c chiu sng vi 4 n hunh quang trong 480 gi. Lng nh nhau 2 chng trn c vo mi mi trng v nui cy trong cng iu kin. Phng php phn tch: s sinh trng ca t bo c nh lng bng sc huyt k 5 ngy. S tiu th glucose c tnh bng glucose peroxidase theo Ogbonna (1997). Sn lng sinh khi v nng cui cng c m t theo Ogbonna v Tanaka (1996). Nng t bo cui cng : X= Xf X0 , Xf ,X0 trong tng ng l lng t bo 480 gi v 0 gi. Sn lng sinh khi: Yield= dx/ds vi dx= X- X0, ds= S0- S vi S0, S tng t l lng c cht ban u v cui cng. Kt qu: Hnh 17 v 18 ch ra c im tc sinh trng ca mi h thng nui cy n ca to hp Euglena gracilis v Chlorella sorokiniana. Kt qu cho thy sau 480h

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Sn xut Protein n bo

nui cy, nng ca Euglena trong c 3 loi nui cy t 8.8*105 t bo/ml, 1.64*105 t bo/ml v 37.2*105 t bo/ml, trong khi Chlorella t ti 27.9*105 t bo/ml, 33.7*105 t bo/ml v 44.8*105 t bo/ml

Hnh 17: Tc sinh trng ca Euglena gracilis trong h thng nui cy t dng, d dng v hn dng

Hnh 18: Tc sinh trng ca C. sorokiniana trong h thng nui cy t dng, d dng v hn dng Hnh 19 v 20 ch ra c im tc sinh trng ca h thng nui cy hn hp ca to Euglena gracilis v Chlorella sorokiniana. Nng ca Euglena trong c 3

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Sn xut Protein n bo

loi nui cy t 16.1*105 t bo/ml, 27.3*105 t bo/ml v 47.7*105 t bo/ml tng ng trong t dng, d dng v hn hp, trong khi Chlorella t ti 39.4*105 t bo/ml, 50.7*105 t bo/ml v 61*05*105 t bo/ml tng ng nh th.

Hnh 19: Tc sinh trng ca Euglena gracilis trong h thng nui cy kp.

Hnh 20: Tc sinh trng ca C. sorokiniana trong h thng nui cy kp Nh vy, c th thy r rng l trong h thng nui cy n v kp, tc sinh trng ca to Chlorella l cao hn hn. Trong cc iu kin nui cy tnh ring cho

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Sn xut Protein n bo

mi loi th kiu nui cy t dng mang li nng sut thp nht trong khi nui cy kiu hn dng li cho kt qu cao nht. Vic nui cy kep 2 chng em li hiu qu ln nht cho s sinh trng ca mi loi. 4. 2.2.2. ng dng ca SCP Chlorella Gii c :Nh c lp mng th(sporopolleine), lng chlorophylle cao, chlorella c th hp thu c cc kim loi nng, thuc tr su, thuc dit cn trng v cc ha cht bo v thc vt hay ca polychlorinated biphenyls (PCBs) l nhm ha cht c cho l gy ra cc bt thng v gen c, c dng trong hng trm ng dng cng nghip v thng mi, chng hn nh sn xut cht cch in, cht lm do trong nha, sn v cao su, cht nhum mu... nh vy gip c th o thi c t ny ra ngoi, thc y qu trnh bi tit. Mt s nghin cu cho thy rng b sung Chlorella c tc ng tch cc i vi vic gim ca nng dioxin trong sa m v n cng c th c tc dng tt i vi tr ang b bng cch tng lng IgA trong sa m. Chlorophylle: Lm sch v cung cp oxy: Chlorella rt giu Chlorophylle, thnh phn gip cung cp oxy cho cc m v lm sch ng rut. Chlorophyll c tc dng tt vi cc loi bnh thiu mu khc nhau, c tc dng c ch s pht trin ca vi khun, tng cng sa cha cc m b ph hy v bo v khi tc nhn gy ung th. Chlorophyll cn c tc dng gip tiu ha tt v lm p da. C.G.F(Chlorella grow factor)- Nhn t sinh trng ca Chlorella :Nm trong nhn ca Chlorella, C.F.G l mt hn hp c to thnh t cc vitamin, cc nucleoit (ADN,ARN) v cc axit amin. C.F.G l c s di truyn ca Chlorella. Tng sc khng t nhin: Vi C.F.G, Chlorella tham gia vo qu trnh lm tng bch huyt bo T( thuc h min dch ca c th). Tng kh nng chu ng v s do dai: C.F.G c th coi l ngun tp trung nng lng: cc th nghim cc vn ng vin th thao cho thy ch vi mt liu dng duy nht, tc dng kh nng chu ng v dai sc c th duy tr trong 10 ting sau . Hiu qu probiotic v cn bng h vi khun rut: S lng vi khun Lactobaccilius tng ln nh s c mt ca C.F.G. Do vy hiu qu probiotic( li ch v sc kho do vi khun c ch c nui cy mang li) c pht huy. Vitamin v khong cht: Tng cng sc sng: trong phi c bit k n vai tr ca vitamin B6, B12 v pht pho c tc dng cng c h thn kinh v ci thin gic ng. Hm lng st cao trong To lc gip tng cng chc nng to mu. Do c nhiu si Xenlul( cht x) nn gip tiu ho tt duy tr s kho mnh ca ng rut.

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Sn xut Protein n bo

Cha bnh: Chlorella c cha mt loi hormon kch thch s ti sinh ca cc t bo. Cc bc s Nht Bn cha tr thnh cng nhiu ca vim lot d dy ch vi Chlorella vi mt loi thuc chng d axit. Chlorella c tc dng lm gim huyt p. iu c bit l n khng lm gim huyt p bnh thng m ch pht huy tc dng ny nhng trng hp huyt p cao, lm gim cholesterol xu v tng cng cholesterol tt. Chlorella c tm thy c c tnh chng khi u khi lm thc n cho chut. Mt nghin cu khc tm thy tng cng chc nng mch mu chut cng cao huyt p liu ung cholera. Chlorella c cha cc axit amin cn thit nh Lysine, Threonine Rt quan trng cho tr nht l tr thiu sa m. Hm lng khong cht v cc vi lng phong ph c th phng trnh cc bnh thiu mu do thiu dinh dng mt cch hiu qu, v cng l ngun b sung dinh dng rt tt cho tr li n. Chlorella c cha cht chng lo ha nh carotene, vitamin E, gamma linoleic axit. Nhng cht ny c kh nng loi b cc gc t do thng qua tc dng chng Oxy ha, lm chm s lo ha ca t bo, ng thi st, canxi c nhiu trong to lc va d hp th va c tc dng phng v h tr iu tr cc bnh thng gp ngi gi nh thiu mu, xp xng. S b sung To lc tiu cu vo sn phm to xon Spirulina lm tng thm rt nhiu ln tc dng ty c c th do cc kim loi nng, Dioxin, thuc bo v thc vt hay cc ha cht c hi khc cng nh tc dng h tr iu tr cac bnh him ngho nh ung th cng nh cc bnh v ri lon chuyn ha nh tiu ng, bo ph, huyt p cao, m mu cao hay gan nhim m. To lc khi c hp thu vo c th c tc dng: Tng cng interferon, lm sch mu, gan thn v rut, kch thch sinh sn t bo hng cu, tng oxy cho t bo no, tr tiu ha, kch thch qu trnh sa cha cc m; gip tng PH mu t trng thi kim hn; gip gi cho tri tim hot ng bnh thng; gip tng cng sn phm ca cc khu h sinh vt trong ng tiu ha.

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Sn xut Protein n bo

PHN III. KT LUN

Protein n bo loi protein c sn xut t sinh khi vi sinh vt, t lu c nghin cu v s dng trn khp th gii. T mt s cng trnh nghin cu c nhc n v phn tch trn y, ta c th thy rng, cng ngh sn xut protein n bo ngy cng c pht trin hon thin, to c lng sinh khi cao hn, thnh phn cht dinh dng c cht lng cao hn, c chp nhn rng ri hn v tt cho sc khe ca con ngi. Khng ch dng li mc tiu p ng ngun thc n protein b sung cho con ngi v cc loi ng vt, protein n bo thu c t sinh khi cc loi vi sinh vt ngy cng c nhiu li ch khc phc v cho nhiu mt ca cuc sng con ngi: cha bnh, lm p,, bn cnh , cng ngh sn xut protein n bo cn c nhiu ng dng trong bo v mi trng, tit kim nhin liu, nguyn liu cng nh gp phn m bo cho s pht trin bn vng ca nhiu quc gia trn th gii.

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TI LIU THAM KHO:

Sn xut Protein n bo

1. Single cell Protein: Production and Process A.T. Nasseri, S. Rasoul-Amini, M.H. Morowvat and Y.Ghasemi 2. Single cell Proteins from Fungi and Yeasts U.O.Ugalde and J.I. Castrillo 3. Use of a Cellulase Derepressed Mutant of Cellulomonas in the Production of a Single-Cell Protein Product from Cellulose E.V.Hitchner and J.M.Leatherwood 4. Production of Single Cell Protein from Yeast using Papaya Extract Medium C. Maragatham and A. Panneerselvam. 5. Production of Single-Cell Protein from Ram Horn Hydrolysate Esab Baaran KURBANOLU 6. Batch Production of Protein from Ethane and Ethane-Methane Mixtures - B. Volesky and J. E. Zajic. 7. Production of fungal single cell protein using Rhizopus oligosporus grown on fruit wastes - Mahnaaz Khan, Shaukat Saeed Khan, Zafar Ahmed and Arshiya Tanveer. 8. Mixed cultivation of Euglena gracilis and Chlorella sorokiniana: a production method of algae biomass on a large scale. - Emmanuel T. Friday, Ejoba Rapheal, Nwalo F.O, Ayodele S.M. 9. Utilization of anaerobically digested distillery effluent for the production of Spirulina platensis (ARM 730) - Rajeev Kaushik, Radha Prasanna and H C Joshi 10. A review on culture, production and use of Spirulina as food for humans and feeds for domestic animals and fish- M. Ahsan B. Habib, Mashuda Parvin 11. Production of biomass and nutraceutical compounds by Spirulina platensis under different temperature and nitrogen regimes- Luciane Maria Colla, Christian Oliveira Reinehr, Carolina Reichert, Jorge Alberto Vieira Costa 12. Micro-algae as a source of protein- E.W. Becker 13. Studies on mass cultivation of Chlorella vulgaris and effective harvesting of biomass by low-cost methods- N. Mohan, P. Hanumantha Rao, R. Ranjith Kumar, S. Sivasankaran and V. Sivasubramanian.

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14. http://www.eplantscience.com/index_files/biotechnology/Microbial%20biotechnol ogy/Single%20Cell%20Protein%20%28SCP%29%20and%20Mycoprotein/biotec h_scp_production_of_algal_biomass.php#cc 15. http://en.wikipedia.org/wiki/Chlorella

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