so sánh tác dụng thủy phân Đậu hũ bằng phức hệ enzyme của bacillus subtilis sp....
DESCRIPTION
upTRANSCRIPT
-
B GIO DC V O TO
TRNG I HC S PHM TP. H CH MINH
------- -------
TRNG TH NH HIU
SO SNH TC DNG THY PHN
U H BNG PHC H ENZYME
CA Bacillus subtilis sp. v Bacillus natto sp.
CH BIN THC UNG CHC NNG
LUN VN THC S VI SINH VT HC
Thnh ph H Ch Minh 2010
-
LI CM N
C c thnh qu nh ngy hm nay, ti xin chn thnh gi li cm n su sc n:
Cn b hng dn: TS. L Chin Phng tn tnh hng dn, truyn t kin thc chuyn mn
cng nh nhng kinh nghim sng qu bu trong sut qu trnh thc hin lun vn.
Ban gim hiu trng HSP TP HCM, qu thy c b mn Vi sinh vt ging dy, hng dn
ti c c nn kin thc nh ngy hm nay.
Cc anh ch em phng th nghim Bin i sinh hc - Vin Sinh hc Nhit i TP HCM to mi
iu kin thun li nht cho ti trong sut qu trnh thc hin lun vn.
Cc bn hc vin cao hc nin kha 2007- 2010 ngnh Vi sinh vt gip , ng gp kin cho
ti trong thi gian thc hin lun vn.
Cui cng ti xin cm n ba m v nhng ngi thn yu ng vin, gip ti hon thnh
lun vn tt nghip ny.
Mt ln na ti xin chn thnh cm n!
TP.HCM, thng 08 nm 2010
Trng Th Nh Hiu
-
LI CAM OAN
Ti xin cam oan nhng s liu trong lun vn ny l trung thc, kt qu th nghim c lp li ti thiu 3
ln v cha ai cng b.
Tc gi lun vn
Trng Th Nh Hiu
-
DANH MC K HIU V CC CH VIT TT
BsTC Bacillus subtilis sp. phn lp t chao
Bn Bacillus natto phn lp t sn phm nattospes
CP Mi trng c peptone
C i chng
VHT n v hot tnh
MT Mi trng
NB Nutrient Broth
NA Nutrent agar
PGA Potato glucose agar
OD Tr s mt quang
TCA Trichloroacetic
TKm Lactobacillus sp. phn lp t m cm ln men
PTHQ Phng trnh hi quy
-
M U
1. T VN
u nnh l mt trong nhng loi cy trng ph bin trn th gii, n c xem l ngun
dinh dng tuyt ho ca nhn loi bi n cha y cc dng cht cn thit nh protein, lipid,
cacbohydrat v cc hot cht hu c khc. c bit, n c xem l dng thc phm ng u v
hm lng protein c ngun gc thc vt. Khng ch vy, trong nhiu nghin cu chng minh
u nnh cn c tc dng gim chlolesterol trong mu, gip ngn nga cc bnh v tim mch, ngn
nga bnh ung th, bnh tiu ng v cc ri lon tin mn kinh ph n [23]
Thng 10 nm 1999, c quan kim tra dc phm v thc phm Hoa K (FDA) a ra kt
lun: S dng 25 gam protein u nnh (c trong 65-70 gam u nnh) trong khu phn n hng
ngy c th lm gim nguy c mc cc bnh l tim mch. [25]
Tuy c gi tr dinh dng v chc nng cao nhng trn thc t nc ta hin nay u nnh
li xp vo nhm c sc cnh tranh yu trong nn kinh t quc dn. [24]
Mt trong nhng l do chnh l hin nay cc sn phm t u nnh phn ln l cc sn phm
truyn thng, thng mn (tng cha 18-20% NaCl, chao c 7-9% NaCl) khng dng c mt
lng ln trong mt khu phn; t c sn phm mi c gi tr cng ngh sinh hc v thc phm
chc nng (dng vi sinh vt c vai tr probiotic) ch bin thc phm. [23]
Trong thi gian gn y, trn th trng trong nc c xut hin mt s sn phm nh
Biosubtil, Bioascimil, Anti-bio, dng bt, ng gi nh, mi gi t 1-3 gam, c cha khong 108-
109 vi khun Bacillus subtilis v vi khun lactic, c tc dng h tr tiu ha. T nm 2007 c sn
phm Nattospes cha enzyme nattokinase t vi khun Bacillus natto, theo qung co l enzyme duy
nht c kh nng lm tan si huyt (fibrin) ngn cn s to huyt khi gy chng tai bin mch mu
no, nhi mu c tim. nc ta c sn phm chao, theo TS. L Chin Phng [20] c cha vi
khun Bacillus subtilis c tc dng tng t v c th so snh c vi Bacillus natto. V vy,
chng ti tin hnh ti So snh tc dng thy phn u h bng phc h enzyme ca Bacillus
subtilis sp. v Bacillus natto sp. ch bin thc ung chc nng.
2. MC CH NGHIN CU
- Xc nh c chng vi khun no c phc h enzyme thy phn u h tt hn ch bin thc
ung chc nng c tc dng h tr tiu ha v c vai tr probiotic.
- Xy dng c quy trnh sn xut thc ung chc nng t u h.
- p ng xu hng gim tiu th cht m v cht bo ng vt thay bng u nnh c ngun gc
thc vt.
- Gp phn a u nnh ra khi nhm c cnh tranh yu trong nn kinh t quc dn.
3. NT MI CA TI
- S dng vi khun Bacillus subtilis sp. (sau khi so snh vi Bacillus natto sp.) thy phn u h
ch bin thc phm chc nng, l tc nhn probiotic th nht.
-
- Dng vi khun lactic (l tc nhn probiotic th 2) c ch hot ng vi khun Bacillus va
bo qun thc phm m khng dng ha cht.
- Bc u to sn phm mi: thc ung chc nng t u h thy phn ln men lactic.
-
CHNG 1. TNG QUAN
1.1. TNG QUAN V U NNH
1.1.1. Lch s pht trin
u nnh c xem l mt trong nhng cy trng c nht ca nhn loi. Nhiu nghin cu
chng minh rng u nnh c ngun gc t Trung Quc. N c trng trn nhiu loi t khc
nhau v c phm vi kh hu rng khp, tri di t vng nhit i Brazil n hn o Hokkaido y
tuyt nm pha Bc Nht Bn. Vi s pht trin ca khoa hc k thut, con ngi tm thy vai
tr ca u nnh trong nhiu lnh vc, c bit trong lnh vc thc phm. V vy, u nnh ngy
cng lan rng khp th gii. [36]
1.1.2. Cy u nnh
Cy u nnh c tn khoa hc l Glycine max (L) Merrill, thuc:
Hnh 1.1. Cy u nnh u nnh l cy bi nh, cao trung bnh di 1m, c lng ton thn, c 3 cht hnh bu dc,
bng trng hoc tm, tri c nhiu lng vng c t 3-5 ht.
Cy u nnh l cy ngn ngy, pht trin tt vng nhit i, a sng, a nhit, chu hn,
trng vo cui ma xun, u ma h. [6]
L cy thc phm c hiu qu kinh t, d trng, sn phm t n rt a dng.
Ngoi ra, cy u tng cn c tc dng ci to t, tng nng sut cc cy trng khc nh
hot ng c nh N2 ca Rhizobium cng sinh trn r cy h u.
1.1.3. Ht u nnh
1.1.3.1. Hnh thi, cu trc [10]
* Hnh dng: ht u nnh c nhiu hnh dng khc nhau nh hnh trn, bu dc, trn di,
trn dt, chy di.
Gii: Plantae
Ngnh: Magnoliophyta
Lp Magnoliopsida
B Fabales
H Fabaceae
Phn h: Faboideae
Chi Glycine
Loi G. max
-
* Mu sc: vng, xanh, nu hoc en v cc mu trung gian nhng mu vng l tt nht nn
c trng v s dng nhiu.
* Cu trc ht: gm 3 phn chnh l v ht, phi v t dip.
- V: l lp ngoi cng ca ht u nnh, c nhiu loi mu sc v l yu t cho vic xc
nh ging u nnh, c tc dng bo v phi mm.
- Phi: l r mm, l phn sinh trng ca ht khi ny mm.
- T dip: gm hai l mm tch tr dinh dng cho ht.
Bng 1.1. Thnh phn cc cht c trong ht u nnh (% khi lng) [15]
1.1.3.2. Thnh phn ha hc
So vi cc nguyn liu thc phm chnh hin nay, u nnh thc s giu dinh dng, va c
ngun protein cao nht trong cc loi u va l dc liu qu.
u nnh c hm lng dinh dng thay i rng ph thuc vo ging v iu kin trng
trt nhng nhn chung chng cha 35-40% protein, 15-25% cht bo, 15-30% carbohydrate; cc
mui khong Ca, Fe, Mg, P, K, Na, S; cc vitamin A, B1, B2, D, E, F; cc enzyme, sp, nha,
cellulose v khong 5% tro. [29]
Theo s liu phn tch ca cng ty Ajinomoto, Thi Lan 1994, hm lng dinh dng ca
ht u nnh Vit Nam nh bng 1.2.
Bng 1.2. Hm lng dinh dng ca ht u nnh Vit Nam [20]
Ch tiu
n v
Ht u
nnh
ng Nai
Ht u
nnh
An Giang
Kh u
nnh p
cng
nghip
Kh u
nnh p
th cng
Protein th
Lipid th
Acid linoleic
Lysin
Methionin
%
%
%
%
%
35,56
17,10
8,00
2,05
0,54
38,06
18,00
8,45
0,39
0,59
47,81
1,50
0,60
2,66
0,61
42,94
3,00
1,40
2,72
0,57
Thnh phn % khi lng
ton ht
Protein
(%)
Lipid
(%)
Carbohydrate
(%)
Tro
(%)
Ht u nnh
T dip
V ht
Phi
100
90
8,0
2,0
40,0
43,0
8,8
41,1
20,0
23,0
1,0
11,0
35,0
29,0
86,0
43,0
4,9
5,0
4,3
4,4
-
Systine
Treonin
Arginin
Phenilalanin
Valin
Leucin
Isoleucin
Serin
%
%
%
%
%
%
%
%
0,61
1,35
2,11
1,49
1,62
2,68
1,51
1,73
0,72
1,53
2,62
1,76
1,74
3,03
1,67
2,05
0,72
1,69
3,03
1,83
1,85
3,39
1,65
2,34
0,75
1,67
2,85
1,95
1,84
3,32
1,77
2,27
Isoflavones trong u nnh dao ng t 4.39 mg/g n 15.58 mg/g, c kh nng phng chng
nhiu loi bnh ung th v bnh tim mch.
Ngoi ra, u nnh cng rt giu cht khong v nhiu cht x.
Bng 1.3. Thnh phn khong trong u nnh
Thnh phn T l (%)
Calci 0.16 0.47
Phospho 0.41 0.82
Mangan 0.22 0.24
Km 0.37 g/kg
St 90 150 mg/kg
Bng 1.4. Thnh phn vitamin ca u nnh
Thnh phn T l (%) Thnh phn T l (%)
Thiamin 11 17.5 % Inositoe 2300 mg%
Riboflavin 3.4 3.6 % Vitamin A 0.18 2.43 %
Niacin 21.4 23 mg/g Vitamin E 1.4 mg%
Pyridoxin 7.1 12 mg/g Vitamin K 1.9 mg%
Biotin 0.8 mg/g Vitamin B1 0.54 mg%
Pantothenic acid 13 21.5 mg/g Vitamin B2 0.29 mg%
Folic acid 1.9 mg/g Vitamin PP 2.3 mg%
Enzyme quan trng ca u nnh l lipoxygenase, c bit n l lipoxydase xc tc phn ng
oxy ha acid bo khng bo ha bi O2, gy mi u nnh.
Enzyme urease c nhiu u nnh sng, phn hy ure thnh amoniac, l mt hp cht c
vi c th ngi do khng nn n u nnh sng. [15]
1.1.4. Cng dng ca u nnh [37]
-
u nnh c cha rt nhiu protein, 8 loi acid amin thit yu v l ngun cung cp calci,
cht x, st v vitamin nhm B. Cc hp cht isoflavon v ha tho (phytochemicals) khc trong
u nnh c kh nng phng nga v tr liu mt s bnh nh: au tim, tai bin mch mu no, ung
th v, ung th kt trng (Theo Vin Ung th Quc gia Hoa K, Vin i hc Havard, Vin i
hc Alabama. Minnesota, Iowa v Helsinki, Phn Lan). Nhng ha tho u nnh gm:
- Protease inhibitors: ngn nga s tc ng ca mt s gene di truyn gy nn chng ung
th, bo v t bo c th khi tc hi ca mi trng sng.
- Phytates: ngn cn tin trnh gy bnh ung th kt trng v ung th v, c kh nng tiu
dit cht lm t bo b ung th v phc hi nhng t bo b h hi.
- Phytosterols: nga cc bnh tim mch bng cch kim sot lng cholesterol mu, gim s
pht trin cc bu ung th kt trng v chng ung th da.
- Saponins: hot ng nh cht chng oxy ha bo v t bo c th, trc tip ngn cn s
pht trin ung th kt trng v lm gim lng cholesterol trong mu.
- Phenolic acid: l mt ha tho chng oxy ha v phng nga cc DNA b t bo ung th
tn cng.
- Lecithin: l mt ha tho lm tng tr nh bng cch nui dng tt cc t bo thn kinh,
lm chc cc tuyn, ti to cc m t bo c th, ci thin h thng tun hon, b xng v tng
cng sc khng.
- Omega-3 fatty acid: l cht bo khng bo ha c kh nng lm gim lng cholesterol xu
ng thi lm tng lng cholesterol tt trong mu.
- Isoflavones (phytoestrogens): l mt ha tho hot ng ging estrogen, c kh nng chng
li cc tc nhn gy nn chng ung th lin h n hormon.
1.1.5. Mt s sn phm t u nnh trn th gii
1.1.5.1. Nhng sn phm u nnh phng Ty
Hnh 1.2. Mt s sn phm u nnh phng ty
Nhng sn phm ny, c pht trin ti Ty phng trong nhiu thp nin qua bi nhng k
thut cao cp bao gm:
Defatted soy flour and grits cha t 50 n 52 % protein, c tch du trong qu trnh xay
nghin ht u thnh bt, nhiu x.
-
Soy protein concentratres cha khong 70 % protein, thng c dng lm gi tht bm,
thc n sng v thc n cho tr s sinh.
Soy protein isolates c cha t 90 n 95% protein, khong 19% methionine b mt trong tin
trnh bin ch.
Textured soy proteins cha khong 52% protein, c to si di p sut v nhit cao t
sn phm defatted soy flour, sau thm mu v gia v ging nh mi v thc phm c ngun
gc tht.
Soy flour l loi protein u nnh n gin nht, n c xay, sng lc v khng c cht tinh
bt nn c dng nh l mt loi thc phm n king.
Du u nnh khng c protein nhng rt giu cht bo khng bo ha v linoleic acid,
lecithin c trong loi du u nnh cha lc rt tt cho no.
Thc phm u nnh n nhanh (Fast food)
S ngi n chay bng thc phm u nnh cng ngy cng gia tng ti Hoa K nn cc nh
ch to thc phm bin ch thc phm u nnh thnh nhng loi thc n nhanh, tin nghi v b
dng nh Vons, Lucky, Hughs v.v
1.1.5.2. Nhng sn phm u nnh phng ng
Nhng s hiu bit v u nnh thc s bt u ng phng v tr thnh mt phn ca
nn vn ha Chu . H ch bin thnh nhng thc n nh l mt ngh thut ca gia nh v
tng a phng. Nhng thc phm ny bao gm u h, sa, nc tng, m cn, tu h ky,
tempeh, miso, natto..v..v..
* Tempeh l bnh lm t u nnh v bt go ln men, ni ting ti Indonesia, cha 9%
protein u nnh, nhiu cht x v t cht bo hn u h v sa u nnh.
Hnh 1.3. Sn phm tempeh
* Miso l hn hp ln men gm u nnh, bt go, mui
v nc, rt ph thng ti Nht Bn v Trung Hoa.
C su loi miso khc nhau nhng mi th u c cha
khong 13% protein, 13% mui, bo qun trong t lnh.
-
Hnh 1.4. Cc loi miso
* Nc tng (soy sauce) rt ph thng Chu .
Lm t hn hp u nnh, go np, mui, nm mc v
nc; t 3 n 12 thng ty theo loi.
Vit nam, c nhiu loi tng nh tng Tu, tu v
yu, tng Bn Hng Yn, tng C hay tng Nam
n. Tng Vit Nam khng kh trng theo phng php
Pasteur. Hnh 1.5. Sn phm tng
* Chao (soya cheese) l sn phm c ln men t u ph nh
cc loi nm mc: Actinormucor elegans, M.hiemalis, M.sivticus,
M.subtilis thy phn u h thnh cc cht dinh dng n gin,
d hp thu. [15]
Hnh 1.6. Sn phm chao
* Natto l mt loi thc phm ln men truyn thng ca ngi Nht Bn, c hai loi: natto si
(s dng vi khun ln men) v natto mui (s dng nm mc ln men). u nnh luc chn
vi Bacillus natto 40oC t 14 18 gi thnh nhng ht u c mu nu, nht cao v c mi
nng. Theo kinh nghim ca nh sn xut, nht cng cao th cht lng natto cng tt v v cng
ngt. [15]
Hnh 1.7. Hnh sn phm natto
* u h v thnh phn dinh dng [3]
u h rt ph thng cc nc Chu . c lm
t u nnh, d n, d ch bin v cn c xem nh
dc phm, c tc dng ngn nga nhiu bnh tt.
C ba loi l loi loi cng (firm tofu), loi mm
(soft tofu) v loi u h la (silken tofu).
-
Hnh 1.8. Sn phm u h
Bng 1.5. Thnh phn dinh dng u h
Thnh phn dinh dng u h cng u h mm u h la
Calories 120 86 72
Protein (g) 13 9 9,6
Cht bo (g) 6 5 2,4
Cht bo bo ha (g) 1 1 0
Carbohydrates (g) 3 2 3,2
Calci (mg) 120 130 40
Natri (mg) 9 8 76
Cholesterol (mg) 0 0 0
St (mg) 8 7 1
X (mg) 1 0 0
% calories t protein 43 39 53
% calories t carbohydrate 10 9 17
% calories t cht bo 45 52 30
Ngun: Composition of Foods: Legumes and Legume Products. United States Department of
Agriculture, Human Nutrition Information Service, Agriculture Handbook 8-16. Revised 12-1986.
1.2. KHI QUT V THC UNG CHC NNG
1.2.1. nh ngha [41]
* Thc phm: tt c cc cht hoc cha ch bin nhm s dng cho con ngi gm
n, ung, nhai, ngm, ht v tt c cc cht c s dng sn xut, ch bin hoc x l thc
phm, nhng khng bao gm m phm hoc nhng cht ch c dng nh dc phm.
* Thc phm chc nng
Cho n nay cha c mt t chc quc t no a ra nh ngha y v thc phm chc
nng, mc d c nhiu Hi ngh quc t v khu vc v thc phm chc nng. Gn y cc nh
ngha v thc phm chc nng c a ra nhiu hn v c xu hng gn thng nht vi nhau.
+ Hip Hi thng tin thc phm quc t (IFIC), nh ngha: Thc phm chc nng l thc
phm mang n nhng li ch cho sc kho vt xa hn dinh dng c bn.
+ Hip Hi nghin cu thc phm Leatherhead (chu u): Thc phm chc nng l thc
phm c ch bin t thc n thin nhin, c s dng nh mt phn ca ch n hng ngy v
c kh nng cho mt tc dng sinh l no khi c s dng.
-
+ B Y t Vit Nam: Thng th s 08/TT-BYT ngy 23/8/2004 v vic Hng dn vic
qun l cc sn phm thc phm chc nng a ra nh ngha: Thc phm chc nng l thc
phm dng h tr chc nng ca cc b phn trong c th ngi, c tc dng dinh dng, to
cho c th tnh trng thoi mi, tng sc khng v gim bt nguy c gy bnh.
Khi qut li c th a ra mt nh ngha nh sau: Thc phm chc nng l thc phm
(hoc sn phm) dng h tr (phc hi, duy tr hoc tng cng) chc nng ca cc b phn
trong c th, c tc dng dinh dng, to cho c th tnh trng thoi mi, tng sc khng v gim
bt nguy c bnh tt.
Nh vy, khi nim thc ung chc nng nm trong khi nim thc phm chc nng.
1.2.2. Khi qut v probiotic
1.2.2.1. Khi nim
Probiotic l cc vi khun sng khi c cung cp vi liu lng thch hp s em li li ch
sc khe cho vt ch. (Theo nh ngha ca FAO v WHO 2001)
1.2.2.2. Vai tr [26]
- Tng thnh bo v min dch, mt s c kh nng kch thch min dch c hiu v khng
c hiu.
- Ngn cn s pht trin ca cc tc nhn gy bnh trong ng rut nh: Staphylococcus,
Salmonella, Yersinia, Clostridia.
- C kh nng xm chim ng rut, bm vo mng nhy rut.
- C kh nng chu c acid d dy, chu c mui mt.
- Phng v cha mt s bnh tiu ha: tiu chy, to bn, ung lot d dy,
- Hn ch s hnh thnh cc khi u nh lm gim hot ng enzyme tit ra t vi khun c hi
gp phn to ra cc cht gy ung th v gim nguy c ung th rut.
- Probiotic c kh nng sn xut ra lactase gip tiu ha lactose.
- Ti thit lp h vi sinh c ch nhanh chng sau khi dng khng sinh.
1.2.2.3. Cc yu cu i vi vi sinh vt probiotic [33]
Vi sinh vt trong ch phm probiotic mun t hiu qu mong mun cn phi p ng ng
mt s yu cu:
- Phi c nh danh chnh xc, ng tn chng loi vi sinh vt.
- C tr ngi, khng c, khng gy bnh.
- s lng tr liu: khong 108-109 t bo trong mt ngy.
- C kh nng tn ti khi i qua d dy, ni c mi trng acid, pH t 1-4 .
- Chu ng c khi qua rut non, ni dch mt ton phn tit ra cao nht.
- Sinh ra mt s cht khng sinh, tng cng sc khng cho c th.
- Di truyn n nh.
-
- Khng chuyn gen khng khng sinh sang nhng vi sinh vt khc.
- Khng c mang vo c th nhng vi sinh vt gy bnh.
- C kh nng cnh tranh v ln t vi nhng vi sinh vt gy bnh v bm vo nim mc
ng tiu ha.
1.2.2.4. Cc chng vi sinh vt thng dng lm probiotic
Hin nay, cc chng vi khun c s dng vi vai tr l cc probiotic ch yu thuc
Lactobacillus v Bifidobacterium, cn Enterococus v Streptococus cng c s dng nhng t
hn. Bn cnh nhng vi khun cn c nm men Saccharomyces boulardii cng c xem l
probiotic.
Bng 1.6. Nhng vi sinh vt c s dng lm probiotic ngi
(Taylor & Francis, 2004)
1.2.2.5. Mt s loi sn phm mi t u nnh hin nay trn th trng
* Sa chua u nnh (soy yogurt)
Sa chua u nnh c lm ging nh sa chua t sa b. Sa tit trng c ln men
vi Acidophilus, Bifida hoc vi cc canh trng ph hp v c ln men cho ti khi canh trng
chuyn t sa u nnh sang dng sa chua, c v tng t nh sa chua t sa b. [30]
Nm 1981, ti Nht ln u tin sn xut soy yogurt s lng ln. [28]
* Nc mm chay u nnh
Hin nay, c rt nhiu nghin cu sn xut nc mm chay, nh theo L Minh Hong -
Trng H An Giang, s dng Bacillus subtilis sp. ln men u h to sn phm nc mm
chay,
Ngoi vic s dng u nnh trong thc phm, cn c mt s ch phm c bit ng ch
c bn ti cc Health Food Store gm:
Chng Lactobacillus Chng Bifidobacterium Cc vi sinh vt khc
L. acidophilus
L. amylovocus
L. casei
L. crispatus
L. gallinarum
L. gasseri
L. johnsonii
L. paracaseii
L. plantarum
L. reuteri
B. adolescentis
B. laimalis
B. bifidum
B. brevis
B. infantis
B. lactis
B. longum
Propionibacterium
freudenreichii
Enterococcus faecalis
Lactococus lactis
Leuconostoc mesenteroides
Streptococus thermophilus
Saccharomyces cerevisiae
Saccharomyces boulardii
-
- Soy Biotic tr cc triu chng kh chu ca ph n vo thi k tt kinh.
- Magic 851 gip tng s hot ng ca h min nhim v ngn nga ung th vi cc cht c
c t u nnh . [37]
1.3. CC GING VI SINH VT S DNG
1.3.1. Bacillus subtilis v Bacillus natto
1.3.1.1. H thng phn loi [40]
Gii: Prokaryota
Ngnh: Protophyta
Lp: Schyzomycetales
B: Eubacteriales
H: Bacillaceae
Ging: Bacillus
Loi: Bacillus subtilis
Bacillus natto
1.3.1.2. Phn b [20]
B.subtilis v B.natto c mt khp mi ni trong t nhin, chng c nhiu trong rm c nn
cn gi l trc khun rm c. Chng cn phn b trn b mt cc loi ht v cc sn phm c
ch bin t cc loi ht . Trong bt m, B.subtilis chim khong 75-95% vi khun to bo t.
Trong cc sn phm thc phm truyn thng nh mm, tng, cm m (cm ln men chua) cng
c mt ca chng v c vai tr ng k trong qu trnh bin i sinh hc.
1.3.1.3. Hnh thi [8],[31]
Bacillus l trc khun nh, thng, c kch thc 0.5-2.5 x 1.2-10m, thng xp thnh cp
i hay chui ngn, hai u t bo trn hoc hi vung. Bo t hnh bu dc c kch thc 0.9
0.6m, khng phn b theo mt nguyn tc cht ch no - lch tm hoc gn tm nhng khng
chnh tm.
L trc khun Gram dng, hiu kh, khi cn non di ng bng tin mao, v gi, tin mao
rng nn mt kh nng di ng.
Khun lc kh hoc nht, v mu hay c mu trng xm nht, hi nhn hay to thnh lp
mng mn lan trn b mt thch, c mp nhn, mp li lm nhiu hay t, bm cht vo mt thch.
-
Hnh 1.9. Vi khun B.subtilis Hnh 1.10. Vi khun B.natto
1.3.1.4. Cu trc [37]
Nh hu ht cc vi khun Gram dng khc, cu trc b mt ca Bacillus kh phc tp v c
cc c tnh kt dnh v chng chu iu kin khc nghit cao. B mt t bo c cu to bi cc
lp gic mc, lp b mt c tnh cht protein (S-layer), mt vi lp lt peptidoglycan v cc protein
trn mt ngoi ca mng t bo.
Hnh 1.11. B mt vi khun Bacillus
(C=Capsule, S=S-layer, P=Peptidoglycan)
S-layer
Hin din trong mt s thnh vin ca ging Bacillus. Chc nng cha c xc nh r rng
nhng dng nh c lin quan n tnh kt dnh ca vi khun.
Gip mc (capsules)
Thnh phn ha hc ca lp v nhy cc vi khun khc nhau cng khc nhau, thng l
c to nn t cc polysaccharide, nitrogen, phosphorite v c th c c polypeptide nhng thnh
phn ch yu l nc (98%), c nhim v nh mt hng ro thm thu bo v t bo chng li
qu trnh kh.
Vch t bo
Vch t bo rt mng (100-200A0), trong sut khng mu. Vch t bo c tnh cht n hi
v c bn rt ln, c th chu c p sut cao. Thnh phn ha hc ch yu l glucid, mt s
cht bo, protide, cc acid amin v chng thay i ty loi vi khun, ty mi trng sng.
Tin mao (flagella)
Hu ht cc vi khun to bo t hiu kh u di ng nh vo cc tin mao l cc si lng rt
mnh mc trn nhng phn xc nh ca t bo vi khun, c cu trc l protid. Chiu di tin mao
thng bng chiu di t bo nhng cng c th di hn ty loi vi khun. S lng v v tr tin
mao cc t bo vi khun khc nhau mi loi. Chng c th mc mt u, hai u hoc mc
xung quanh,
1.3.1.5. S hnh thnh bo t [5]
-
Mt trong nhng c im quan trng ca B.subtilis v B.natto l kh nng to bo t trong
nhng iu kin nht nh. Bo t c tnh khng nhit, khng bc x, khng ho cht, khng p sut
thm thu. Trong thi k ngh, bo t vi khun trng thi sng n (cryptobiosis). c nhng
chng c v vic duy tr sc sng 200-300 nm ca bo t vi khun Bacillus.
Cc t bo sinh bo t khi gp iu kin thc n hoc c tch ly cc sn phm trao i cht
c hi s bt u thc hin qu trnh hnh thnh bo t. V mt hnh thi, c th chia qu trnh hnh
thnh bo t ra thnh cc giai on:
- Hnh thnh nhng bi cht nhim sc.
- T bo phn ct khng i xng, to ra mt vng nh gi l tin bo t.
- Tin bo t hnh thnh hai lp mng, tng cao tnh khng bc x.
- Kt thc vic hnh thnh o bo t.
- Kt thc to v bo t, bo t thnh thc, bt u c tnh khng nhit.
- Bo nang v ra; bo t thot ra ngoi.
1.3.1.6. H enzyme [13], [27]
* H enzyme protease
- Cc enzyme protease (peptid hydrolase) xc tc cho qu trnh thy phn lin kt peptid
trong cc peptid hoc protein.
- B. subtilis v B.natto c kh nng tng hp protease trung tnh v kim.
- Chng tng hp protease ngoi phn (exoprotease) phn gii protein v cc c cht cao
phn t khc c trong mi trng dinh dng thnh cc dng phn t thp vi sinh vt d hp th.
y l chc nng r nht ca protease ngoi bo.
- Theo Otrosko v tp th (1977) th glucose, saccharose, mantose, fructose, socbit l
ngun cacbon tt nht cho qu trnh tng hp protease B.natto var.amylolyquefaciens 795.
* H enzyme amylase
- B. subtilis, B.natto c kh nng to mt lng ln - amylase.
- Amylase ca B. subtilis v B.natto khng c cc lin kt sulfihidril v c kh nng phn
gii tinh bt nhanh gp 22,5 ln so vi - amylase nm mc.
- - amylase ca vi khun B. subtilis cng nh ca B.natto thy phn tinh bt to ra cc
dextrin c mch di khong 6-8 gc glucose. Cc dextrin ny li b phn gii tip tc theo s :
G6 G1 + G5 G7 G1 + G6 hay G2 + G5 (t)
G8 G2 + G6 hay G3 + G5 (t)
- Sn phm cui cng ca s thy phn c cht bi amylase l glucose v maltose. vi
khun t l ny l 1: 5.
- pH ti thch cho dextrin ha v ng ha ca - amylase l t 5,66,2.
1.3.1.7. Cng dng [19],[27]
-
- To khng sinh: subtilin, eumycin, bacillin, bacillomin chng c nhiu loi vi trng gy
bnh.
- Ngi ta cng thng thu - amylase chu nhit cao, dng trong cng nghip dt tch
h tinh bt trn vi t B. subtilis.
- Ch phm amylase t B. subtilis c thay th i mch ny mm (malt) lm tc nhn
ng ha tinh bt trong sn xut ru, bia t nguyn liu c bt.
- B.subtilis v B.natto c kh nng to mt lng ln -amylase ngoi bo. -amylase ca
chng bn nhit hn v phn gii tinh bt nhanh hn 2 2.5 ln so vi enzyme ca nm mc.
1.3.2. Vi khun lactic
1.3.2.1. Tng quan v vi khun lactic [5],[3]
L vi khun Gram dng, khng to bo t, k kh tu , hu ht khng di ng. T bo
dng cu khun (ng knh 1m) hay trc khun (kch thc 1 x 2-3m). Tn ti dng t bo
n, kt i, kt t (tetracoccus) hay kt thnh chui. * Theo kha phn loi Bergey, vi khun
lactic thuc:
Gii: Bacteria
Ngnh: Firmicutes
Lp: Bacilli
B: Lactobacillales
H: Lactobacillaceae
Ging: Lactobacillus
Pesiococcus
H: Enterococcaceae
Ging: Enterococcus
H: Leuconoscaceae
Ging: Leuconostoc
H: Streptococcaceae
Ging: Streptococcus
Lactococcus
* Cn c vo kiu ln men, ngi ta chia vi khun lactic thnh hai nhm [8]:
- Vi khun lactic ng hnh
Tham gia vo qu trnh ln men lactic ng hnh m sn phm chnh to thnh l acid
lactic (90-98% tng sn phm ln men), c th c mt t cc sn phm ph nh: ethanol, acid acetic,
acetoin, CO2.
iu ny c ngha quan trng trong cng nghip sn xut acid lactic v cng nghip thc
phm, c bit l trong cng nghip sa nh sn xut sa chua, phomai. Chng ng vai tr quan
trng cng vi vi khun propionic trong vic to hng cho cc loi phomai rn.
-
- Vi khun lactic d hnh
Tham gia vo qu trnh ln men lactic d hnh m sn phm ln men ngoi acid lactic cn
c hng lot cc sn phm khc vi t l kh cao nh sau: acid lactic 40%, acid acetic 10%, cc cht
kh 20%.
Nhiu sn phm nn vic tch v c lp cc sn phm khc nhau rt tn km nhng cng
c vai tr quan trng trong ch bin thc phm. Vi khun lactic ln men d hnh c th lm cho sn
phm c mi v thm ngon v c trng hn.
1.3.2.2. Lactobacillus [31]
T bo hnh que ngn, kch thc 0.5 1.2 x 1 10m, i khi c
dng hnh cu kt chui ngn.
L vi khun Gram dng, khng to bo t, him khi di ng
bng lng roi, k kh khng bt buc.
Khun lc c kch thc 2-5mm, li, m c, khng nhum mu.
Hnh thc dinh dng l ha dng hu c, khng kh c
nitrate, khng lm tan gelatin, khng c catalase cng nh cytochrome.
Hnh 1.12. Lactobacillus
Nhit ti u cho s pht trin l 30 40oC.
1.3.2.3. ng dng ca vi khun lactic [5], [32]
* Bacteriocin
Ln men thc phm bng vi khun lactic l phng php bo qun thc phm c xa nht
da vo s i khng ca cc vi sinh vt khc nhau. Tuy nhin, phng php ny mi nhn c s
ch ca cc nh khoa hc, c bit l vic s dng cc vi khun lactic khc nhau.
Mt trong nhng c im quan trng ca vi khun lactic l kh nng to cc hot cht
khng khun - bacteriocin c kh nng km hm s pht trin ca vi khun gy bnh hay gy h
hng thc phm nh Listeria, Clostridium, Staphylococcus, Bacillus spp., Enterococcus spp..
Bng 1.7. c tnh ca mt vi loi bacteriocin
Bacteriocin Vi sinh vt sn xut c im
Nisin Lactococcus lactis subsp.
lactis ATCC 11454
Ph khng khun rng, c sinh ra vo
cui chu k tng trng.
Pediocin A Pediococcus pentosaceus
FBB61, L-7230
Ph khng khun rng.
Pediocin AcH Pediococcus acidilactici H Ph khng khun rng.
Leucocin Leuconostoc gelidum Ph khng khun rng, c sinh ra vo
u chu k tng trng.
Helveticin L.helveticus 481 Ph khng khun hp.
-
Carnobacteriocin Carnobacterium piscicola
LV17
Ph khng khun hp, c sinh ra vo
u chu k tng trng.
* Probiotic
Vi khun lactic c ch s dng nhiu trong vai tr probiotic, c nhiu loi c s dng
nh trnh by phn 1.2.2 ca lun vn ny.
* Mt s ng dng khc
- ng dng trong cng nghip ch bin sa sn xut sa chua, kefir, phomai, koumis
- ng dng trong mui chua rau qu: bp ci mui, da chut mui,
- ng dng trong chua thc n gia sc dng trong chn nui.
- ng dng trong cng nghip sn xut acid lactic v cc loi mui lactate.
- Acid lactic c ng dng trong ngnh thuc da, dt, cng nghip tng hp cht do, cng
nghip thc phm.
- Lactate calci l loi dc phm b sung calci di dng d hp thu, lactate st dng cha
bnh thiu mu, lactate ng dng lm dung mi,
-
CHNG 2
VT LIU V PHNG PHP NGHIN CU
2.1. VT LIU
2.1.1. Nguyn liu
* u h: u h Vina Tofu
Hnh 2.1. u h Vina Tofu
* Sa u nnh khng ng Vfresh
Ngun: cng ty c phn sa Vit Nam Vinamilk.
Dng lm mi trng tng sinh cc chng ging vi sinh vt trong ti.
* ng aspartam
Ngun: cng ty c phn dc phm dc liu Pharmedic - TP.HCM.
Dng b sung vo sn phm to v ngt.
* Hmg liu (hng phomai)
Ngun: do cng ty FRAGRANCE OILS (International) LMD. ca Anh sn xut, c mua
ti ca hng Ton Hng, s 451 An Dng Vng, Phng 14 - Qun 5, chuyn cung cp tinh du,
hng liu v bt mu thc phm.
2.1.2. Ging vi sinh vt
Do phng th nghim Cng ngh bin i Sinh hc - Vin Sinh hc nhit i TP.HCM cung
cp.
- Bacillus subtilis sp. c phn lp t chao, k hiu BsTC.
- Bacillus natto sp. phn lp t sn phm Nattospes ca Nht Bn, c k hiu l Bn.
- Lactobacillus sp. phn lp t m cm ln men, k hiu l TKm.
2.1.3. Dng c v thit b th nghim
Do Vin Sinh hc nhit i TP.HCM cung cp.
-
- Dng c th nghim bao gm: ng nghim, becher, erlen, ng ong, pipet, a Petri, buret,
phu lc, giy lc, .
- Thit b th nghim nh t cy, knh hin vi, my lc, t hp, cn in t, b n nhit, my
Kjeldahl bn t ng, my Soxhlet, my o quang ph,
2.1.4. Mi trng dng trong thc nghim
- Mi trng gi ging BsTC v Bn.:
Mi trng thch nc mm (CP: c peptone).
- Mi trng nhn ging BsTC v Bn.
Mi trng dch nc mm (CP: c peptone).
Mi trng sa u nnh.
- Mi trng gi ging Lactobacillus sp.: mi trng c chua (N2)
- Mi trng nh tnh enzyme.
Mi trng thch Czapek Dox + 1% tinh bt nh tnh enzyme amylase.
Mi trng thch Czapek Dox + 1% casein nh tnh enzyme protease.
Mi trng thch Czapek Dox + 1% fibrin nh tnh enzyme protease.
(Thnh phn cc mi trng c trnh by r phn ph lc ca lun vn ny).
2.2. PHNG PHP NGHIN CU
2.2.1. S nghin cu
Trong ti ny, chng ti tin hnh kho st hot tnh h enzyme (amylase, protease) ca
BsTC v Bn trong iu kin khc nhau ca cc yu t nh hng nh thi gian, nhit , pH. Sau ,
ti u ha iu kin cc yu t nh hng n hot tnh cc enzyme ca hai chng ging vi khun
trn. T kt qu thu c, s chn ra mt chng ging ti u hn thy phn u h to sn phm
thc ung chc nng. Ni dung nghin cu c th ca ti c trnh by s sau:
-
Hnh 2.2. S nghin cu
2.2.2. Phng php nghin cu hnh thi cc chng vi sinh vt c trong ti (vi khun
Bacillus subtilis sp., Bacillus natto sp., Lactobacillus sp.)
2.2.2.1. Phng php nhum Gram [14]
* Nguyn tc
Da trn kh nng bt mu ca t bo vi thuc tm kt tinh (crystal violet) v iod m vi
khun chia lm 2 nhm khc nhau l:
Xc nh thnh phn ha hc trong nguyn liu u h
Xy dng ng cong sinh trng. Xc nh sinh khi ti a ca BsTC, Bn trong mi trng CP v trong mi trng sa u nnh.
Kho st hot tnh enzyme amylase, protease trn c cht u ca BsTC v Bn tng yu t thi gian, nhit , pH ri ti u ha thc nghim. T chn chng ti u.
Kho st qu trnh ln men
Xc nh t l ging (BsTC hoc Bn)
Thi gian thy phn u h.
Xc nh cc ch s dinh dng (m, ng, lipid, )
Kho st qu trnh bo qun
Xc nh t l ging TKm
Hm lng ng saccharose
Thi gian bo qun
Hon thin sn phm
Kho st ph gia: ng aspartam, hng
nh gi cm quan sn phm
Xc nh cc ch s dinh dng (m, ng tng, lipid, )
Xy dng quy trnh ch bin thc ung chc nng t u h
-
Nhm th nht vn gi nguyn mu tm ca thuc nhum, khng b ra tri khi x l bng
cn do c lp v t bo dy to bi peptidoglycan. Vi sinh vt thuc nhm ny l Gram dng.
Loi th hai c lp v t bo mng hn (do t peptidoglycan hn) v c bao bc bi mt
mng mng nn c mu hng. Vi sinh vt nhm ny l Gram m.
* Cch tin hnh
- Lau nh sch tiu bn bng giy mm, h qua n cn.
- Dng bt lng ghi tn mu, v vng trn pha di mt lam nh du vt khun pha
trn lame.
- Nh dung dch NaCl 9 ln gia vng trn.
- Dng que cy kh trng ngui ly mt t sinh khi vi khun ha vo git NaCl 9
trn lame.
- Dn mng thnh vt bi, h nhanh trn ngn la n cn.
- t tiu bn ln thanh thy tinh ch U, trn thau nha.
- t ming giy lc ln vng pht knh.
- Nh dung dch crystal violet thm t ht giy lc, yn t 30 giy - 1 pht, ra tri
thuc nhum d vi nc.
- Nh dung dch Lugol, 30 giy, ra li nh nhng vi nc.
- Ty mu bng cn 96o t 15-20 giy: gi phin knh gc nghing nh v cn thn nh
git cn cho cn chy ngang qua vt bi cho n khi khng thy vt thuc nhum chy
theo. Ngay lp tc ra vt bi li vi nc. Thi gian kh mu trong bc ny ng vai
tr quyt nh kt qu ca qu trnh nhum.
- Ph hon ton vt bi vi safranin, yn trong 30 giy, ra vi nc.
- Thm kh phin knh vi giy thm. Khi phin knh kh hon ton, quan st di knh
hin vi vi vt knh du x100.
2.2.2.2. Phng php nhum bo t [14]
Vi thuc nhum Fuchsin, HCl 0,5%, H2SO4 1% v xanh methylen
- Lm vt bi trn mt phin knh sch v kh t nhin.
- Nh vi git HCl 0,5% ln vt bi, h nng trn ngn la n cn cho n bc hi trong
hai pht ri ra vi nc.
- Nhum vt bi vi thuc nhum Fuchsin, qua ming giy lc, h nng cho n bc hi
trong vng 5 pht.
- Ra vt bi bng nc
- Ty mu bng dung dch H2SO4 1% trong 2 pht.
- Ra vt bi bng nc.
- Nhum vt bi bng xanh methylen trong 5-15 pht
- Ra li vi nc v kh t nhin.
-
- Quan st di knh hin vi vi vt knh du (x100). Bo t s mang mu , t bo sinh
dng mang mu xanh.
2.2.3. Phng php nghin cu c im sinh l vi khun
2.2.3.1. Phng php nghin cu c im sinh l Lactobacillus sp.
* Phng php nh tnh acid lactic
Nguyn tc
Khi tc dng vi acid lactic, thuc th Ufermen s i t xanh tm sang vng. Quan st s i
mu thuc th xc nh c kh nng to acid lactic ca vi khun.
Cch tin hnh
Chun b khong 5ml mi trng dinh dng, cy vi khun phn lp vo. Nui cy
nhit phng khong mt ngy. Sau cho dch ln men phn ng vi thuc th Ufermen.
Chun b cc ng nghim cha cc thnh phn nh sau:
- ng 1: 3 ml dch mi trng, 3 ml thuc th Ufermen.
- ng 2: 3 ml dch ln men, 3 ml thuc th Ufermen.
- ng 3: 3 ml acid lactic 98 %, 3 ml thuc th Ufermen.
Quan st v nhn xt s i mu ca thuc th.
* Phng php nh lng acid lactic [31]
Nguyn tc
Acid tng c o bng cch chun vi NaOH 0,1M; thm 1-2 git phenolphtalein 1%
lm cht ch th. Kt qu % acid lactic c tnh theo cng thc:
% acid lactic =
90: trng lng phn t acid lactic (g)
D: pha long
Cch tin hnh
Cn 10 g mu ha tan vo 90 ml nc ct. Thm 1 2 git phenolphtalein 1%. Sau chun
bng NaOH 0,1N cho n khi xut hin mu hng nht, bn mu trong 1 pht.
therner: mt Therner tng ng mt ml NaOH 0.1M dng chun 100 ml
nguyn liu.
2.2.3.2. Phng php nghin cu c im sinh l vi khun BsTC v Bn
* Xy dng ng cong tng trng trong mi trng CP [23]
Kho st ng cong sinh trng ca vi khun l mt cng vic quan trng trong cng
nghip ln men. N cho bit c ti thi im no th cht lng ging vi khun cho vo c cht
ln men l tt nht.
VNaOH x NaOH 0,1M x 90 x1 00
mmu x 100 x D
-
Nguyn tc
S tng trng vi sinh vt l s gia tng s lng t bo vi sinh vt trong qun th. Tc
tng trng l s tng trng ca vi sinh vt trong mt n v thi gian. Theo di s pht trin ca
vi sinh vt theo thi gian ta c th xc nh c ng cong tng trng ca chng.
Dng c v mi trng
Dng c: a Petri, ng nghim nc ct, u tp 1ml c hp kh trng, micropopet 1
ml.
Mi trng: mi trng thch c pepton (CP), mi trng CP lng.
Cch tin hnh
- Vi khun c nui (tng sinh) trong mi trng CP lng t ng ging gc.
- Khong 4 gi ly mu cy trang trn mi trng thch c pepton (CP agar).
- m khun lc xc nh mt t bo (CFU/ml) c trong mu.
+ Pha mu thnh nhiu pha long bc 10 lin tip sao cho c pha long vi mt t
bo thch hp xut hin cc khun lc ring l trn mt thch vi s lng ln hn ch sai
s khi m v tnh ton.
+ S lng khun lc ti u c ngh bi cc c quan c uy tn nh FDA, AOAC l 30
300 khun lc trn a.
+ Tnh kt qu
Mt t bo vi sinh vt trong mu ban u tnh t s liu ca pha long Di c tnh
theo cng thc l:
Mi (CFU/ml) = Ai * Di/V
Ai: l s khun lc trung bnh trn a
Di: l pha long th i.
V: l dung tch huyn ph t bo cho vo mi a (ml).
- V th ng cong sinh trng gia log CFU/ml - trc tung v thi gian - trc honh.
* Xy dng ng cong tng trng trong mi trng sa u nnh [23]
Thc hin xy dng ng cong tng trng ca BsTC v Bn trong mi trng sa u nnh
tng t nh mi trng CP nhng khc l mi trng nui (tng sinh) vi khun l mi trng
sa u nnh.
2.2.4. Phng php nghin cu c im sinh ha
2.2.4.1. Phng php nghin cu c im sinh ha ca vi khun Lactobacillus sp.
* Th nghim oxydase [23]
Nguyn tc
-
Th nghim ny xc nh s hin din h enzyme oxidase vi sinh vt. Quan trng nht ca
h thng ny l cytochrome oxidase trong chui truyn in t vi O2 l cht nhn in t sau cng
cc loi hiu kh hay k kh ty . S loi cytochrome trong t bo thay i ty tng loi vi sinh
vt. Hot tnh cytochrome oxidase c pht hin nh thuc th p-phenylendiamin. Khi c
cytochrome c kh trong t bo, thuc th ny b oxy ha thnh indolphenol c mu xanh dng.
Cch tin hnh
Cy sinh khi chng thun ln ng thch nghing nutrient agar, 370C trong 24-48 gi.
Chuyn mt t sinh khi t khun lc vo nc mui sinh l v trng, lc u. Dng pipette
nh mt git dung dch trn vo a giy th hot tnh oxydase. Quan st v ghi nhn s xut hin
mu xanh dng trong vi pht.
c kt qu
Th nghim oxidase dng tnh (+) khi xut hin mu xanh dng m, nu khng c s
xut hin ca mu xanh ny th phn ng l m tnh (-).
* Th nghim catalase [23]
Nguyn tc
Cc vi sinh vt hiu kh v k kh ty cha chui chuyn in t c cytochrome u c
catalase (tr cc Streptococcus spp.). Enzyme ny l mt trong cc thnh vin ca h thng cc
enzyme c vai tr bo v t bo khi cc tn thng bi cc dn xut c tnh cao ca oxy phn t
trong t bo hiu kh v k kh ty . Cc vi sinh vt ny c kh nng bin dng nng lng theo
phng thc h hp vi oxy l cht nhn in t cui cng trong chui truyn in t to H2O2.
Catalase thy phn hydrogen peroxide thnh H2O v O2, ngn cn s tch t cc phn t c c tnh
cao ny trong t bo. S thy phn hydrogen peroxide s gii phng oxy c ghi nhn qua hin
tng si bt kh.
Cch tin hnh
Dng que cy v trng ly mt t sinh khi t khun lc (tt nht l khun lc khng qu 24
gi) t ln 1 lam knh sch. Nh 1 git dung dch oxy gi (H2O2) 3% ph ln khun lc. Ghi nhn
s si bt nu c.
c kt qu
Nu thy xut hin bt kh l catalase (+).
* Th nghim nitratase [23]
Nguyn tc
Cc vi sinh vt c kh nng kh nitrate tng hp h enzyme nitratase xc tc s kh nitrate
thnh nitrite v nit phn t. y l cch bin dng nng lng theo cch h hp k kh. Sn phm
-
cui cng ca s kh ny c th l nitrite, amoniac, nit phn t, ty thuc vo tng loi vi sinh
vt v iu kin mi trng. Nitrite c to ra t nitrate s phn ng vi sulphanilamide v N-
napthylethyleneiamine hydrocloride pH acid cho hp cht c mu hng.
Cch tin hnh
Cy sinh khi chng thun vo ng nghim c 200 l nc mui sinh l v trng, lc u.
Chuyn a giy nitrate vo ng nghim trn, 370C qua m.
Chuyn a giy tm thuc th tm nitrate vo ng nghim trn.
c kt qu
Phn ng (+) c mu hng xut hin. Ngc li, khng chuyn mu l (-).
2.2.4.2. Phng php nghin cu c im sinh ha ca vi khun Bacillus [31]
* Phn ng nitratase
Tng t nh 2.2.4.1.
* Phn ng protease
Nguyn tc
Vi sinh vt tit protease phn gii casein thnh polypeptide v acid aimin.
Cch tin hnh
Tng sinh vi khun Bacillus trn mi trng sa u nnh trong 24 gi. c mt l trn mi
trng NB thch a c b sung 1% casein. Nh dch tng sinh Bacillus vo, 370C trong 24 gi.
c kt qu
Nh thuc th Folin pha long 5 ln ln trn b mt thch. Kim tra vng phn gii
casein.
* Phn ng Fibrin
Nguyn tc
Vi sinh vt tit protease phn gii fibrin thnh polypeptide v acid amin.
Cch tin hnh
Tng sinh vi khun BsTC v Bn trn mi trng sa u nnh trong 24 gi. c mt l trn
mi trng NB thch a c b sung 1% fibrin. Nh dch tng sinh BsTC v Bn vo, 37oC trong
24 gi.
Phng php thu si fibrin
Dng 5ml huyt heo: b sung 0,5% kalioxalat (K2C2O4.H2O), thm 150ml NaCl 0,8% tip
theo thm 5 ml dung dch CaCl2 (2,5g/100ml nc ct). 10-15 pht. Lc qua giy lc.
Ra mu trn giy bng NaCl 0,8% n khi si fibrin khng cn mu (trc khi ra bng
NaCl 0,8%, kha van phu li yn khong 10 pht cho mi ln ra).
em sy kh hon ton nhit 400C. Sau em i nghin mn.
-
c kt qu
Nh Folin pha long 5 ln ln b mt thch. Kim tra vng phn gii fibrin.
Phn ng (+): mi trng xung quanh vi khun xut hin mu xanh.
Phn ng (-): khng bt mu vi thuc th Folin.
* Phn ng amylase
Nguyn tc
Mt s vi sinh vt c enzyme -amylase thuc nhm hydrolase phn gii lin kt glycozide
to thnh dextrin v maltose. V vy, di tc dng ca -amylase th tinh bt b phn gii thnh
dextrin khng cho phn ng mu xanh tm vi iod.
Cch tin hnh
Tng sinh vi khun trn mi trng sa u nnh trong 24 gi. c mt l trn mi trng
starch agar, 370C trong 24 gi.
Nh iod ln trn b mt mi trng quan st ng knh vng phn gii.
c kt qu
Phn ng (+): mi trng xung quanh vi khun khng mu, trong sut, l vng phn gii
tinh bt.
Phn ng (-): mi trng xung quanh vi khun c mu xanh tm.
2.2.5. Phng php kho st h enzyme ca vi khun BsTC v Bn
2.2.5.1. Phng php xc nh hot tnh h enzyme ca BsTC v Bn
* Phng php xc nh hot tnh enzyme amylase [18]
Nguyn tc
Hot tnh enzyme amylase c xc nh theo phng php Smith v Roe (1966). Hot tnh
amylase biu th kh nng amylase xc tc thy phn tinh bt n dextrin trong 1 pht 50oC v
c th hin bng s n v ca enzyme trong mt gam mu.
Khi phn ng thy phn tinh bt xy ra, lng tinh bt cn li cha b phn hy s to phn
ng vi iod v c o bng my so mu quang hc.
Dng c v ha cht
- My o quang ph.
- B n nhit.
- Dung dch m phosphate (pH 6,2)
+ Dung dch mononatri orthophosphate 0,2M: 27,8 g Na2HPO4 ha tan, dn nc n 1000
ml (dung dch a).
+ Dung dch dinatri hydrophosphate: 53,05 Na2HPO4.7H2O hoc 71,1 g Na2HPO4.12H2O
ha tan dn nc n 1000 ml (dung dch b).
+ chnh pH = 6,2: 81,5 ml dung dch a + 18,5 ml dung dch b.
-
- Dung dch tinh bt 1%: 1 g tinh bt tan, cho vo bnh nh mc 100 ml, thm 50 ml nc
ct, lc u trong b cch thy 10 15 pht, n khi tinh bt tan hon ton, thm 10 ml dung dch
m phosphate 6,2. Thm nc ct cho 100 ml.
- Dung dch Lugol: 0,5 g Iod + 5 g KI, thm nc ct cho 200 ml.
- Dung dch HCl 1N: ha tan 85 ml dung dch HCl m c trong nc ct cho va 1 lt.
- Dung dch NaCl 3%: cn 3 g NaCl ha tan vi nc ct cho 100 ml.
Tin hnh
Dng cc ng nghim c nh du sn v tin hnh cho vo cc ng nghim cc dung dch
v ha cht
ng chun ng th
1 ml nc ct 1 ml dch enzyme cc mu
1 ml m phosphate pH 6.2 1 ml m phosphate pH 6.2
1 ml dung dch tinh bt 1% 1 ml dung dch tinh bt 1%
0,5 ml dung dch NaCl 3% 0,5 ml dung dch NaCl 3%
em 50oC trong 30 pht, sau cho mi ng 1 ml dung dch HCl 1N km hm s hot
ng ca enzyme. Cho nc ct n 10 ml mi ng. Cho tip mt git dung dch Lugol vo mi
ng. em o mt quang bc sng 595 nm.
n v hot tnh ca enzyme amylase c tnh theo cng thc:
UI=mta
LCba
**
**)(
a: mt quang hc ca ng chun.
b: mt quang hc ca ng th.
C: lng tinh bt ban u tham gia phn ng (10mg).
L: h s pha long
m: trng lng (g) hoc th tch (ml) mu.
* Phng php xc nh hot tnh enzyme protease [11]
Nguyn tc
Dng casein lm c cht, xc nh hot tnh phn gii protein ca enzyme protease trn c s
nh lng sn phm to thnh trong phn ng bng phn ng mu vi thuc th Folin.
Da vo th chun tnh lng tyrosin tng ng vi lng sn phm thy phn di
tc dng enzyme.
Dng c ha cht
- ng nghim. - HCl 0,1M.
-
- B n nhit. - NaCl 2M.
- My o quang ph. - NaOH 0,1N.
- Giy lc. - H3PO41/30M.
- Dung dch trichloroacetic 5%. - Na2CO3 0,4M.
- Dung dch m phosphat pH = 6,2. - Ca(CH3COOH)2 0,2M.
- Tyrosin tinh khit. - Dung dch casein 1%.
- Thuc th Folin.
Tin hnh
Dng ng chun
Pha dung dch tyrosin cc nng khc nhau: 10, 20, 30, 40, 50 g tyrosin /1 ml HCl
0,1M. Thm 5 ml Na2CO3 0,4M vo 1 ml dung dch tyrosin (cc nng khc nhau trn) v thm
1 ml thuc th Folin pha long 5 ln vo dung dch hn hp. Sau khi trn u n nh dung
dch 370,50C trong 20 pht.
o hp th ca dung dch bc sng 660 nm (ghi nhn kt qu AS10, AS20, AS30, AS40,
AS50.)
ng i chng: dng 1ml HCl 0,1M thay cho tyrosin, o hp th ca dung dch bc
sng 660 nm (ghi nhn kt qu ny AS0).
Dng ng chun theo hm lng g tyrosin v hp th:
5
5040302010
050040030020010 SSSSSSSSSS AAAAAAAAAAF
nh lng enzyme trong mu
Cho 1 ml dung dch c cht casein vo ng nghim, nhit 370,50C trong 15 pht. Sau
thi gian , cho 1ml dung dch enzyme vo, lc u, hn hp ny nhit 370,50C trong 60
pht. Sau cho vo hn hp ny 2 ml dung dch trichloroacetic (TCA) 5%. n nh nhit trong
25 pht, sau lc dung dch ny qua giy lc loi ta. Cho 5ml dung dch Na2CO3vo 1 ml dch
lc. Cho thm thuc th Folin pha long 5 ln vo hn hp, yn 370,50C trong 20 pht.
Khi xut hin mu xanh, em o hp thu bc sng 660 nm.
Mu i chng: ly 1 ml nc ct thay cho 1 ml dung dch enzyme v tin hnh cc bc
tng t mu th nghim vi cng iu kin.
Tnh kt qu
Mt n v hot tnh enzyme protease chnh l lng enzyme to amino acid tng ng
100 g tyrosin trong 1 ml dch lc trong iu kin th nghim.
Hot tnh enzyme protease:
VHT/g hoc ml dung dch enzyme= (A60 A0)*F*n*1/100
A60: hp th ca mu.
-
A0: hp th ca mu i chng.
F: h s tng quan gia hm lng tyrosin v hp thu bc
sng 660nm trn ng chun.
n: h s pha long mu.
1/100: h s chuyn i.
2.2.5.2. Phng php kho st cc yu t nh hng n hot tnh h enzyme ca BsTC v Bn
C ba yu t cn kho st: thi gian, nhit v pH v y l mt trong nhng yu t c nh
hng nhiu n hot tnh h enzyme ca vi khun.
* Yu t thi gian
- Cy 1% (v/w) vi khun BsTC , Bn hot ha trong mi trng sa u nnh vo c cht
u h ti cng thi im.
- nhit phng ln lt theo cc mc thi gian: 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40
gi.
- Xc nh hot tnh enzyme cc mc thi gian trn.
* Yu t nhit
- Cy 1% (v/w) vi khun BsTC , Bn hot ha trong mi trng sa u nnh vo c cht
u h ti cng thi im.
- mu cc nhit : 15, 20, 25, 30, 35, 40, 450C trong 24 gi, pH c cht.
- Xc nh hot tnh enzyme cc mc nhit trn.
* Yu t pH
- Cy 1% (v/w) vi khun BsTC , Bn hot ha trong mi trng sa u nnh vo c cht
u h ti cng thi im.
- mu nhit phng trong 24 gi vi pH ln lt l 2, 3, 4, 5, 6, 7, 8.
- Xc nh hot tnh enzyme cc mu trn.
2.2.5.3. Ti u ha iu kin nh hng n hot tnh enzyme ca BsTC v Bn [1]
Chng ti b tr th nghim theo quy hoch thc nghim vi ba yu t thi gian, nhit , pH
v y l mt trong nhng yu t quan trng nh hng n hot tnh enzyme ca BsTC , Bn.
Yu t khng i trong qu trnh kho st l c cht u h v t l ging cy 1% (v/w).
S th nghim ti u 23 = 8, ngoi ra cn c thm 3 th nghim tm kim tra ngha cc
h s ca phng trnh hi quy.
Ba yu t cn kho st:
- X1 l yu t thi gian vi cc gii hn c xc nh sau kho st trn.
- X2 l yu t nhit vi cc gii hn c xc nh sau khi kho st trn.
- X3 l yu t pH vi cc gii hn c xc nh sau khi kho st trn.
-
Hm mc tiu Y l hot tnh enzyme c tnh sau o OD595 (amylase) hoc OD660
(protease).
Bng 2.1. B tr quy hoch thc nghim
Mu th nghim X1 X2 X3 Y
1 + + +
2 + + -
3 + - +
4 + - -
5 - + +
6 - + -
7 - - +
8 - - -
9 0 0 0
10 0 0 0
11 0 0 0
Ghi ch: (-): mc di; (0): tm; (+): mc trn.
-
2.2.6. Quy trnh cng ngh ln men u h ch bin thc ung chc nng
Hnh 2.3. Quy trnh cng ngh thy phn u h lm thc ung chc nng
Gii thch quy trnh
Nhn ging
Sau khi so snh hot tnh enzyme ca BsTC v Bn, chn vi khun c hot tnh enzyme ti u
hn thy phn u h to sn phm thc ung chc nng.
Dng que cy ly vi khun c chn t ng ging cy vo 10 ml mi trng sa u
nnh thanh trng, nui cy trong 24 gi nhit phng.
La chn nguyn liu
Cht lng u h c nh hng rt ln n cht lng, c bit l gi tr cm quan ca sn
phm.
Yu cu ca u h:
Nhn ging trong mi trng sa u nnh
Ging vi khun c chn
u h
Hp thanh trng (1000C, 15 pht)
Ln men
Xay nhuyn
Cy ging
Cy Lactobacillus sp.
Ph gia
Sn phm
-
- Trng, mm, mn.
- Mi thm.
- V bo.
- m bo v sinh v n nh cht lng.
Thanh trng
Hp thanh trng 1000C trong 15 pht nhm tiu dit v c ch mt phn cc vi sinh vt c
trong u h chun b cy ging vo cho qu trnh ln men.
Xay nhuyn
Dng my xay sinh t gia nh ( v trng) xay u h ti nhuyn trong thi gian 3 pht.
Cy ging
Cy ging phi thc hin trong t cy c v trng bng cch lau cn, chiu n cc
tm. Ht ging c tng sinh t mi trng sa u nnh cho vo u h xay nhuyn vi t
l c kho st
- T l ging kho st: 0.5%, 1%, 2% (v/w).
Ln men
Qu trnh ln men c thc hin iu kin kho st.
- Kho st thi gian ln men: 15, 18, 21, 24, 27 gi.
Cy Lactobacillus sp.
Cy vi khun Lactobacillus sp. c tng sinh t mi trng sa u nnh cng vi lng
ng saccharose c kho st vo u h c ln men nhm mc ch bo qun v to v
chua c trng v theo nguyn tc Lactobacillus sp. s s dng ng saccharose lm ngun dinh
dng v ln men to acid lactic.
- Kho st t l ging Lactobacillus b sung vo: 0%,1%, 2% (v/w).
- Kho st lng ng saccharose b sung: 0%, 1%, 2% (v/w)
- Kho st iu kin bo qun: nhit phng v t lnh (10-200C).
- Theo di thi gian bo qun.
B sung ph gia
Nhm tng gi tr cm quan ca sn phm ta cn b sung mt s ph gia vi nng ph
hp sau khi kho st.
- V mu sc, sn phm c mu trng ca u h rt c gi tr cm quan nn khng b sung
ph gia to mu.
- V mi, sn phm c mi u h ln men nn chng ti chn hng phomai c mi thm
bo nh u h.
Kho st t l hng b sung: 10/00, 20/00, 3
0/00, 40/00(v/w).
-
- V v, chng ti s dng ng aspartam, l loi ng to v ngt nhng khng cha calo.
Kho st t l ng aspartam b sung: 0,350/00, 0,70/00, 1,05
0/00, 1,40/00 (v/w) tng ng 1,
2, 3, 4 gi thnh phm ng aspartam (35 mg/gi).
2.2.7. Phng php phn tch
2.2.7.1. Xc nh m [3]
Nguyn tc
Lm bay hi ht nc trong thc phm. Cn trng lng thc phm trc v sau khi sy kh,
t tnh ra phn trm nc c trong thc phm.
Dng c
- T sy iu chnh nhit .
- Cn phn tch.
- Bnh ht m.
- a Petri.
Cch tin hnh
- Cn a sy kh.
- Cn 10 g mu chun b sn, nghin nh cho vo a Petri.
- Cho tt c vo t sy, sy nhit 100 1050C, sy kh n trng lng khng i,
thng ti thiu l 6 gi.
- Sy xong, em lm ngui bnh ht m (25 30 pht) v em cn cn phn tch vi
chnh xc n 0.0001 g.
- Cho vo li t sy 100 1050C trong 30 pht, ly ra ngui bnh ht m v cn nh trn
cho ti trng lng khng i. Kt qu gia hai ln cn lin tip khng c cch nhau qu 0.5 mg
cho mi gam cht th.
Tnh kt qu
m theo phn trm (X) c xc nh bng cng thc:
X = (G1 G2) * 100 / (G1 G)
G: trng lng a Petri.
G1: trng lng a Petri v mu trc khi sy.
G2: trng lng a Petri v mu sau sy ti trng lng khng i.
2.2.7.2. nh lng protein bng phng php Kjeldahl [12]
Nguyn tc
- V c ha m bng H2SO4 m c thnh amon sulphate ((NH4)2SO4)). Mui ny em tc
dng vi kim mnh nh NaOH s gii phng NH3.
H2SO4 m c
-
Cht m (NH4)2SO4
Xc tc, nhit
(NH4)2SO4 + NaOH 2 NH3 + 2 H2O + Na2SO4
- Sau , lng NH3 c hi nc li cun sang mt bnh tam gic c cha mt lng tha
H3BO3. M H3BO3 t phn ly.
H3BO3 HBO2 + H2O
- Khi ct m, NH3 bay sang s phn ng vi HBO2
NH4OH + HBO2 NH4+ + BO2- + H2O
BO2- l mt base mnh, v vy dung dch ca bnh hng s chuyn t mu tm sang mu
xanh l m. Lng BO2- c to thnh tng ng vi lng NH3 b y ra trong qu trnh ct
m. Xc nh lng BO2- bng cch chun ngc vi HCl 0,25N. Giai on chun kt thc
khi dung dch chuyn t mu xanh l m sang mu tm .
BO2- + H+ HBO2
Ha cht v thit b
- My Kjeldahl bn t ng ca hng Prolabo
- Burret 25 ml
- Bp nung
- Bnh chng ct
- H2SO4 m c
- NaOH 32%
- HCl 0,25N
- Cht xc tc: l hn hp ca 60 g selenium + 75 g CuSO4+ 865 g K2SO4
- Hn hp ch th mu:
+ Ha tan 0,264 g methyl trong 250 ml cn tuyt i.
+ Ha tan 1,28 g bromo cresol blue trong 50 ml cn tuyt i.
Trn u hai dung dch trn , b sung n 1 lt bng cn tuyt i.
- Dung dch acid boric 4% vi ch th mu: ha tan 80 g acid boric trong 1800 ml nc ct,
un nng mt cht, ngui b sung 25 ml hn hp ch th mu. B sung n 2 lt bng nc ct.
Cch tin hnh
- V c ha mu
+ Nguyn tc: s v c ha cht m l s v c ha tt c cc cht m d bt c dng
no (hu c, protein, v c) thnh hp cht v c l amoniac sulphate (NH4)2SO4) m c v cht
xc tc.
-
+ Thc hnh: cn 100 mg mu nghin nh, 1 g cht xc tc cho vo ng ph mu + 5 ml
H2SO4 m c, ni b thu kh v bt u ph mu. Khi thi gian ph mu kt thc ng ph mu
ngui. B sung 50 ml nc ct, trn u v ngui, lp vo my chng ct.
+ Chng ct
Lp ng ph mu vo my chng ct, b sung 80 ml NaOH 32%. Khi ng qu trnh chng
ct, dch chng ct chuyn sang bnh tam gic c cha sn 20 ml dung dch acid boric 4% c ch th
mu.
Ngng chng ct khi dch chng ct ra khng cn NH3 (th bng giy qu).
Chun bng HCl 0,25N. Ngng chun khi xut hin mu pht .
Tnh kt qu
Thng qua lng HCl 0,25N ta bit c lng acid boric kt hp vi NH3 v do vy bit
c NH3 gii phng t mu.
1 ml HCl 0,25N tng ng vi 0,0035 g nit hu c.
% Nit tng s =C
VHCl*100*0035.0
VHCl 0.25N : th tch HCl 0,25N dng chun .
C (g): trng lng mu em xc nh.
2.2.7.3. Phng php xc nh m amoniac [12]
Nguyn tc
Trong nguyn liu cha m thng c mt loi m di dng v c (mui amon hay cc
amin d bay hi). y l dng m to thnh do s phn hy ca protein (b ln men thi hoc qu
trnh phn hy cacboxyl ca acid amin) c bn cht l base, v th v kha cnh dinh dng th loi
m ny khng cn cho c th. Nu c n hm lng cao th nguyn liu c nh gi l km
cht lng.
Do loi m ny c tnh kim yu nn di tc dng yu ca MgO th nhng loi m trn s
b ui ra khi dung dch, chng s b li ko theo hi nc n mt bnh cha, c sn mt lng
tha acid. Sau em nh phn lng acid d, cho php chng ta xc nh c loi m amoniac
ny.
2 (NH4)+ + Mg(OH)2 2 NH3 + 2 H2O + Mg
2+
2 NH3 + H2SO4 (NH4)2SO4.
Ha cht v thit b
- My Kjeldahl bn t ng ca hng Prolabo.
- Burret 25 ml.
- MgO khan.
-
- Dung dch H2SO4 0,1N.
- Dung dch NaOH 0,1N.
- Phenolphtalein 1%.
Cch tin hnh
Cn 1 g nguyn liu hay sn phm ha tan vo 10 ml nc ct cho vo ng ph mu. Cho
vo 5 g MgO. Lp ng ph mu vo my chng ct. Dch chng ct a vo mt bnh tam gic
cha 10 ml H2SO4 0,1N + 3 git phenolphtalein 1%. Chng ct ti khi khng cn NH3 (th bng
giy qu).
nh phn lng H2SO4 0,1N bng NaOH 0,1N, ta s bit c lng acid phn ng vi
NH3.
Lm mt mu i chng vi 10 ml nc ct.
Tnh kt qu
Gi V0 l th tch NaOH 0,1N th khng.
Vt l th tch NaOH 0,1N th tht.
V = V0 - Vt l lng NaOH 0,1N tng ng vi lng NH3 phng thch c hp
th trong H2SO4 0,1N.
S g m amoniac trong 100 g mu:
V . 0,0014 . 100 = V . 0,14 (g/ 100 g)
Vi 0,0014 l s gam m amoniac tng ng vi 1 ml NaOH 0,1N.
2.2.7.4. Phng php xc nh m formol [12]
Nguyn tc
Phng php ny cho php xc nh m c trong acid amin, peptid. Nh chng ta bit,
phn t amin hay peptid c mt u COOH v u kia l NH2. Formol s tc dng ln u
NH2 to thnh hp cht metylen.
V th sn phm l hp cht c cha nhm metylen, u COOH c tnh acid nn ta c th
nh phn bng NaOH, t gin tip cho php ta xc nh c lng NH2 (tiu biu cho cht
m trong nguyn liu).
Dng c v thit b
- Dng c thng thng ca phng th nghim.
- Dung dch formol pha long (t l 1:1) trung ha bng NaOH 0,1N: ly 50ml dung
dch formol pha long , thm vo vi git phenoltalein 1%, chun bng NaOH 0,1N cho n
khi dung dch c mu hng nht.
- Dung dch NaOH 0,1N.
- Cht ch th mu phenolphtalein 1% trong cn.
-
Cch tin hnh
- Cn 2 g mu ti, thm 10 ml nc ct, lc qua giy lc, chnh nc n 20 ml.
- Ht 10 ml dch lc vo bnh tam gic, thm vo 10 ml dung dch formol pha long
trung ha bng NaOH 0,1N + vi git phenoltalein 1%, lc u phn ng xy ra hon ton. Sau
, nh phn bng NaOH n khi dung dch chuyn sang mu hng nht, ly mu chun l mu
ca formol sau khi trung ha.
- Thc hin 3 ln th tht, 3 ln th khng (thay 10 ml dch lc bng 10 ml nc ct)
Tnh kt qu
Gi: V0 l th tch NaOH 0,1N trung bnh ca 3 ln th khng.
Vt l th tch NaOH 0,1N trung bnh ca 3 ln th tht.
V = Vt - V0 : lng NaOH trung ha nhm COOH.
T suy ra s mol NaOH c trong V (ml):
)(10**1000
1.0** 4 molXVXV
Vi: X l h s chnh l dung dch NaOH 0,1N.
- S mol NaOH tng ng vi s nhm COOH, tng ng vi s nhm NH2, tng ng
vi s mol NH2 l:
V . X . 10-4 (mol)
- S gam m c trong 10 ml dung dch nguyn liu pha long 10 ln l:
14 . V . X . 10-4 (mol)
- S gam m c trong 10 ml dung dch nguyn liu cha pha long l:
14 . V . X . 10-4 . 10 = 14 . V . X . 10-3 (gam)
- S gam m c trong 1 lt nguyn liu:
)/(**141000*10*
10
**14 3 lgXVXV
2.2.7.5. Phng php xc nh ng tng [11]
Nguyn tc
S nh phn hm lng ng tng s ha tan da trn phn ng mu c trng bi ng
v nhiu cht hu c vi s hin din ca H2SO4. S chun xc ca kt qu ph thuc :
- sch ca dng c..
- tinh khit ca thuc th nht l H2SO4.
- Nhit phi c nh trong sut thi gian phn ng.
Dng c v ha cht
- Bnh nh mc 500 ml, 100 ml, 50 ml.
-
- Erlen 50 hay 100 ml.
- Ci chy s.
- Phu lc.
- ng nghim.
- ng ht.
- Quang ph k.
- Cn 960, cn 800.
- Phenol 5%.
- H2SO4 m c.
- Dung dch ng saccharose 0,1%: 500 mg saccharose sy kh 60oC, ha tan trong 50
ml nc ct.
Cch tin hnh
- Cch trch ng: ly 2 g nguyn liu ti nghin nh cho vo cc thy tinh 50 ml v
thm 10 ml cn 96o. un si trn ni cch thy cho si 3 ln. Khuy u bng a thy tinh. Sau
ngui, lc qua giy lc khng tro.
- Sau , cho 10 ml cn 80o vo cc ng b, khuy u un si 2 ln trn bp cch thy.
ngui lc tip tc, chit rt nh vy khong 2 ln, xong b ln lc v ra 2 3 ln bng cn 80o
(ra tng t mt).
- Cn qua lc c cho bay hi nhit phng hoc trn l cch thy un nh. Sau khi
cn bay hi ht, mu c th lu trong bnh ht m.
- Cn kh trong cc c pha long thnh 50 ml vi nc ct (bnh nh mc). Nu c cn
th lng xung. Khi em lm hin mu, dung dch ny c th pha long thnh 5 10 ln ty nng
nhiu hay t.
- Thc hin phn ng mu: ht 1 ml ng cho vo ng nghim ri thm vo 1 ml dung
dch phenol 5%. Sau , cho chnh xc 5 ml dung dch H2SO4 m c vo ng nghim. 10 pht
ri lc u, gi trn ni cch thy 10 20 pht 25 30oC xut hin mu. Mu bn vng trong
vi gi. Xc nh cng mu trong quang ph k 490 nm.
Xy dng ng chun
Ly 7 bnh nh mc 100 ml ri cho vo theo th t 1, 2, 3, 4, 5, 6, 7 ml dung dch ng
saccharose mu 0,1% cho nc ct ti vch nh mc. T mi bnh ly ra 1ml cho vo ng ri
nhum mu bng phenol 5% v H2SO4 m c nh trn. Trong mi ng nghim s cha tng
ng 10, 20, 30, 40, 50, 60, 70 g saccharose. So mu bc sng 490 nm, sau v th mu.
Lm thm mt ng th vi 1 ml nc ct.
Tnh kt qu
-
Tr s mt quang (OD) ca nhng ng i chng sau khi tr i tr s ca ng th s xc
nh c mt ng cong mu.Vi dung dch cn xc nh nng , ta cng tr i ng th ri
chiu ln ng cong mu suy ra nng x trong ng, t tnh % lng ng trong mu.
2.2.7.6. Phng php xc nh ng kh [2]
Nguyn tc
Trong mi trng kim, ng kh kali ferixianua thnh kali peroxia. Vi s c mt ca
gelatin, kali ferixianua kt hp vi st sulphate to thnh mt cht c mu xanh bn.
Cng mu c xc nh trn my so mu. Phng php ny cho php xc nh lng
ng rt thp trong dung dch (0,01 0,1 mg/ml).
Dng c v ha cht
- Dung dch kali ferixianua (K3Fe(CN)6): ha tan 1,65 K3Fe(CN)6v 10 g NaNO3 trong 1 lt
nc ct. Bo qun dung dch trong l thy tinh mu nu.
- Dung dch st sulphate (Fe2(SO4)3): ha tan 1 g Fe2(SO4)3 trong 10 ml H2SO4 m c.
t t dung dch vo nc ct c sn trong bnh nh mc, pha long thnh 1000 ml.
- Gelatin 10%.
- Trn ln dung dch Fe2(SO4)3 v Gelatin 10% theo t l 20 : 1. Hn hp dung dch ch s
dng trong ngy.
Cch tin hnh
Ly 1 g nguyn liu ha tan trong 20 ml nc ct, lc ly dch lc (dch ng), ra b lc
sau hiu chnh n mc 50 ml. Cho vo ng nghim 2 ml dch chit ng v 2 ml dung dch
kali ferixianua, khuy u, un trn ni cch thy si 15 pht. Sau ngui, thm vo ng
nghim 4 ml dung dch Fe2(SO4)3 khuy u v dn n mc 20 ml. o cng mu bc sng
710 nm.
Tnh kt qu
Da vo mt quang (OD) i chiu vi th chun, tnh lng ng.
2.2.7.7. Phng php xc nh hm lng lipid th [3]
Nguyn tc
Nguyn liu c lm kh, sau trch ly lipid ra khi nguyn liu bng ether du ha hoc
ether ethylic trn my Soxhlet v xc nh khi lng cht bo.
Dng c v ha cht
- My Soxhlet.
- Giy lc.
- Ether du ha ether ethylic.
Cch tin hnh
-
- Giy lc c sy 105oC n trng lng khng i.
- Nguyn liu v sn phm c nghin nh, sy 105oC n trng lng khng i.
- Cn chnh xc 2- 5 g nguyn liu kh tuyt i cho vo giy lc v gi li cho kn khng
mu ri ra ngoi. giy lc c cha mu vo tr chit ca my Soxhlet. Chit bng ether du ha
hoc ether ethylic. Sau khi chit ht lipid (khong 8 gi), ly ti mu ra, cho bay ht dung mi, sy
kh n trng lng khng i.
Tnh kt qu
Hm lng lipid th c tnh theo cng thc
X=c
ba 100*)(
X : hm lng lipid %.
a : trng lng giy lc v mu trc khi chit (g).
b : trng lng giy lc v mu sau khi chit (g).
c : trng lng mu ly phn tch lipid.
2.2.7.8. Phng php xc nh peroxide [3]
Nguyn l
Ch s peroxid l s g iot gii phng ra bi peroxid c trong 100 g mu.
R1 - CH - CH - R2 + 2 KI + 2 CH3COOH
O O
R1 - CH2 CH - R2 + 2 CH3COOK + H2O + I2
O
I2 + 2 Na2S2O3 2 NaI + Na2S4O6.
Nguyn liu v ha cht
- Acid acetic.
- Dung dch KI bo ha (pha dng ngay), lc k y nt cn thn.
- Dung dch Na2S2O3 0,01N.
- Dung dch ha tinh bt 1%.
- Cloroform.
Cch tin hnh
Ly 1 g mu kh tuyt i, ng i chng 1 ml nc ct. Mi bnh cho 10 ml dung dch hn
hp acid acetic : Cloroform (2:1), 1 ml dung dch KI bo ha (mi pha), cho erlen vo ch ti 10
pht. Cho thm vo erlen 0,5 ml ch th h tinh bt 1% chun iod to thnh bng dung dch
Na2S2O3 (n khi mt mu nu).
Tnh kt qu
-
Ch s peroxide
P=C
fba 100*01269.0**)(
a : s ml dung dch Na2S2O3 dng chun mu tht.
b : s ml dung dch Na2S2O3dng chun mu th khng .
c : trng lng mu..
f : h s hiu chnh ca dung dch Na2S2O3 0,01N.
0,01269: s gam iod tng ng vi 1 ml dung dch Na2S2O3 0,01N.
100: h s quy chun 100 g cht bo.
2.2.7.9. nh lng bo t Bacillus subtilis sp. trong sn phm bng phng php m khun
lc [17]
Nguyn tc
Cy mt th tch xc nh mu huyn ph vi sinh vt cy ln mi trng thch c thnh phn
c cht c trng v thch hp cho loi vi sinh vt cn nh lng pht trin. Sau mt khong thi
gian nui cy, trn b mt thch s xut hin cc khun lc. Da vo s khun lc m c, th
tch mu cy v h s pha long, ta c th suy ra s khun lc c trong 1 ml mu kho st ban
u.
Cch tin hnh
- Xc nh bo t c trong sn phm
Nhum bo t: bo t ca Bacillus subtilis sp. c mu hng, thng nm gia t bo c mu
xanh.
- Kh trng mu theo kiu Pasteur l bin php hp nng nguyn liu mt ln nhit thp
hn 100oC. Nhit v thi gian kh trng theo kiu Pasteur ty thuc vo sc khng ca vt
liu i vi nhit .
un nng dch t bo thu c t cc th nghim trn nhit 80oC trong 15 pht.
- Pha long dch huyn ph bo t cc nng thch hp 10-1, 10-2, 10-3 Tin hnh cy
0,1 ml dch mu cc pha long khc nhau vo cc a Petri mi trng NA c sn.
t cc a thch va cy nhit phng v trong 24 gi.
- Kt thc thi gian , ly cc a thch ra, tin hnh m khun lc v tnh s lng bo t
trong 1 ml mu.
Th nghim lp li 2 ln.
Tnh kt qu
Mt t bo c tnh theo cng thc sau:
C (CFU/ml) = N
n1V1f1 + + niVifi
-
Trong : C (CFU/ml): s t bo vi khun trong 1 ml mu.
N (CFU): tng s khun lc m trn cc a.
ni: s a cy nng pha long th i.
Vi(ml): th tch mu cy trong mi a.
fi: pha long th i.
2.2.8. Phng php nh gi cm quan sn phm
2.2.8.1. Phng php m t mi - v [4]
Mc ch
Xc nh nng hng, lng ng aspartam b sung vo thc ung chc nng u h
nhm ci thin mi v ca nguyn liu ban u, tng gi tr cm quan.
C s
Phng php m t mi - v (flavor profile) c ngun gc t nhm t vn Arthur D. Little
vo cui nhng nm 1940. Vo thi im , nhm ny cung cp mt cng c chung nh gi
tnh cht c trng hng - v ca mt thc phm v thay th phng php s dng chuyn gia th
nm bng mt hi ng gm cc thnh vin c hun luyn.
Khng ging cc phng php thc nghim hin i, phng php lun ca flavor profile s
dng mt qu trnh ng thun. Nhng nh gi ring bit ca nhm bt u bng lm vic c
lp vi nhau nhng sau li tp hp li trong mt nhm kt thc flavor profile.
Cch tin hnh
- T chc 3 nhm, mi nhm 5 ngi.
- Chun b sn phm
+ nh gi hng (hng phomai): chun b cc mu th vi nng hng khc nhau, c
k hiu nh bng 2.2.
Bng 2.2. K hiu mu th hng
T l hng 0/00
(v/w)
10/00 20/00 3
0/00 40/00
K kiu mu A B C D
+ nh gi v (ng aspartam): chun b cc mu th vi nng ng khc nhau, c k
hiu nh bng 2.3.
Bng 2.3. K hiu mu th v
T l ng 0/00 (mg/g)
0.350/00
(1 gi)
0.70/00
(2 gi)
1.050/00
(3 gi)
1.400/00
(4 gi)
-
K kiu mu A B C D
- Th sn phm, mi thnh vin lm vic c lp, ghi li cm nhn ca tng sn phm v
chn ra sn phm va ming.
2.2.8.2. Phng php php th cho im th hiu [22]
Mc ch
Tm hiu mc hi lng, a thch ca ngi s dng i vi sn phm thc ung chc
nng t u h thy phn ln men lactic.
Nguyn tc
Ngi th c mi th sn phm nhng thay vo vic nh gi cng ca mt tnh cht
cm quan no h s o mc a thch, hi lng ca mnh i vi sn phm bng thang im
9 c nh ngha trc thng qua cc thut ng m t cp hi lng, a thch:
1. Cc k khng thch
2. Rt khng thch
3. Khng thch
4. Tng i khng thch
5. Khng thch cng khng ght
6. Tng i thch
7. Thch
8. Rt thch
9. Cc k thch
Cch tin hnh
- Chun b mu th.
- Chn i tng th: 100 ngi khu ph 1 - P.Hip Bnh Phc - Q. Th c - Tp.HCM
vi y cc la tui, gii tnh v mc thu nhp.
- Th v cho im sn phm theo thang 9 dim vi mc nh gi nh trn.
2.2.9. Phng php x l s liu
2.2.9.1. Phng php x l s liu quy hoch thc nghim
Mc ch
- Xy dng phng trnh hi quy.
- Kim tra phng trnh hi quy c tng thch vi thc nghim hay khng.
Cch tin hnh
Phng trnh hi quy c dng:
Y = b0 + b1X1 + b2X2 + b3X3 + b12X1X2 + b13X1X3 + b23X2X3 + b123X1X2X3.
-
kim nh tnh ngha ca cc h s trong phng trnh hi quy v s tng thch ca
phng trnh hi quy vi thc nghim, ta tm phng sai ti hin th nghim ti tm.
- Gi tr trung bnh ca 3 th nghim ti tm:
y =
yu3
u= 1
3
- Phng sai ti hin
sth2 =
3
u = 1
(yu - y )2
n0 - 1 sth
- Phng sai cc h s:
sbj = sthN
- Cc h s trong phng trnh hi quy c xc nh nh sau:
bj = 1N
N
i = 1xyi
- nh gi mc ngha ca cc h s theo tiu chun Student.
+ Tra bng tp(f) = t0.05(2) = 4.3 khi chn p=0.05
+ Tnh cc gi tr t theo cng thc sau:
tj = | |bjsbj
So snh cc gi tr t vi tp(f) xc nh phng trnh hi quy tng ng.
- nh gi mc tng thch ca phng trnh theo tiu chun Fisher.
+ Tnh cc gi tr ca y theo phng trnh hi quy xc nh ^
y
s2tt =
i = n
i = 1
(yi - ^
yi ) /n-l
+ Tnh F
F = s2
tt
s2th
+ Gi tr bng ca tiu chun Fisher vi mc ngha p = 0.05, cc bc t do f1 = 3; f2 = 2 l F1-
p(f1, f2) = 19.2.
-
Nu Ftnh < F1-p(f1, f2) th phng trnh hi quy tng quan vi thc nghim, cn ngc li th
khng.
2.2.9.2. Phng php x l s liu nh gi cm quan bng ton hc thng k
Trong th nghim nh gi cm quan, tin hnh trn mu gm 100 ngi th.
Mc ch
- Xc nh mc nh gi sn phm theo thang im c xy dng.
- Kim tra kt qu thu c c tin cy khng.
Cch tin hnh
- Lp bng phn phi th nghim
im 1 2 3 4 5 6 7 8 9
S ngi
- Tnh cc tham s
+ Trung bnh cng
-
X =
1f
i = 9
i = 1
Xifi
Vi Xi : im s; fi l s ngi cho im Xi.
+ Sai s trung bnh cng
m = S/
f
Vi S l mc phn tn quanh gi tr trung bnh
S =
f
1 i = 9
i = 1
fi(Xi - -
X)
+ H s bin thin Cv%: phn nh mc bin thin trong nhiu tp hp c X khc nhau.
Cv% = S-
X
*100%
Cv% = 0%-10% : dao ng nh, ng tin cy.
Cv% = 10%-30%: dao ng trung bnh, tin cy.
Cv% = 30%-100%: dao ng ln, t tin cy.
- V th.
-
CHNG 3. KT QU V BIN LUN
3.1. KT QU NGHIN CU C IM SINH HC CA CC CHNG TRONG TI
3.1.1. Bacillus subtilis sp. (BsTC) v Bacillus natto sp. (Bn)
3.1.1.1. c im hnh thi
Hnh 3.1. Khun lc ca BsTC (tri) v Bn (phi)
Hnh 3.2. T bo v bo t ca BsTC (tri) v Bn (phi)
Bng 3.1. c im hnh thi vi khun BsTC v Bn
c im BsTC Bn
Hnh dng khun lc
Khun lc to; mu trng c, bm
vo mt thch; b mt khun lc
hi nhn, nht.
Khun lc to; trng c, bm
vo mt thch; b mt c th
nhn hoc trn.
Hnh dng t bo
Hnh que, ng ring r hay kt
chui. T bo c mang bo t
hnh trng; bo t nm gia t
bo sinh dng.
Hnh que, ng ring r hay
kt chui. T bo c mang
bo t hnh trng; bo t nm
gia t bo sinh dng.
Kch thc t bo 1 - 1,5 m 1 - 1,5 m
Bo t
-
Nhum Gram Gram dng Gram dng
Nhn xt
Nh vy, v mt hnh thi th Bacillus subtilis sp. khng khc g so vi Bacillus natto sp., do
nu da vo tiu ch ny th khng phn bit hai chng vi khun trn.
3.1.1.2. c im sinh l
ng cong sinh trng ca BsTC v Bn trn mi trng CP
Bng 3.2. Kt qu mt t bo theo thi gian
Thi gian (gi) BsTC (logCFU/ml) Bn (logCFU/ml)
0 3.64 2.85
4 4.71 3.42
8 5.98 5.16
12 7.46 6.23
16 8.15 6.77
20 8.27 7.19
24 8.33 7.78
28 8.39 7.96
32 8.28 8.19
36 7.92 8
40 7.47 6.92
0
1
2
3
4
5
6
7
8
9
0 4 8 12 16 20 24 28 32 36 40
Thi gian (gi)
log
CF
U/m
l
th 3.1. ng cong sinh trng ca BsTC v Bn trn mi trng CP
Nhn xt
-
T ng cong sinh trng ca vi khun BsTC v Bn trn mi trng CP, chng ti nhn
thy:
- Pha lag ca c hai ng cong u ko di trong khong t 0-2 gi u, s lng t bo
tng ln khng ng k. di pha lag tng i ngn, chng t cc t bo vi khun cn tr, nhanh
chng thch nghi vi mi trng CP. Sau , vi khun bt u sinh trng v pht trin theo ly
tha do vi khun thch nghi vi mi trng v trong mi trng giu cht dinh dng. Mt t
bo t cc i 28 gi BsTC khong 2,43.108 v 32 gi Bn khong 1,56.108.
- Pha n nh ko di t 20-32 gi, trong giai an ny sinh sn vn tip tc din ra mt s
t bo trong mi trng nui cy.
- Sau 32 gi, mt t bo gim dn do mi trng ngy cng cn kit cht dinh dng,
ng thi cc sn phm trao i cht ca chng sinh ra cng c ch li s pht trin ca vi khun.
Kt lun
Mt t bo t cc i BsTC cao v sm hn Bn: 28 gi BsTC khong 2,43.108 v 32
gi Bn khong 1,56.108.
ng cong sinh trng ca BsTC v Bn trn mi trng sa u nnh
Bng 3.3. Kt qu mt t bo theo thi gian
Thi gian (gi) BsTC (logCFU/ml) Bn (logCFU/ml)
0 7.84 7.63
4 7.86 7.78
8 8.00 7.94
12 8.56 8.02
16 8.78 8.23
20 8.98 8.63
24 9.23 9.01
28 9.45 9.24
32 9.40 9.31
36 9.34 9.29
40 9.06 8.87
-
0
1
2
3
4
5
6
7
8
9
10
0 4 8 12 16 20 24 28 32 36 40
Thi gian (gi)
log
CF
U/m
l
th 3.2. ng cong sinh trng BsTC v Bn trn mi trng sa u nnh
Nhn xt:
- Pha lag ca BsTC khong t 0-8 gi ngn hn ca Bn khong t 0-16 gi.
- Mt t bo cc i ca BsTC 28 gi, ca Bn l 32 gi.
Kt lun: Chng ti kt thc qu trnh tng sinh vi khun trong mi trng sa u nnh sau
24 gi nui cy (i vi BsTC ) v 28 gi (i vi Bn).
3.1.1.3. c im sinh ha
Phn ng nitratase dng tnh
(1): (-) H2O
(2): (+) Pseudomonas aeruginosa
(3): (+) BsTC
(4): (+) Bn
Hnh 3.3. Th nghim nitratase
Kh nng thy phn casein
Hnh 3.4. Phn ng thy phn casein ca BsTC (tri) v Bn (phi)
1 2 3 4
-
Kh nng thy phn fibrin
Hnh 3.5. Phn ng thy phn fibrin ca BsTC (tri) v Bn (phi)
Kh nng thy phn tinh bt
Hnh 3.6. Phn ng thy phn tinh bt ca BsTC (tri) v Bn (phi)
Nhn xt
Kt qu cho thy c BsTC v Bn u c kh nng thy phn protein, tinh bt. y l c im
quan trng ng dng trong cng ngh thc phm, c th l thy phn u h lm thc ung chc
nng. Thm vo, chng c kh nng thy phn fibrin lm tng thm gi tr probiotic v c tc dng
tt trong cc bnh tim mch.
3.1.2. Lactobacillus sp.
3.1.2.1. c im hnh thi
Hnh 3.7. Khun lc Lactobacillus sp. Hnh 3.8. T bo Lactobacillus sp.
Bng 3.4. c im hnh thi Lactobacillus sp.
Hnh dng khun lc Khun lc nh trn, mu trng c
Hnh dng t bo Hnh que, ng ring r
Kch thc t bo 0,5-1 m
-
Nhum Gram Gram dng
3.1.2.2. c im sinh l
Kh nng lm ng t sa
(+): ng t sa sau cy Lactobacillus sp. vo sa u
nnh 24 gi.
(C): sa u nnh c hp tit trng, khng cy
ging vi khun.
Hnh 3.9. Kh nng lm ng t sa ca Lactobacillus sp.
Kt qu nh tnh acid lactic
ng 1: 3 ml dch mi trng, 3 ml thuc th Ufermen.
ng 2: 3 ml dch ln men, 3 ml thuc th Ufermen.
ng 3: 3 ml acid lactic 98 %, 3 ml thuc th Ufermen.
Hnh 3.10. Kt qu nh tnh acid lactic
3.1.2.3. c im sinh ha
* Oxydase m tnh
1: Lactobacillus sp.(-)
2: (-) E.coli
3: (+) Pseudomonas
4: H2O
Hnh 3.11. Th nghim oxydase
* Catalase m tnh, khng c hin tng si bt
Staphylococcus aureus dng tnh Vi khun lactic m tnh Hnh 3.12. Th nghim catalase
*
1 2 3
1 2 3 4
++
-
Th nghim nitratase: Nitratase m tnh
1: (-) H2O
2: (+) Pseudomonas.
3: (-) Lactobacillus acidophilus
4: Lactobacillus sp.
Hnh 3.13. Th nghim nitratase
* Kh nng ln men glucose
(1): C (-): mi trng phenol red broth
(2): C (+): E.coli
(3): Lactobacillus sp.
Hnh 3.14. Th nghim kh nng ln men glucose
3.2. KT QU NH LNG H ENZYME CA BsTC v Bn
3.2.1. Kt qu kho st hot tnh chung enzyme amylase ca BsTC v Bn
3.2.1.1. Kt qu kho st hot tnh amylase ca BsTC v Bn theo thi gian
Bng 3.5. S bin thin hot tnh amylase ca BsTC v Bn theo thi gian
Thi gian (gi)
BsTC Bn OD VHT/gMT OD VHT/gMT
0 1.044 0.0126 1.05 0.0123 4 1.019 0.0139 1.012 0.0143 8 1.000 0.0149 0.911 0.0195 12 0.988 0.0155 0.902 0.02 16 0.848 0.0228 0.875 0.0214 20 0.771 0.0267 0.848 0.0228 24 0.623 0.0344 0.723 0.0292 28 0.495 0.041 0.761 0.0273 32 0.595 0.0357 0.605 0.0354 36 0.732 0.0288 0.677 0.0316 40 0.835 0.0235 0.932 0.0184
1 2 3 4
1 2 3
-
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0 4 8 12 16 20 24 28 32 36 40
Thi gian (gi)
Ho
t
tn
h (
V
HT
/gM
T)
th 3.3. S bin thin hot tnh amylase ca BsTC v Bn theo thi gian
Nhn xt
- Hot tnh enzyme amylase ca c hai chng tng khng ng k trong thi gian u, tng r
16 - 32 gi v t cc i 28 gi (BsTC ) v 32 gi (Bn), sau gim dn.
- Nhn chung, hot tnh enzyme amylase ca Bn thp hn, bin ng hn v thi gian t cc
i lu hn so vi BsTC.
3.2.1.2. Kt qu kho st hot tnh amylase ca BsTC v Bn theo nhit
Bng 3.6. S bin thin hot tnh enzyme amylase ca BsTC v Bn theo nhit
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
15 20 25 30 35 40 45
Nhit
Ho
t tn
h (
VH
T/gM
T)
Nhit (0C)
BsTC Bn OD VHT/gMT OD VHT/gMT
15 0.066 0.0194 0.109 0.0261 20 0.051 0.0301 0.105 0.0276 25 0.041 0.0373 0.086 0.0346 30 0.037 0.0401 0.074 0.0391 35 0.04 0.038 0.076 0.0384 40 0.043 0.0358 0.081 0.0365 45 0.053 0.0287 0.123 0.0209
-
th 3.4. S bin thin hot tnh amylase ca BsTC v Bn theo nhit
Nhn xt
Nhn vo th, ta thy hot tnh enzyme amylase ca hai chng ging theo nhit tng
ng nhau, chng c hot tnh cao nht vo khong 300C, tuy nhin hot tnh ca Bn thp hn BsTC
nhng khng ng k.
3.2.1.3. Kt qu kho st hot tnh enzyme amylase ca BsTC v Bn theo pH
Bng 3.7. S bin thin hot tnh enzyme amylase ca BsTC v Bn theo pH
pH BsTC Bn
OD VHT/gMT OD VHT/gMT 2 0.722 0.014 0.844 0.0051 3 0.722 0.014 0.69 0.0163 4 0.721 0.0141 0.622 0.0213 5 0.631 0.0206 0.576 0.0247 6 0.41 0.0346 0.54 0.0273 7 0.498 0.0303 0.553 0.0263 8 0.692 0.0162 0.594 0.0233
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
2 3 4 5 6 7 8
pH
Ho
t
tn
h (
V
HT
/gM
T)
th 3.5. S bin thin hot tnh enzyme amylase ca BsTC v Bn theo pH
Nhn xt
Trong iu kin pH = 2 (dch v d dy), c hai chng ging u c kh nng phn gii tinh
bt ca u h nhng khng cao, chng t cc i khi pH = 6. 3.2.1.4. Ti u iu kin nh
hng n hot tnh chung enzyme amylase ca BsTC v Bn
T kt qu trn, chng ti tin hnh lm quy hoch thc nghim, chn cc yu t nh hng
vi cc gii hn v min kho st nh bng 3.8.
Bng 3.8. iu kin th nghim c chn
Yu t Cc mc Khong
-
Hm mc
tiu Y l hot tnh
enzyme
amylase thu c
sau o OD595.
Phng trnh biu din mi quan h c dng Y = f (X1, X2, X3)
Thit lp ma trn th nghim v thu c kt qu nh bng 3.9.
Bng 3.9. Ma trn v kt qu quy hoch thc nghim cc yu t nh hng n hot tnh
chung enzyme amylase ca BsTC v Bn
Mu th nghim X1 X2 X3 Y
BsTC Bn
1 + + + 0.0421 0.024
2 + + - 0.0392 0.02
3 + - + 0.0343 0.016
4 + - - 0.0352 0.018
5 - + + 0.0278 0.018
6 - + - 0.0322 0.015
7 - - + 0.029 0.013
8 - - - 0.0325 0.014
9 0 0 0 0.0427 0.027
10 0 0 0 0.0438 0.029
11 0 0 0 0.0431 0.03
Y = 0.034 + 0.0037 X1+ 0.0013X2 - 0.00074X3 + 0.0017X1X2 + 0.001238X1X3 (1)
Y = 0.01725 + 0.00225X1 + 0.002X2 (2)
Ghi ch: (-): mc di; (0): tm; (+): mc trn.
Sau khi gii bi ton quy hoch thc nghim v tnh cc h s phng trnh hi quy, ngha
ca cc h s c kim tra theo tiu chun Student vi p = 0.05, s bc t do f = 3-1 = 2, chng ti
thu c phng trnh hi quy (1) ca chng BsTC v phng trnh hi quy (2) ca Bn..
Kim tra s tng thch phng trnh hi quy vi thc nghim theo tiu chun Fisher vi
Ftnh = 6.0516 (BsTC) , Ftnh = 5.128 (Bn); F0.95(5.2) = 19.296.
Do Ftnh ca c hai phng trnh hi quy v hot tnh chung amylase BsTC v Bn u b hn
F0.95(5.2) nn c hai phng trnh hi quy u tng thch vi thc nghim.
Mc di (-) Tm (0) Mc trn (+) bin thin
BsTC Bn BsTC Bn BsTC Bn
X1 : thi gian 24 28 28 32 32 36 4
X2 : nhit 25 25 30 30 35 35 5
X3 : pH 5 5 6 6 7 7 1
-
3.2.2. Kt qu kho st hot tnh chung enzyme protease ca BsTC v Bn
3.2.1.1. Kt qu kho st hot tnh enzyme protease ca BsTC, Bn theo thi gian
Bng 3.10. S bin thin hot tnh protease ca BsTC v Bn theo thi gian
Thi gian (gi)
BsTC Bn OD VHT/gMT OD VHT/gMT
0 0,081 2.236 0.079 2.116 4 0,092 2.957 0.093 3.028 8 0,11 4.35 0.099 3.467 12 0,135 6.724 0.122 5.426 16 0,148 8.159 0.133 6.515 20 0,168 10.638 0.139 7.151 24 0,183 14.502 0.14 7.259 28 0,203 15.761 0.149 8.276 32 0,176 11.86 0.176 11.721 36 0,132 8.99 0.159 9.482 40 0,109 4.963 0.143 7.591
0
2
4
6
8
10
12
14
16
18
0 4 8 12 16 20 24 28 32 36 40
Thi gian (gi)
Ho
t
tn
h (
V
HT
/gM
T)
th 3.6. S bin thin hot tnh protease ca BsTC v Bn theo thi gian
Nhn xt
Hot tnh protease ca c hai chng ging u tng dn trong khong t 12-32 gi, sau
gim dn. Vi BsTC, hot tnh protease t cc i 28 gi cn Bn th 32 gi. Nhn chung, hot
tnh protease ca BsTC cao hn ca Bn.
3.2.1.2. Kt qu kho st hot tnh enzyme protease ca BsTC, Bn theo nhit
Bng 3.11. S bin thin hot tnh protease ca BsTC v Bn theo nhit
Nhit (0C)
BsTC Bn OD VHT/gMT OD VHT/gMT
15 0.548 25.036 0.556 27.229 20 0.580 34.122 0.629 49.654 25 0.685 69.805 0.636 52.033 30 0.733 89.114 0.655 58.691 35 0.731 88.272 0.634 51.349
-
40 0.675 66.018 0.619 46.324 45 0.647 55.852 0.525 19.022
0
10
20
30
40
50
60
70
80
90
100
15 20 25 30 35 40 45
Nhit
Ho
t
tn
h (
V
HT
/gM
T)
th 3.7. S bin thin hot tnh protease ca BsTC v Bn theo nhit
Nhn xt
C hai chng ging u cho hot tnh protease cao nht 300C nhng hot tnh chung
protease ca BsTC cao hn ng k so vi Bn.
3.2.1.2. Kt qu kho st hot tnh enzyme protease ca BsTC v Bn theo pH
Bng 3.12. S bin thin hot tnh enzyme protease ca BsTC v Bn theo pH
pH BsTC Bn
OD VHT/gMT OD VHT/gMT 2 0.028 0.04 0.031 0.087 3 0.029 0.055 0.032 0.105 4 0.061 0.964 0.032 0.105 5 0.07 1.37 0.06 0.923 6 0.081 1.957 0.068 1.274 7 0.079 1.843 0.072 1.47 8 0.071 1.419 0.057 0.805
0
0.5
1
1.5
2
2.5
2 3 4 5 6 7 8
pH
Ho
t
tn
h (
V
HT
/gM
T)
th 3.8. S bin thin hot tnh enzyme protease ca BsTC v Bn theo pH
-
Nhn xt
Trong iu kin pH thay i th hot tnh protease cng thay i theo, t gi tr cc i pH
= 6 i vi BsTC v pH = 7 i vi Bn.
3.2.1.4. Ti u iu kin nh hng n hot tnh chung enzyme protease ca BsTC v Bn
T kt qu kho st tng yu t, chng ti tin hnh lm quy hoch thc nghim kho st
hot tnh protease chung cc yu t.
Cc gii hn v min kho st ca cc yu t nh bng 3.13.
Bng 3.13. iu kin th nghim c chn
Hm mc tiu Y l hot tnh enzyme protease thu c sau o OD660.
Phng trnh biu din mi quan h c dng Y = f (X1, X2, X3)
Thit lp ma trn th nghim v thu c kt qu nh bng 3.14.
Bng 3.14. Ma trn v kt qu quy hoch thc nghim cc yu t nh hng n hot
tnh chung enzyme protease ca BsTC v Bn
Mu th nghim X1 X2 X3 Y
BsTC Bn
1 + + + 1.19 2.01
2 + + - 2.72 2.07
3 + - + 1.92 2.12
4 + - - 3.78 2.141
5 - + + 1.95 1.058
6 - + - 2.206 1.534
7 - - + 1.95 1.649
8 - - - 2.272 1.708
9 0 0 0 4.579 2.206
10 0 0 0 4.461 2.344
11 0 0 0 4.45 2.549
Y = 2.2485 + 0.154X1 - 0.232X2 - 0.496X3 - 0.2155X1X2 - 0.3515X1X3 (3)
Yu t
Cc mc Khong
bin thin Mc di (-) Tm (0) Mc trn (+)
BsTC Bn BsTC Bn BsTC Bn
X1 : thi gian 24 28 28 32 32 36 4
X2 : nhit 25 25 30 30 35 35 5
X3 : pH 5 6 6 7 7 8 1
-
Y = 1.78625 + 0.299X1 (4) Ghi ch: (-): mc di; (0): tm; (+): mc trn.
Sau khi gii bi ton quy hoch thc nghim v tnh cc h s phng trnh hi quy, ngha
ca cc h s c kim tra theo tiu chun Student vi p = 0.05, s bc t do f = 3-1 = 2, chng ti
thu c phng trnh hi quy (3) ca chng BsTC , (4) ca Bn..
Kim tra s tng thch phng trnh hi quy vi thc nghim theo tiu chun Fisher vi
Ftnh = 2.768 (BsTC) , Ftnh = 4.558 (Bn); F0.95(5.2) = 19.296. Do Ftnh < F0.95(5.2) nn c hai phng
trnh hi quy u tng thch vi thc nghim.
Kt lun chung
Qua kt qu kho st hot tnh h enzyme ca hai chng BsTC v Bn, chng ti nhn thy gn
nh c hai hot tnh amylase v protease ca BsTC c phn tri hn so vi Bn trong iu kin xt
ring tng yu t cng nh khi thit lp ma trn quy hoch thc nghim cc yu t nh hng n
hot tnh h enzyme ca hai chng vi khun trn. iu ny c gii thch, c l BsTC l chng
ging c phn lp t chao Vit Nam nn c kh nng thch nghi vi iu kin mi trng th
nghim ti Vit Nam cao hn so vi chng Bn c phn lp t sn phm nattospes ca Nht Bn.
Do vy, chng ti chn BsTC thy phn u h to sn phm thc ung chc nng.
3.3. KT QU CH BIN SN PHM
3.3.1. Kt qu kho st t l ging v thi gian thy phn u h hp l
Bng 3.15. Kt qu kho st t l ging BsTC v thi gian thy phn u h
Thi gian
c im
T l ging
0.5% 1% 2%
15 gi - C cht
- BsTC (logCFU/g)
Cha nhuyn
7.826
Cha nhuyn
9.127
Hi nhuyn
9.428
18 gi
- C cht
- BsTC (logCFU/g)
Cha nhuyn
Nhim
Nhuyn, bo, thm
9.505
Nhuyn, bo,
hi ng
9.790
21 gi
- C cht
- BsTC (logCFU/g)
Nhuyn
Nhim
Nhuyn, hi
ng, nng
9.77
Nho, cht,
nng
10.04
24 gi
- C cht
- BsTC (logCFU/g)
Hi nho,
ng, nng
Hng
Nho, ng,
nng
10.11
Rt nho, rt
ng, nng
27 gi - C cht
- BsTC (logCFU/g)
Hi, to kh,
mu en
Rt ng,
nng
Rt ng, nng