1

Chng 1
M U

1.1. t vn
Thc phm c b sung vi sinh vt sng (probiotic) c nhiu nghin cu
xc nh l c li cho sc khe con ngi v ng vt v hin vn ang c cc
chuyn gia trn nhiu lnh vc c lin quan nghin cu to ra nhiu ch phm sinh
hc c li (Ross v ctv, 2005). V vy thc phm cha probioic ang c ngi tiu
dng v c nh sn xut quan tm. Hin nay, cc sn phm cha probiotic khng dng
li vic ch tn ti mt ging vi sinh vt no m cn l hn hp ca nhiu ging
vi sinh vt probiotic nhm to ra li ch rng ln hn trn mt liu dng.
Lactobacillus acidophilus l vi khun c li thng tr trong ng
rut gip gi cn bng cho h vi sinh vt ng rut, c ch s pht trin
ca cc vi sinh vt c hi (Kligler v Cohrssen, 2008), h tr iu tr mt s chng
bnh lin quan n tiu ha (Frank v Roelof, 1993), sinh ra enzyme lactase gip c
th hp thu ng sa (Monatalto v ctv, 2006).
Bacillus subtilis l mt trong nhng vi sinh vt quan trng trong vic kch thch
h thng min dch t nhin, c dng iu tr mt s chng bnh ln quan n
tiu ha (Kahl, 1999), sinh ra mt s enzyme c li cho h tiu ha nh amylase,
protease,
Saccharomyces cerevisiae gip ci thin t l tiu ha cc cht dinh dng
(Ghasemi v ctv, 2006), tng kh nng p ng min dch (Asli v ctv, 2007), cnh
tranh v tr bm dnh, cnh tranh dinh dng vi vi sinh vt gy bnh trong ng
rut.
S nui cy ba ging vi sinh vt k trn trn cng mt mi trng l cn
thit, gip n gin ha qu trnh nui cy, thun tin cho vic thu sinh khi to sn
phm probiotic c mt c L. acidophilus, B. subtilis, S. cerevisiae v sn phm ny c
c tc dng l tc dng cng gp ca c ba ging probiotic trn.
2
Trn c s chng ti thc hin ti: Nghin cu nui cy ng thi
Lactobacillus acidophilus, Bacillus subtilis, Saccharomyces cerevisiae trn canh
trng MRS ci tin.
1.2. Mc ch
Mc ch: nui cy ng thi L. acidophilus, B. subtilis, S. cerevisiae trn canh
trng MRS ci tin nhm t mt mi chng > 10
6
CFU/ml dch nui cy ng
dng thu sinh khi sn xut sn phm probiotic cha c ba loi vi sinh vt c li trn.
t c mc ch trn, ti thc hin nhng ni dung sau:
1. Xc nh ng cong sinh trng ca ba chng vi sinh vt th nghim
khi tng sinh chung trn canh trng MRS.
2. Th nghim sng lc cc yu t nh hng n mt vi sinh vt nui
cy theo thit k Plackett Burman.
3. Ti u ha qu trnh nui cy s dng phng php RSM-CCD.
4. nh gi mc tng thch ca gii php ti u bng thc nghim.

3


Chng 2
TNG QUAN TI LIU

2.1. Probiotic
Probiotic c FAO/ WTO (2001) nh ngha l: Cc vi sinh vt sng m khi
dng vi s lng s to ra li ch v sc khe trn vt ch. Hin nay thut ng
probiotic c s dng ch nhng ch phm vi sinh vt sng hu ch khi c
a vo c th ng vt v con ngi thng qua thc n hoc nc ung to nn
nhng nh hng c li cho vt ch.
Vic con ngi s dng vi sinh vt sng nhm tng cng sc khe khng phi
l mi. T lu, con ngi bit tiu th cc thc phm cha vi sinh vt sng c li
chng hn nh cc sn phm ln men: sa chua, chao, rau qu ln men cha chng
y bng, n khng tiu, to bn, au bng tiu chy d khng bit n tc ng ca
h vi sinh vt c li trong sn phm. Nm 1930 cc nh khoa hc i hc Havard
chng minh rng cc vi khun ng rut ng mt vai tr quyt nh trong qu trnh
tiu ha, gip tiu ha thc n, cung cp mt s vitamin v cc cht dinh dng khc
nhau m c th vt ch khng t sn xut ra c (Fuller, 1992).
Trong nhng nm gn y, cc nh nghin cu quan tm n kh nng khng vi
khun gy bnh ca vi sinh vt probiotic thay th cho vic s dng khng sinh v
h tr vic s dng khng sinh trong iu tr bnh, phng nga v lm chm qu trnh
pht trin ca bnh ung th, gim cholesterol mu.
C nhiu ging vi sinh vt c nui cy v s dng vi vai tr l probiotic
nhng ch yu thuc chng Lactobacillus v Bifidobacterium; ngoi ra cn c mt s
loi thuc Enterococcus, Streptococus, Saccharomyces, Bacillus, (Tannock, 2001).
Trong hin nay L. acidophilus, B. subtilis, S. cerevisiae l cc dng chnh c p
dng (Chen v ctv, 2009). Vamanu (2002) cho rng cc sn phm probiotic thu c
vi sinh khi hn hp (vi khun v nm men) c th ng mt vai tr quan trng trong
4
iu tr cc bnh khc nhau, li th ca cc sn phm ny l khng cn s h tr ca
thuc v khng h c tc dng ph.
Hiu qu ca probiotic ph thuc vo loi vi sinh vt s dng, liu dng,
tui, tnh trng sc khe ca ngi dng. Liu dng thng c khuyn co l 5 - 10
t t bo sng trn mt liu dng hng ngy cho tr em v 10 - 20 t cho ngi ln
(Kligler v Cohsen, 2008). Hin nay, khoa hc cng chng minh mc lm
sng l probiotic c dng vi liu lng thch hp c tc dng tch cc i vi sc
khe nh tng cng s vi sinh vt c li trong ng rut, tng kh nng tiu ha
thc n do sinh vitamin v emzym ngoi bo, c bit l enzym phn gii ng
lactose. Theo Desmond v ctv nm 2000 th probiotic cn gip tng cng h min
dch t nhin chng li nhng bnh nhim trng ng tiu ha v ri lon tiu ha do
s dng khng sinh. Cn theo cng b ca Kligler v ctv nm 2008 th probiotic cn
c hiu qu tr nhng vn lin quan n tiu ha nh: tiu chy, to bn, d ng
thc n; iu ny trng vi nghin cu ca Kim vo nm 2006. Nm 2000 Brady
cng cng s cho rng ngoi tc dng tt n h tiu ha probiotic cn c hiu qu
trong ngn nga v lm chm s pht trin ca mt s bnh ung th nh: ung th rut
kt, bnh vim rut, vim i trng, Tamayo cng nh Liong cng cc cng s ca
mnh trong nm 2008, OKeefe trong nm 2009 cng nghin cu v c cng kt
lun nh trn. Liong v Shah trong nghin cu vo nm 2005 v 2008 cho rng
probiotic cn c hiu qu gim cholesterol huyt thanh v gim huyt p mu khi c
s hin din ca prebiotic - cht kch thch s sinh trng ca li khun.
Hin nay, ngoi vic dng probiotic nh ch phm h tr tiu ha b sung vo
ch n ung, probiotic cn c dng nh mt thuc cha bnh, tuy nhin cha c
bng chng xc thc no v hiu qu ca cch dng ny nn vn c WHO v FDA
quan tm khuyn kch nghin cu.
Khi ni v tc ng ca probiotic, Kligler (2008) cho rng tc dng c li ca
probiotic ch th hin khi chng vn cn sng khi vo n rut non, cc ging vi sinh
vt khc nhau th c nhng li ch v kiu tc ng khc nhau nhng ch yu c cc
tc ng nh:
Cnh tranh v tr bm dnh v cnh tranh dinh dng vi cc vi sinh vt c hi
hay cnh tranh loi tr. Cc vi sinh vt probiotic c ng v tng dn s trong rut,
5
kha cht cc v tr th cm v ngn cn s bm dnh ca cc vi sinh vt khc nh
Escherichia coli, Salmonella,... Mt s nm men probiotic (S. cerevisiae ; S.
boulardii) khng ch tranh v tr bm dnh vi cc vi khun khc m cn c th y cc
vi khun c roi ra khi v tr bm dnh trn nim mc rut (Kligler v Cohsen, 2008).
Tuy nhin, cnh tranh dinh dng l phng thc cnh tranh khc lit nht, v s sinh
si vi s lng ln ca mt loi vi sinh vt no l mt mi e da nghim trng
i vi cc loi khc v ngun c cht cho s pht trin.
Vi sinh vt probiotic sinh ra cht khng khun sinh hc, l cc cht km hm vi
khun nh lactoferrin, lysozym, hydrogen peroxide cng nh mt s axit hu c khc.
Cc cht ny gy tc ng bt li ln vi khun c hi ch yu l do s gim thp pH
trong rut (Conway, 1996), ng thi cng kch thch h thng min dch t nhin ca
vt ch.
Ngoi ra, cc vi sinh vt probiotic cn c th sinh ra cc cht c li cho vt ch
nh enzyme, vitamin. Enzyme c li cho tiu ha thng c vi sinh vt to ra l
amylase, protease, c bit l enzyme lactase hy ng lactose gip tr chng khng
dung np lactose (Gilliland, 1990); cc vitamin thng l vitamin nhm B v K (Rossi
v ctv, 2011) v mt s cht khc gip tng h s hp th thc n ca vt ch, ci
thin kh nng gim hp thu khong.
Tuy vy, khng phi vi sinh vt c li no cng c th lm probiotic, vic la
chn cc chng vi sinh vt c chc nng probiotic l rt phc tp. Tiu chun u tin
i vi vi sinh vt lm probiotic l phi an ton cho vt ch, c kh nng sng st
trong sut qu trnh sn xut, bo qun v tr nn chim u th trong ng tiu ha
vt ch. Cc tiu chun la chn ny c hp l ha thng qua cc th nghim in
vitro, t s tuyn chn c cc chng c tim nng nh l ngun probiotic. Cc
tiu chun la chn nh sau:
Mt l khng gy bnh, c li i vi vt ch, d nui cy, c th sng st v
n nh trong sut qu trnh sn xut ch phm.
Hai l c tnh bm dnh trn b mt ng tiu ha hoc cc t bo biu m.
Cc chng probiotic phi bm dnh c vo thnh rut non v sinh si ny n y.
y l mt trong nhng ch tiu quan trng v vi sinh vt c bm dnh th mi tn ti,
6
pht trin trong ng tiu ha v c thi gian phi lu pht huy tc dng ca
mt probiotic.
Ba l c hot tnh khng khun chng li cc vi khun gy bnh. La chn
c cc chng c kh nng sn sinh cc cht khng khun l c tnh quan trng
trong pht trin probiotic. Cc chng probiotic cn c hot tnh c ch vi khun gy
bnh nh E. coli, Salmonella v Campylobacteria (Klaenhammer v Kullen, 1999).
Bn l c t l sng cao khi qua h tiu ha vn c axit thp, enzyme v mui
mt. Khoang ming v d dy ca vt ch l ni c mi trng axit pH t 2 - 3 v c
mt cc enzym tiu ho (amylase, protease, lysozym,). Ngoi ra, cc chng
probiotic phi c kh nng tn ti v pht trin vi nng mui mt 2%, hay c
mt s ging nm men, Bacillus v Lactobacillus c kh nng khng li mui mt
(Klaenhammer v Kullen, 1999). Hin nay th trng a vo s dng cc b bc
bo v hay b sung cc cht prebiotic tng kh nng sng xt ca vi sinh vt lm
probiotic.
Cui cng l c kh nng khng li khng sinh v khng sinh cng l mt vn
nh hng n kh nng sng ca vi sinh vt probiotic (Klaenhammer v Kullen,
1999).
2.2. Lactobacillus acidophilus
Phn loi:
B: Bacilli
H: Lactobacillaceae
Ging: Lactobacillus
Loi: Lactobacillus acidophilus
L. acidophilus c xem l vi khun thn thin thng tr trong ng
rut, ming v m o ca ngi; thuc nhm vi khun Gram
+
, khng sinh bo
t, l loi vi sinh vt d dng, k kh hoc vi hiu kh; ln men c 15 loi ng
khc nhau trong c glucose, galactose, lactose, fructose, sucrose, (Wheater,
1955). L vi khun ln men lactic ng hnh theo EMP, sinh ra cc cht hu c c vai
tr l khng sinh. Sinh sn bng cch nhn i.
L. acidophilus l vi khun vi hiu kh nn pht trin c trn mi trng
khng c hoc c t oxy (Klaenhammer v Kullen, 1999); pH tng trng tt l t 4,5
7
n 7, ti u pH 6 (Riaz, 2010). Nhit pht trin trong khong 20 - 48
0
C, pht
trin ti u 37
0
C.
L. acidophilus l loi c nhu cu dinh dng rt phc tp, ngoi ngun cabon
v nit L. acidophilus cn c nhu cu tuyt i i vi mt s axit amin v cht
khong. Trong mi trng MRSA k kh sau 48 gi 37
0
C xut hin khun lc
nh, hnh cu ra dp c mu vng nht. Trn mt mi trng PDA L. acidophilus pht
trin rt yu, ch to ra cc chm nh li ti mu trng.
Theo nghin cu ca Nguyn V Tng Vy v ctv nm 2007 th L. acidophilus
chu c mi trng axit trong 2 gi pH 1 v 2 vi t l rt thp nhng pH 3 v 4
l 41 - 65%; khng vi t nht l 3 khng sinh, c th chu c ampicillin (32
g/ml), streptomycin (32 g/ml) hn 10 gi. Hood v Zottola (1988) cho rng sinh
khi L. acidophilus pH 4,0 khng gim ng k sau 2 gi.
L. acidophilus c xem l vi khun thn thin trong ng rut, thng lin
quan n s c ch ca cc loi vi sinh vt gy bnh, gip h tr tiu ha, c bit l
vi nhng ngi khng dung np c ng lactose (Hove v ctv, 1999; Vrese v
ctv, 2001; Monatalto v ctv, 2006). L. acidophilus c lin quan n vic ngn nga
tiu chy c lin quan n s dng khng sinh, nhim virut hay tiu chy du lch do
n phi thc phm nhim khun, ci thin chng nhim trng m o, gim kh nng
ung th rut kt (Gilliland v ctv, 1990; Havenaar v ctv, 1992; Frank v Roelof,
1993). iu tr triu chng ca hi chng rut kch thch v bnh vim rut (Bhatia v
ctv, 1989; Kligler v Cohrssen, 2008) tng p ng min dch (Perdigon, v ctv,
1990), gim mc cholesterol trong huyt thanh bng cch phn gii mui mt
(Gilliland, 1990; Frank v Roelof, 1993), iu tr vim da d ng tr em (Kligler v
Cohrssen, 2008). Theo Sanders v Klaenhammer (2001) L. acidophilus c bit n
l vi khun sn xut khng sinh t nhin v c th hn ch c 27 loi vi khun,
trong c 11 loi gy bnh in hnh nh Staphylococcus aureu, Salmonella,
Clostridium, E. coli (Kligler v Cohrssen, 2008).
Liu dng c khuyn co 100 triu n 35 t t bo sng/ ngy (Kligler v
Cohsen, 2008)

8
2.3. Bacillus subtilis
Phn loi:
B: Bacillales
H: Bacillaceae
Ging: Bacillus
Loi: Bacillus subtilis
Vi khun B. subtilis thuc nhm vi khun bt buc trong ng rut, chng
c phn b hu ht trong t nhin: t, nc, bi, c kh, nhng ch yu nhiu
nht l trong t. B. subtilis thuc nhm trc khun, Gram
+
, c kh nng sinh ni bo
t, hiu kh hoc k kh ty nghi (Dahl, 1999); ln men c cc loi ng: glucose,
maltose, manittol, saccharose, xylose, arabinosse. B. subtilis c khng nguyn H v O,
cu trc khng nguyn dng D v L - glutamic axit; sn sinh khng sinh subtitracin c
tc dng c ch vi khun G
+
, G
-
.
Sinh sn bng cch nhn i hoc ny mm do s nt
bo t, c kh nng chu nhit, chu m (T Minh Chu, 2000).
B. subtilis l vi khun hiu kh nhng cng c th pht trin trong mi trng
thiu oxy. pH pht trin t 7 n 7,4; ti u pH 7,2. Nhit pht trin t 30 45
o
C,
ti u 37
0
C. Dinh dng cn C, H, O, N v mt s nguyn t vi lng khc. B.
subtilis khng pht trin c trong mi trng MRSA ym kh; trn b mt mi
trng PDA to vt loan mu trng, rng ca khng u trn mt thch, ra dp nht
mu xung quanh.
B. subtilis l vi khun chu c axit d dy v cht dch tiu ha trong ng
rut. Nguyn V Tng Vy v ctv (2007) ch ra rng B. subtilis chu c mi trng
axit trong 2 gi pH 1; 2; 3 v 4 ln lt l 3; 9; 70 v 85%, chu c mi trng c
0,5 - 1% v 1,5 - 2% mui mt trong 10 gi ln lt l 2% v 1%. B. subtilis khng
vi t nht l 4 khng sinh, c th chu c ampicillin (32 g/ml) hn 10 gi,
streptomycin (2 mg/ml) hn 5 gi.
y l loi vi sinh vt c h enzyme phong ph nhng quan trng nht l
amylase v protease, l hai loi enzyme thuc h thng men tiu ha nn c tc dng
h tr tiu ha thc n. B. subtilis c th c ch s pht trin ca E. coli, Salmonella
v c dng trong iu tr tiu chy, vim rut cp v mn tnh, ri lon tiu ha, tr
em i ngoi phn sng, vim i trng.
9
B. subtilis t lu c s dng sn xut nhiu loi enzyme v s dng c t
bo sng trong nhiu qu trnh sn xut v ch bin thc phm nh mt vi sinh vt an
ton (Kahl, 1999).
2.4. Saccharomyces cerevisiae
Phn loi:
B: Saccharomycetales
H: Saccharomycetaceae
Ging: Saccharomyces
Loi: Saccharomyces cerevisiae
S. cerevisiae thuc nhm vi sinh vt khng thng tr trong ng rut nhng
rt ph bin trong t nhin. L loi nm men hu dng nht c s dng t lu i
ln men sinh ra cn v sn xut bnh m.
S. cerevisiae ln men c hu ht cc loi ng tr lactose v cellobiose. Tuy
nhin, kh nng tng trng ca S. cerevisiae trn cc loi ng khc nhau l khc
nhau (Chen, 2009). H enzyme ca nm men cng kh phong ph bao gm: saccarase,
catalase, invertase, amylase, nhng khng c enzym phn hy protein. S. cerevisiae
sinh sn ch yu bng cch ny chi nhng cng c th sinh sn hu tnh.
S. cerevisiae l loi c th pht trin trong iu kin hiu kh hay k kh, iu
kin hiu kh sinh trng tng sinh khi nhanh, iu kin ym kh ch yu ln men to
cn; pH pht trin trong khong 4 - 6,8, ti u pH 5,8; nhit pht trin trong
khong t 27 35
o
C, ti u 32
0
C. Ngun dinh dng carbohydrate chnh l cc loi
ng, thch hp nht l ng fructose, c th dng ngun nit v c ln hu c, s
c mt ca biotin v mt s nguyn t vi lng nh st, canxi, magi kch thch pht
trin ca S. cerevisiae. S. cerevisiae trong mi trng MRSA ym kh khng pht
trin c, trn b mt mi trng PDA to khun lc mu trng c, trn nh cao, b
mt bng.
Theo Nguyn V Tng Vy v ctv (2009), S. cerevisiae c th sng trong mi
trng axit ca d dy trong 3 gi, mt s chng thuc S. cerevisiae c kh nng thch
nghi tt trong mi trng cha pancreatine ca rut non v mi trng c cha mui
mt. Kh nng bm dnh rut non ca S. cerevisiae t ti a 80,88% (H L Qunh
Chu v ctv, 2010). Khi sng trong h tiu ha S. cerevisiae kch thch tiu ha, hp
10
thu cht c v cnh tranh v tr bm dnh, cnh tranh dinh dng vi vi sinh vt gy
bnh nh E. coli, Shigella, Salmonella (Ghasemi v ctv, 2006; Asli v ctv, 2007),
Sthaphylococcus aureus (Vamanu, 2002). c bit, S. cerevisiae pht trin nhanh
trong mt thi gian ngn, tuy nhin khng c tr lu di v d loi b khi ng rut.
2.5. Cc nghin cu c lin quan
Nguyn Thanh Thong (2007) trong nghin cu ca mnh ch ra rng khi
nui phi hp B. subtilis v S. cerevisiae trn mi trng r ng ti u th vn gi
c tc tng trng tng ng vi trn mi trng tt u ca mi ging.
Hosoi v ctv (2000) cho rng B. subtilis gip tng kh nng tng trng v kh
nng tn ti ca Lactobacilli.
Benjamas v ctv (2003) th cho rng S. cerevisiae c nhng tc ng tch cc
n vic gim lng axit lactic dn n hiu ng tch cc ln s tng trng ca
Lactobacillus, bi lng axit do Lactobacillus sinh ra khi sinh trng lm gim pH,
n mt lc no s l tc nhn km hm i vi chnh n, cn do S. cerevisiae sinh
ra s trung ha mt phn axit gi pH mi trng nui cy khng gim qu nhanh nn
ko di thi gian tng trng ca Lactobacillus.
Chng ti chn mi trng MRS lm mi trng nui cy th nghim v mi
trng ny c y cht dinh dng cn thit, pH nm trong khong sinh trng
tng sinh khi ca c ba chng trn; chng ti thc hin ti u ha mi trng t
mt t bo ca mi chng l ti a v tng ng gia ba chng.

11


Chng 3
VT LIU V PHNG PHP NGHIN CU

3.1. Thi gian v a im nghin cu
Thi gian tin hnh: 3/2011 - 7/2011
a im thc hin: Phng th nghim vi sinh thuc Vin Cng ngh Sinh hc -
Thc phm, Trng i hc Cng Nghip TP. H Ch Minh.
3.2. Vt liu v phng tin th nghim
Chng vi sinh L. acidophilus, B. subtilis, S. cerevisiae c phn lp v bo
qun ti Vin Cng ngh Sinh hc - Thc phm, Trng i hc Cng Nghip
TP.HCM.
Mi trng nui cy vi sinh: Mi trng gi ging, hot ha ging v m vi
sinh vt c trnh by bng 3.1. Thnh phn mi trng c trnh by ti ph lc
1.
Bng 3.1: Mi trng nui cy vi sinh vt
Mi trng
Vi sinh vt
Gi ging Hot ha ging m
L. acidophilus MRSA MRSB MRSA
B. subtilis NA NB PDA
S. cerevisiae Thch Sabouraud Sabouraud PDA

Ha cht s dng: cn 90
0
, pepton, D - glucose, HCl 10%, NaOH 10%.
Thic b: Ni hp tit trng BK75
T m hiu SHELAB
T cy v trng ESCO
pH k Sartorius - Docu
12
Cn k thut 2 s l Sartorius TE 612
T sy hiu SHELAB
Dng c: nhit k, pipettman, a petri, ng nghim, que cy trn, que trang,
n cn, erlen 150 ml, erlen 250 ml, ng ong, u tp v cc iu kin thng thng
khc ca ca phng th nghim vi sinh.
ti thc hin quy hoch thc nghim bng phng php p ng b mt -
thit k cu trc c tm (RSM - CCD), phn mm Design - Expert 7.0.0 c s dng
thit k th nghim v s l s liu.
3.3. Phng php th nghim
3.3.1. K thut nui cy
T mi trng gi ging sau 24 gi dng que cy trn ly mt khun lc ring
l cy vo erlen 150 ml cha 50 ml mi trng hot ha ging, L. acidophilus c
37
0
C; B. subtilis, S. cerevisiae nui cy lc 90 vng/ pht nhit phng 30 2
0
C.
Dng que cy vng ly mt vng vi khun t mi trng hot ha ging cy
vo erlen 150 ml cha 50 ml mi trng th nghim MRS. Th nghim c nhit
30
0
C c iu chnh bi t m, nhit di 30
0
C c t trong phng my lnh c
nhit k theo di.
3.3.3. K thut m s lng vi sinh vt
Vi tt c cc nng pha long c dng m khun lc, dng pipettman
vi u tp v trng ht 0,1 ml dch nui cy cy ln a petri chun b sn mi
trng. Vi L. acidophilus s dng phng php a ym kh, dch mu ln a
trc ri mi mt lp 5 ml mi trng thch lng ln, sau khi lp mi trng ny
ng hn th tip mt lp 5 ml mi trng thch lng ln tip to mi trng k
kh, ngui. Vi hai ging cn li dng phng php trang a, dch nui cy ln
mi trng chun b sn mt ngy trc v trang u bng que trang v trng.
nhng a petri ny trong t m 37
0
C trong 24 gi em ra m, 24 gi sau em ra
kim tra li. Ch s dng s liu ca nhng a c s lng t 25 n 300 khun lc
ca mi loi vi sinh vt. Khun lc c trng trn mi trng thch m c th hin
hnh 3.1.
13
h

Hnh 3.1: A: mi trng PDA, (a) - khun lc B. subtilis; (b) - khun lc S. cerevisiae
B: khun lc L. acidophilus trn MRS Agar.

Cng thc tnh mt t bo:

Trong :
A l mt t bo (CFU/ ml)
N l tng s khun lc m c
n
i
l s a c m nng pha long i
V l s ml dch mu cy vo mi a
f
i
l nng pha long c s khun lc c chn m.
3.4. Ni dung thc hin
3.4.1. Th nghim 1: Kho st ng cong sinh trng ca ba chng vi sinh vt
khi tng sinh chung trn canh trng MRS
Mc ch: lp ng cong sinh trng ca ba chng vi sinh vt nghin cu
c c nhn nh s b v s sinh trng ca ba chng khi nui cy chung trn canh
trng MRS. y l c s chn yu t th nghim v phm vi nghin cu cc th
nghim tip theo.
B A
14
T mi trng hot ha ging dng que cy vng cy mt vng vi khun vo
mi trng th nghim, th nghim c thc hin trn 50ml mi trng, nhit
37
0
C vi ba ln lp li.
3.4.2. Th nghim 2: Thit k sng lc yu t th nghim theo Plackett - Burman
Mc ch: tm ra yu t th nghim nh hng nht n ch tiu theo di, qua
loi b bt cc yu t th nghim. Kt qu thu c th nghim 2 s c dng lm
yu t th nghim th nghim 3 tip theo.
Plackett - Burman l thit k chuyn bit cho th nghim t 2 - 31 yu t, m
mi yu t l khc nhau hn hai cp . Thit k ny c s dng khi gi nh s
vng mt ca hai yu t tng tc, kim tra chc chn cho s loi b yu t tc
dng rt t hoc khng c tc ng n ch tiu theo di. Kt qu s a ra 3 hoc 4
yu t c mc nh hng nht n ch tiu theo di.
Th nghim c thc nghim mc mt yu t, tc l lm cc th nghim ch
c mt yu t thay i nhm xc nh ngha ca cc yu t nghin cu, xc nh
phng hng v mc nh hng ca mi yu t trong cc th nghim tip theo ca
qu trnh ti u ha.
Sau khi thc hin th nghim thm d, xc nh mc nh hng n mt
vi sinh vt nui cy, chng ti chn cc yu t th nghim vi phm vi kho st ca
cc yu t theo di nh bng 3.2. Ma trn quy hoch thc nghim theo thit k
Plackett - Burman vi t hp 12 th nghim khc nhau c thit lp bi 5 yu t th
nghim mc thp (1) v mc cao (1). B tr th nghim c thc hin nh bng
3.4; yu t th nghim v ch tiu theo di c trnh by trong bng 3.2 v 3.3.
Bng 3.2: Cc yu t s dng trong Plackett - Burman
Yu
t
Tn yu t n v
Phm vi
nghin cu
Mc
-1 1
X1 pH 5,8 - 6,5 5,8 6,5
X2 Nhit 0C 30 - 37 30 37
X3 Thi gian gi 12 - 18 12 18
X4 Pepton b sung % (w/v) 0 - 10 0 10
X5 D - glucose b sung % (w/v) 0 - 20 0 20
15

Bng 3.3: Ch tiu theo di trong ti
Ch tiu theo di Tn n v
Y1 L. acidophilus CFU /ml
Y2 B. subtilis CFU /ml
Y3 S. cerevisiae CFU /ml
Bng 3.4: Bng ma trn theo thit k Plackett - Burman
Nghim thc X1 X2 X3 X4 X5
1 1 1 -1 1 1
2 -1 1 1 -1 1
3 1 -1 1 1 -1
4 -1 1 -1 1 1
5 -1 -1 1 -1 1
6 -1 -1 -1 1 -1
7 1 -1 -1 -1 1
8 1 1 -1 -1 -1
9 1 1 1 -1 -1
10 -1 1 1 1 -1
11 1 -1 1 1 1
12 -1 -1 -1 -1 -1
o = 0,05
3.4.3. Th nghim 3: Thit k ti u ha mi trng nui cy theo RSM - CCD
Mc ch: trn c s kt qu thc hin ca th nghim 2 mc 3.4.2, chng ti
tin hnh quy hoch thc nghim, kt qu thu c l phng n sn xut ti u nht
tha cc iu kin a ra trc. Phng php quy hoch thc nghim c s dng l
phng php hm p ng b mt - thit k cu trc c tm (RSM CCD).
i vi vic ti u mi trng nui cy vi sinh vt bng RMS c miu t bi
Giang Th Kim Lin (2009), qu trnh gm hai giai on c bn:
Giai on u: Thc nghim mt yu t. Tc l lm cc th nghim ch c mt
yu t thay i nhm xc nh ngha ca cc yu t nghin cu, xc nh phng
hng v mc nh hng ca mi yu t trong cc th nghim tip theo ca qu
trnh ti u ha.
Giai on th hai: Tm mt tng quan ti u cho cc yu t quan trng nht
(c ngha nht) trn c s c nh cc yu t cn li gim bt chi ph th nghim.
16
Phng n cu trc c tm (CCD) l mt trng hp c bit trong nhm cc
m hnh quy hoch thc nghim. N c cu thnh t ba thnh phn:
- Nhn: l mt phng n tuyn tnh vi 2
k
nh ca mt hnh khi u
trong khng gian k chiu. Nu k > 5, c th gim bt s th nghim bng cch s dng
phng n yu t tng phn 2
k-1
.
- 2k im sao ( * ) nm trn cc trc ta ca khng gian yu t. Cc ta
ca cc im sao l ( o, 0, 0,...,0), (0, o, 0,...,0),..., (0, 0,...,0, o). o l khong
cch t tm phng n n im sao gi l cnh tay n sao. Cc im sao l cn thit
m rng khng gian nghin cu kho st tc ng ca mt yu t n c th tm
c cc c lng ca h s b
ii
trong phng trnh hi quy bc 2.
- n
0
th nghim tm phng n tm phng sai ti hin.
Cnh tay n sao o v s th nghim n
o
tm c chn ph thuc vo tiu
chun ti u, thng l phng n trc giao hay phng n quay.
Phng trnh hi quy ( m hnh ha) c biu din bng phng trnh sau:
y = b
0
+ b
1
x +b
2
x
2
+ b
3
x
3 +
b
12
x
1
x
2 +
b
23
x
2
x
3
+ b
13
x
1
x
3
+ b
11
x
1
2
+ b
22
x
2
2
+
b
33
x
3
2

Trong :
b
0:
l h s t do
b
1
, b
2,
b
3
: l h s bc 1
b
11
, b
22
, b
33
: l h s bc 2
b
12,
b
23,
b
13
:

h s ca tng cp yu t.
x
1
, x
2
, x
3
: l cc bin
y : l ch tiu theo di
Ta c:

=
=
N
i
i ij j
y x
N
b
1
1

Nu dng phng trnh hi quy y hn c k n cc hiu ng tng tc hai bin:
3 2 23 3 1 13 2 1 12 3 3 2 2 1 1 0
x x b x x b x x b x b x b x b b y + + + + + + =
Th:

=
=
N
i
i ij j
y x
N
b
1
1

17

=
=
N
i
i i l j jl
y x x
N
b
1
) (
1

Phng sai ca cc h s c tnh nh sau:
N
SS
SS
PE
bj
=
Tin hnh: th nghim c b tr theo thit k Central Composite. Th nghim
c b tr nh bng 3.6; yu t X5 c dng mc 10% trong tt c cc th nghim.
Central Composite l thit k th nghim m trong mi yu t s a dng
hn 5 cp : cng alpha (+o) v tr alpha (-o), cng mt (1) v tr mt (- 1) v im
trung tm (0). Nu yu t phn loi c thm vo, thit k trung tm tng hp s
c nhn i cho mi s kt hp ca cc cp yu t phn loi.
Kt qu sng lc cc yu t th nghim theo thit k Plackett Burman thc
hin mc 3.4.2 cho thy phm vi nghin cu cc yu t th nghim thc hin l
hp l. Cc yu t th nghim v ch tiu theo di dng trong RSM - CCD c trnh
by bng 3.5 v bng 3.3.
Bng 3.5: Cc yu t s dng trong RSM - CCD
Yu
t
Tn n v
Phm vi
nghin cu
Mc
-o -1 0 1 o
X1 pH 5,45 - 6,85 5,45 5,8 6,15 6,5 6,85
X2 Nhit
0
C 26,5 - 40,5 26,5 30 33,5 37 40,5
X3 Thi gian gi 9 - 21 9 12 15 18 21
X4
Pepton b sung % (w/v)
- 5 - 15 -5
0 5 10 15

Sau khi s l phn tch s liu, chng trnh s a ra cc thng s cho tng
bin hm t gi tr cc i, sau s phn tch a ra phng n sn xut ti u
theo yu cu t ra.
3.4.4. Chn phng n sn xut ti u - kim tra bng thc nghim
Sau khi phn tch phn mm s a ra cc phng n tha yu cu t ra bao
gm gi tr ca ch tiu theo di t cc i, cc yu t theo di nm trong phm vi
18
nghim cu. Sau khi chn c phng n sn xut ti u chng ti tin hnh kim
tra bng thc nghim trn 1000 ml mi trng vi 3 ln lp li, mc ch kim tra la
nh gi li s tng thch gia l thuyt m hnh vi thc t sn xut.
Cc s liu thc hin theo thit k Plackett - Burman v RSM - CCD c nhp
vo chng trnh Design Expert 7.0.0 v tin hnh phn tch.

Bng 3.6: K hoch b tr th nghim ti u ha mi trng nui cy theo thit k
Central Composite
Nghim
thc
X1 X2 X3 X5
Nghim
thc
X1 X2 X3 X5
1 -1 -1 -1 -1 16 1 1 1 1
2 1 -1 -1 -1 17 -o 0 0 0
3 -1 1 -1 -1 18 o 0 0 0
4 1 1 -1 -1 19 0 -o 0 0
5 -1 -1 1 -1 20 0 o 0 0
6 1 -1 1 -1 21 0 0 -o 0
7 -1 1 1 -1 22 0 0 o 0
8 1 1 1 -1 23 0 0 0 -o
9 -1 -1 -1 1 24 0 0 0 o
10 1 -1 -1 1 25 0 0 0 0
11 -1 1 -1 1 26 0 0 0 0
12 1 1 -1 1 27 0 0 0 0
13 -1 -1 1 1 28 0 0 0 0
14 1 -1 1 1 29 0 0 0 0
15 -1 1 1 1 30 0 0 0 0
o= 0,05

19


Chng 4
KT QU V THO LUN

4.1. Kt qu th nghim 1 - Kho st ng cong sinh trng
ng cong sinh trng ca L. acidophilus, B. subtilis, S. cerevisiae c th
hin trn hnh 4.1. Khi nui cy kt hp, L. acidophilus khng c pha tim pht v mi
trng hot ha ging cng l MRSB, t cc i gi th mi tm; B. subtilis vo
pha tng trng t gi nui cy th su, t cc i gi th mi lm; S. cerevisiae
vo pha tng trng t gi nui cy th su, t cc i gi th mi tm. Tuy
nhin, S. cerevisiae im tng trng cc i l thp, khng c s t bin v s
lng trong pha tng trng, im cc i ch t 5,1 (log CFU/ml).

Hnh 4.1: th din t ng cong sinh trng ca L. acidophilus, B. subtilis,
S. cerevisiae khi nui cy kt hp trn canh trng MRS.
Khi nui cy trn mi trng MRS nhit 37
0
C l iu kin tng trng ti
u ca L. acidophilus nn vi khun ny pht trin mnh. Tuy nhin, vi nhng iu
kin nh trn khng l iu kin cc thun cho s pht trin ca B. subtilis nhng vi
0
1
2
3
4
5
6
7
8
9
10
0 5 10 15 20 25 30
L. acidophilus
B. subtilis
S.cerevisiae
l
o
g

c
e
l
l

(
C
F
U
/
m
l
)

hour
20
khun ny vn tng trng tt, iu ny c th gii thch l do s tc ng qua li ca
B. subtilis vi hai chng cn li. S. cerevisiae pht trin chm, dc ca ng cong
sinh trng thi im pha tng sinh khng ln c th gii thch nh sau: iu kin
mi trng (pH = 6,5; nhit 37
0
C) l khng ph hp cho s pht trin ca S.
cerevisiae, cng vi s pht trin nhanh chng ca L. acidophilus v B. subtilis khin
cho s cnh tranh v dinh dng ca S. cerevisiae km i. Nh vy c th thy rng:
pH mi trng, nhit nui cy v dinh dng u l nhng yu t nh hng n
s pht trin ca cc vi sinh vt th nghim.
T mc ch t c mt vi sinh vt ln nht v tng ng gia cc vi
sinh vt nui cy, chng ti xc nh nhim v ca ti u ha mi trng nui cy l
phi tng mt t bo ca S. cerevisiae bng cch ci tin mi trng, iu chnh
iu kin nui cy; ng thi, phi duy tr s pht trin tt ca L. acidophilus v B.
subtilis.
4.2. Kt qu th nghim 2 - Thit k sng lc yu t th nghim theo Plackett -
Burman
Kt qu m s lng mt vi sinh vt thc hin theo thit k Plackett
Burman c tnh by bng 4.1, kt qu ny c nhp vo phn mm Design
Expert phn tch. Kt qu phn tch l mc nh hng ca yu t th nghim n
ch tiu theo di v phng sai mt yu t theo thit k Plackett Burman sai
bit o = 0,05%. Kt qu phn tch mc nh hng ca yu t th nghim n ch tiu
theo di c th hin bng 4.2, kt qu phn tch ANOVA c th hin ph lc
3.1.
Ti kt qu phn tch mc nh hng ca ch tiu theo di n yu t th
nghim, nu kt qu cho mc nh hng c gi tr dng c ngha l yu t th nghim
lm tng mc ca ch tiu theo di, ngc li, nu mc nh hng cho gi tr m c
ngha l yu t th nghim lm gim mc ca ch tiu theo di. Kt qu bng 4.2
cho thy cc yu t th nghim X1, X2, X3, X4 cho mc nh hng dng i vi c
ba ch tiu theo di Y1, Y2, Y3 vi gi tr p < 0,05; yu t X5 cho mc nh hng m
i vi c ba ch tiu theo di vi gi tr p < 0,05, iu ny c ngha l ng glucose
b sung lm gim mt t bo ca vi sinh vt nui cy th nghim. Trong bng phn
21
tch ANOVA, chng ti tr trng n mc ngha v mt thng k (gi tr p) i vi
tng yu t th nghim, gi tr ny c th hin bng 4.2.
Bng 4.1: Kt qu th nghim sng lc theo Plackett-Burman
Nghim
thc
Yu t th nghim Ch tiu theo di
X 1 X 2 X 3 X 4 X 5 Y1 Y2 Y3
1 1 1 1 -1 1 4,89 x 10
11
7,23 x 10
7
4,754 x 10
8

2 -1 1 1 1 -1 9,758 x 10
10
1,193 x 10
7
1,548 x 10
8

3 1 -1 -1 1 1 1,99 x 107 1,815 x 10
7
1,193 x 10
5

4 -1 1 1 -1 1 6,327 x 10
10
5,236 x 10
7
2,609 x 10
8

5 -1 -1 1 1 -1 5,086 x 10
8
4,9 x 10
5
2,45 x 10
6

6 -1 -1 -1 -1 1 3,761 x 10
6
1,906 x 10
7
1,545 x 10
6

7 1 -1 1 -1 -1 3,636 x 10
9
4,012 x 10
6
2,7 x 10
5

8 1 1 -1 -1 -1 1,834 x 10
7
7,5 x 10
7
1,572 x 10
5

9 1 1 -1 1 -1 3,182 x 10
6
3,12 x 10
6
8,35 x 10
5

10 -1 1 -1 1 1 1,996 x 10
6
1,9 x 10
5
1,868 x 10
6

11 1 -1 1 1 1 5,154 x 10
10
2,413 x 10
6
2,031 x 10
6

12 -1 -1 -1 -1 -1 3,039 x 10
6
2,4 x 10
5
4,654 x 10
5


Bng 4.2: Kt qu phn tch mc nh hng ca cc yu t th nghim n ch tiu
theo di theo Plackett Burman
Yu
t
Y1 Y2 Y3
Mc nh
hng
p
Mc nh
hng
p
Mc nh
hng
p
X1 6,382 x 10
10
0,012 1,513 x 10
7
0,0021 9,196 x 10
6
0,0024
X2 9,902 x 10
10
0,005 2,843 x 10
7
0,027 1,467 x 10
8
0,003
X3 1,176 x 10
11
0,006 4,635 x 10
6
0,0005 1,482 x 10
8
0,0002
X4 8,369 x 10
10
0,02 1,160 x 10
7
0,0033 9,603 x 10
7
0,0028
X5 -6,771 x 10
10
0,026 -3,110 x 10
7
0,039 -9,722 x 10
7
0,0012
22
T kt qu phn tch ANOVA v kt qu nh gi mc nh hng ca tng yu
t th nghim ln ch tiu theo di chng ti nh gi yu t th nghim vi phm vi
nghin cu nh trn l ph hp v c ngha v mt thng k (p < 0,001) (xem ph
lc 3.1). Vi mc tiu l ch tiu theo di (mt vi sinh vt) ti a nn chng ti
chn cc yu t c mc tng tc dng v gi tr tng tc ln, gi tr p nh (s liu
c ngha v mt thng k) thit lp cc th nghim tip theo. Nh vy cc yu t
th nghim c chn s dng th nghip tip theo l pH (X1), nhit (X2), thi
gian (X3) v pepton b sung (X4); D - glucose (X5) c c nh mc trung tm l
10% do c mc nh hng m ln c ba ch tiu theo di nhng khng th loi b do
s gy thiu ht ngun carbohydrates trong mi trng.
4.3. K qu th nghim 3 - Thit k ti u ha mi trng nui cy theo RSM -
CCD
Kt qu m mt vi sinh vt khi thc hin th nghim theo thit k RSM
CCD c th hin bng 4.3, kt qu ny c nhp vo phn mm Design Expert
tin hnh phn tch. Trong phn nh gi ca chng trnh a ra, phng trnh bc
ba khng s liu thc hin, phng trnh bt hai hon ton tng thch. Do
chng ti chn phng trnh bc hai tin hnh phn tch.

23
Bng 4.3: Kt qu ma trn quy hoch thc nghim theo RSM - CCD
Th
nghim
Yu t th nghim Ch tiu theo di
X 1 X 2 X 3 X 4 Y1 Y2 Y3
1 -1 -1 -1 -1 2,037 x 10
6
1,541x 10
6
8,714 x 10
5

2 1 -1 -1 -1 3,041 x 10
6
1,026 x 10
6
5,067 x 10
5

3 -1 1 -1 -1 3,067 x 10
6
1,134 x 10
7
2,941 x 10
5

4 1 1 -1 -1 8,136 x 10
6
5,395 x 10
7
1,47 x 10
4

5 -1 -1 1 -1 4,343 x 10
7
4,133 x 10
7
2,97 x 10
8

6 1 -1 1 -1 7,545 x 10
7
5,189 x 10
7
4,852 x 10
7

7 -1 1 1 -1 8,656 x 10
7
1,346 x 10
8
9,727 x 10
7

8 1 1 1 -1 3,084 x 10
8
4,09 x 10
8
2,975 x 10
7

9 -1 -1 -1 1 3,561 x 10
6
1,907 x 10
6
2,766 x 10
6

10 1 -1 -1 1 5,479 x 10
6
2,4 x 10
6
7,687 x 10
5

11 -1 1 -1 1 3,545 x 10
6
1,968 x 10
6
5,517 x 10
5

12 1 1 -1 1 1,233 x 10
7
6,880 x 10
7
2,436 x 10
5

13 -1 -1 1 1 1,118 x 10
8
7,809 x 10
7
6,245 x 10
8

14 1 -1 1 1 3,068 x 10
8
3,232 x 10
8
7,095 x 10
7

15 -1 1 1 1 2,794 x 10
8
6,798 x 10
7
5,609 x 10
8

16 1 1 1 1 5,294 x 10
7
4,023 x 10
8
5,663 x 10
7

17 -o 0 0 0 7,092 x 10
6
3,37 x 10
4
8,348 x 10
7

18 o 0 0 0 3,324 x 10
7
1,324 x 10
8
2,286 x 10
5

19 0 -o 0 0 2,855 x 10
7
3,404 x 10
4
2,091 x 10
7

20 0 o 0 0 4,767 x 10
7
1,11 x 10
8
2,114 x 10
5

21 0 0 -o 0 8,51 x 10
5
5,673 x 10
6
5,858 x 10
4

22 0 0 o 0 4,914 x 10
8
5,219 x 10
8
5,136 x 10
8

23 0 0 0 -o 1,263 x 10
7
4,095 x 10
6
1,224 x 10
8

24 0 0 0 o 1,099 x 10
7
3,616 x 10
6
7,246 x 10
6

25 0 0 0 0 4,045 x 10
7
2,586 x 10
8
1,038 x 10
8

26 0 0 0 0 3,809 x 10
7
2,042 x 10
8
1,418 x 10
8

27 0 0 0 0 2,677 x 10
7
1,436 x 10
8
7,543 x 10
7

28 0 0 0 0 2,655 x 10
7
3,456 x 10
8
7,654 x 10
7

29 0 0 0 0 4,908 x 10
7
3,501 x 10
8
1,054 x 10
7

30 0 0 0 0 1,91 x 10
8
3,431 x 10
8
1,535 x 10
7

24
4.3.1. Kt qu phn tch thc nghim theo cu trc c tm cho L. acidophilus
Kt qu phn tch ANOVA c trnh by bng 4.4. Sau khi phn tch
ANOVA, phng trnh hi quy c dng nh l mt m hnh tin on s lng
vi sinh vt. Phng trnh tin on mt t bo L. acidophilus c th hin nh sau:

Bng 4.4: Kt qu phn tch ANOVA thc nghim cho L. acidophilus
SV
SS df MS F
TN
P
Nghim thc
5,3428 x 10
17
8 6,679 x 10
16
19,58 < 0,0001
pH
2,4568 x 10
16
1 2457 x 10
16
7,203
**
0,014
Nhit
2,1446 x 10
16
1 2,145 x 10
16
6,288
**
0,02
Thi gian
2,995 x 10
17
1 2,995 x 10
17
87,81
***
< 0,0001
Pepton b sung
2,1539 x 10
16
1 2,154 x 10
16
6,315
**
0,02
pH x Thi gian
2,9072 x 10
16
1 2,907 x 10
16
8,524
**
0,008
Nhit x Thi gian
2,6672 x 10
16
1 2,667 x 10
16
7,82
**
0,011
Thi gian x Pepton
b sung
3,1062 x 10
16
1 3,106 x 10
16
9,107
**
0,007
Thi gian x Thi gian
8,0422 x 10
16
1 8,042 x 10
16
23,58
***
< 0,0001
Sai bit
5,1286 x 10
16
21 3,411 x 10
15


Tng cng
6,059 x 10
17
29 4,068 x 10
15


R hi quy 0,8818
R hiu chnh 0,8368
R tin on 0,7705
o = 0,05. S khc bit:

rt c ngha (
***
),

kh c ngha (
**
), c ngha (
*
), khng c ngha (NS).
H s hi quy (R
2
) tnh c l 0,8818. iu ny th hin rng c 88,18 % s
liu thc nghim tng thch vi s liu tin on theo m hnh. Thng thng, gi tr
R
2
ln hn 0,75 th hin m hnh tng thch vi thc nghim. Gi tr R
2
tin on l
0,7705 ph hp vi R
2
iu chnh l 0,8368 ( lch 0,0663 < 0,2). T l tn hiu so
vi nhiu l 16,548 > 4 ch ra rng tn hiu y .
th b mt p ng (xem hnh 4.2 v hnh 4.3) th hin s tng tc ca
tng cp yu t, t th ny c th xc nh c gi tr ti u ca tng yu t lm
cho hm p ng cc i. M hnh d on s lng L. acidophilus ti a t c
25
l 4,354 x 10
8
CFU/ml ( v mt l thuyt vi 88,18 %) gi tr cc yu t: pH = 6,5;
nhit 37
0
C; thi gian 18 gi; pepton b sung 10 %. T th chng ti nhn thy
trong phm vi cc yu t kho st, s lng L. acidophilus tng khi pH, thi gian,
nhit , lng pepton b sung tng; c s tng tc gia pH v nhit , nhit v
thi gian, thi gian v pepton b sung.

Hnh 4.2: th biu din nh hng ca cc yu t th nghim n mt ca
L. acidophilus.
26

Hnh 4.3: th 3D biu din nh hng ca nhit v pH n mt ca
L. acidophilus.
4.3.2. Kt qu phn tch thc nghim cu trc c tm cho B. subtilis
Kt qu phn tch ANOVA c trnh by bng 4.5. Sau khi phn tch
ANOVA, phng trnh hi quy c dng nh l mt m hnh tin on s lng
vi sinh vt. Phng trnh tin on mt t bo B. subtilis c th hin nh sau:

H s hi quy (R
2
) tnh c l 0,8358. iu ny th hin rng c 83,58% s
liu thc nghim tng thch vi s liu tin on theo m hnh. Thng thng, gi tr
R
2
ln hn 0,75 th hin m hnh tng thch vi thc nghim. Gi tr R
2
tin on l
0,7013 ph hp vi R
2
iu chnh l 0,7726 ( lch 0,0713 < 0,2). T l tn hiu so
vi nhiu l 12,729 > 4 ch ra rng tn hiu y .
th b mt p ng (xem hnh 4.4 v hnh 4.5)th hin s tng tc ca tng
cp yu t, t th ny c th xc nh c gi tr ti u ca tng yu t lm cho
hm p ng cc i. M hnh d on s lng B. subtilis ti a t c l 4,268
x 10
8
CFU/ml ( v mt l thuyt vi 83,58%) gi tr cc yu t: pH = 6,49; nhit
34,55
0
C; thi gian 18 gi; pepton b sung 5,41%. T th chng ti nhn thy trong
phm vi cc yu t kho st, s lng B. subtilis t l thun vi thi gian; cn khi pH,
27
nhit , lng pepton b sung tng th s lng B. subtilis ch tng n mt mc
no th bt u gim; c s tng tc gia pH v thi gian nui cy.
Bng 4.5: Kt qu phn tch ANOVA thc nghim cho B. subtilis
SV SS df MS F
TN
p
Nghim thc
5,9497 x 10
17
8 7,4372 x 10
16
13,3129 < 0,0001
pH
6,3931 x 10
16
1 6,3931 x 10
16
11,4439
**
0,0028
Nhit
3,1577 x 10
16
1 3,1577 x 10
16
5,65235
*
0,027
Thi gian
2,396 x 10
17
1 2,396 x 10
17
42,8896
***
< 0,0001
Pepton b sung
2,4193 x 10
15
1 2,4193 x 10
15
0,43306
NS
0,5176
pH x Thi gian
3,5638 x 10
16
1 3,5638 x 10
16
6,37934
*
0,0197
pH x pH
7,2213 x 10
16
1 7,2213 x 10
16
12,9265
**
0,0017
Nhit x Nhit

8,0026 x 10
16
1 8,0026 x 10
16
14,3251
**
0,0011
Pepton x Pepton
1,2335 x 10
17
1 1,2335 x 10
17
22,0809
*
0,0001
Sai bit
1,1732 x 10
17
21 5,5864 x 10
15


Tng cng
7,1229 x 10
17
29

R hi quy 0,8358
R hiu chnh 0,7726
R tin on 0,7013
o = 0,05. S khc bit:

rt c ngha (
***
),

kh c ngha (
**
), c ngha (
*
), khng c ngha (NS).


Hnh 4.4: th biu din nh hng ca nhit v pH n s lng ca B. subtilis.
28

Hnh 4.5: th 3D biu din nh hng ca nhit v pH n s lng ca B.
subtilis.
4.3.3. Kt qu phn tch thc nghim cu trc c tm cho S. cerevisiae
Kt qu phn tch ANOVA c trnh by bng 4.6. Sau khi phn tch
ANOVA, phng trnh hi quy c dng nh l mt m hnh tin on s lng
vi sinh vt. Phng trnh tin on mt t bo S. cerevisiae c th hin nh sau:

H s hi quy (R
2
) tnh c l 0,8406. iu ny th hin rng c 84,06% s
liu thc nghim tng thch vi s liu tin on theo m hnh. Thng thng, gi tr
R
2
ln hn 0,75 th hin m hnh tng thch vi thc nghim. Gi tr R
2
tin on l
0,6811 ph hp vi R
2
iu chnh l 0,7898 ( lch 0,1087 < 0,2). T l tn hiu so
vi nhiu l 14,091 > 4 ch ra rng tn hiu y .
th b mt p ng (xem hnh 4.6 v hnh 4.7) th hin s tng tc ca
tng cp yu t v t th ny c th xc nh c gi tr ti u ca tng yu t lm
cho hm p ng cc i. M hnh d on s lng S. cerevisiae ti a t c
l 5,003 x 10
8
CFU/ml (v mt l thuyt vi 84,06%) gi tr cc yu t: pH = 5,8;
nhit 37
0
C hoc 30
0
C; thi gian 18 gi; pepton b sung 10%. T th chng ti
29
nhn thy trong phm vi cc yu t kho st, s lng S. cerevisiae t l thun vi thi
gian, lng pepton b sung; t l nghch vi pH, c s tng tc gia pH v thi gian,
pH v pepton b sung, thi gian v pepton b sung.
Bng 4.6: Kt qu phn tch ANOVA thc nghim cho S. cerevisiae
SV SS df MS F
TN
p
Nghim thc
7,146 x 10
17
7 1,021 x 10
17
16,5707 < 0,0001
pH
9,923 x 10
16
1 9,923 x 10
16
16,1083
***
0,00064
Thi gian
3,282 x 10
17
1 3,282 x 10
17
53,2762
***
< 0,0001
Pepton b sung
1,565 x 10
16
1 1,565 x 10
16
2,54017
NS
0,125
pH x Thi gian
1,174 x 10
17
1 1,174 x 10
17
19,0653
***
0,0002
pH x pepton b sung
3,455 x 10
16
1 3,455 x 10
16
5,60895
*
0,027
Thi gian x pepton
b sung
4,387 x 10
16
1 4,387 x 10
16
7,12217
*
0,014
Thi gian x thi gian
7,561 x 10
16
1 7,561 x 10
16
12,2739
**
0,002
Sai bit
1,355 x 10
17
22 6,16 x 10
15


Tng cng
8,501 x 10
17
29

R hi quy 0,8406
R hiu chnh 0,7898
R tin on 0,6811
o = 0,05. S khc bit:

rt c ngha (
***
),

kh c ngha (
**
), c ngha (
*
), khng c ngha (NS).

Hnh 4.6: th 3D biu din nh hng ca Pepton b sung v thi gian n mt
ca S. cerevisiae.
30

Hnh 4.7: th biu din nh hng ca yu t th nghim n mt ca
S. cerevisiae.
4.4. Chn phng n sn xut ti u - kim tra bng thc nghim
Kt qu phn tch RSM CCD ca Design - Expert c 29 gii php c tm
thy (xem bng 4.7) tha yu cu mt vi sinh vt nui cy t cc i vi c ba
chng, cc yu t th nghim trong phm vi kho st. Trong 29 gii php ny, xt v
mt ca mi chng th gii php th 29 cho gi tr hm cc i c L. acidophilus
v B. subtilis l ln hn c, gii php th 20 cho gi tr hm cc i cho S. cerevisiae
31
l ln hn nhng gii php cn li. Xt v tng mt vi sinh vt th t gii php th
1 n gii php th 15 tng mt vi sinh vt > 9,6 x 10
8
(CFU/ml) vi sai lch
gia cc gii php l 0,024 x 10
8
, mt ny l ln hn so vi cc gii php cn li.
Phng n ti u nht c chn phi tha mn hai yu cu, th nht l tng mt vi
sinh vt phi ln m bo li ch kinh t, th hai l sai lch mt phi nh
m bo rng mt t bo gia ba chng tng ng nht vi nhau. i chiu vi
yu cu trn, chng ti nhn thy gii php th 7 v 8 tha mn vi tng mt t bo
l 9,619 x 10
8
(CFU/ml), sai lch mt l 0,193 x 10
8
. Tuy nhin gii php th 8
c lng pepton b sung l 9,69% t hn gii php th 7 (10%) hay gii php th 8 t
tn km mi trng hn gii php th 7. Vy gii php ti u nht chng ti chn
a vo sn xut thc t l gii php th 8 vi cc thng s nh sau: pH = 6,11; nhit
36,84
0
C ~ 37
0
C; thi gian nui cy 18 gi; 9,69% pepton b sung; 10% D
glucose b sung.
Sau khi chn c phng n sn xut chng ti tin hnh kin tra bng
thc nghim vi ba ln lp li trn 1000 ml mi trng. Vic kim tra bng thc
nghim ny nhm nh gi xem s d on ca m hnh c ph hp vi thc t hay
khng. Kt qu c trnh by bng 4.8, th b mt p ng c th hin hnh
4.8 v hnh 4.9. So snh mt vi sinh vt thc nghim m c vi gii php sn
xut th 8, chng ti nhn thy mt t bo vi sinh vt ca mi chng v tng mt
t bo vi sinh vt u ln hn so vi gii php th 8, nh vy chng t phng n
c chn ph hp vi thc nghim. Mt t bo ca mi chng u t mc 10
8
(CFU/ml), tng cng c ba chng t 10
9
(CFU/ml) nh vy t mc yu cu ti t
ra. Phng n sn xut c chn c th a vo sn xut thc t.
Bng 4.8: Kt qu kim tra thc nghim
Vi sinh vt
Mt (CFU/ml)
T l vi
m hnh Ln 1 Ln 2 Ln 3
Trung
bnh
SD
L.
acidophilus
4,906x10
8
4,636x10
8
4,489x10
8
4,667x10
8
2,115x10
7
136,038%
B. subtilis 3,672x10
8
3,577x10
8
3,473x10
8
3,574x10
8
0,995x10
7
120,094%
S.
cerevisiae
3,941x10
8
3,227x10
8
3,673x10
8
3,614x10
8
3,607x10
7
112,751%
Tng 11,864x10
8

123,339%
32
Kt qu thc nghim cho kt qu mt L. acidophilus t 4,667 x 10
8
(CFU/ml) nh
vy l thp hn so vi mt trn mi trng MRS ca nhng nghin cu trc y,
nghin cu ca Sivakumar (2010) l 8,3 x 10
8
(CFU/ml), Rooj (2010) l 8 x 10
8
(CFU/ml). Kt qu thc nghim cho kt qu mt B. subtilis t 3,574 x 10
8
(CFU/ml) sau 18 gi nui cy l cao hn so vi nghin cu ca Korsten (1996) l 1,8
x 10
8
(CFU/ml) sau 24 gi nui cy lc trn mi trng STD v nghin cu ca
Th Bch Thy (2006) l 2,51,8 x 10
8
(CFU/ml) trn mi trng c hot protase cao
sau 24 gi. Kt qu thc nghim cho kt qu mt S. cerevisiae t 3,614 x 10
8
(CFU/ml) sau 18 gi nui cy l thp hn so vi nghin cu ca Pasternakiewicz
(2006) l 2,04 x 10
9
(CFU/ml) trn mi trng c b sung canxi sau 36 gi nui cy
lc v nghin cu ca Donev (2002) l 1,8 x 10
9
(CFU/ml) sau 30 gi nui cy. Tng
mt vi sinh vt l 1,186 x 10
9
(CFU/ml) tuy khng cao nhng vn mc chp nhn
c. Mt tch cc ca phng php ny l tit kim mi trng cng nh din tch
mui cy, ngoi vic thun tin cho vic thu sinh khi cng lc c ba chng, gim thi
gian thu sinh khi ca B. subtilis v S. cerevisiae, hay ni cch khc, vic nui cy kt
hp ny gip n gin qu trnh nui cy v tit kim chi ph sn xut.

Hnh 4.8: th biu din nh hng ca cc ch tiu theo di n mt ti a ca
ba chng vi sinh vt.
33

Hnh 4.9: th 3D biu din s tng tc ca cc yu t th nghim n mt ti
a ca c ba chng vi sinh vt.

Bng 4.7: Cc gii php ti u theo RSM - CCD
Tn Mc tiu
Gii hn
di
Gii hn trn
pH trong phm vi nghin cu 5,8 6,5
Nhit trong phm vi nghin cu 30 37
Thi gian trong phm vi nghin cu 12 18
Pepton b sung trong phm vi nghin cu 0 10
L. acidophilus ti a 8,509 x 10
5
5,294 x 10
8

B. subtilis ti a 3,370 x 10
4
5,219 x 10
8

S. cerevisiae ti a 1,470 x 10
4
6,245 x 10
8

Gii
ph
p
pH
Nhit

(
0
C)
Thi
gian
(gi)
Pept
on
b
sung
(%)
Mt (CFU/ml)
Tng mt

(CFU/ml)
SD mt

(x10
8
) L.
acidophil
us
B. subtilis
S.
cerevisiae
1 6,11 36,66 18 10 3,426x10
8
2,904x10
8
3,312x10
8
9,642x10
8

0,274
2 6,11 36,74 18 10 3,455x10
8
2,909x10
8
3,279x10
8
9,643x10
8

0,278
3 6,11 36,68 18 9,99 3,445x10
8
2,925x10
8
3,279x10
8
9,649x10
8

0,265
34
4 6,11 36,81 18 9,99 3,471x10
8
2,898x10
8
3,274x10
8
9,643x10
8

0,291
5 6,11 36,67 18 9,93 3,435x10
8
2,946x10
8
3,253x10
8
9,634x10
8

0,247
6 6,11 36,32 18 10
3,482x10
8
2,895x10
8
3,249x10
8
9,626x10
8

0,296
7 6,12 36,34 18 10
3,409x10
8
3,023x10
8
3,187x10
8
9,619x10
8

0,193
8 6,11 36,84 18 9,69
3,347x10
8
3,036x10
8
3,236x10
8
9,619x10
8

0,193
9 6,11 36,12 18 10 3,326x10
8
2,996x10
8
3,29x10
8
9,612x10
8

0,268
10 6,08 36,97 18 9,83 3,424x10
8
2,822x10
8
3,391x10
8
9,637x10
8

0,338
11 6,14 36,86 18 9,99 3,552x10
8
2,997x10
8
3,074x10
8
9,623x10
8

0,300
12 6,05 36,37 18 9,91 3,245x10
8
2,796x10
8
3,583x10
8
9,624x10
8

0,394
13 6,07 35,77 18 10 3,172x10
8
2,904x10
8
3,509x10
8
9,585x10
8

0,303
14 6,05 35,88 18 10 3,166x10
8
2,847x10
8
3,582x10
8
9,595x10
8

0,368
15 6,11 36,56 17,97 10 3,387x10
8
2,931x10
8
3,241x10
8
9,559x10
8

0,232
16 6,17 35,65 18 10 3,374x10
8
3,241x10
8
2,913x10
8
9,528x10
8

0,237
17 6,24 36,79 18 10 3,746x10
8
3,245x10
8
2,541x10
8
9,532x10
8

0,605
18 6,11 34,69 18 9,94 3,034x10
8
3,113x10
8
3,252x10
8
9,399x10
8

0,189
19 6,22 37 17,98 9 3,577x10
8
3,375x10
8
2,496x10
8
9,448x10
8

0,574
20 5,95 35,75 18 10 2,916x10
8
2,431x10
8
4,173x10
8
9,52 10
8

0,899
21 6,2 34,4 18 10 3,178x10
8
3,344x10
8
2,766x10
8
9,288x10
8

0,297
22 6,08 37 18 7,23 3,042x10
8
3,248x10
8
2,944x10
8
9,234x10
8

0,155
23 6,2 34,12 18 9,99 3,127x10
8
3,346x10
8
2,743x10
8
9,216x10
8

0,305
24 6,22 33,91 18 10 3,12x10
8
3,367x10
8
2,659x10
8
9,146x10
8

0,359
25 6,11 37 18 6,45 2,983x10
8
3,397x10
8
2,683x10
8
9,063x10
8

0,358
26 6 37 18 6,48 2,763x10
8
3,017x10
8
3,179x10
8
8,959x10
8

0,209
27 6,22 33,33 18 10 2,999x10
8
3,309x10
8
2,666 10
8
8,974x10
8

0,321
28 5,84 37 18 7,21 2,526x10
8
2,194x10
8
4,112x10
8
8,832 10
8

1,025
29 6,48 35,8 18 8,84 3,885x10
8
3,895x10
8
1,118 10
8
8,898x10
8

1,600


35


Chng 5
KT LUN V KIN NGH

5.1. Kt lun
Sau khi hon thnh cc th nghim, chng ti rt ra c cc kt lun v nhn
xt nh sau:
Khi nui cy ng thi trn canh trng MRS nhit 37
0
C s pht trin ca
L. acidophilus v B. subtilis l tt nhng s pht trin ca S. cerevisiae l thp, khng
tng ng vi hai chng cn li.
Kt qu sng lc theo ma trn Plackett Burman xc nh c s nh
hng ca cc yu t pH, nhit , thi gian nui cy v lng pepton b sung ln mt
vi sinh vt nghin cu.
Qu trnh nui cy ti u theo thit k RSM - CCD c 29 phng n t
c mt vi sinh vt ln nht v tng ng gia ba chng, phng n mi trng
ti u c chn ngoi thnh phn mi trng MRS cn c thnh phn nh sau:
Pepton b sung: 9,69 %
D glucose b sung 10 %
Vi iu kin nui cy:
pH: 6,11
Nhit : 36,84 ~ 37
0
C
Thi gian: 18 gi
Mt vi sinh vt (CFU/ml) thc hin theo phng n t c vi cc chng
L. acidophilus, B. subtilis, S. cerevisiae ln lt l 4,667 x 10
8
; 3,574 x 10
8
; 3,614 x
10
8
, tng mt ba chng trong mi trng nui cy t 11,684 x 10
8
(CFU/ml).

36
5.2. Kin ngh
ti nguyn cu ca chng ti gp phn n gin ha qu trnh nui cy vi
sinh vt thu sinh khi s dng vo sn xut sn phm probiotic. Tuy nhin, trong
khun kh ca thi gian tin hnh ti, cc kt qu ca chng ti mi ch l bc
u, cn c nhiu hn ch. Chng ti c mt s ngh sau:
Nghin cu nh hng ca cc nguyn t vi lng n s tng trng
ca cc chng vi sinh vt nhm tng s lng vi sinh vt nui cy.
Kim tra tnh i khng vi cc vi sinh vt gy bnh ch th.
Kim tra hot tnh enzyme ca cc vi sinh vt .
M rng quy m sn xut, thu sinh khi ng dng sn xut cc sn phm
probiotic.

37

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41

PH LC

Ph lc 1: Thnh phn mi trng nui cy vi sinh vt.

1. NB (Nutrient Borth)
Peptone 5,0 g
Cao tht 3,0 g
Nc ct 1000 ml
pH sau tit trng 7,2 0,2; hp tit trng 121
0
C /15 pht
2. NA (Nutrient Agar)
Mi trng NB thm 15 20 gram agar cho 1000 ml mi trng.
3. Sabouraud
D-glucose 40,0 g
Pepton 20,0 g
Nc ct 1000 ml
Hp tit trng 121
0
C/ 20 pht
4. MRSB (Man, Rogosa and Sharpe)
Pepton 10 g
Cao nm men 10 g
Cao tht 5 g
Glucose 20 g
Tween 80 1 ml
Phosphate dipotassique 2 g
Natri acetate 5 g
Citrate triammonique 2 g
Magie sulfate 200 mg
Mangan sulfate 500 mg
Nc ct 1000 ml
Agar (nu cn) 15 20 g
pH sau tit trng 6,5 0,2; hp tit trng 121
0
C/ 15 pht
42
5. PDA (Potato D - glucose Agar)
Khoai ty 200 g
D - glucose 20 g
Agar 15 20 g
Khoai ty ct 2 3 cm, un si 30 pht, lc v nh mc cho th tch; hp
tit trng 121
0
C /30 pht

Ph lc 2: Kt qu m s lng vi sinh vt
Bng s 1: S lng vi sinh vt kho st theo thi gian
Thi
gian
S lng trung bnh
(CFU/ ml)
log10
trung bnh s lng

L.
acidophilus
B. subtilis S. cerevisiae
L.
acidophilus
B.
subtilis
S.
cerevisiae
3 3158,3333 4836,3636 166,1905 3,4995 3,6845 2,2206
6 17000,0 16000,0 260,9523 4,2304 4,2041 2,4166
9 322727,2727 47333,3333 1204,7619 5,5088 4,6752 3,0809
12 4690909,091 238636,3636 3571,4285 6,6713 5,3777 3,5528
15 346363636,4 103300000 11904,7619 8,5395 8,0 4,0757
18 1877272727 170952381 75238,0952 9,2735 8,2329 4,8764
21 2761904762 228181818,2 157500 9,4412 8,3583 5,1972
24 2445454545 208636363,6 141363,6364 9,3883 8,3194 5,1503


43
Ph lc 3: Kt qu phn tch ANOVA
Ph lc 3.1. Kt qu phn tch phng sai ANOVA ca ma trn Plackeet
Burman
Bng 1: Kt qu phn tch phng sai ANOVA ca ma trn Plackeet Burman cho
L. acidophilus

Bng 2: Kt qu phn tch phng sai ANOVA ca ma trn Plackeet Burman cho
B. subtilis

44
Bng 3: Kt qu phn tch phng sai ANOVA ca ma trn Plackeet Burman cho
S. cerevisiae


45
Ph lc 3.2. Kt qu phn tch phng sai ANOVA ca thit k RSM CCD
Bng 4: Kt qu phn tch phng sai ANOVA ca thit k RSM - CCD cho L.
acidophilus


46
Bng 5: Kt qu phn tch phng sai ANOVA ca thit k RSM - CCD cho B.
subtilis

47


48
Bng 6: Kt qu phn tch phng sai ANOVA ca thit k RSM - CCD cho S.
cerevisiae


49