B? GIO D?C V O T?O TR?NG ?I H?C C?N TH VI?N NGHIN C?U V PHT TRI?N CNG NGH? SINH H?

? TI NGHIN C?U KHOA H?C C?P TR?NG

KH?O ST CC Y?U T? ?NH H?NG ?N KH? NNG T?O HEMIHEMICELLULOSE C?A VI KHU?N acetobacter xylinum TRONG MI TR?NG S?A LN MEN ACID LACTIC

CN B? H?NG D?N SINH VIN TH?C HI?N TS. NG TH? PHNG DUNG NGUY?N MINH HI?U L?P: CNSH K34 MSSV: 3082594

C?n Th, Thng 12/2012

PH?N K? DUY?T CN B? H?NG D?N SINH VIN TH?C HI?N

TS. Ng Th? Phng Dung

Nguyn Minh Hi?u

DUY?T CUA H?I ?NG B?O V? LU?N VN

C?n Th, ngy

thng 05 nm 2012

CH? T?CH H?I ?NG

L?I C?M T? -----------(((----------Xin chn thnh c?m n Ban Gim Hi?u Tr?ng ?i h?c C?n Th, Ban Gim ?c Vi?n Nghin c? ?n Cng ngh? Sinh h?c ? t?o m?i i?u ki?n cho ti th?c hi?n ? ti ny. Xin chn thnh c?m n cha m?, anh ch? v ng?i thn ? lun bn c?nh ?ng vin, gip ? r?nh th?c hi?n ? ti ny. Thnh th?t c?m n qu? Th?y C Vi?n Nghin c?u v Pht tri?n Cng ngh? Sinh h?c ? truy? h?ng ki?n th?c qu? bu cho ti trong su?t th?i gian h?c t?p t?i Tr?ng. Chn thnh c?m n Th?y Hu?nh Xun Phong ? t?n t?nh h?ng d?n, truy?n ?t nh?ng kinh ng m qu? bu v gip ? t?o m?i i?u ki?n cho ti hon thnh ? ti ny ng th?i gian quy ?n Thnh th?t bi?t n anh Nguy?n Ng?c Th?nh, cn b? ph?ng th nghi?m Cng ngh? Sinh h?c ( CNSH) Th?c Ph?m Cu?i cng ti xin c?m n ch? L? Hu?nh Lin Hng l?p CNSH K32TT, Nguy?n Lam Anh, H Thi l?p CNSH K34; Nguy?n Vn Nguy?n l?p CNSH K35; Ng Thanh Ph l?p CNSH tin ti?n K35 ? ?ng in, gip ?, trao ?i v ng gp ? ki?n cho ti th?c hi?n ? ti ny. Knh chc qu? vi nhiu s?c kh?e, h?nh phc v thnh ?t. Xin trn tr?ng c?m n! Nguy?n Minh Hi?u

TM L?C Th?ch s?a ?c ln men b?i vi khu?n Acetobacter v Lactobacillus bulgaricus, Streptoc occus thermophilus trong mi tr?ng n?c d?a b? sung s?a ti. Th?c ph?m ny ch?a hemihemic llulose nn giu ch?t x, r?t t?t cho h? tiu ha ?ng th?i gip vi khu?n c l?i pht tri?n ?a ln men chua cung c?p nhi?u lo?i vitamin c?n thi?t cho s?c kh?e nh vitamin B6, B 12, K, niacin, acid folic ?ng th?i sinh bacteriocin gip tng c?ng h? mi?n nhi?m, ?c c h? s? pht tri?n c?a vi khu?n c h?i. ? ti Kh?o st cc y?u t? ?nh h?ng ?n kh? nng t? icellulose c?a vi khu?n Acetobacter xylinum trong mi tr?ng s?a ln men acid lactic nh ?m kh?o st cc y?u t? ?nh h?ng ?n ch?t l?ng s?n ph?m th?ch s?a bao g?m: t? l? nguyn li u (t? l? s?a ti v n?c d?a), t? l? gi?ng vi sinh v?t, hm l?ng ?ng b? sung, pH ban ? t ?, th?i gian ln men v ?nh h?ng c?a CaCl?, (NH?)?SO?, (NH?)?HPO? ? t?m ra ?c i?u k ?i u nh?t cho qu tr?nh ln men th?ch s?a. K?t qu? nghin c?u cho th?y quy tr?nh ln men th?ch s?a cho kh?i l?ng th?ch cao v gi tr? c?m quan t?t c?n ?c ti?n hnh v?i t? l? ngu li?u (s?a ti/n?c d?a) l 1/3, t? l? gi?ng ch?ng l: Acetobacter/Lactobacillus bulgaric us, Streptococcus thermophilus=2/1 (m?t s? c? hai gi?ng ch?ng l 108 tb/ml), hm l?ng ?ng b? sung l 12% (w/v) saccharose, pH=5 v ln men ? nhi?t ? ph?ng (28-32C) trong th gian 7 ngy. T? kha: Acetobacter, Lactobacillus bulgaricu, Streptococcus thermophilus, bac terial hemicellulose, th?ch s?a.

M?C L?C TOC \o "1-3" \h \z \u HYPERLINK \l "_Toc311323283" M?C L?C PAGEREF _Toc311323283 \h ii HYPERLINK \l "_Toc311323284" PH?N I. M? ?U PAGEREF _Toc311323284 \h 1 HYPERLINK \l "_Toc311323285" PH?N II. T?NH H?NH NGHIN C?U TRONG V NGOI N?C C LIN QUA

PH?M VI C?A ? TI PAGEREF _Toc311323285 \h 3 HYPERLINK \l "_Toc311323286" 2.1. Thnh ph?n ha h?c v gi tr? dinh d?ng c?a n?c d?a PAGEREF _Toc311323286 \h 3 HYPERLINK \l "_Toc311323287" 2.2 Sa va cac san ph?m cua sa (Kiu Hu Anh, 1999) 23287 \h 5 HYPERLINK \l "_Toc311323288" 2.3. Vi khu?n acid lactic PAGEREF _Toc311323288 \h 9 HYPERLINK \l "_Toc311323289" 2.3.1. Gi?i thi?u v? vi khu?n acid lactic PAGEREF _Toc311323289 \h 9 HYPERLINK \l "_Toc311323290" 2.3.2. Lactobacillus bulgaricus v Streptococcus thermo philus PAGEREF _Toc311323290 \h 11 HYPERLINK \l "_Toc311323291" 2.4. Vi khu?n Acetobacter PAGEREF _Toc311323291 \h 13 HYPERLINK \l "_Toc311323292" PH?N III. PHNG TI?N V PHNG PHP NGHIN C?U PAGEREF _T 23292 \h 18 HYPERLINK \l "_Toc311323293" 3.1. Phng ti?n PAGEREF _Toc311323293 \h 18 HYPERLINK \l "_Toc311323294" 3.1.1. Th?i gian v ?a i?m ti?n hnh th nghi?m PAGEREF _Toc311323294 \h 18 HYPERLINK \l "_Toc311323295" 3.1.2. Nguyn li?u PAGEREF _Toc311323295 \h 18 HYPERLINK \l "_Toc311323296" 3.1.3. D?ng c? v thi?t b? PAGEREF _Toc311323296 \h 18 HYPERLINK \l "_Toc311323297" 3.1.4. Ha ch?t PAGEREF _Toc311323297 \h 18 HYPERLINK \l "_Toc311323298" 3.2. Phng php PAGEREF _Toc311323298 \h 19 HYPERLINK \l "_Toc311323299" 3.2.1. S ? ln men t?ng qut PAGEREF _Toc311323299 \h 19 HYPERLINK \l "_Toc311323300" 3.2.2. Gi?i thch quy tr?nh PAGEREF _Toc3113 23300 \h 19 HYPERLINK \l "_Toc311323301" 3.2.3. Cc th nghi?m th?c hi?n PAGEREF _Toc3113 23301 \h 20 HYPERLINK \l "_Toc311323302" PH?N IV. N?I DUNG V K?T QU? ?T ?C PAGEREF _Toc3113 23302 \h 26 HYPERLINK \l "_Toc311323303" 4.1. K?t qu? ty l nguyn li?u c kh? nng ln men cho s?n hach sa t?t. PAGEREF _Toc311323303 \h 26 HYPERLINK \l "_Toc311323304" 4.2. ?nh h?ng c?a t? l? vi khu?n ?n qu tr?nh ln men thac sa. PAGEREF _Toc311323304 \h 27 HYPERLINK \l "_Toc311323305" 4.3. ?nh h?ng c?a hm l?ng ?ng v pH ban ?u ?n qu tr?n PAGEREF _Toc311323305 \h 28 HYPERLINK \l "_Toc311323306" 4.4. ?nh h?ng c?a nhi?t ? ? v th?i gian ln men ?n qu l th?ch s?a. PAGEREF _Toc311323306 \h 31 HYPERLINK \l "_Toc311323307" PH?N IV. K?T LU?N V ? NGH? PAGEREF _Toc311323307 \h 33 HYPERLINK \l "_Toc311323308" 5.1. K?t lu?n PAGEREF _Toc311323308 \h 33 HYPERLINK \l "_Toc311323309" 5.2. ? ngh? PAGEREF _Toc311323309 \h 34 HYPERLINK \l "_Toc311323310" TI LI?U THAM KH?O PAGEREF _Toc311323310 \h 36 HYPERLINK \l "_Toc311323311" PH? L?C 1. M?T S? HINH ANH LIN QUAN ?N ? TI PAGEREF _Toc311323311 \h 39 HYPERLINK \l "_Toc311323312" PH? L?C 2. CC PHNG PHP PHN TCH PAGEREF _Toc311323 40 HYPERLINK \l "_Toc311323313" 2.1. Cng th?c mi tr?ng nui c?y Acetobacter PA _Toc311323313 \h 40 HYPERLINK \l "_Toc311323314" 2.2. o pH PAGEREF _Toc311323314 \h 40 HYPERLINK \l "_Toc311323315" 2.3. o Brix PAGEREF _Toc311323315 \h 40 HYPERLINK \l "_Toc311323316" 2.4. o hm l?ng acid t?ng PAGEREF _Toc311323316 \h 40 HYPERLINK \l "_Toc311323317" 2.5. Quan st vi khu?n b?ng knh hi?n vi PAGEREF _T 23317 \h 40 HYPERLINK \l "_Toc311323318" 2.6. ?m m?t s? vi khu?n b?ng bu?ng ?m h?ng c?u PA _Toc311323318 \h 41 HYPERLINK \l "_Toc311323319" PH? L?C 3. B?NG S? LI?U K?T QU? TH NGHI?M PAGEREF _Toc311323319 \h 42

HYPERLINK \l 23320 \h 42 HYPERLINK \l 23321 \h 44 HYPERLINK \l 23322 \h 45 HYPERLINK \l 49 HYPERLINK \l 23324 \h 52 HYPERLINK \l 23325 \h 52 HYPERLINK \l 23326 \h 53 HYPERLINK \l 23327 \h 54 HYPERLINK \l 23328 \h 55 DANH SCH B?NG

"_Toc311323320" 3.1. "_Toc311323321" 3.2. "_Toc311323322" 3.3. "_Toc311323323" 3.4.

S? li?u k?t qu? th nghi?m 1 S? li?u k?t qu? th nghi?m 2 S? li?u k?t qu? th nghi?m 3 S? li?u th nghi?m 4

PAGEREF _Toc3113 PAGEREF _Toc3113 PAGEREF _Toc3113

PAGEREF _Toc311323323 \h PAGEREF _Toc3113 PAGEREF _Toc3113 PAGEREF _Toc3113 PAGEREF _Toc3113 PAGEREF _Toc3113

"_Toc311323324" PH? L?C 4. B?NG S? LI?U TH?NG K "_Toc311323325" 4.1. K?t qu? th?ng k th nghi?m 1 "_Toc311323326" 4.2. K?t qu? th?ng k th nghi?m 2 "_Toc311323327" 4.3. K?t qu? th?ng k th nghi?m 3 "_Toc311323328" 4.4. K?t qu? th?ng k th nghi?m 4

TOC \o "1-3" \h \z \u HYPERLINK \l "_Toc311446479" B?ng 1. Thnh ph?n dinh d?ng c?a n?c d?a PAGEREF _Toc311446479 \h 3 HYPERLINK \l "_Toc311446480" B?ng 2. Thnh ph?n vitamin c?a n?c d?a PAGEREF _Toc3114 46480 \h 3 HYPERLINK \l "_Toc311446481" B?ng 3. Thnh ph?n khong ch?t c?a n?c d?a PAGEREF _Toc311446481 \h 4 HYPERLINK \l "_Toc311446482" B?ng 4. Thnh ph?n acid amin c?a n?c d?a PAGEREF _Toc311446482 \h 4 HYPERLINK \l "_Toc311446483" Bang 5. Thanh phn cua sa bo (%) PAGEREF _Toc311446 5 HYPERLINK \l "_Toc311446484" Bang 6. Ham lng vitamin cua sa PAGEREF _Toc311446 8 HYPERLINK \l "_Toc311446485" B?ng 7. Hm l?ng ?ng v pH ban ?u trong ln men th?ch s?a PAGEREF _Toc311446485 \h 22 HYPERLINK \l "_Toc311446486" B?ng 8. Nhi?t ? v th?i gian ln men th?ch s?a PAGEREF _Toc311446486 \h 24 HYPERLINK \l "_Toc311446487" B?ng 9. Hm l?ng acid t?ng, ? Brix v kh?i l?ng hemicellul e t?o thnh sau khi ln men v?i cc hm l?ng ?ng b? sung v cc m?c pH khc nhau PA 46487 \h 30 HYPERLINK \l "_Toc311446488" B?ng 10. Hm l?ng acid t?ng, ? Brix v kh?i l?ng hemicellu se t?o thnh sau khi ln men ? cc nhi?t ? v th?i gian khc nhau PAGEREF _Toc311446 32 DANH SCH H?NH

TOC \o "1-3" \h \z \u HYPERLINK \l "_Toc311447152" Hinh 1. S tao thanh phc h cas casein PAGEREF _Toc311447152 \h 7 HYPERLINK \l "_Toc311447153" H?nh 2. S ? sinh t?ng h?p hemicellulose (Chawla et al., 2008) PAGEREF _Toc311447153 \h 16 HYPERLINK \l "_Toc311447154" H?nh 3. S ? quy tr?nh ln men t?ng qut PAGEREF _Toc3114 47154 \h 19 HYPERLINK \l "_Toc311447155" H?nh 4. Kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc t? l? nguyn li?u khc nhau PAGEREF _Toc311447155 \h 26 HYPERLINK \l "_Toc311447156" H?nh 5. Kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc t? l? vi khu?n khc nhau PAGEREF _Toc311447156 \h 28 HYPERLINK \l "_Toc311447157" H?nh 6. Kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc hm l?ng ?ng b? sung v cc m?c pH khc nhau. PAGEREF _Toc311447157 \h 29 HYPERLINK \l "_Toc311447158" H?nh 7. Kh?i l?ng hemicellulose t?o thnh khi ln men ? cc nhi?t ? v th?i gian khc nhau PAGEREF _Toc311447158 \h 32

HYPERLINK \l "_Toc311447159" H?nh 8. S ? quy tr?nh ln men th?ch s?a 47159 \h 34 T? VI?T T?T A. xylinum tb/ml CNSH CS DAP GK GLc-1-P GLc-6-P LAB NAD+ xy-ha NADH NXB PGM SA UGP lase UDPGlc

PAGEREF _Toc3114

Acetobacter xylinum T? bo/ml Cng ngh? Sinh h?c Hemicellulose synthase (NH4)2HPO4 Diammonium Phosphate Glucokinase Glucose-1-phosphate Glucose-6-phosphate Vi khu?n acid lactic Nicotinamide adenine dinucleotide d?ng o Nicotinamide adenine dinucleotide d?ng kh? Nh xu?t b?n Phosphoglucomutase (NH4)2SO4 amonium sulfate Uracil diphosphste glucose pyrophosphory Uracil diphosphste glucose

PH?N I. M? ?U Th?ch d?a (Nata de coco) l m?t lo?i th?c n ph? bi?n, c ngu?n g?c t? Philippin, ?c t?o ra t? s? ln men n?c d?a b?i vi khu?n Acetobacter xylinum. y l m?t trong s? cc lo? th?c ph?m thng m?i ?u tin ?ng d?ng t? hemicellulose vi khu?n. Do b?n ch?t ha h?c l h micellulose nn s?n ph?m ny ch?a hm l?ng ch?t x cao, do r?t t?t cho h? tiu ho, kc ?ng ru?t gip i?u h?a bi ti?t t?t hn ?ng th?i t?o mi tr?ng cn b?ng ? vi khu?n c n trong ru?t, i?n h?nh l vi khu?n acid lactic (LAB). S?n ph?m th?ch d?a l m?t mn n tr g mi?ng dai, trong su?t v r?t ngon ?ng th?i c?ng l m?t trong nh?ng h?ng i ? a d?ng h n ph?m t? cy d?a. S?a chua l th?c n b? d?ng v?i s? hi?n di?n c?a nhm vi khu?n sinh acid lactic. Th eo The Lancet - t? bo c?a Hi?p H?i Y Khoa Anh Qu?c, vi khu?n sinh acid lactic tro ng s?a ln men c th? s?n xu?t h?p ch?t c tc d?ng ngn ch?n ho?t ?ng c?a cc enzyme tham a vo s? h?nh thnh ung th trong ?ng ru?t, ngn ng?a ung th ru?t gi v ru?t th?ng. Ngo ?t khng ung th do vi khu?n s?n xu?t c?ng gi?m nguy c ung th ? nh?ng ni khc trong c t M?t trong nh?ng gi?ng quan tr?ng nh?t c?a vi khu?n acid lactic l Lactobacillus b ulgaricu, Streptococcus thermophilus c kh? nng s?n xu?t ra bacteriocin h?p ch?t c tn h nng ?c ch? s? pht tri?n c?a cc vi khu?n gy b?nh v ?c ?ng d?ng trong b?o qu?n th?c m. Bn c?nh , vi khu?n lactic c?n ?c dng ? s?n xu?t cc ch? ph?m sinh h?c cho ng?i v ?c bi?t, LAB c nhi?u trong thnh ph?n men tiu ha, LAB g?n vo thnh ru?t, pht tri?n v l?i vi khu?n gy b?nh qua cc c ch? nh: canh tranh v? tr bm dnh v?i vi khu?n gy h?i et al., 2009) thay ?i pH ?ng ru?t (Nagendra, 2000), ti?t cc ch?t c tnh khng khu?n v sinh, tc ?ng khng enterotoxin v kch thch h? mi?n nhi?m (Perdigon et al., 2002). Hi?n ay, n?c ta s?n xu?t kho?ng 150,8 ngn t?n s?a chua m?i nm ( HYPERLINK "http://dunghan gviet.vn" http://dunghangviet.vn, 28/09/2011); v theo ?i di?n B? Cng Thng t?i h?i th?o g ngh? ti?t trng UHT trong ngnh ch? bi?n v ng gi th?c ph?m ngy 28/9/2011, tiu th? s ?m s?a b?nh qun 20 nm qua c?a Vi?t Nam ? tng nhanh chng (tng trn 30 l?n t? nm 1990 kg/ng?i/nm ?n nm 2010 l 14.4kg/ng?i/nm). Theo trang http:// HYPERLINK "http://www.n ceuticalsworld.com" http://www.nutraceuticalsworld.com ? a ngy 8/2/2010, t? l? tiu th? ?a chua ? khu v?c chu Thi B?nh Dng ?c d? on s? tng ?n 5,2 t? t?n vo nm 2012. V?i nh?ng l?i ch trn, ? c nhi?u nghin c?u v? vi khu?nAcetobacter xylinum v nh khu?n lactic ?c th?c hi?n. Lapuz v c?ng s? (1967) v Ramos (1977) ? nghin c?u cc y?u ?nh h?ng ?n qu tr?nh ln men Nata de coco. Qu tr?nh ln men s?a chua calci ? h? tr? r? v ph?ng ch?ng b?nh lo?ng xng ?c Vng Tr?ng Ho v T?ng Th? M nghin c?u vo nm 2 y Savadogo nghin c?u v? ho?t tnh khng khu?n c?a vi khu?n lactic t? s?a ln men, ng ? t m ?c 8 ch?ng vi khu?n l Lactobacillus fermentum, Pediococcus spp., Leuconostoc mese nteroides subsp. meseteroides, Lactococcus trong s?a ? Burkina Faso, Pakistan. H

YPERLINK "https://cyber.bu.u-bordeaux1.fr/cgi-bin/nph-proxy.cgi/000011A/http/web 5s.silverplatter.com/webspirs/doLS.ws?ss=Benito-M-J+in+AU" \t "wsr" Benito, M.J v n h?ng c?ng s? c?a ng ? phn l?p v tuy?n ch?n nh?ng ch?ng vi khu?n lactic d?a trn kh? nn ln men glucose, sinh acid lactic v acid axetic, kh? nng phn gi?i protein, lipid vbac eriocin c?a cc ch?ng vi khu?n ny nm 2007. Nghin c?u v? ?nh h?ng c?a ngu?n c ch?t v k ln men ?n qu tr?nh h?nh thnh hemicellulose vi khu?n c?a Nguy?n Thy Hng ?c cng b? . Tuy nhin, v?n cha c nhi?u nghin c?u v? s? tng tc c?a 2 nhm vi khu?n ny trong nh? i?n khc nhau. V? th? ? ti: Kh?o st cc y?u t? ?nh h?ng ?n kh? nng t?o hemihemicellu a vi khu?n Acetobacter xylinum trong mi tr?ng s?a ln men acid lactic ra ?i v?i mong mu?n t? k?t qu? kh?o st v nghin c?u cc y?u t? ?nh h?ng ?n s? t?o thnh hemihemicellul trong qu tr?nh ln men s? rt ra ?c nh?ng i?u ki?n t?i u cho vi?c t?o thnh hemilhem ulose trong mi tr?ng s?a ln men lactic. Qua c th? p d?ng nh?ng i?u ki?n ny ? ngh s?n xu?t th?c t? s?n ph?m ln men t? Acetobacter xylinum trong mi tr?ng s?a v?a ? t? o nn m?t s?n ph?m c l?i cho s?c kh?e, v?a gp ph?n a d?ng ha cc s?n ph?m ln men t? s?

PH?N II. T?NH H?NH NGHIN C?U TRONG V NGOI N?C C LIN QUAN ?N PH?M VI C?A ? TI 2.1. Thnh ph?n ha h?c v gi tr? dinh d?ng c?a n?c d?a N?c d?a ch?a nhi?u d?ng ch?t c?n thi?t cho c th?: ?ng, ?m, vitamin nhm B, ch?t ?ng oxy ha, ch?t khong (kali, natri, magie, s?t, ). N c r?t nhi?u cng d?ng trong ch? b ?n th?c ph?m. N?c d?a kh dng ? kho th?t, s?n xu?t n?c mu, ln men r?u, th?ch d?a,... ?a ti ?c dng lm mi tr?ng nui c?y m v? c ch?a ch?t i?u h?a sinh tr?ng. Ngoi ra h?c u?ng ch?a nhi?u ch?t dinh d?ng. S?a b? ti pha ch? v?i n?c d?a l lo?i th?c u?ng h? p d?n v c tc d?ng ch?a b?nh ?ng ru?t, tiu ch?y, nn m?a ? ch?ng m?t n?c,... M?t s? thnh ph?n dinh d?ng, khong ch?t, acid amin, vitamin c trong n?c d?a ?c nh by l?n l?t ? B?ng 1, 2, 3 v 4. B?ng 1. Thnh ph?n dinh d?ng c?a n?c d?a Gi tr? (g/100g) Carbohydrate Ch?t dinh d?ng Ch?t x ?ng t?ng s? Lipid t?ng s? N?c Protein Tro 3,71 1,10 2,61 0,20 94,99 0,72 (Ngu?n: USDA National Nutient Database For Standard Reference, Release 19, 2006) 0,39 B?ng 2. Thnh ph?n vitamin c?a n?c d?a Acid pantothenic Gi tr? Vitamin (mg/100g) B1 (Thiamin) B2 (Riboflavin) B6 (Pyridoxin) C (Acid ascorbic) Niacin 0,043 0,030 0,057 0,032 2,400 (Ngu?n: USDA 0,080 National Nutient Database For Standard Reference, Release 19, 2006) B?ng 3. Thnh ph?n khong ch?t c?a n?c d?a CaGi tr? (mg/100g) Ch?t khong Cu Fe K Mg Mn Na

24,000 0,040 0,290 250,000 25,000 0,142 105,000 20,000 1,000 (Ngu?n: USDA National Nutient Database For Standard Reference, Release 19, 2006) 0,100 B?ng 4. Thnh ph?n acid amin c?a n?c d?a Acid amin(g/100g) Gi tr? aspartic Acid glutamic Alanine Argine Cystein Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine 0,070 0,165 0,037 0,118 0,014 0,034 0,017 0,028 0,053 0,032 0,013 0,037 0,030 0,037 0,026 0,008 0,022 (Ngu?n: USDA National Nutient Database For Standard Reference, Release 19, 2006) 0,044

P Se Zn

2.2 Sa va cac san ph?m t? sa (Kiu Hu Anh, 1999) Sa co thanh phn dinh dng cao, cha nhiu nc va giau cac mui khoan tose) va cac vitamin. Mt lit sa nng 1032 g bao gm: 902 g nc va 130 g cht kh c cha 49 g l m ng kinh 1-8 ?m gm glyceride va cac cht tan trong lipid nh cholesterol va K); 37,2 g protein (chu yu la casein); 9 g cht khoang (gm phosphate, citrate, KCl va MgCl2). Bang 5. Thanh phn cua sa bo (%) Caseinlng (%) Ham Thanh phn Cht beo

Cht tro ng Protein Protein nhu 3,0 3,7 0,7 5,0 3,6 > Protein 0,6 Vao nm 1877, Hammersten phn bit ba loai protein co trong sa, o la casei n va lactoglobulin. tach tng loai protein ny, sa a vt vang c pha loan in se ng lai, trong khi dich ng sa (whey) vn trong dung dich. c im cid yu. V sau ngi ta a phat hin ra rng thanh phn protein cua sa phc ta ac protein cua dich ng sa va cac loai casein. Cac thanh phn chu yu cua ?-lactoglobulin, ?-lactalbumin, serumalbumin, cac globulin min dich IgG1, IgG2, IgA, IgM va cui cung la mt s lng nho cac enzyme. Casein la thanh phn chu y cac loai sau y: * ?s1-casein: co bn dang A, B, C, D, trong o ang chu y la loai B vi ai 199 acid amin, trong lng phn t 23 kDal. Khi co mt ion Ca2+ mc co tr dang mui Ca2+ khng tan. * ?-casein: co nhiu dang, trong o dang A2 co c im: la mt chu?i pep trong lng phn t 24,5 kDal, bi kt tua khi co mt Ca2+ nng co trong s ein se co dang hoa tan. * ?-casein: co hai dang, dang B co c im: la mt chu?i peptide dai 169 phn t 18 kDal, co cha mt b phn carbohydrate ham lng thay i nhng trung b tosemin, 2,4% acid N-acetylneuramic, gn vao chu?i peptide qua Thr133. ?-casein la th anh phn duy nht cua casein vn nm di dang hoa tan khi co mt cac ion Ca2+ va ?-casein vi ?-casein a ngn nga s kt tua cua sa khi co mt ion Ca2+ trong bc nh?t trong vic tao thanh va duy tri cac phc h casein bn vng va trong sa. * ?-casein: y la san phm thuy phn cua ?-casein, c tao thanh nh ca casein thu c nh s phn ct cac gc acid amin 1-28, ?2-casein va ?3-casein c amin 1-105 va 1-107. * ?-casein: chu yu bao gm cac oan ?s1-casein, ?-casein c tao thanh nh asmin bo. Chi co khoang 10% phn casein tn tai di dang cac n phn t. Chung th y l c?>c?>c?s1. Tuy nhin, phn chinh thi c kt hp lai thanh cac phc h loat cac thng s nh co th thy Hinh 1.

Hinh 1. S tao thanh phc h casein v micelle casein > Carbohydrate ng chu yu trong sa la lactose, chim 4-6% trong lng cua sa. Dang C12H22O11.H2O. ngot cua lactose thp hn nhiu so vi fructose, glucose hay sacch e va mt s ng amin khac cung nh cac oligosaccharide co mt trong sa vi s > Lipid Cht beo cua s?a cha 95-96% triglycerol. Ham lng tng i cao cua cac a acid butyric, la c im c trng cua sa. Ham lng cac acid amin b?o hoa t phospholipid chim 0,8-1% trong sa, con cac sterol, chu yu la cholesterol chim t bo cua sa phn b ng u trong tng sa. ng kinh cua cac tiu cu s h bi mt lp mang gm phospholipid va mt lp protein kep, ca hai chim khoang > Cac acid hu c Acid citric la acid chim u th trong sa. Trong khi bao quan, acid nay bi do tac dung cua vi khun. Cac acid khac nh acid lactic, acid acetic la nhng san

actose. S co mt cua acid orotic, mt san phm trung gian trong s sinh tng hp e la mt c trng i vi sa. > Vitamin Sa cha tt ca cac loai vitamin vi s lng khac nhau (Bang 6). Trong tan trong m c gi lai nh kem, trong khi cac vitamin hoa tan trong nc con l ich ng sa. Bang 6. Ham lng vitamin cua sa Ham lng (mg/l) A (Retinol) Vitamin Acid folic Acid pantothenic B1 (Thiamin) B2 (Riboflavin) B6 (Pyridoxin) B12 (Cyanocobalamin) Biontin C (Acid ascorbic) D (Canxiferol) E (Tocopherol) 0,300 Nicotinamide 0,050 3,000 0,400 1,700 0,500 0,050 0,040 20,000 0,001 1,400 1,000 > Enzyme Sa cha nhiu loai enzyme, m?t s? enzyme co kh? nng gip phat hin sa a chi ra kiu va mc un nong. Cac hydrolase a c xac inh la: amylase, lip tase. Cac cht kim ham protease cung co mt trong sa. Cac oxydoreductase quan trong trong sa la: aldehyde dehydrogenase, lactoperox ydase va catalase. M?t s? s?n ph?m ln men c?a s?a: s?a chua, kefir, phomai, 2.3. Vi khu?n acid lactic 2.3.1. Gi?i thi?u v? vi khu?n acid lactic Nhm vi khu?n quan tr?ng nh?t trong ln men chua th?c ph?m ni chung v ln men chua s?a ni ring l nhm LAB. LAB l vi khu?n Gram dng, oxydase m tnh, catalase m tnh, ?c h?nh c?u, khng t?o bo t?. Cc vi khu?n ny ch? y?u t?o lactate khi ln men carbohydra te (bao g?m glucose v lactose). Cc gi?ng LAB quan tr?ng l Lactobacillus, Streptococ cus, Lactococcus, Leuconostoc, Pediococcus, Bifidobacterium v Carnobacterium. T?t c? cc LAB ?u l? thu?c vo carbohydrate nh l ngu?n cung c?p nng l?ng. Chng khng c ins (cytochromes, catalase) nhng chng c th? sinh tr?ng trong i?u ki?n c oxy. a s? ch vi khu?n k? kh khng b?t bu?c (Abee et al., 1999). LAB c yu c?u v? dinh d?ng cao, nhng kh? nng t?ng h?p acid amin v vitamin r?t h n ch?. Khng thnh vin no c?a loi ny c th? pht tri?n trong mi tr?ng ngho mu?i kho e v mu?i amonium. H?u h?t chng yu c?u m?t s? l?ng l?n vitamin (riboflavin, thiamine, acid folic, biotin v nh?ng vitamin khc), acid amin, purine v pyridine (Abee et al. , 1999). LAB yu c?u nhi?u dinh d?ng khc nhau, nn chng k?t h?p r?ng r?i v?i nh?ng mi tr? dinh d?ng nh s?a, th?c v?t nhng chng t ?c t?m th?y trong ?t v n?c. Mi tr?ng s?n chng l s?a, nh?ng ni s?n xu?t s?a (Lactobacillus brevis, L. bulgaricus, L. casei, L . fermentum,...) v trong mng nh?y ru?t c?a ng?i v ?ng v?t (Lactobacillus acidophilus, Bifidobacterium, Enterococcus faecalis, Streptococcus salivarius, Streptococcus bovis, Streptococcus pyogenes, Streptococcus pneumoniae,...) (Abee et al., 199 9). LAB c m?t s? l?ng l?n h? th?ng v?n chuy?n ? tch l?y dinh d?ng t? mi tr?ng. Nh? h? th?ng ny ?nh v? ? mng t? bo ch?t. Qu tr?nh trao ?i ch?t chnh l ?ng phn, chu t

e phosphate. ? vi khu?n ny, NADH ?c oxy-ha thnh NAD+. T? con ?ng ny, LAB chuy?n ?i te thnh lactate v?i vi?c s? d?ng lactate dehydrogenase. C hai ki?u trao ?i ch?t car bohydrate l ln men ?ng h?nh (homofermentative) v ln men d? h?nh (heterofermentative) (Abee et al., 1999). LAB ln men ?c monosaccharide v disaccharide. Nhng khng ph?i t?t c? cc vi sinh ny ?u s? d?ng ?c b?t k? lo?i disaccharide no. M?t s? chng khng ln men ?c sacchar s? l?i khng s? d?ng ?c maltose. Cc LAB khng ln men ?c tinh b?t v cc polysaccharid c loi Lactobacillus delbrueckii l ?ng ha ?c tinh b?t). M?t s?, ch? y?u l cc vi kh en lactic d? h?nh, s? d?ng ?c pentose v acid citric (Lng ?c Ph?m, 2002). Cc loi LAB c kh? nng r?t khc nhau t?o thnh acid lactic trong mi tr?ng v nh h?u ?ng acid c?ng khc nhau. a s? cc tr?c khu?n lactic ?ng h?nh t?o thnh acid cao hn o?ng 2-3,5%) lin c?u khu?n (kho?ng 1%). Cc tr?c khu?n ny c th? pht tri?n ? pH 3,8-4,0 . C?u khu?n khng th? pht tri?n ?c ? mi tr?ng ny. Ho?t tnh ln men m?nh nh?t c?a tr? ? vng pH 5,5-6,0 (Lng ?c Ph?m, 2002). LAB ch?u ?c ? tr?ng thi kh h?n, b?n v?ng v?i CO2 v c?n etylic, nhi?u loi v?n s? g ?c trong mi tr?ng c 10-15% c?n ho?c cao hn; m?t s? tr?c khu?n b?n v?i NaCl (t?i 7-1 %) (Lng ?c Ph?m, 2002). Cc LAB a ?m c nhi?t ? sinh tr?ng t?i a l 25-35C, cc loi a nhi?t c nhi?t a l 40-45C, a l?nh pht tri?n ? nhi?t ? tng ?i th?p (5C ho?c th?p hn). Khi gia n -80C, h?u h?t chng b? ch?t sau 10-30 pht (Lng ?c Ph?m, 2002). M?t s? lo?i c kh? nng t?o thnh mng nh?y. M?t s? khc l?i c kh? nng ?i khng v? o?i sinh v cc vi sinh v?t gy b?nh ho?c lm th?i r?a th?c ph?m. Nh v?y, ngoi kh? nng t thnh acid lactic cc loi ny c sinh ra cc h?p ch?t c ho?t tnh khng khun (c?n g?i l ). Nh?ng ch?t khng khun ny khng dng trong y h?c m ch? dng trong b?o qu?n th?c ph?m qu? kh? quan (Lng ?c Ph?m, 2002). 2.3.2. Lactobacillus bulgaricus v Streptococcus thermophilus 2.3.2.1. Lactobacillus bulgaricus Gii : Bacteria Nganh: Firmicutes Lp: Bacilli B: Lactobacillales Ho: Lactobacillaceae Chi: Lactobacillus Loai: L. delbrueckii ( HYPERLINK "http://en.wikipedia.org/wiki/Lactobacillus_bulgaricus" http://e n.wikipedia.org/wiki/Lactobacillus_bulgaricus) L. bulgaricus l vi khu?n ln men i?n h?nh, pht tri?n t?t ? nhi?t ? 45 50oC trong mi g c ? acid cao. Loi ny c th? t?o ra kh?i s?a ?n 2,7% acid lactic t? ?ng lactose (Ng th? Xun, 2011). 2.3.2.2. Streptococcus thermophilus Gii : Bacteria Nganh: Firmicutes Lp: Cocci B: Lactobacillales Ho: Streptococcaceae Chi: Streptococcus Loai: S.salivarius (Ngu?n : http://en.wikipedia.org/wiki/Streptococcus_thermophilus) S. thermophilus pht tri?n t?t nh?t ? nhi?t ? 50oC v sinh s?n t?t ? nhi?t ? 37 40oC. c?ng l vi khu?n lactic ch?u nhi?t ln men i?n h?nh, c th? ch?u ?c nhi?t un nng ?n 6 ng 30 pht nhng ch? pht tri?n ?c trong mi tr?ng acid th?p hn L.bulgaricus. (Nguy?n t , 2011) Hai loi vi khu?n trn thu?c lo?i vi khu?n hi?u kh v ch?u ?c mi tr?ng c ? a (pH = 4 4.5). Vi?c c?y h?n h?p hai loi vi khu?n ny cho k?t qu? sinh acid lactic t?t hn l c h? s? d?ng t?ng loi. L.bulgaricus c kh? nng th?y phn casein thnh m?t s? acid amin do ?o i?u ki?n cho S.thermophilus pht tri?n. ? giai o?n ?u, pH c?a s?a thch h?p cho loi Streptococcus ho?t ?ng chi?m u th? v ?m b?o cho qu tr?nh ln men lactic ?c b?t ?u. Ho?t ? c?a cc enzyme phn h?y casein c?a Lactobacillus kch thch s? pht tri?n c

a Streptococcus, ? acid tng ln. pH c?a s?a thay ?i lm cho Streptococcus kh pht tri?n ny Lactobacillus s? pht tri?n v lm ? acid tng ln ?n khi ?t cn b?ng gi?a hai loi ?m ? ?c ?n ?nh. L.bulgaricus v S.thermophilus l 2 lo?i vi khu?n ?c FDA cng nh?n v khi cho 2 k u?n ny vo trong n?c s?a ?m, chng s? b?t ?u qu tr?nh ln men v lm s?a t? d?ng l?ng s g ?c, t?o ra v? chua. y l cc probiotic r?t ph? bi?n, r?t c l?i cho s?c kh?e: - Ti?t ra cc ch?t khng khu?n. - Tc ?ng ln m bi?u b? ru?t. - Tc ?ng mi?n d?ch. - Tc ?ng ?n vi khu?n ?ng ru?t. - M?t s? vai tr? khc ?i v?i c th? : ch?ng d? ?ng, ch?ng ung th, gi?m n?ng ? cholester ol, c tc d?ng nh khng sinh. (Ngu?n : http://vi.wikipedia.org/wiki/Probiotic) 2.3.2.3. S? ln men lactic Ln men lactic l qu tr?nh chuy?n ho k? kh t?o acid lactic d?i tc d?ng c?a vi khu?n ctic, co hai dang: ln men lactic ng hinh va ln men lactic di hinh. - Ln men lactic ng hinh la qua trinh ln men ma san phm acid lactic tao ra ln men va mt lng nho acid acetic, acetone, diacetyl,... - Ln men lactic di hinh la qua trinh ln men ma san phm tao ra ngoai acid la phm phu khac nh: acid succinic (khoang 20%), ru ethylic va acid acetic (khoang ai khi (khoang 20%),... i khi khng co cac khi ma thay vao o la s hinh tha 2.4. Vi khu?n Acetobacter 2.4.1. ?c i?m gi?ng vi khu?n Acetobacter Gii : Bacteria Nganh: Proteobacteria Lp: Alpha Proteobacteria B: Rhodospirillales Ho: Acetobacteraceae Chi: Acetobacter (Theo http://en.wikipedia.org/wiki/Acetobacter, 09/04/2011) Vi khu?n Acetobacter l cc tr?c khu?n Gram m, khng sinh bo t?. Nhng theo Gonzal al. (2005) v nghin c?u c?a Kaderel et al. (2008) th? Acetobacter c?n c d?ng h?nh e lip. Ngoi ra, Maal et al. (2009) c?n pht hi?n ra nh?ng d?ng Acetobacter h?nh c?u t ? cho th?y s? a d?ng v? h?nh d?ng c?a loi vi khu?n ny. a s? cc nghin c?u v? Acetobacter ?u cho th?y gi?ng vi khu?n ny c nh?ng ?c i?m g nh: Gram m, hi?u kh b?t bu?c, catalase dng v oxidase m, chng c th? x?p thnh chu ng?n, c khi ?ng ring l?, kch th?c t? 0,6-0,8 1,0-4,0 ?m. Acetobacte c kh? nng oxy anol thnh acid acetic, oxy ha lactate v acetate thnh CO2 v H2O. y c?ng l ?c i?m ?i Gluconobacter. Tuy nhin, trong m?t s? tr?ng h?p Gram c th? bi?n ?i (Gram variable ) (Gonzal et al., 2005 v Kaderel et al., 2008). Trong mi tr?ng c ?y ? ch?t dinh d?ng v oxy cung c?p lin t?c. Sau 12 gi?, l?n kh?i m chng sinh ra c th? tng g?p 17 tri?u l?n so v?i ban ?u (Nguy?n ?c L?ng, 2002). Tuy nhin, ty theo t?ng loi m c s? khc nhau v? kch th?c, kh? nng ch?u n?ng ? d, C loi chuy?n ?ng ?c (c tim mao n ho?c chu mao) v c loi khng chuy?n ?ng (k g ch?u ?c n?ng ? acid cao, m?t s? v?n pht tri?n ? pH = 3,2. C loi sinh t?ng h?p ?c in B1, B2, B12. C loi th? c?n vitamin nh l ngu?n dinh d?ng trong mi tr?ng ? sinh tr ?c Ph?m, 2002). Vi khu?n acetic khng nh?ng oxy ha ?c r?u etylic thnh acid acetic m c?n oxy ha propionic thnh acid propionic, r?u butylic thnh acid butyric, nhng chng khng oxy ha b?c cao v r?u methylic (Nguy?n ?c L?ng, 2002). Trong mi tr?ng ? r?u etylic (5-13%) th? s?n ph?m ch? y?u l acid acetic, n?u n?n g ? r?u th?p hn cc vi khu?n acetic oxy ha tri?t ? r?u t?o thnh CO2 v n?c (Lng 2.4.2. M?t s? loi Acetobacter c ? ngh?a trong th?c t? ?n nay c ?n 20 loi ?c bi?t ?n, chng ?u c th? s? d?ng vo s?n xu?t. Tuy nhi d?i y c ? ngh?a kinh t?, ng ch ? hn c? (Nguy?n ?c L?ng, 2002). - Acetobacter aceti: tr?c khu?n ng?n, khng sinh bo t?, khng chuy?n ?ng, cc t? b th?ng lin k?t v?i nhau t?o thnh chu?i di. Chng ch?u ?c n?ng ? c?n cao kho?ng 11%, rong mi tr?ng ?y ? i?u ki?n sinh tr?ng, chng c th? t?o ra 6% acid acetic. Nhi?t ? cho loi ny pht tri?n l 34C. Chng b?t mu v?i iod, chuy?n sang mu vng khi nhu?m. - Acetobacter pasteurianum: tr?c khu?n ng?n, khi nhu?m v?i iod chng b?t mu x

anh. Kh? nng ch?u ?c ? c?n y?u hn Acetobacter aceti. Trong i?u ki?n mi tr?ng pht t u?n l?i, chng c kh? nng t?o ra 5-6% acid acetic. - Acetobacter orleaneuse: h?nh thi c?ng gi?ng nh Acetobacter aceti v Acetobac ter pasteurianum nhng kch th?c nh? hn nhi?u. ?c bi?t hai ?u t? bo th?ng nh? l?i. Nh r?ng h?p ng?i ta l?n l?n vi khu?n ny v?i Clostridium. Trong d?ch n?c tri cy chng th ?o ra m?t vng r?t m?ng trn b? m?t. Vng vi khu?n th?ng r?t ch?c, t? bo s? chuy?n sang mu vng khi nhu?m v?i iod. Chng ch?u ?c ? c?n ln ?n 12% v c th? tch l?y ?c 9,5% - Acetobacter xylinum: ?c s? d?ng r?ng r?i trong cng nghi?p th?c ph?m v? hi?u su?t ln men t?o hemicellulose cao v ?n ?nh. A. xylinum c ngu?n g?c t? Philippines. A. xylinum sinh tr?ng ? pH ? 5, nhi?t ? t? 28-32oC v c th? tch l?y 4.5% acid acetic t rong mi tr?ng. Tuy acid acetic l s?n ph?m sinh ra trong qu tr?nh ho?t ?ng c?a vi khu? n, nhng khi chng v?t qu m?c cho php, chng s? quay ng?c tr? l?i lm ?c ch? ho?t ?ng khu?n. A. xylinum th?ng ?c g?p cng v?i n?m men trong n?m ch , c?n g?i l thu? hoi s i s?n ph?m gi?i kht b? d?ng theo cch lm c?a ng?i Trung Hoa. l m?t lo?i n?c chua c g ? pha n?c gi?i kht trong cc gia ?nh ng?i Trung Hoa, trn m?t n?c c m?t vng vi si nui s?ng b?ng n?c ch v ?ng (Nguy?n Thy Hng, 2007). - Acetobacter schiitzenbachii: vi khu?n gram m, h?nh que nhng kch th?c nh? hn c loi ? nu ? trn, khng chuy?n ?ng. Chng c th? tch t? ?c 11-12% acid acetic. Chng l?p vng nhng khng ch?c. Ng?i ta th?ng s? d?ng loi ny vo s?n xu?t gi?m. - Acetobacter curvum: c th? t?o ra 10-11% acid acetic. Nhi?t ? thch h?p ? chng pht tri?n l ? 35-37C. Chng c th? t?o ra vng r?t ch?c trn b? m?t. - Acetobacter suboxydans: vi khu?n ny c th? tch t? ?c l?ng acid r?t l?n, n?u tr ng mi tr?ng ta cho thm m?t l?ng nh? cc ch?t dinh d?ng c?n thi?t, v d? glucose vi khu ny c th? chuy?n ha hon ton c?n thnh acid acetic, l?ng acid acetic t?o ra c th? ln h? c?n sau 48 gi? ln men. Nhi?t ? thch h?p ? vi khu?n ny pht tri?n l 28-30C. Trong nh ln men c?n thng kh lin t?c v? vi khu?n ny c?n l?ng oxy r?t l?n ? chuy?n ha c?n t id acetic v cho qu tr?nh pht tri?n. Chng th?ng ?c dng ? s?n xu?t vitamin C. 2.4.3. S? ln men t?o th?ch c?a Acetobacter xylinum trn mi tr?ng n?c d?a H?nh 2. S ? sinh t?ng h?p hemicellulose (Chawla et al., 2008) Qu tr?nh t?ng h?p hemicellulose tr?i qua r?t nhi?u giai o?n v?i s? tham gia c?a nh?ng enzyme v ph?c h?p nh?ng protein xc tc, i?u h?a. ?u tin l qu tr?nh phosphoryl ucose thnh glucose-6-phosphate (GLc-6-P) v?i s? tham gia c?a enzyme xc tc glucokina se (GK). Ti?p ?n glucose-1-phosphate (GLc-1-P) s? h?nh thnh b?i s? ?ng ha GLc-6-P do enzyme phosphoglucomutase xc tc. Qu tr?nh bi?n ?i ti?p t?c di?n ra, GLc-1-P chuy?n h?a thnh UDPGLc b?i enzyme UDPGLc pyrophosphorylase. Sau , hemicellulose synthase xc tc qu tr?nh polymery ha glucose thnh chu?i glucan ?-1-4 v m?t chu?i m?i d?i d?ng c?u trc chu?i (ribbon-like structure) c?a hng trm, hng ngn nh?ng chu?i hemicellulose n. ?nh qu tr?nh t?ng h?p hemicellulose c?n x?y ra hng lo?t cc qu tr?nh ?ng ha khc, tuy qu tr?nh t?ng h?p hemicellulose x?y ra ?c l?p v?i cc qu tr?nh ny (Chawla et al., 200 8).

PH?N III. PHNG TI?N V PHNG PHP NGHIN C?U 3.1. Phng ti?n 3.1.1. Th?i gian v ?a i?m ti?n hnh th nghi?m - Th?i gian: t? thng 06 nm 2011 ?n thng 12 nm 2011. - ?a i?m: ph?ng th nghi?m Cng ngh? Sinh h?c Th?c ph?m, Vi?n Nghin c?u v Pht tr Cng ngh? Sinh h?c, Tr?ng ?i h?c C?n Th. 3.1.2. Nguyn li?u - D?ng vi khu?n Acetobacter xylinum mua t? Trung tm chuy?n giao cng ngh? (S? Khoa H?c Cng Ngh?, B?n Tre). - Vi khu?n Lactobacillus bulgaricus, v Streptococcus thermophilus trong s?a chua Vinamilk. - N?c d?a kh mua ? ch? Xun Khnh. - S?a ti ti?t trng khng ?ng c?a Vinamilk mua ? siu th? Coop-Mart. 3.1.3. D?ng c? v thi?t b? - ?u cone (cone vng v cone xanh), pipet, ?ng nghi?m, ?ng ong, ?a petri, que c?y , lame, lamelle, b?nh tam gic 250 ml, brix k?, cn phn tch, knh hi?n vi quang h?c, my l?c, n?i h?p ti?t trng, pH k?, t? c?y v trng, t? l?nh, t? ? v m?t s? d?ng c? v thi?t b? khc th?ng dng trong ph?ng th nghi?m. 3.1.4. Ha ch?t

- Glucose, saccharose, acid acetic, ethanol, (NH4)2SO4 (SA), (NH4)2HPO4 (D AP), Agar, CaCl2 - Ha ch?t dng trong phn tch acid t?ng: NaOH 0.8M, phenolphtalein. - Mi tr?ng th?ch MRS agar (De Man, Rogosa and Sharpe). - Mi tr?ng l?ng MRS broth.

3.2. Phng php 3.2.1.S ? ln men t?ng qut

H?nh 3. S ? quy tr?nh ln men t?ng qut 3.2.2. Gi?i thch quy tr?nh - N?c d?a ?c ch?a trong l? v ? qua m nh?m h? pH xu?ng 4.0 v b? sung saccharose - Ti?n hnh kh?trng mi tr?ng? ph?i ch?? 115C trong 15 pht ? tiu di?t cc v ng mi tr?ng v ? ngu?i. - Mi tr?ng ? ngu?i ?c b? sung s?a. - Ch?nh pH mi tr?ng b?ng n?c d?a cha ? nh?m t?o cc gi tr? pH thch h?p cho th m. - Sau cho mi tr?ng vo cc h? th?y tinh ckch th?c, th?tch nhnhau, ch?ng etobacter v vi khu?n lactic trong s?a chua, ?y l?i b?ng n?p v em ln men trong t? ? 30 trong v?ng 7 ngy. Trong th?i gian ny c?n ? yn, khng nn khu?y ?ng ? trnh ?nh h?ng hemicellulose ang h?nh thnh. - Trong qutr?nh th?c hi?n? ti, t? l? nguyn li?u (t? l? s?a ti v n?c d?a), m u?n ch?ng vo, t? l? saccharose b? sung, pH mi tr?ng c?ng nh nhi?t ? v th?i gian ? tu heo b? tr c?a t?ng th nghi?m c? th?. Nguyn li?u s? ?c ch?n thng qua th nghi?m 1, m?t vi khu?n ch?ng vo mi tr?ng s? ?c ch?n thng qua th nghi?m 2, n?ng ? saccharose b? s pH ban ?u c?a mi tr?ng s? ?c ch?n thng qua th nghi?m 3, nhi?t ? v th?i gian ln me n thng qua th nghi?m 4. K?t thc m?i th nghi?m s? li?u s? ?c x? l? b?ng chng tr?nh S APHICS Plus v3.0, ch?n ra i?u ki?n t?t nh?t ln men. 3.2.3. Cc th nghi?m th?c hi?n a) Th nghi?m 1: Kh?o st ?nh h?ng c?a ty l nguyn li?u ?n qu tr?nh ln men th?ch > M?c ch th nghi?m: ch?n ty l nguyn li?u thch h?p cho qu tr?nh ln men th? > B? tr th nghi?m: th nghi?m ?c b? tr hon ton ng?u nhin v?i ngu?n nguyn l trong i?u ki?n b? sung n?c d?a v?i t? l? l?n l?t l 4/1, 3/1, 2/1, 1/1, 1/2, 1/3, v 1/ 4. Th nghi?m c 7 nghi?m th?c, ?c b? tr v?i 1 nhn t? v 3 l?n l?p l?i. T?ng c?ng c 7 v? th nghi?m. > Phng php ti?n hnh th nghi?m: * Chu?n b?: 600 ml n?c d?a, 600 ml s?a ti, vi khu?n Acetobacter v s?a chu a, h? th?y tinh 50 ml, ?ng ong 100 ml v 25 ml,... * Ti?n hnh th nghi?m: n?c d?a mua v? ch?a trong b?c, ?c ? qua m, l?c ? b ch?t. N?c d?a sau khi l?c ?c kh? trng ? 115C trong 15 pht v ? ngu?i. N?c d?a ? n sung s?a theo t? l? b? tr th nghi?m v cho vo cc h? th?y tinh ? kh? trng (m?i h? 49 m ung d?ch n?c d?a-s?a), ch?ng gi?ng 0,5 ml Acetobacter m?t s? 108 tb/ml v 0,5ml vi khu?n lactic m?t s? 108 tb/ml, ?y h? l?i b?ng n?p (khng ?y kn qu) v ??nhi?t? ph?n

rong th?i gian ln men c?n ? yn, khng nn khu?y ?ng d?ch ln men ? trnh ?nh h?ng l?p ulose ang h?nh thnh. > Ch? tiu theo d?i: * Cn kh?i l?ng th?ch t?o thnh. * o ? Brix c?a s?n ph?m ln men. * pH c?a s?n ph?m ln men. b) Th nghi?m 2: Kh?o st ?nh h?ng c?a t? l? vi khu?n Acetobacter xylinum v vi khu? n lactic ?n qu tr?nh ln men thach sa. > M?c ch: xc ?nh t? l? gi?ng ch?ng thch h?p ? ln men th?ch s?a. > B? tr th nghi?m: d?a vo k?t qu? c?a th nghi?m 1 ? ti?n hnh ln men. V?i m?t ban ?u l 108 c?a Acetobacter v s?a chua gi?ng ?c ch?ng nh sau: * Nghi?m th?c 1: 1ml Acetobacter v 3ml vi khu?n lactic * Nghi?m th?c 2: 1ml Acetobacter v 2ml vi khu?n lactic * Nghi?m th?c 3: 1ml Acetobacter v 1ml vi khu?n lactic * Nghi?m th?c 4: 2ml Acetobacter v 1ml vi khu?n lactic * Nghi?m th?c 5: 2ml Acetobacter v 2ml vi khu?n lactic * Nghi?m th?c 6: 2ml Acetobacter v 3ml vi khu?n lactic * Nghi?m th?c 7: 3ml Acetobacter v 1ml vi khu?n lactic * Nghi?m th?c 9: 3ml Acetobacter v 2ml vi khu?n lactic * Nghi?m th?c 10: 3ml Acetobacter v 3ml vi khu?n lactic Th nghi?m c 9 nghi?m th?c, ?c b? tr v?i 1 nhn t? v 3 l?n l?p l?i. T?ng c? c 93 = 27 n v? th nghi?m. > Phng php ti?n hnh th nghi?m: t? l? nguyn li?u ch?n ? th nghi?m 1 ?c dng th nghi?m ny. Cc b?c chu?n b? mi tr?ng ln men, th?i gian v nhi?t ? ln men tng ?m 1. Nhng vi khu?n Acetobacter v vi khu?n lactic ?c ch?ng vo theo t? l? b? tr th ng m. > Ch? tiu theo d?i: * Kh?i l?ng th?ch t?o thnh. * ? Brix c?a s?n ph?m ln men. * pH c?a s?n ph?m ln men. c) Th nghi?m 3: Kh?o st ?nh h?ng c?a hm l?ng ?ng v pH ban ?u ?n qu tr?nh ln > M?c ch: xc ?nh l?ng ?ng v pH ban ?u thch h?p cho qu tr?nh ln men th?c > B? tr th nghi?m d?a vo k?t qu? c?a cc th nghi?m 1 v 2 ? ti?n hnh ln men. ?m ?c l?p l?i 3 l?n, m?i l?n c 25 nghi?m th?c d?a trn 5 m?c pH (nhn t? H) v 5 hm l? (nhn t? B) khc nhau. T?ng c?ng c 553 = 75 n v? th nghi?m. * Nhn t? H g?m H1: pH 4,0; H2: pH 4,5; H3: pH 5,0; H4: pH 5,5. * Nhn t? B g?m B1: khng b? sung ?ng; B2: b? sung 3% (w/v); B3: b? sung 6% (w/v); B4: b? sung 9% (w/v); B5: b? sung 12% (w/v). B?ng 7. Hm l?ng ?ng v pH ban ?u trong ln men th?ch s?a H4B5 H4B4 H4B3 H4B2 H4B1 H3B5 H3B4 H3B3 H3B2 H3B1 H2B5 H2B4 H2B3 H2B2 H2B1 H1B5 H1B4 H1B3 H1B2 H1B1l?ng saccharose ti?n hnh th 32 5,0 Khng Hm4,5 pH 4,0b?>sung ?ng b? sung (%w/v) nghi?m: N?c d?a ?c ? qua m, l?c v ?c chia lm 5 1 9 6 5,5 Phng php m?i ph?n 720 ml), ?c b? sung saccharose theo t? l? l?n l?t l 3, 6, 9, 12% (w/v) v khn b? sung saccharose. Sau em kh? trng ? 115C, trong 15 pht v ? ngu?i. N?c d?a ? n -40C ?c b? sung s?a theo t? l? ? ?c ch?n ? th nghi?m 1. Cho h?n h?p d?a v s?a vo c y tinh ? kh? trng (m?i h? 49 ml), a pH mi tr?ng v? cc m?c ? c?n b? tr b?ng n?c d? n, ch?ng gi?ng vi khu?n Acetobacter v lactic theo t? l? ? ?c ch?n ? th nghi?m 2, ?y l i b?ng n?p v ??nhi?t? ph?ng 6 ngy. > Ch? tiu theo d?i: * Kh?i l?ng th?ch t?o thnh. * ? Brix c?a s?n ph?m ln men. * pH c?a d?ch ln men. d) Th nghi?m 4: Kh?o st ?nh h?ng c?a nhi?t ? ? v th?i gian ln men ?n qu tr?nh l h?ch s?a. > M?c ch: xc ?nh nhi?t ? v th?i gian ph h?p cho qu tr?nh ln men th?ch s?a. > B? tr th nghi?m d?a vo k?t qu? c?a cc th nghi?m 1, 2, v 3 ? ti?n hnh ln nghi?m ?c l?p l?i 3 l?n, g?m 20 nghi?m th?c d?a trn 2 nhn t? nhi?t ? (nhn t? G) v th gian ln men (nhn t? H). * Nhn t? nhi?t ? ln men: nhi?t ? ph?ng (28-32C); 25C; 30C; 35C. * Nhn t? th?i gian ln men: ngy th? 6 ln men, ngy th? 7 ln men, ngy th? 8 en, ngy th? 9 ln men, ngy th? 10 ln men. T?ng c?ng ta c: 5x5x3= 75 n v? th nghi?m.

B?ng 8. Nhi?t ? v th?i gian ln men th?ch s?a Nhi?t ? ph?ngmen (ngy) Th?i gianln men (C) ln N10/35 N10/30 N10/25 N10/P 10 N9/35 N9/30 N9/25 98/P N8/35 N8/30 N8/25 87 N7/35 N7/30 N7/25 N7/P N6/35 N6/30 N6/25 66/P 35 30 25 (28-32C) N9/P > Phng php ti?n hnh th nghi?m: N?c d?a ?c ? qua m, sau ?c b? sung sa l? ? ?c ch?n ? th nghi?m 3 v ?c kh? trng ? 115C, trong 15 pht v ? ngu?i. N?c d ung s?a theo t? l? ? ?c ch?n ? th nghi?m 1. Sau cho mi tr?ng vo cc h? th?y tinh (m?i h? 49 ml), ch?ng gi?ng vi khu?n Acetobacter v s?a chua theo t? l? ? ?c ch?n ? t h nghi?m 2, ?y l?i b?ng n?p v ??cc nhi?t? c?n b? tr. K?t qu? th nghi?m ?c thu ? 6, 7,8, 9, 10. > Ch? tiu theo d?i: * Cn kh?i l?ng th?ch t?o thnh. * o ? Brix c?a s?n ph?m ln men. * pH c?a d?ch ln men. e) Th nghi?m 5: Kh?o st ?nh h?ng c?a s? b? sung CaCl2, SA v DAP ?n qu tr?nh t?o he ihemicellulose trong mi tr?ng s?a ln men > M?c ch: T?m ra i?u ki?n t?i u cho vi?c h?nh thnh hemicellulose khi b? sung CaCl2, SA v DAP. > Cch th?c hi?n: T? k?t qu? cc th nghi?m 1, 2 v 3, ti?n hnh th nghi?m b? sung CaCl2 0.2g/l (theo L H?c V?), SA 0.1g/l (theo Nguy?n Th? Thy Hng) v DAP 0.1g/l (theo Nguy?n Th? Thy Hng): h?i l?ng CaCl2 b? sung theo Vng Tr?ng Ho v T?ng Th? M (2002), b? sung SA v DAP theo n Ph H?a (1992). Th nghi?m ?c ti?n hnh v?i cc nghi?m th?c (NT) nh sau: NT1: khng b? sung NT2: SA NT3: DAP NT4: CaCl2 NT5: SA-DAP NT6: SA-CaCl2 NT7: DAP-CaCl2 NT8: SA-DAP-CaCl2 Th nghi?m g?m 8 nghi?m th?c, v?i 1 nhn t? v ?c th?c hi?n 3 l?n l?p l?i. > Ch? tiu theo d?i - o ? brix v pH c?a s?n ph?m ln men. - Cn kh?i l?ng hemicellulose t?o thnh. K?t lu?n: ?nh h?ng c?a CaCl2, SA v DAP ?n kh? nng t?o hemicellulose.

PH?N IV. N?I DUNG V K?T QU? ?T ?C 4.1. K?t qu? ty l nguyn li?u c kh? nng ln men cho s?n ph?m thach sa t?t. Nguyn li?u s? d?ng trong ln men ?nh h?ng r?t l?n ?n ch?t l?ng s?n ph?m t?o thnh. K i c m?t t? l? ln men thch h?p th? kh?i l?ng th?ch t?o ra s? nhi?u v hemicellulose ?c o thnh s? c ch?t l?ng t?t. Theo L Th? Lin (2010) th? hi?u qu? ln men t?o hemicellulose khi ln men v?i n?c d? a ?c ? cao hn so v?i khi ln men v?i n?c d?a khng ?c ?. Trong giai o?n ph?i ch?, n?c d?a cha qua ln men t? nhin ch?a ch? y?u l saccharose, c?n nhi?u acid acetic ? i?u ch?nh pH. Nh v?y, khi n?c d?a ?c ? th? hm l?ng saccharo ?m, hm l?ng glucose tng v pH mi tr?ng gi?m, hm l?ng acid acetic b? sung vo t, hem e ?c t?ng h?p ?c nhi?u. N?u n?c d?a ?c ? tr?c khi ti?n hnh ln men p d?ng vo trong th?c t? th? r?t c ? n ong s?n xu?t v? c th? ch? ?ng ?c ngu?n n?c d?a ? nhi?u ni, ?ng th?i do s? hi?n di?n cc vi sinh v?t c trong n?c d?a lm cho pH gi?m gip ti?t ki?m ?c acid acetic ? i?u H ban ?u, n?c d?a s? khng b? vng khi n?u m c?n cho hi?u qu? ln men t?o hemicellulose ao. Theo Budhiono et al. (1999), saccharose ?c t?m th?y trong n?c d?a ti nhng ch? c g ose t?m th?y trong n?c d?a ? ? v ng k?t lu?n hemicellulose ?c t?o thnh ch? y?u do s? ng glucose. Vi khu?n acid lactic c?ng phn gi?i lactose thnh glucose nn t nhi?u c?ng ?nh h?ng ? qu tr?nh t?o hemicellulose trong qu tr?nh ln men s? d?ng mi tr?ng l nguyn li?u d?a. K?t qu? th nghi?m kh?o st ?nh h?ng c?a t? l? nguyn li?u ?n qu tr?nh ln men ?c t ? H?nh 4.

H?nh 4. Kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc t? l? nguyn li?u khc nhau Ghi ch: Cc s? li?u bi?u th? trn cc c?t trong bi?u ? l gi tr? trung b?nh c?a 3 l?n l? ?i. Cc gi tr? c cng k? t? th? hi?n s? khc bi?t khng c ? ngh?a th?ng k ? ? tin c?y K?t qu? th nghi?m cho th?y vi khu?n s? d?ng ? ln men th?ch s?a khng t?o th?ch kh i trong mi tr?ng ch? c s?a, v? vi khu?n sinh acid lactic khng c kh? nng t?o hemicellu ose. Qu tr?nh ln men t?o th?ch x?y ra khi trong mi tr?ng t?n t?i n?c d?a ho?c c? n?c ?a v s?a. Kh?i l?ng hemicellulose ?t gi tr? cao nh?t khi t? l? n?c d?a/s?a ti l 3/1 h 4). Theo k?t qu? th?ng k v?i ? tin c?y 95% ? t? l? nguyn li?u l 3/1 th? kh?i l?ng h emicellulose ?c t?ng h?p cao nh?t v khc bi?t c ? ngh?a v?i t?t c? cc nghi?m th?c c?n ?i. Do t? l? ny ?c ch?n ? ti?n hnh th nghi?m ti?p theo. 4.2. ?nh h?ng c?a t? l? vi khu?n Acetobacter v vi khu?n lactic ?n qu tr?nh ln men tha h sa. T? k?t qu? th nghi?m ch?n l?c t? l? nguyn li?u, cc d?ng vi khu?n Acetobacter, La ctobacillus bulgaricus v Streptococcus thermophilus ?c ch?ng theo t? l? b? tr th nghi ?m v?i m?t s? ban ?u l 108 tb/ml. T?t c? cc nghi?m th?c ?c ? ? nhi?t ? v th?i gian g g nh ? th nghi?m 1. K?t qu? ?c tr?nh by ? H?nh 5:

H?nh 5. Kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc t? l? vi khu?n khc nhau Ghi ch: Cc s? li?u bi?u th? trn cc c?t trong bi?u ? l gi tr? trung b?nh c?a 3 l?n l? ?i. Cc gi tr? c cng k? t? th? hi?n s? khc bi?t khng c ? ngh?a th?ng k ? ? tin c?y Kh?i l?ng hemihemicellulose ?t gi tr? cao nh?t khi t? l? Acetobacer/vi khu?n aci d lactic ch?ng vo d?ch ln men l 2/1 (19,66g), khc bi?t khng c ? ngh?a th?ng k v?i ng m th?c ch?ng t? l? Acetobacter/vi khu?n acid lactic l 3/1 (18,25g) v t? l? 1/2 (17 ,36g). Tuy nhin ta ch?n t? l? 2/1 ? ti?n hnh th nghi?m ti?p theo v? s? ti?t ki?m ?c g ?ng nn c l?i cho vi?c s?n xu?t. 4.3. ?nh h?ng c?a hm l?ng ?ng v pH ban ?u ?n qu tr?nh ln men pH v hm l?ng ?ng b? sung ban ?u cho qu tr?nh ln men r?t quan tr?ng, n?u qu t qu cao ?u ?c ch? qu tr?nh ln men. pH v hm l?ng ?ng b? sung ph h?p th? qu tr?nh ra t?t, c?n ng?c l?i th? qu tr?nh ln men s? khng t?t ho?c khng x?y ra (Theo gio tr?nh th?c t?p Tr?n V? Phng v Cao Ng?c i?p). ?ng l c ch?t c?a qu tr?nh ln men nn ?nh h?ng nhi?u ?n hi?u su?t ln men, nn llulose t?o ra cao hay th?p s? ph? thu?c vo hm l?ng ?ng ?c s? d?ng trong d?ch ln me i hm l?ng ?ng th?p th? khng ? ngu?n th?c n cho vi khu?n d?n ?n hi?u su?t ln men th khi hm l?ng ?ng cao, th? hi?u su?t ln men c?ng th?p do ?ng ?c chuy?n ha thnh gl . L?ng gluconic acid tch t? ngy cng nhi?u s? lm gi?m pH xu?ng th?p hn so v?i m?c pH t i thch cho vi khu?n Acetobacter (pH t? 4,0-6,0) pht tri?n t?t nh?t ? t?o hemicellul ose v vi khu?n lactic (c th? pht tri?n ? pH 3,8-4,0 v ln men m?nh nh?t ? pH 5,5-6,0) ln men chua s?a. Mt yu t khac cung anh hng ln n qua trinh ln men la p ?t c?a vi khu?n, v? v?y l?ng hemicellulose t?o ra cao hay th?p ph? thu?c vo pH c?a

qu tr?nh ln men. Th nghi?m s? d?ng t? l? nguyn li?u ?c ch?n l?c t? th nghi?m 1 v t? l? vi khu?n h?ng vo d?ch ln men t? th nghi?m 2. D?ch ln men ?c b? sung saccharose v?i cc hm l? l?t l 3, 6, 9 v 12% (w/v) v khng b? sung saccharose v i?u ch?nh pH l?n l?t v? 4,0; 5,0 v 5,5 . T?t c? cc nghi?m th?c ?c ? ? nhi?t ? mi tr?ng xung quanh trong 6 ngy. K?t qu? ?nh by ? B?ng 9.

B?ng 9. Hm l?ng acid t?ng, ? Brix v kh?i l?ng hemicellulose t?o thnh sau khi ln v?i cc hm l?ng ?ng b? sung v cc m?c pH khc nhau *Ghi 13.36bcd 12,73 10.92cde 8,67 12.21cd 9,01 10 8,677 5,5 15.71b 12,8 24.31a 14,8 0,44 11.56cd 14,3 11,87 0,64 5,0 16.08b 11,64 10.87de 11,2 13,2 0,48 12 0,36 10,47 4,5 8.07e 12% 11.99cd 11,67 0,56 9% 13.86bc 10,93 0,52 6% 8? 0,60 0f 11,47 0,58 0% ch: 4,0 l?ng hemicellulose ngh?a th?ng (w/v)) Kh?iBrix S? khc Hm l?ng acid 3% bi?t ? (g/50 ml) pH ban ?u?ngt?ng (% (w/v)) sung (%k ch? c ? ngh?a theo c?t. Cc gi tr? trung b?nh cn 0f saccharose b? ? t? th? hi?n s? khc bi?t khng c ? ngh?a th?ng k ? ? tin c?y 95%. Masaoka et al. (1992) k?t lu?n n?u hm l?ng ?ng b? sung vo mi tr?ng ban ?u qu ng gluconic acid sinh ra cng nhi?u, hi?u qu? t?ng h?p hemicellulose c?ng gi?m. Th eo Toda et al. (1997) hm l?ng gluconic acid sinh ra trong su?t qu tr?nh ln men s? tng sau s? gi?m tr? l?i, theo pH mi tr?ng ban ?u gi?m c?ng s? tng tr? l?i. Masaoka e (1992) c?ng ? c?p gluconic acid c?n ?c chuy?n ha thnh ch?t khc trong qu tr?nh ln m h v?y, v?i hm l?ng ?ng b? sung ban ?u l 9% (w/v) th? khng cao nn l?ng gluconic ac khng nhi?u, chng ? nhanh chng chuy?n ha thnh ch?t khc. Cc nghi?m th?c khng b? sung v b? sung 3% (w/v) ?ng khng ph h?p ? ln men ? t?t c pH nn qu tr?nh ln men t?o th?ch khng x?y ra. C?n nghi?m th?c c hm l?ng ?ng b? sun nhng pH khng ph h?p th? qu tr?nh ln men c?ng khng x?y ra nh nghi?m th?c H3B3 (b? su 6% ?ng v pH 4,5). Nghi?m th?c 9% (w/v) ?ng v pH 5,0 cho hi?u qu? ln men t?o th?ch cao v khc bi?t c gh?a th?ng k so v?i cc nghi?m th?c c?n l?i ? m?c ? ngh?a 5% (H?nh 6 v B?ng 9). ?ng t h?i s?n ph?m ln men ? nghi?m th?c ny mang nh?ng ?c i?m c?m quan t?t hn (c mi thm c? chua, v? ng?t v?a ?) so v?i s?n ph?m t?o thnh ? cc nghi?m th?c khc. Do nghi?m th?c ?c ch?n ? ti?n hnh th nghi?m ti?p theo. Hm l?ng acid t?ng v ? Brix sau ln men ? B?ng 9 cho th?y khng c s? thay ?i l?n gi nghi?m th?c c cng hm l?ng ?ng b? sung ban ?u. K?t qu? cho th?y khi n?c d?a ?c ? qua m c?ng cho hi?u qu? t?t nh?t, i?u ny r?t c h?a v? khng c?n ph?i b? sung nhi?u ho ch?t ? i?u ch?nh pH. Ngoi ra, vi?c ? qua m c?n ?o i?u ki?n thu?n l?i cho s?n xu?t v? ph?i m?t nhi?u th?i gian ? thu l?ng n?c d?a v c hu?n b?. 4.4. ?nh h?ng c?a nhi?t ? ? v th?i gian ln men ?n qu tr?nh ln men th?ch s?a. Thi nghim nay c tin hanh vi cac iu kin thch h?p a c khao sat l?n l?t l 25, 30, 35C v ? nhi?t ? ph?ng (28-32C) trong 6, 7, 8, 9 v 10 ngy. K?t qu ?nh by ? B?ng 10. B?ng 10. Hm l?ng acid t?ng, ? Brix v kh?i l?ng hemicellulose t?o thnh sau khi ln n ? cc nhi?t ? v th?i gian khc nhau *Ghi 14,2 6,5 22,72a 10,84de 14,97 25 10 14,3 10,93 20,01ab 14,1 1,08 10.25de 14,77 0,84 9 14,4 1,24 17,14bcd 10,47 10,49de 14,27 1,12 5.86e 10,2 0,80 8 14,7 13,98cd 14,23 22,07a 11,8 1,16 12,9 0,96 7 14 1,32 35 11,83cd 12.2 1,04 30 11,2de 14,6 1,28 0f 11,87 1,00 ? hemicellulose 65 Kh?i ch: S? ? l?ngacid t?ng (% HmBrix lnkhc bi?t ? ngh?a Nhi?tgianph?ngmen (ngy) (g/50 th?ng Th?il?ngln men (C) (w/v)) ml) k ch? c ? ngh?a theo c?t. Cc gi tr? trung b?nh cn 2 ? t? th? hi?n s? khc bi?t khng c ? ngh?a th?ng k ? ? tin c?y 95%. Ngoi cc nhn t? kh?o st ? cc th nghi?m tr?c th? nhi?t ? v th?i gian ln men c?n r?t l?n ?n qu tr?nh ln men. Trong nghin c?u s?n xu?t th?ch s?a, ngoi ?c i?m c?m qua ?a s?n ph?m tng t? nh ?c i?m c?a s?a ln men lactic th? kh?i l?ng hemicellulose ?c trong s?n ph?m l quan tr?ng nh?t. Kh?i l?ng hemicellulose ?t gi tr? cao ? cc nghi?m th?c N8/30 (ln men trong 8 ngy ? 30C), N9/30 (ln men trong 9 ngy ? 30C), N7/P (ln me rong 7 ngy ? nhi?t ? ph?ng), v ?t gi tr? cao nh?t ? nghi?m th?c N10/P (ln men trong 1 ngy ? nhi?t ? ph?ng) (H?nh 7 v B?ng 10). Tuy kh?i l?ng th?ch t?o thnh th?p hn nhng nghi?m th?c N7/P (ln men trong 7 ngy nhi?t ? ph?ng) khc bi?t khng c ? ngh?a th?ng k v?i nghi?m th?c N10/P (ln men trong 1 ngy ? nhi?t ? ph?ng), l nghi?m th?c cho kh?i l?ng hemicellulose cao nh?t ? ? tin c?y 95%. K?t qu? phn tch th?ng k c?ng cho th?y nghi?m th?c N7/P c l?ng hemicellulose t?o thnh c?ng khng khc bi?t ? ngh?a v?i cc nghi?m th?c N8/30 v N9/30. So v?i nghi?m th?c N8/30 v N9/30 th? nghi?m th?c N7/P khng c?n trang thi?t b? ? i?u ch?nh nhi?t ?, c?n so v?i nghi?m th?c N10/P th? s? ti?t ki?m ?c th?i gian ln men nn se mang tinh kinh t

hn khi a vo s?n xu?t v? ti?t ki?m c trang thi?t b? v thi gian tao ra san ph men 7 ngy ? nhi?t ? ph?ng l nghi?m th?c thch h?p nh?t v c kh? nng ?ng d?ng cao khi s xu?t ? qui m nng h?. 4.5 ?nh h?ng c?a ch?t b? sung ?n qu tr?nh ln men th?ch s?a. Th nghi?m ny ?c ti?n hnh d?a trn cc i?u ki?n c?a th nghi?m 1, 2, 3, 4. Ta ti ? sung SA (0.1g/l), DAP (0.1g/l), CaCl2 (0.2g/l) theo b? tr th nghi?m. K?t qu? ?c tr ?nh by ? h?nh 8.

H?nh 8. Kh?i l?ng th?ch t?o thnh khi ln men v?i cc ch?t b? sung khc nhau. D?a vo bi?u ? ta th?y nghi?m th?c c b? sung SA v CaCl2 c khc bi?t ? ngh?a v? m? th?ng k so v?i cc nghi?m th?c c?n l?i. Ch? vi?t th?m nhe

PH?N IV. K?T LU?N V ? NGH? 5.1. K?t lu?n Qua qu tr?nh nghin c?u, cc k?t qu? ?c rt ra nh sau: t? l? nguyn li?u s?a v n? men t?t nh?t l 3/1, t? l? vi khu?n ch?ng vo d?ch ln men ? ln men t?t nh?t l 2% Acetob cter v 1% vi khu?n acid lactic (m?t s? 108 tb/ml), hm l?ng ?ng b? sung l 9% (w/v) sac harose v i?u ch?nh pH v? 5,0 tr?c khi ln men. Qu tr?nh ln men t?t di?n ra ? nhi?t ? ng trong th?i gian 7 ngy. Rt ra ?c quy tr?nh ln men th?ch s?a ?c rt ra nh sau:

H?nh 8. S ? quy tr?nh ln men th?ch s?a 5.2. ? ngh? > Kh?o st ?nh h?ng c?a cc lo?i ?ng b? sung khc nhau. > ? hon thi?n s?n ph?m th?ch s?a c?n ti?p t?c nghin c?u k? hn v? t? l? ph?i ch? n guyn li?u v ?ng, cch b?o qu?n s?n ph?m,... ki?m tra cc ch? tiu vi sinh v l? ha,...

TI LI?U THAM KH?O

Ti?ng vi?t Kiu Hu Anh, 1999. Giao trinh Vi sinh v?t h?c Cng nghi?p. NXB Khoa hoc va Ky thu 05-109. L Th? Lin, 2010. Nghin c?u cc y?u t? t?o hemicellulose c?a Acetobacter trong s?n xu ?t th?ch d?a. Lu?n vn T?t nghi?p ?i h?c ngnh Cng ngh? Sinh h?c. Lng ?c Ph?m, 1998. Cng ngh? Vi sinh v?t. NXB Nng Nghi?p, trang 224-227. Lng ?c Ph?m, 2002. Vi sinh v?t h?c v an ton v? sinh th?c ph?m. NXB Nng Nghi?p, H N?i rang 58-81 v 91. Nguy?n ?c L?ng, 2002. Vi sinh v?t h?c Cng nghi?p. NXB ?i h?c Qu?c gia Thnh ph? H? Ch inh, trang 307-312. Nguy?n Thy Hng, 2007. ?nh h?ng c?a ngu?n c ch?t v ki?u ln men ?n nng su?t v ch?t ellulose vi khu?n. T?p ch Khoa h?c HQGHN, Khoa h?c T? nhin v Cng ngh? 24 (2008) 205-2 10. Ti?ng anh Abee, T. and R. Nout, 1999. Food fermentation part 1, Department of Food Technol ogy and Nutritional Sciences, Wageningen Agriculture University. Barefoot, S.F. and T.R. Klaenhammer, 1983. Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Appl Environ Microbiol. J un;45(6):1808 1815. Bruce, F., 2005. Coconut Cures. Piccadilly Books, Ltd, ISBN 0-941599-60-4. Buck, B.L., M.A. Azcarate-Peril and T.R. Klaenhammer, 2009. Role of autoinducer2 on the adhesion ability of Lactobacillus acidophilus. HYPERLINK "/content/bsc/ jam%3Bjsessionid=9d7akabl9toob.alice"Journal of Applied Microbiology, Volume 107,Num ber 1, July , pp. 269-279 (11). Budhiono, A., B. Rosidi, H. Taher and M. Iguchi, 1999. Kinetic aspects of bacter ial hemicellulose formation in nata-de-coco culture system. Carbohydrate Polymer s 40:137-143. Chawla, P.R., I.B. Bajaj, S.A. Survase and R.S. Singhal, 2008. Microbial Hemicel lulose: Fermentative Production and Applications. Food technol. Biotechnol 47:10 7-124. Gonzal Angel, Jose Manuel Guillamon, Albert Mas and Montse Poblet, 2005. Application of molecular methods for routine identification of acetic acid bacteria. Intern ational Journal of Food Microbiology 108:141-146. Kaderel, T.T., T. Miyamoto, R.K. Oniang`o, P.M. Kutima1 and S.M. Njoroge, 2008. Isolation and Identification of the Genera Acetobacter and Gluconobacter in Coco nut Toddy (mnazi). African Journal of Biotechnology 7:2963-2971. Maal, K.B and S. Shafiee, 2009. Isolation and Identification of an Acetobacter S train from Iranian White-Red Cherry with High Acetic Acid Productivity as a Pote ntial Strain for Cherry Vinegar Production in Food and Agriculture Biotechnology

. World Academy of Science, Engineering and Technology 54:201-204. Masaoka, S., T. Ohe and N. Sakota, 1992. Production of Hemicellulose from Glucos e by Acetobacter xylinum. Juornal of Fermentation and Bioengineering 75:18-22. Nagendra, P.S., E.V.L. Warnakulasuriya, L.B. Margaret and S.A.K. William, 2000. Survival of Lactobacillus acidophilus and Bifidobacterium bifidum in commercial yoghurt during refrigerated storage, Department of Food Technology, Victoria Uni versity of Technology, PO Box 14428, MMC, Melbourne, Victoria 3000, Australia. Perdigon, G., G.C. Maldonado, J.C. Valdez and M. Medici, 2002. Interaction of la ctic acid bacteria with the gut immune system, European Journal of Clinical Nutr ition, vol.56 Suppl 4. Russell, N.J. and G.W. Gould, 2003. Food preservatives (second edition). TP371.2 .F67 2003. Toda, K., T. Asakura, M. Fukaya, E. Entani and Y. Kawamura. 1997. Hemicellulose Production by Acetic Acid-Resistant Acetobacter xylinum. Juornal of Fermentation and Bioengineering 84:228-231. USDA National Nutient Database For Standard Reference. Release 19. 2006. Trang web http:// HYPERLINK "http://www.nlm.nih.gov/medlineplus/druginfo/natural/patient-ac idophilus.html" www.nlm.nih.gov/medlineplus/druginfo/natural/patient-acidophilus.h tml (24/03/2011). HYPERLINK "http://suckhoecongdong.vn/News/modules.php?name=News&op=viewst&sid=33 " http://suckhoecongdong.vn/News/modules.php?name=News&op=viewst&sid=33 (23/03/201 1). HYPERLINK "http://en.wikipedia.org/wiki/Lactobacillus_acidophilus" http://en.wiki pedia.org/wiki/Lactobacillus_acidophilus (09/04/2011). http://en.wikipedia.org/wiki/Acetobacter (09/04/2011). HYPERLINK "http://www.dairycouncilofca.org/Milk-Dairy/"http://www.dairycouncilofca. org/Milk-Dairy/ (2000) (09/04/2011). PH? L?C 1. M?T S? HINH ANH LIN QUAN ?N ? TI

H?nh 9. H?nh d?ng kh?n l?c vi khu?n Actobacter

H?nh 10. knh hi?n vi quang h?c (2), n?i kh? trng nhi?t ?t (4), t? l?nh (4), pH k? ( 5), t? ? (6)

H?nh 11. Mi tr?ng ang ln men (7), th?ch s?a thnh ph?m (8) PH? L?C 2. CC PHNG PHP PHN TCH 2.1. Cng th?c mi tr?ng nui c?y Acetobacter

> Glucose 10 g > DAP 2 g > SA 8 g > N?c d?a kh 1.000 ml > Acid acetic (i?u ch?nh ?n khi pH = 4,5) (Budhiono et al., 1999) > Agar 40 g 2.2. o pH Gi tr? pH u?c o b?ng pH k?. 2.3. o Brix Gi tr? Brix ?c o b?ng Brix k?. 2.4. o hm l?ng acid t?ng > Chu?n b? dung d?ch NaOH 0,8M v dung d?ch phenoltalein (40 mg phenoltalein/25 ml ethanol 75%). > Bm vo ?ng nghi?m ? ch?a 1 ml m?u c?n o acid t?ng 2-3 gi?t dung d?ch phenoltalei n ? chu?n b?, ti?p t?c bm NaOH 0,8M vo (m?i l?n 25 l) v l?c ?u. Bm cho t?i khi mu h i?n di?n b?n trong dung d?ch th? ng?ng l?i. Ghi l?i s? l?n bm NaOH. > Cng th?c tnh hm l?ng acid t?ng (% (w/v)): Hm l?ng acid t?ng = 0,12(s? l?n bm NaOH 1) 2.5. Quan st vi khu?n b?ng knh hi?n vi > Nh? 1 gi?t n?c c?t v trng ln lame. > Kh? trng kim c?y trn ng?n l?a n c?n v ? ngu?i. > Dng kim c?y l?y m?t t khu?n l?c r?i tr?i ?u ln gi?t n?c trn lame. Dng lamelle gi?t n?c b?ng cch ? m?t c?nh c?a lamelle ti?p xc v?i lame m?t gc 45o r?i h? t? t? v n ? nhng sao cho trong m?u khng c b?t kh. > Quan st m?u v?t d?i knh hi?n vi i?n t? v ch?p h?nh. 2.6. ?m m?t s? vi khu?n b?ng bu?ng ?m h?ng c?u > Dng n?c ? kh? trng pha lo?ng m?u. ? pha lo?ng th? no ? khi ?m, s? t? bo trong khng qu 15 t? bo. > Dng ?ng ht chuy?n vi sinh v?t vo h?p ?m sao cho khng c b?t kh. > ?t bu?ng ?m ln bn knh hi?n vi, sau ti?n hnh ?m t? bo trong 4 l?n ? 4 gc. n, ?m l?n l?t t? con th? nh?t ?n con th? 16. Ch? ?m t? bo n?m bn trong con v n n?m 2 c?nh lin ti?p cng chi?u. Ghi s? l?ng t? bo ?m ?c trong 4 l?n. ?m c? 2 bu?n l?y gi tr? trung b?nh. Sau khi dng xong bu?ng ?m v l knh ph?i ?c em i r?a nga > S? l?ng t? bo trong 1 ml m?u d?ch ?c tnh b?ng cng th?c: EMBED Equation.3 Trong : N: S? t? bo trong 1 ml m?u. a: T?ng s? t? bo trong 4 vung l?n. 0,016: Th? tch c?a 4 vung l?n. n: ? pha lo?ng. 103: S? chuy?n mm3 thnh ml (1.000 mm3 = 1 ml). PH? L?C 3. B?NG S? LI?U K?T QU? TH NGHI?M 3.1. S? li?u k?t qu? th nghi?m 1 B?ng 11. S? li?u kh?i l?ng hemicellulose t?o thnh sau khi ln men v?i cc t? l? ng uyn li?u khc nhau 4/0 4/1 3/1 2/1 1/1 1/2 1/3 3.85 2.38 1/4 0/4 Kh?i nguyn li?u (n?c (g/50 ti) L?n l?p T? l?l?ng hemicellulosed?a/s?aml) 92.5 60.64 70.83 5.9 80.37 0.3 .36 3.2 3.45 .92 6.3 6.51 3.65 .06 4.6 1.08 .64 .28 .99 2.26

B?ng 12. S? li?u hm l?ng acid t?ng v ? Brix sau khi ln men v?i cc t? l? nguyn li khc nhau 4.93 0.56 4/0 4,3 4,47 4/1 4,7 0,52 3/1 4,80 0,48 2/1 4,5 4,77 1/1 4,73 1/2 4,9 4,8 4,70 4,4 0,56 1/3 4,87 4,6 1/4 5,0 3,48 5,4 2,84 5,20 5,2 0,76 1,72 0/4 ? l?ng Hm l?p acid t?ng trung b?nh L?nBrix trung b?nh (% (w/v)) (% (w/v)) T? l? nguyn li?u (s?a ti/n?c d?a) 0,60

3.2. S? li?u k?t qu? th nghi?m 2 B?ng 13. S? li?u kh?i l?ng hemicellulose t?o thnh sau khi ln men v?i cc t? l? vi khu?n khc nhau T? l? vi khu?n ch?ng vo d?ch ln men 3/1 3/2 3/3 2/1 2/2 2/3 1/1 1/2 35.46 24.55 1.14 1/3 l?ng Kh?iB?ng hemicellulose l?ng acid L?n l?p 14. S? li?u (Acetorbacter/lactic)hm (g/50 ml) t?ng v ? Brix sau khi ln men v?i cc t? l? vi khu?n 86.11 8.26 8.02 8.48 6.65 7.1 7.07 6.03 6.25 5.19 9.34 0.22 8.24 7.09 7.45 1.25 1 0.5 3.09 4.15 3.26 6.55 9.42 hc nhau

3/2 3/3 2/1 4,50 0,52 2/2 4,9 4,77 0,56 2/3 4,2 4,63 4,5 1/1 4,73 4,8 1/2 4,4 3,48 2,72 4,53 4,6 0,60 1,60 1/3 ? l?ng Hm l?p acid t?ng vo b?nh L?nBrix khu?n ch?ngtrungd?ch (% (w/v)) T? l? vitrung b?nh (% (w/v)) ln men) 0,36 4,8 3/1 2 0,60 4,5 4,50 3 0,36 4,2

3.3. S? li?u k?t qu? th nghi?m 3. B?ng 15. S? li?u kh?i l?ng hemihemicellulose t?o thnh g ?ng b? sung v cc m?c ? pH khc nhau. Hm l?ng ?ng b? sung % (w/v) pH ban ?u 1 4,5 Kh?i l?ng hemicellulose (g/50 ml) L?n l?p 5 3 2 0 3 3 0 5,5 1,5 5 4 3 2 0,2 5,5 61.35 4,5 33.58 25.75 12.26 5 41.46 01.87 5,5 92.94 54.47 4,5 4.44 3.4 22.05 15.17 8.72 9.79 4.8 8.35 0.94 5,5 12 50.51 4,5 4.54 30.8 21.2 10.75 8.16 6.94 5.17 5.02 5.24 6.58 6.42 5,5 32.94 25.02 12.12 B?ng 16. S? li?u hm l?ng acid t?ng v ? Brix sau khi v cc m?c ? pH khc nhau. 0,56 5 4,3 0 4,43 0,52 1 4,5 3 2,48 4,00 4,0 0,60 4,60 ? l?p L?n l?ng acid b? trung b?nh pH Brix trung b?nh (% % (w/v) Hmban ?u?ngt?ngsung(w/v)) (% (w/v)) 5,5 7,5 5 7,33 8,0 0,48 7,00 7,0 0,56 4 3 2,60 4,50 4,5 0,52 1,48 0 5,5 0,44 5 6 0,48 4,5 10,00 10,0 0,64 4,36 3,72 2,60 7,00 7,0 0,52 1,48 0 5,5 12,3 12,37 5 12,4 9 12,47 0,44 4,5 12,50 12,5 0,60 4 3,48 2,60 10,00 10,0 0,36 1,36 0 5,5 3,36 12,0 2,60 12,33 12,5 0,48 1,48 0 0,44 5 12 15,00 0,40 1 4,5 3,36 15,0 2,48 15,03 15,1 0,56 4,60 0 5,5 3.4. 3,60 2,48 15.00 15,0 0,48 1,36 S? li?u th nghi?m 4 0 B?ng 17. S? li?u kh?i l?ng hemihemicellulose t?o thnh ian v nhi?t ? khc nhau. 35 30 33.48 21.78 1hi?t 65 Kh?i ? hemicellulose L?n l?ngln Nhi?tgianph?ngmen (ngy) (g/50 ml) Th?il?p ln men (C) 74.92 9.28 83.12 N3.1 0.34 4.54 1.17 5.7 .02 5.37 0.09 .71 .46 2.41 .84 5.75 9.35 0.21 1.07 35 30 Nhi?t 31.62 2.45 10.69 ? 18. S? 25 B?ng ph?ng li?u hm l?ng acid t?ng v ? Brix sau khi 08.17 91.99 6.19 84 4.89 3.94 7.21 2.01 2.72 3.44 .6 0.84 5.07 0 9.88 khc nhau Nhi?t ? ln Th?i gianln men (ngy) 1,32 35 1,04 30 10,40 1,28 Nhi?t 3,96 10,6 2,84 10,20 10,0 1,00 1,20 ? 45 ? l?ng Hm l?p acid L?n (C) ph?ng t?ng trung b?nh menBrix trung b?nh (% (w/v)) (% (w/v)) 0,32 ,44

sau khi ln men v?i cc hm l?

ln men v?i cc hm l?ng ?ng

sau khi ln men ? cc th?i g

ln men ? cc th?i gian v nhi?

8,60 10,20 9,8 1,08 0,84 7,84 1,24 10,00 1,00 1,12 10,27 0,80 6,32 35 10,13 30 10,0 10,07 10,2 1,16 Nhi?t 3,08 2,20 10,40 10,4 0,96 1,96 ? ph?ng 55 0,72 10,2 10,27

2 0,72 10,4

3 0,96 10,2

Nhi?t ? ph?ng 1 1,32 1,16 10,2 10,13

2 1,08 10,0

3 1,08 10,2

30 1 0,96 1,00 10,0 10,00

2 1,08 10,0

3 0,96 10,0

35 1 1,32 1,32 10,4 10,20

2 1,32 10,2

3 1,32 10,0

3.5 S? li?u th nghi?m 5. B?ng 19. S? li?u kh?i l?ng hemicellulose t?o thnh khi ln men c b? sung c ch?t khc nha . B?ng ?i SA-CaCl2-DAP DAP-CaCl2 SA-CaCl2 SA-DAP CaCl2 DAP 3,91 2,4 1,26 20. SA l?ng Kh?i b? L?n Ch?ngS?hemicellulose (g/50 t?ng Ch?tl?p sungli?u hm l?ng acidml) v ? Brix sau khi ln men ? cc c ch?t khc nhau. 8,99 5,68 6,53 7,72 ,35 ,08 ,2 ,6 ,8 ,92 ,03 ,52 ,11 ,96 ,61 ,86 ,05 ,28 ,04 ,88 ?i 4,8 SA-CaCl2-DAP DAP-CaCl2 0,52 SA-CaCl2 0,56 SA-DAP CaCl2 4,4 DAP 3,48 2,72 4,2 0,60 1,60 SA Ch?ng ? l?ng Hm l?p acid t?ng vo b?nh L?nBrix khu?n ch?ngtrungd?ch (% (w/v)) T? l? vitrung b?nh (% (w/v)) ln men) 0,36 PH? L?C 4. B?NG S? LI?U TH?NG K 4.1. K?t qu? th?ng k th nghi?m 1 B?ng 19. Phn tch phng sai kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc t? uyn li?u khc nhau

??

B?ng 20. Ki?m ?nh LSD kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc t? l? ng uyn li?u khc nhau SHAPE \* MERGEFORMAT 4.2. K?t qu? th?ng k th nghi?m 2 B?ng 21. Phn tch phng sai kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc t? i khu?n khc nhau SHAPE \* MERGEFORMAT B?ng 22. Ki?m ?nh LSD kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc t? l? vi khu?n khc nhau SHAPE \* MERGEFORMAT

4.3. K?t qu? th?ng k th nghi?m 3 B?ng 23. Phn tch phng sai kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc h b? sung v cc m?c pH khc nhau SHAPE \* MERGEFORMAT B?ng 24. Ki?m ?nh LSD kh?i l?ng hemicellulose t?o thnh khi ln men v?i cc hm l?ng b? sung v cc m?c pH khc nhau SHAPE \* MERGEFORMAT 4.4. K?t qu? th?ng k th nghi?m 4 B?ng 25. Phn tch phng sai kh?i l?ng hemicellulose t?o thnh khi ln men ? cc th?i v nhi?t ? khc nhau SHAPE \* MERGEFORMAT B?ng 26. Ki?m ?nh LSD kh?i l?ng hemicellulose t?o thnh khi ln men ? cc th?i gian v nhi?t ? khc nhau SHAPE \* MERGEFORMAT

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\* MERGEFORMAT i PAGE \* MERGEFORMAT 50