B GIO DC V O TO

TRNG I HC NNG LM TP.HCM

B MN CNG NGH SINH HC

KHA LUN TT NGHIP

KHO ST S SINH TRNG CA SACCHAROMYCES SP.

TRN MI TRNG CM GO, R NG
V MT S YU T NH HNG
N SC SNG CA CHNG
TRONG CH PHM

Ngnh hc: CNG NGH SINH HC

Nin kho: 2003-2007
Sinh vin thc hin: VNG TH HNG VI

Thnh Ph H Ch Minh
-09/2007-
 B GIO DC V O TO
TRNG I HC NNG LM TP.HCM
B MN CNG NGH SINH HC

KHA LUN TT NGHIP

KHO ST S SINH TRNG CA SACCHAROMYCES SP.

TRN MI TRNG CM GO, R NG
V MT S YU T NH HNG
N SC SNG CA CHNG
TRONG CH PHM

Gio vin hng dn: Sinh vin thc hin:

TS. NGUYN NGC HI VNG TH HNG VI

Thnh Ph H Ch Minh
-09/2007-
 LI CM N

Vi tt c lng knh trng, em xin chn thnh cm n Ban Gim Hiu

Trng i Hc Nng Lm Thnh Ph H Ch Minh, Ban Ch Nhim B Mn
Cng Ngh Sinh Hc, cng tt c qu Thy C truyn t kin thc cho em
trong sut qu trnh hc ti trng.

Em xin chn thnh cm n Ts. Nguyn Ngc Hi ht lng hng dn dy

d, ng vin, quan tm, ng h em hon thnh kho lun.

Em xin chn thnh cm n Ts. L Anh Phng, BSTY. Nguyn Th Kim

Loan gip , to iu kin thun li cho em trong sut qu trnh hon thnh
kho lun.

Em xin chn thnh cm n Phng Vi Sinh, Khoa Chn Nui Th Y cho

php v to mi iu kin thun li cho em hc tp v nghin cu ti phng.

Ti xin cm n cc bn lp CNSH 29, Th Y 28 chia s cng ti nhng

vui bun trong thi gian hc cng nh ht lng h tr, gip ti trong thi gian
thc tp.

Sinh vin thc hin

Vng Th Hng Vi
 TM TT

ti Kho st s sinh trng ca nm men Saccharomyces sp. trn mi

trng cm go, r ng v mt s yu t nh hng ti kh nng sng ca chng
trong ch phm c tin hnh ti phng Thc Tp Vi Sinh khoa Chn Nui Th
Y Trng i Hc Nng Lm TP.HCM, thi gian t thng 3 n thng 8 nm
2007. ti c thc hin vi mc ch tm hiu mt s iu kin trong quy trnh
sn xut sinh khi ca nm men Saccharomyces sp. nhm ng dng sn xut ch
phm sinh hc s dng cho gia sc, gia cm.
Kt qu chng ti ghi nhn c:
Chng ti phn lp c t dch qu nho chng nm men thuc loi
S. cerevisiae v t ch phm sinh hc chng nm men thuc loi S. boulardii. C 2
chng u c kh nng sinh trng tt trn mi trng r ng 60B v mi trng
cm go. Tuy nhin, mi trng cm go thch hp cho s sinh trng ca loi
S. boulardii hn mi trng r ng 60B. Thu hoch S. boulardii vo thi im 60
gi sau khi nui cy s cho kt qu s lng t bo cn sng trong ch phm nhiu
hn 36 gi v 48 gi. Ngc li, loi S. cerevisiae sinh trng trn mi trng r
ng 60B mnh hn trn mi trng cm go. V thi im thu hoch
S. cerevisiae thch hp nht l 36 gi sau khi nui cy.
Vitamin C v cht nn (cm go, bt m) khng nh hng n sc sng ca
nm men trong ch phm (sau 22 ngy sn xut). Vic nui cy chung vi khun
sB. subtilis khng nh hng n s lng t bo nm men S. cerevisiae.
 ABSTRACT

The study on the growth of Saccharomyces sp. on rice bran medium, sugar
cane molasses medium and some factors influence on their vitality power for the
purpose of producing probiotic. Our topic is done from March to August, 2007 at
Microbiology - Infectious Diseases Department, Husbandry and Veterinary Faculty,
Nong Lam University, Ho Chi Minh city.
S. boulardii was isolated from probiotic and S. cerevisiae was isolated from
grapes. They grew well both on rice bran medium and sugar cane molasses medium.
Experimental results showed S. boulardii grew better on rice bran than on
sugar cane molasses medium. The best time for harvesting S. boulardii biomass was
60 hours after culturing. But, S. cerevisiae grew better on sugar cane molasses
medium and the best time for harvesting their biomass was 36 hours after culturing.
The rate acid ascorbic and background substance (wheat flour or rice bran)
didnt show the influence to yeast vitality.
No considerable oscillations of S. cerevisiae biomass occurred in co-culture
with Bacillus subtilis.
 MC LC
CHNG TRANG
Trang ta
Li cm n iii
Tm tt iv
Abstract v
Mc lc vi
Danh sch cc hnh ix
Danh sch cc bng x

Chng 1. PHN M U .......................................................................................1

1.1. t vn ............................................................................................................1
1.2. Mc ch ti ....................................................................................................2
1.3. Yu cu ti ......................................................................................................2
Chng 2. TNG QUAN ...........................................................................................3
2.1. c im chung ca nm men .............................................................................3
2.2. Vai tr ca nm men ............................................................................................4
2.3. Cc hnh thc sinh sn ca nm men ..................................................................5
2.3.1. Sinh sn v tnh .........................................................................................5
2.3.1.1. Sinh sn v tnh bng hnh thc ny chi ..........................................5
2.3.1.2. Sinh sn v tnh bng hnh thc phn chia t bo ..............................5
2.3.2. Sinh sn hu tnh .......................................................................................6
2.3.2.1. Sinh sn hu tnh bng bo t ti (ascospore) ...................................6
2.3.2.2. Sinh sn hu tnh bng bo t bn (ballistospore) .............................6
2.4. c im sinh l, sinh ha ca nm men ............................................................7
2.5. c im ca ging Saccharomyces ...................................................................9
2.5.1. Saccharomyces cerevisiae .......................................................................10
2.5.2. Saccharomyces boulardii ........................................................................10
2.6. S sinh trng v pht trin ca nm men ........................................................11
2.6.1. Giai on thch nghi ................................................................................11
2.6.2. Giai on logarit ......................................................................................11
2.6.3. Giai on n nh ....................................................................................12
2.6.4. Giai on thoi ha .................................................................................12
2.7. Cc yu t nh hng n s sinh trng ca nm men trong iu kin
nui cy thu sinh khi t bo ....................................................................................12
2.7.1. Mi trng nui cy ................................................................................12
2.7.2. Nhit ...................................................................................................14
2.7.3. pH ca mi trng ..................................................................................14
 2.7.4. Tc sc kh v khuy trn ..................................................................14
2.8. C s ca vic s dng nm men trong sn xut v ch bin thc n ..............15
2.9. Nhng nghin cu v ng dng nm men trong chn nui ..............................18
2.9.1. nc ngoi ...........................................................................................18
2.9.2. trong nc ...........................................................................................19
Chng 3. NI DUNG V PHNG PHP NGHIN CU ...............................21
3.1. Thi gian v a im thc hin ti ..............................................................21
3.1.1. Thi gian .................................................................................................21
3.1.2. a im ..................................................................................................21
3.2. Vt liu th nghim ............................................................................................21
3.2.1. Mu kho st ...........................................................................................21
3.2.2. Thit b v dng c ..................................................................................21
3.2.3. Ha cht ..................................................................................................21
3.2.4. Mi trng nui cy ................................................................................21
3.3. Ni dung nghin cu .........................................................................................22
3.4. Phng php nghin cu ...................................................................................22
3.4.1. Phn lp nm men ...................................................................................22
3.4.1.1. Mu ch phm sinh hc v men bnh m .........................................22
3.4.1.2. Mu u v nho .............................................................................22
3.4.2. Th nghim 1: Kho st s pht trin ca cc chng phn lp c
trong mi trng r ng 60B..........................................................................23
3.4.2.1. Mc ch...........................................................................................23
3.4.2.2. Thng s c nh ..............................................................................23
3.4.2.3. Ch tiu theo di ...............................................................................23
3.4.2.4. B tr th nghim ..............................................................................23
3.4.3. Th nghim 2: Kho st nh hng ca mi trng nui cy v
thi gian thu hoch ln s sinh trng ca nm men Saccharomyces .............24
3.4.3.1. Mc ch...........................................................................................24
3.4.3.2. Thng s c nh ..............................................................................24
3.4.3.3. Ch tiu theo di ...............................................................................24
3.4.3.4. B tr th nghim ..............................................................................25
3.4.4. Th nghim 3: Kho st nh hng ca acid ascorbic (vitamin C)
v cht nn ln thi gian sng ca nm men trong ch phm ..........................25
3.4.4.1. Mc ch...........................................................................................25
3.4.4.2. Thng s c nh ..............................................................................25
3.4.4.3. Ch tiu theo di ...............................................................................26
3.4.5. Th nghim 4: Kho st nh hng ca mi trng nui cy,
cht nn v thi gian thu hoch ln sc sng ca nm men trong ch phm ...26
3.4.6. Th nghim 5: Kho st nh hng ca vi khun Bacillus subtilis
ln s sinh trng ca nm men Saccharomyces cerevisiae trong iu kin
nui cy chung ..................................................................................................26
3.4.6.1. Mc ch...........................................................................................26
3.4.6.2. Thng s c nh ..............................................................................26
 3.4.6.3. Ch tiu theo di ...............................................................................27
3.4.6.4. B tr th nghim ..............................................................................27
3.4.7. Phng php m s t bo nm men bng bung m hng cu .........27
3.4.8. Phng php m s t bo sng ( s khun lc trn a thch) ............28
Chng 4. KT QU V THO LUN ................................................................30
4.1. Th nghim 1: Kho st s sinh trng ca cc chng phn lp trong
mi trng r ng 60B...........................................................................................30
4.2. Th nghim 2: Kho st nh hng ca mi trng nui cy v thi gian
thu hoch ln s sinh trng ca nm men Saccharomyces ....................................33
4.2.1. Saccharomyces boulardii ........................................................................33
4.2.2. Saccharomyces cerevisiae .......................................................................35
4.3. Th nghim 3: Kho st nh hng ca acid ascorbic (vitamin C) v
cht nn ln sc sng ca nm men trong ch phm................................................37
4.3.1. Saccharomyces boulardii ........................................................................38
4.3.2. Saccharomyces cerevisiae .......................................................................38
4.4. Th nghim 4: Kho st nh hng ca mi trng nui cy, cht nn v
thi gian thu hoch ln sc sng ca nm men trong ch phm ..............................39
4.4.1. Saccharomyces boulardii ........................................................................39
4.4.2. Saccharomyces cerevisiae .......................................................................42
4.5. Th nghim 5: Kho st nh hng ca vi khun Bacillus subtilis ln
s sinh trng ca nm men Saccharomyces cerevisiae trong iu kin
nui cy chung ..........................................................................................................43
Chng 5. KT LUN V NGH .....................................................................47
5.1. Kt lun ..............................................................................................................47
5.2. ngh...............................................................................................................47
TI LIU THAM KHO .........................................................................................49
PH LC ..................................................................................................................51
 DANH SCH CC HNH

HNH TRANG
Hnh 4.1: Phn lp nm men t ch phm sinh hc Ultra Levure 31
Hnh 4.2: Phn lp nm men t dch qu nho 31
Hnh 4.3: Hnh thi S. boulardii 32
Hnh 4.4: Hnh thi S. cerevisiae 32
Hnh 7.1: Bnh nui cy nm men 51
Hnh 7.2: Khun lc Saccharomyces sp. trn mi trng Sabouraud 51
Hnh 7.3: Ch phm sinh hc cha nm men Saccharomyces 51
 DANH SCH CC BNG

BNG TRANG
Bng 2.1: Thnh phn ho hc ca nm men 17
Bng 2.2: Thnh phn acid amin ca nm men 17
Bng 2.3: Thnh phn khong ca nm men 18
Bng 2.4: Thnh phn vitamin ca nm men 18
Bng 4.1: S lng t bo nm men trong 1 ml dch nui cy 30
Bng 4.2: S lng t bo S. boulardii trong 1 ml dch nui cy 33
Bng 4.3: S lng t bo S. boulardii trong 1 g ch phm 34
Bng 4.4: S lng t bo S. cerevisiae trong 1 ml dch nui cy 35
Bng 4.5: S lng t bo S. cerevisiae trong 1 g ch phm 36
Bng 4.6: S lng t bo S. boulardii trong 1 g ch phm 38
Bng 4.7: S lng t bo S. cerevisiae trong 1 g ch phm 38
Bng 4.8: S lng t bo S. boulardii trong 1 g ch phm 41
Bng 4.9: S lng t bo S. cerevisiae trong 1 g ch phm 41
Bng 4.10: S lng t bo S. cerevisiae trong 1 ml dch nui cy 43
Bng 4.11: S lng t bo S. cerevisiae trong 1 g ch phm 44
Bng 7.1: S lng t bo nm men trong 1 ml dch nui cy 48
Bng 7.2: S lng t bo S. boulardii trong 1 ml dch nui cy 48
Bng 7.3: S lng t bo S. boulardii trong 1 g ch phm 48
Bng 7.4: S lng t bo S. cerevisiae trong 1 ml dch nui cy 49
Bng 7.5: S lng t bo S. cerevisiae trong 1 g ch phm 49
Bng 7.6: S lng t bo S. boulardii trong 1 g ch phm 50
Bng 7.7: S lng t bo S. cerevisiae trong 1 g ch phm 50
Bng 7.8: S lng t bo S. cerevisiae trong 1 ml dch nui cy 51
Bng 7.9: S lng t bo S. cerevisiae trong 1 g ch phm 51
Bng ANOVA 7.1 51
Bng ANOVA 7.2 51
Bng ANOVA 7.3 52
Bng ANOVA 7.4 52
Bng ANOVA 7.5 52
Bng ANOVA 7.6 52
Bng ANOVA 7.7 52
Bng ANOVA 7.8 53
Bng ANOVA 7.9 53
Bng ANOVA 7.10 53
Bng ANOVA 7.11 53
 1

Chng 1
PHN M U
1.1. t vn
Trong gii vi sinh vt, khi nhc n nhng ng dng ca nm men trong ch
bin thc phm th Saccharomyces c xem l mt trong nhng ng c vin sng
gi nht. T rt lu, chng c s dng trong ch bin thc phm cho ngi
nh ln men ru, bia, sn xut men bnh m, nc gii kht c cn V trong vi
thp k gn y n c s dng nhiu cho chn nui nh ch bin thc n giu
tinh bt t cc ph ph phm cng nng nghip, sn xut sinh khi giu protein v
vitamin t cc ngun nguyn liu phong ph, r tin hoc s dng trong sn xut
cc ch phm sinh hc dng lm thc n b sung cho vt nui. Vic nghin cu sn
xut qui m nh v va cc ch phm sinh hc t nm men gip phng bnh v
kch thch sinh trng cho gia sc, gia cm ph hp vi thc tin nc ta v
ang c trin khai nghin cu ng dng.
Vi xu hng , vic nghin cu kh nng sinh trng v pht trin ging
nm men Saccharomyces trn cc loi mi trng khc nhau nhm thu c lng
sinh khi cao vi chi ph nui cy thp l cng vic quan trng v cn thit. Tuy
nhin, chng ta cng cn quan tm n thi gian thu hoch v phng php bo
qun kh nng sng ca chng trong ch phm. S d Saccharomyces c s dng
ph bin b sung vo thc n cho vt nui v chng thuc loi nm n bo,
cha ngun protein phong ph, hm lng vitamin cao (c bit l vitamin nhm
B) m thi gian chng sinh sn nhanh li d nui cy v thu hoch.
c s ng ca B Mn Cng Ngh Sinh Hc v s gip ca Khoa
Chn Nui Th Y Trng i Hc Nng Lm Thnh Ph H Ch Minh cng vi s
hng dn tn tnh ca Ts. Nguyn Ngc Hi, chng ti tin hnh thc hin ti
 2

Kho st s sinh trng ca nm men Saccharomyces sp. trn mi trng cm

go, r ng v mt s yu t nh hng ti kh nng sng ca chng trong ch
phm.

1.2. Mc ch ti
Tm hiu mt s iu kin trong quy trnh sn xut sinh khi nm men
Saccharomyces sp. nhm ng dng sn xut ch phm sinh hc s dng cho gia
sc, gia cm.

1.3. Yu cu ti

Phn lp cc chng nm men t 4 ngun mu ch phm sinh hc, men bnh

m, u v nho.
nh gi kh nng sinh trng ca nm men Saccharomyces sp. trn mi
trng cm go v mi trng r ng ma.
Kho st, tm hiu thi gian thch hp thu hoch nm men.
nh gi nh hng ca acid ascorbic (vitamin C) v cht nn (cm go, bt
m) ln sc sng ca Saccharomyces sp. trong ch phm.
nh gi nh hng ca vi khun Bacillus subtilis ln s sinh trng ca
nm men Saccharomyces cerevisiae trong iu kin nui cy chung.
 3

Chng 2
TNG QUAN
2.1. c im chung ca nm men
Nm men l tn chung ch nhng nhm nm c cu to n bo, sng
ring l hoc sng thnh tng m, khng di ng v sinh sn v tnh ch yu bng
hnh thc ny chi.
Chng phn b rng ri trong thin nhin nh trong t, nc, lng thc
thc phm, c bit c nhiu trong cc loi hoa qu chn, ngt.
Hnh dng v kch thc nm men thay i ty theo loi, ging, iu kin
dinh dng v nhiu yu t khc.
Nm men thng c dng hnh trng, hnh bu dc (Saccharomyces
cerevisiae, Saccharomyces ellipsoideus), hnh trn (Candida utilis), hnh ng di
(Pichia), hnh qu da chut (Saccharomyces pastorianus), hnh mt u nhn
(Brettanomyces), hnh tam gic (Trigonopsis) v mt s hnh c bit khc.
Mt s nm men c t bo hnh di, ni tip nhau thnh nhng dng si gi
l khun ty (mycelium) hoc khun ty gi (pseudomycelium). khun ty gi, cc t
bo khng ni lin nhau mt cch cht ch nh khun ty. Khun ty v khun ty
gi thng quan st thy cc ging Endomycopsis, Candida, Trichosporon,
nhiu loi nm ch sinh khun ty gi khi khng c cung cp oxy.
Hnh dng nm men khng n nh, n cn ph thuc vo tui ging v iu
kin ngoi cnh. V d, Saccharomyces thng c hnh bu dc trong mi trng
nui cy giu dinh dng. Trong iu kin ym kh thng c hnh trn, trong iu
kin hiu kh t bo c hnh di hn.
Kch thc t bo nm men thay i rt nhiu ty theo ging, loi v tng
giai on pht trin. Nhn chung, t bo nm men to hn t bo vi khun mt cch
 4

r rt, kch thc trung bnh 3 5 * 10 12 m. Kch thc chiu ngang t bo

nm men ging Torulopsis l 5 8 m, mt s loi men ru l 10 11 m, men
bia l 6 8 m, nm men gia sc thng nh hn.
quan st hnh thi v o kch thc t bo nm men, ngi ta thng s
dng mi trng mch nha dch th vi nng ng khong 10 150B (
Baum, n v thng s dng tnh hm lng ng) hoc mi trng thch
mch nha, thi gian nui cy l 3 ngy 25 300C.
Nm men l sinh vt c nhn tht, cu to t bo c mng t bo, nguyn
sinh cht, nhn, ty th, ribosome, khng bo, mt s th vi (glycogen, lipit,
volutin).

2.2. Vai tr ca nm men

Nm men c kh nng sinh sn nhanh chng, sinh khi ca chng rt giu
protein, lipid v vitamin (c bit l vitamin B). Chng c kh nng ln men cc
loi ng to thnh ru trong iu kin ym kh, cn trong iu kin hiu kh
th chng c kh nng tng nhanh lng sinh khi t bo.
Trong qu trnh trao i cht ca hu ht ging nm men u khng sinh ra
cht c gy hi cho sc kho ca ngi v vt nui nn chng c s dng rng
ri trong cng nghip ch bin thc phm nh sn xut ru, bia, nc gii kht c
cn, lm men bnh m, ch bin cc thc phm sa ln men kefir, johur cho
ngi v thc n gia sc.
Ngi ta cn s dng nm men sn xut protein n bo, sn xut
vitamin, enzyme v c bit loi Saccharomyces cerevisiae ang c s dng
nh mt cng c c lc mang cc DNA ti t hp phc v cho vic sn xut cc
sn phm th h mi ca ngnh cng ngh sinh hc hin i.
Tuy nhin, cng c mt s nm men gy bnh cho ngi v gia sc nh
Candida albican, Cryptoccocus neoforman v gy h hng thc phm nh
Endomycopsis fiibuligera, Saccharomyces bisporus
 5

2.3. Cc hnh thc sinh sn ca nm men

Nm men sinh sn di 2 hnh thc sinh sn v tnh v sinh sn hu tnh.

2.3.1. Sinh sn v tnh

2.3.1.1. Sinh sn v tnh bng hnh thc ny chi

mt giai on nht nh no , trn t bo m xut hin chi v sau
pht trin thnh t bo con. T bo con ln dn, khi t n kch thc ca t bo
m th nh s chuyn ng ca cht lng trong mi trng, n tch khi t bo m.
Trc lc tch ra, gia 2 t bo ny c mi lin kt bn trong rt khng kht. T bo
m v t bo con khc nhau v phm cht, chc nng nn tc sinh sn ca chng
cng khng ging nhau.
Nhng cng c trng hp t bo con khng tch khi t bo m m to
thnh nhng chi nh lin kt vi nhau ngay c khi chng trng thnh, do to
thnh chui t bo, gi l khun ty gi.
Hnh thc ny chi l hnh thc sinh sn ph bin nht nm men, thng
gp ging Saccharomyces, Candida, Torulopsis.

2.3.1.2. Sinh sn v tnh bng hnh thc phn chia t bo

Mt s loi phn i t bo to thnh 2 t bo con bng nhau, ging nh a
s cc vi khun. Lc u cht nhn chia lm 2 phn, sau phn gia t bo xut
hin vch t bo, vch ny ln dn ln v chia t bo m thnh 2 t bo con. Mi t
bo con cha na cht nhn v dn dn 2 t bo tch khi nhau thnh 2 t bo c
lp.
Hnh thc sinh sn ny thng gp nm men c dng si di nh ging
Schizosaccharomyces, Endomyces.
 6

2.3.2. Sinh sn hu tnh

2.3.2.1. Sinh sn hu tnh bng bo t ti (ascospore)

Trong qu trnh nui cy nm men chuyn t ngt t mi trng giu sang
mi trng ngho dinh dng, trong khi vn gi nguyn m, tch t cc hp
cht trung gian, oxy ca khng kh th t bo s sinh bo t nm trong cc ti,
c gi l bo t ti. Bo t ti bn vi tc nhn bn ngoi nh nhit cao, kh
hn, nhng km bn nhit hn so vi bo t vi khun. Chng thng cht 600C,
cn bo t vi khun cht 1200C.
Bo t ti thng c sinh ra trong nhng ci ti nh gi l nang hay ti
(ascus) mi ti cha 1 8 bo t ti (ascospore), thng l 1 4. Bo t ti c kch
thc v hnh dng khc nhau ty tng loi nm men; c th hnh bu dc, bn cu,
hnh thoi
Ti c th c sinh ra theo 1 trong 3 phng thc sau:
Tip hp ng giao (conjugation isogamic): do 2 t bo nm men c hnh
dng v kch thc ging nhau tip hp vi nhau to thnh. Gp nhiu loi trong
ging Schizosaccharomyces, Zygosaccharomyces, Debaryomyces.
Tip hp d giao (conjugation heterogamic): do 2 t bo c hnh dng v kch
thc khng ging nhau tip hp vi nhau to thnh. Gp 1 s loi trong ging
Zygopichia, Nadsodia.
Sinh sn n tnh (pathenogenesis): l qu trnh hnh thnh bo t trc
tip t 1 t bo ring l khng thng qua tip hp. Gp nhiu loi trong ging
Schiwanniomyces, Pichia.
Cc bo t ti sau khi ra khi ti gp iu kin thun li s pht trin thnh
t bo nm men mi. T bo ny li sinh sn theo li ny chi.

2.3.2.2. Sinh sn hu tnh bng bo t bn (ballistospore)

L loi bo t ch thy cc loi trong ging Brullera, Spodiobolus,
Sporoliomyces, Aessaspora. Sau khi hnh thnh, bo t ny c th bn mnh ra pha
i din.
 7

2.4. c im sinh l, sinh ha ca nm men

Nm men l vi sinh vt hiu kh ty nghi, chng h hp nh mt c th hiu
kh bc cao, khi mi trng ht oxy phn t chng chuyn sang h hp k kh, gi
l qu trnh ln men. Khi phn ng ln men bt u pht trin, tc sinh sn ca t
bo nm men b km hm v n mt giai on nht nh hu nh khng cn na.
V c ch sinh hc, y l mt qu trnh s dng khng ht nng lng nhng cht
dinh dng ca mi trng. V qu trnh phn hy 1 phn t gam ng bng cch
ln men ch tit ra khong 28 kcal, trong khi nu oxy ha hon ton 1 phn t
gam ng ta s c 674 kcal. Qu trnh ln men tm gi l qu trnh phosphoryl
ha. V trong khi ln men, cc hp cht ATP, ADP tham gia vo 1 cch tch cc.
Nm men tip nhn thc n bng con ng hp th chn lc trn b mt
ca t bo v sau khuch tn vo bn trong. Mng v lp bao bc nguyn sinh
cht ca t bo ng vai tr mng bn thm ngn cch, iu ho cc cht dinh
dng vo t bo v thi cc sn phm trao i cht ra ngoi. Cc cht dinh dng
sau khi c hp th vo trong t bo s xy ra nhng phn ng ha hc chuyn
ha thnh cc hp cht nh protein, glucid, lipid

Dinh dng cacbon:

Trc ht phi k n cc loi ng, ng glucose c tt c cc loi
nm men s dng. Cc loi nm men dng sn xut men gia sc thuc ging
Candida, Torulopsis c th ng ha c ng pentose. V vy, cc men ny c
th nui cy dch thy phn t g hoc cc ngun giu hemicellulose. Nhng
disacarit (maltose v saccarose) trc khi c nm men s dng phi qua thy
phn s b thnh ng n nh enzyme tng ng ca nm men.
Nh l mt qui lut, trong mi trng c mt hn hp cc ngun cacbon
dinh dng th ngun no cung cp cho nm men sinh trng tt s c s dng
trc. ng glucose v fructose c s dng trc ht, k tip l cc acid bo
(ph thuc vo chng loi nm men v thnh phn ca acid ny), nhng hp cht c
nhiu C trong phn t c s dng sau cng.
 8

Cc acid hu c chim mt v tr quan trng trong trao i cht ca nm

men. Chng c th kch thch hoc c ch sinh trng nm men. Chng c th l
ngun dinh dng cacbon v nng lng duy nht.
S dng hydrocacbon t du m v kh t lm ngun cacbon nui cy nm
men rt c quan tm trong vi thp k gn y. Trong , parafin c th l ngun
cacbon dinh dng d dng i vi mt s chng ca ging Candida v Torulopsis.
Dinh dng nit:
Nm men c kh nng tng hp c tt c cc acid amin, thnh phn
protein trc tip t cc hp cht m v c v cacbon hu c.
a s nm men khng ng ha c nitrate (tr ging Hasenula v Pichia).
Ngun nit v c c nm men s dng tt l cc mui amoni ca acid v
c cng nh hu c. l amoni sulfate, phosphate ri n cc mui acetate,
lactate, malate Trong mi trng c mui amoni, c bit l sulfate, th nm men
s dng gc amoni trc, gc acid cn li s s dng sau hoc t s dng v s lm
mi trng acid ha, gim pH.
Cc ngun nit hu c thng l hn hp cc acid amin, peptit, nucleotit
Trong thc t, ngi ta hay dng cao ng, cao nm men, dch thy phn u tng
lm ngun nit hu c. nm men tiu ha rt tt cc acid amin, cn peptone km
hn v hon ton khng s dng c protein. Cc mui amon c nm men s
dng tt hn cc acid amin. Trong qu trnh nui cy nm men, cc acid amin va
l ngun nit va l ngun cacbon dinh dng. Tuy nhin, nm men ch s dng
c acid amin dng L-acid amin.
thu sinh khi Saccharomyces c tt, trong mi trng nui cy nn c
mt c ngun nit v c v hu c.
Dinh dng cc nguyn t v c:
Trong cc nguyn t v c s dng nui cy nm men, phospho c quan
tm trc ht, sau l kali, magi, lu hunh
Phospho tham gia vo thnh phn quan trng ca t bo (nucleoproteit, acid
nucleic, phospholipit). Cc hp cht phospho ng vai tr xc nh trong cc
 9

phn ng sinh ha, c bit l trong trao i cht hydrocacbon v vn chuyn nng
lng.
Trong phng th nghim vi sinh, ngi ta thng dng mui KH 2PO4 v
K2HPO4 lm ngun P v K. cn trong sn xut, thng dng dch chit t
supephosphat lm ngun P.
Dinh dng cc cht kch thch sinh trng:
cho nm men pht trin v ln men c bnh thng cn phi c cc
vitamin lm cofacto trong nhiu enzyme ca t bo nm men. Nm men c th tng
hp c tt c cc vitamin trong chng mc no , ngoi tr biotin. V vy, trong
mi trng nui cy cn phi b sung vitamin ny.
Nhng nhn t sinh trng c bn i vi nm men khng c sc t l 6
vitamin nhm B: inozit (vit B8), biotin (vit B7), acid pantotenic (vit B3), tiamin (vit
B1), pyridoxyn (vit B6), acid nicotinic (vit B5).
Trong cng nghip, thng dng cc ngun vitamin l cao ng, cao nm
men, nc chit cm, dch thy phn u tng, r ng. Trong cc th nghim
nui cy qui m nh c th dng cc dch chit t gi u, rau ci, c chua, c rt,
khoai ty lm ngun vitamin b sung vo mi trng.

2.5. c im ca ging Saccharomyces

Mt s loi nm men c kh nng ln men ru v bia ln u tin c
Meyen gp thnh 1 ging gi l Saccharomyces vo nm 1939. y l ging c bo
t ti, thng c 1 4 bo t, c khi n 8 bo t. T bo ca chng c hnh cu,
di, elip..., ny chi nhiu pha, khng c khun ty, khng to bo t bn, khng to
vng, khun lc c dng bt nho, hi bng.
y l mt trong s nhng ging nm men c ng dng rng ri, ph bin
nht.
 10

2.5.1. Saccharomyces cerevisiae

c Meyen m t vo nm 1938, t bo c dng hnh trng, bu dc,
kch thc trung bnh 3 6 * 5 12 m, sinh sn bng hnh thc ny chi khng
theo qui lut, c th xut hin tng ci mt, tng i hoc mt chui.
Khun lc c mu trng nht, ra trn, li ln, b mt sng lp lnh, ng
knh 1 2 mm vo ngy th ba.
Pht trin ti u 33 350C trong mi trng cha 10 30% glucose. Nhit
ti thiu l 40C trong 10% glucose v 130C trong 50% glucose, nhit ti a l
38 390C (Jermini and Schmidt-Lorenz, 1987, trch t L Nguyn Bo Trn, 2005).
C kh nng pht trin pH = 1,6 trong HCl; pH = 1,7 trong H 3PO4 v
pH = 1,8 2 trong acid hu c, c sc chu ng ln nht i vi acid benzoic
100 mg/kg pH = 2,5 4 v acid sorbic 200 mg/kg pH = 4 (Juven et al, 1978,
trch t L Nguyn Bo Trn, 2005).
C kh nng ln men ng glucose, galactose, maltose, saccharose, rafinose
v cc dextrin n gin, khng ln men lactose, manitol, khng ng ha nitrate,
khng phn gii tinh bt.
L loi nm men hay c s dng lm men bnh m, ln men ru, bia v
hin cn c s dng lm probiotic phc v cho chn nui gia sc v nui trng
thy sn nh c kh nng chu c acid d dy v mui mt tt, khng t nhin
vi khng sinh.

2.5.2. Saccharomyces boulardii

c Herry Boulard pht hin vo nm 1923 trong v tri cy chn, t bo
c dng hnh trn, bu dc, kch thc 4 6 * 6 9 m, sinh sn bng hnh thc
ny chi v to bo t ti, h hp hiu kh ty nghi.
C kh nng ln men ng glucose, saccharose, rafinose, xylose; khng ln
men lactose, manitol, maltose, khng hnh thnh hp cht phn gii tinh bt.
 11

L loi nm men khng mang mm bnh, c s dng nh mt cht

biotherapeutic ngn cn bnh tiu chy (Surawicz et al.,1989 and 2000, trch t
L Nguyn Bo Trn, 2005).
Ngoi kh nng gip cn bng h vi khun ng rut tng t vi khun
lactic, Saccharomyces boulardii cn c mt u im vt tri l kh nng khng
vi khng sinh v sulfamide (tr khng sinh khng nm).
C kh nng tit ra protease tiu gii c t ca Clostridium difficile, vi
khun gy vim rut kt trng gi cng nh trung ho c ni c t ca E. coli v
Vibrio cholera nn c th dng trong d phng tiu chy cp tnh.

2.6. S sinh trng v pht trin ca nm men

S sinh trng v pht trin ca nm men din bin qua 4 giai on.

2.6.1. Giai on thch nghi

L giai on t lc cy nm men vo mi trng n lc chng bt u sinh
sn. giai on ny, chng cn phi thch nghi vi iu kin mi trng mi.
Trong giai on ny, t bo nm men tri qua nhng bin i ln v hnh
thi v sinh l, kch thc tng ln ng k v chng tr nn nhy cm vi nhng
tc ng bn ngoi. S lng t bo nm men giai on ny l khng tng hoc
tng khng ng k.

2.6.2. Giai on logarit

S lng v sinh khi t bo trong giai on ny tng theo cp s nhn. Kh
nng thch ng vi nhng iu kin khng thun li ca mi trng ngoi tng ln
r rt, ng thi xut hin chc nng ln men ru.
Giai on ny thun tin xc nh nng lng sinh sn, thi gian ny chi,
nhng khng nn nh gi kch thc ca t bo cng nh nhng du hiu khc ca
khun lc. Do trong thi k u ca giai on ny, tc sinh sn ca t bo
thng nhanh hn tc to thnh t bo cht nn kch thc ca t bo c phn
nh i.
 12

2.6.3. Giai on n nh
S lng t bo trong giai on ny khng tng na, c th l do s cn bng
gia s sinh ra v cht i. Song kch thc t bo tng ln r rt. Qu trnh ln men
ru cng bt u.

2.6.4. Giai on thoi ha

S lng t bo gim xung do c hin tng tiu hy. Lng protein v
acid nucleic gim xung, glycogen v treganose hon ton tiu bin.
Nh vy c th thy s lng t bo nm men t cao nht giai on
logarit, song sinh khi t bo li t cao nht giai on n nh v khi lng t
bo giai on ny ln.

2.7. Cc yu t nh hng n s sinh trng ca nm men trong iu kin

nui cy thu sinh khi t bo

2.7.1. Mi trng nui cy

Mi trng nui cy thch hp nht cho nm men cn c ngun
hydratcarbon, ngun nit, phospho, mt s nguyn t vi lng nh K, Na, Mg, Ca
v vitamin.
Dng ng cc lm ngun nguyn liu sn xut sinh khi nm men rt tt.
Bt hoc tinh bt cc loi dng vo mc ch ny trc tin phi thy phn bng
acid hoc enzyme ca mm m, ca vi sinh vt bin cc polysacarit thnh cc
dng ng m nm men ng ha c.
Saccharomyces cerevisiae c kh nng pht trin trn mi trng m ngun
carbon duy nht l tinh bt, pht trin d hn trn mi trng ng vi ngun
nguyn liu c nu chn th tt hn. Mt s ging nm men phn lp t men
thuc bc c kh nng s dng c mi trng ng v mi trng tinh bt hoc
cy trc tip vo thc n sng m vn pht trin tt.
Sau 48 gi nui cy trn mi trng cm, s lng nm men c th t hng
chc triu t bo/ml dch nui cy (Nm men dng trong chn nui ln, 1970).
 13

Mi trng r ng hoc dung dch ng c acid ha vi pH = 4, b

sung thnh phn dinh dng v duy tr nhit 25 300C, lng sinh khi thu
c khong 25 50 g/l (Trn Minh Tm, 2002, trch t Nguyn Th Hng Phng,
2006).
Theo phng vi sinh thuc Vin Cng Nghip Thc Phm, t 1 m3 nc b
ru nui nm men c th thu c 10 15 kg men kh.
Mi trng nc chit gi u cng c s dng nui cy nm men do
trong u cha hm lng protein cao, l ngun thc n tt cho nm men. Ngoi ra,
cn c cc vitamin A1, B1, B2, C, E, K v cc cht kch thch t tng trng khc.
Tuy nhin, vitamin C trong nc chit gi u c th lm hn ch s pht trin ca
nm men.
Ngoi ra, mi trng thy phn t cellulose thc vt (g, v bo, rm r, li
ng, b ma), hay parafin t du m cng c th s dng nui cy nm men.
c bit, nm men c kh nng s dng c mi trng dch kim sulfit
(cht thi ca nh my giy), thnh phn ch yu l ng pentose. Ngi ta tnh
c rng, khong 5 tn bt cellulose dng sn xut giy s thi mt lng dch
kim sulfit cha khong 180 kg ng. Dch ny hp th nhiu oxy nn khi nui
cy nm men c th gim mc cung cp oxy ti 60% so vi bnh thng (Lng
c Phm, 2006).
Hin nay, mi trng c s dng nui cy nm men ph bin nht vn
l mi trng r ng. 90% lng sinh khi nm men dng b sung vo thc n
chn nui trn th gii c sn xut t r ng ma v c ci ng. Thnh phn
chnh ca loi nguyn liu ny l saccharose, khong 35 40%. Trong ng ma
cha cc cht sinh trng (biotin, acid pantotenic, inozit) vi hm lng ln, nhng
li ngho cht khong v cc acid amin. V vy, khi s dng ng ma lm ngun
carbon nui cy nm men cn phi loi b mt phn cc cht sinh trng, ng thi
b sung cc mui khong cn thit v c th phi thm hn hp cc acid amin dng
protein thy phn vo giai on nhn ging. Lng ng trong mi trng khong
2 3%, khng nn nhiu hn hay t hn. Nu lng ng cao s va lng ph v
 14

va to ra nhng sn phm trao i cht khc, gy c ch ngc n qu trnh to

sinh khi. Nu lng ng qu nh s khng ngun carbon cn thit cho s to
sinh khi. Mt im ng lu l h keo v cc cht mu c trong mt r. H keo
c trong mt r c hnh thnh bi protein v pectin, n thng c nht cao v
lm cn tr qu trnh trao i cht ca t bo, gy ra hin tng thoi ha, t bo s
pht trin v sinh sn km, dn n hiu sut sinh khi thu c thp. Cc cht mu
c trong r ng nh hp cht caramen, melanins lm sinh khi nm men c
mu sm, nh hng n cm quan. V th, c bit trong sn xut men bnh m
ngi ta phi x l r ng trc khi nui cy.

2.7.2. Nhit
Mi vi sinh vt u c khong nhit ti u cho s sinh trng v pht
trin ca chng. Vi Saccharomyces cerevisiae, nhit ti u l 28 300C, trn
430C v di 280C th s sinh sn ca nm men chm hoc ngng hn.
300C, nm men hoang di pht trin nhanh hn S. cerevisiae 2 3 ln,
35 380C chng pht trin nhanh hn 6 8 ln.
nhit cao, hot tnh ca nm men gim nhanh; cn nhit thp
khong 20 230C, hn ch c mc tp nhim v kh nng ln men cao, ko
di hn.

2.7.3. pH ca mi trng
pH ti u cho nm men khong 4,5 5,6. pH = 4, tc tch lu sinh khi
gim, pH = 3 3,5 th s sinh sn ca nm men ngng li. Mc hp th cht
dinh dng vo t bo, hot ng ca h thng enzyme, s sinh tng hp protein
u b nh hng bi pH nn cht lng ca nm men s gim i nu pH mi
trng nm ngoi khong 4,5 5,6.

2.7.4. Tc sc kh v khuy trn

Trong qu trnh nui cy, cn gi cho dch men lin tc bo ho oxy ho tan.
Ngng cung cp oxy trong 15 giy s gy nn tc ng m trn hot ng sng ca
 15

t bo nm men. Oxy khng kh di chuyn vo t bo nm men qua 2 giai on: u

tin oxy c ho tan vo mi trng nui cy sau nm men mi hp th oxy
vo trong t bo.
V l thuyt, cn 1,066 kg (0,764 m3) oxy oxy ha 1 kg ng, nhng
thc t ch 1 phn nh oxy bm vo l c nm men s dng, phn cn li b mt
i do cc qu trnh tip xc, nhit , nng , nht ca mi trng (Lao Th
Nga, 1987).
Khi nui cy nm men qui m cng nghip, kch thc ca thit b nui
cy nm men l tin cn thit, nh hng gin tip ln s tng trng ca nm
men.

2.8. C s ca vic s dng nm men trong sn xut v ch bin thc n

Nm men c rt nhiu chng loi khc nhau, a dng trong chuyn ha v
tng hp cc cht hu c, phm vi phn b v iu kin sng nh nhit , m ,
nh sng, khng kh rng nn d dng chn c cc chng c kh nng thch
hp vi qui trnh sn xut v p ng c yu cu i hi ca th trng.
C tc pht trin nhanh, tng gp i khi lng c th th nm men
cn 1 2 gi, vi khun t 20 60 pht, to t 2 6 gi, nm si t 4 12 gi trong
khi g con cn 200 gi, heo con cn 600 gi, b, ngh cn 1500 gi (Nguyn
Ln Dng, 1992).
C kh nng pht trin trn nhiu ngun dinh dng khc nhau cho php
ngi ta s dng cc ngun dinh dng sn c, r tin sn xut nhm tng sn
lng v gim gi thnh sn phm (Nguyn Khc Tun, 1996).
Khng nh nm mc, hu ht nm men khng sinh c t trong mi trng
t nhin v mi trng nhn to, tr 1 s loi gy bnh nh Candida albican
(Nguyn Vn Hng, 1990). Ngoi ra, khi ln men nm men cn to mi thm c
trng cho ru, bia.
 16

Nm men cng nh vi sinh vt d gy t bin bng cc tc nhn vt l, ha

hc, do c th dng k thut di truyn bin i c im sinh hc ca nm
men theo hng c li.
Gi tr dinh dng ca nm men rt ln, c bit l hm lng protein, acid
amin v vitamin nhm B trong nm men rt cao, d tiu ha v hp th
Vi c im sinh l ca nm men d dng thit lp dy chuyn cng ngh
cao khai thc cc sn phm t nm men nhm phc v nhu cu thc tin.
Trong 24 gi 1 con b nng 300 kg c chm sc tt cng ch tng trung
bnh khong 1,1 1,2 kg, trong c khong 120 g protein. Nu ly 300 kg nm
men ging nui trong h thng ln men trong 24 gi th to 25.000 30.000 kg sinh
khi, trong c khong 11.000 13.000 kg protein. R rng ta thy s tch lu
protein nm men cao hn nhiu so vi heo, b, g vi nghn ln v tng t gp
vi trm ln cy u, ng cc (Nm men dng trong chn nui ln, 1970).
Trong nm men c 1 s men tiu ha (amylase, protease) v vitamin (c
bit l vitamin B) xc tin nhanh qu trnh trao i cht, kch thch s thm n ca
gia sc. Nh , cng tc chn nui thu c li nhun cao.
Tuy nhin, sinh khi t bo nm men c nhc im l mc d hm lng
protein cao khong 55 60%, nhng ng thi n cng cha lng acid nucleic
qu cao (10%), iu ny nh hng ti sc kho con ngi. Do , cn c cc bin
php lm gim lng acid nucleic ny. Ngoi ra, mng t bo nm men kh vng
chc nn cn c bin php thch hp ph v mng khi thu protein.
 17

Bng 2.1: Thnh phn ha hc ca nm men (Nm men dng trong

chn nui ln, 1970)

Thnh phn T l %
Protid 44 45%
Glucid 25 35%
Lipid 1,5 5%
Cc cht chit xut v 22 40%
m
Cc cht khang 6 12%

Bng 2.2: Thnh phn acid amin ca nm men (Nm men dng trong
chn nui ln, 1970)
Acid amin T l % Acid amin T l %
Cystin 0,54 Tryptophan 0,68
Histidin 1,19 Frolin 1,59
Phenylalanin 2,01 Glycin 2,10
Serin 2,20 Arginin 2,33
Threonin 2,50 Tyrosin 2,77
Lysin 3,11 Valin 3,30
Isoleucin 3,70 Leucin 3,80
Acid asparaginic 3,97 Alanin 5,78
Acid glutamic 6,74 Methionin 0,60
 18

Bng 2.3: Thnh phn khang ca nm men (Nm men dng trong
chn nui ln, 1970)
Thnh phn T l % Thnh phn T l %
K2 O 28 48 Fe2O3 0,1 7,3
Na2O 0,06 1,9 P2O5 41 59
CaO 1 5,5 SO3 0,4 6
MgO 4 8,1 SiO2 1,6

Bng 2.4: Thnh phn vitamin ca nm men (mg/kg)

Vitamin Hm lng Vitamin Hm lng
(mg/kg) (mg/kg)
D2 (calciferol) 250 B6 (pyridoxyn) 60
B1 (thiamin) 40 B9 (acid folic) 4,2
B2 (riboflavin) 50 H (biotin) 2,0
B5 (nicotinic) 21 100

2.9. Nhng nghin cu v ng dng nm men trong chn nui

2.9.1. nc ngoi
c nhiu cng trnh nghin cu trc ht v bn cht sinh hc, kh nng
sinh ha, phn loi ca 1 s ging nm men c s dng ph bin trong sn xut
sinh khi t bo nh Saccharomyces, Torulopsis v Candida (Cainsnorth G et al,
1962, trch t Nguyn Khc Tun, 1996).
Nhng nghin cu u tin v sinh vt hc trong bnh men ru nc ta
c thc hin vo cui th k 19 bi Calmette v hc tr ca ng vo 1948.
Cc nh khoa hc trn th gii ang hng n nghin cu chn ging cho
qu trnh nng cao cng ngh ln men truyn thng trn mi trng xp lm giu
protein cho cc sn phm giu tinh bt nh sn v cc ph phm nng nghip nh
b da, v chui, b ma(Schoch JJ, 1982, trch t Nguyn Khc Tun, 1996).
Trc y, ngi ta s dng nm si ln men song do vn sinh c t
 19

mycotoxin lm cc nh chn nui e ngi. Do , h ch n nm men v 1 s vi

khun ln men truyn thng nh vi khun lactic (Rose A.H, 1981, trch t Nguyn
Khc Tun, 1996).
Ch phm ezyme vi sinh vt ngy nay c sn xut qui m cng nghip.
cc ch phm ny ngy cng c s dng rng ri, trong chn nui ezyme c
dng s ch thc n, phn hy cc hp cht phc tp, lm tng kh nng tiu ha
cho gia sc.
Gn y, hng dc Biocodex-Montronge (Php) cho ra sn phm men tiu
ha c tn Bioflor 250 c cha 250mg t bo nm men S. boulardii dng phng
tr tiu chy, ri lon tiu ha.

2.9.2. trong nc
Cng tc nghin cu v ng dng nm men trong chn nui nc ta c th
ni c bt u t 1960 tr li y. Bao gm cc lnh vc sn xut sinh khi t
bo, ln men thc n bt ng v sn xut ch phm sinh hc.
Nm 1975, Trn c Trn v Nguyn Cng Xun s dng sinh khi nm
men c sn xut t r ng ca nh my ng Vn im chn nui heo vi
t l b sung 3 4% trong khu phn ca heo 2 5 thng tui t kt qu tt
(Nguyn Khc Tun, 1996).
to ra ging nm men va c kh nng ng ha cao va c kh nng
to sinh khi ln, Vin K Thut Sinh Hc thuc Trung Tm Khoa Hc v Cng
Ngh Quc Gia Vit Nam nghin cu cy chuyn gen amylase c tch t
chng Endomycopsis fibuligera v gy bin np cho Saccharomyces cerevisiae lm
cho n c 2 c tnh ng ha cao v sinh tng hp protein cao dng trong ch
bin thc n (Trng Nam Hi, 1994, trch t Nguyn Khc Tun, 1996).
Hin nay, cc nh chn nui kh quan tm n vic s dng ch phm sinh
hc b sung vo khu phn thc n cho gia sc, gia cm. c trng ca cc ch
phm sinh hc ny l s kt hp ca nhiu chng loi vi sinh vt c ch nh
 20

Bacillus. spp, Lactobacillus. spp, Saccharomyces. spp, cc enzyme amylase,

protease
 21

Chng 3
NI DUNG V PHNG PHP NGHIN CU
3.1. Thi gian v a im thc hin ti

3.1.1. Thi gian

ti c thc hin t thng 03 n thng 08 nm 2007.

3.1.2. a im
Phng thc tp vi sinh khoa Chn Nui Th Y Trng i Hc Nng
Lm TP.HCM.

3.2. Vt liu th nghim

3.2.1. Mu kho st
Ch phm sinh hc, men bnh m, u , nho.

3.2.2. Thit b v dng c

Thit b: t sy, ni hp kh trng autoclave, t lnh, knh hin vi, bung
m hng cu, my li tm, my sc kh, micropipette
Dng c: ng nghim, a petri, que cy, a khuy, bnh tam gic, n cn,
gi ng nghim, lame, lamelle, giy o pH

3.2.3. Ha cht
Ha cht dng trong phn lp v nui cy nm men nh cn 960, nc ct,
agar, KH2PO4, MgSO4, K2HPO4, (NH4)2PO4.

3.2.4. Mi trng nui cy

Mi trng phn lp v gi ging nm men l mi trng Sabouraud.
 22

Mi trng kho st nui cy l mi trng cm go, mi trng r ng

ma, mi trng nc chit gi u.

3.3. Ni dung nghin cu

Phn lp nm men t ch phm sinh hc, men bnh m, u , nho.
Kho st s pht trin ca cc chng c phn lp t 4 mu trn trong mi
trng r ng 60B.
Kho st s pht trin ca nm men trong cc loi mi trng nui cy khc
nhau.
Kho st thi gian thu nhn sinh khi nm men trong cc loi mi trng trn.
Kho st s phi hp nui cy Saccharomyces cerevisiae v vi khun Bacillus
subtilis trn mi trng cm go v mi trng r ng ma.

3.4. Phng php nghin cu

3.4.1. Phn lp nm men

3.4.1.1. Mu ch phm sinh hc v men bnh m

Pha mu vi 1 t nc mui sinh l 90/00 ri em cy ria trn mt thch
Sabouraud.
nhit phng trong vng 24 48 gi.
Chn nhng khun lc nghi ng c mu trng c, nhn bng, ng knh
khong 2 3 mm, b mt hi li, ra trn, mc ri em nhum n xem hnh
thi.

3.4.1.2. Mu u v nho
Chn nhng ch b dp, hi c mi ru ri cy ria trn mt thch
Sabourand.
nhit phng trong 24 48 gi.
Chn nhng khun lc nghi ng c mu trng c, nhn bng, b mt hi
li, ra trn, mc ri em nhum n xem hnh thi.
 23

Sau khi xc nh c l chng nm men thuc ging Saccharomyces th

em cy chuyn sang mi trng thch nghing Sabouraud tng sinh.

3.4.2. Th nghim 1: Kho st s pht trin ca cc chng phn lp c

trong mi trng r ng 60B

3.4.2.1. Mc ch
T cc chng ca 4 ngun mu trn, chn chng c kh nng sinh trng v
pht trin mnh tin hnh cc th nghim tip theo.

3.4.2.2. Thng s c nh
Mi trng nui cy: mi trng mt r ng ma 60B.
Th tch mi trng nui cy: 100ml.
Nhit nui cy: nhit phng.
Thi gian nui cy: 36 gi.
S lng mu ban u.
C sc kh.

3.4.2.3. Ch tiu theo di

S lng t bo nm men trong 1 ml dch nui cy bng phng php m
trc tip trn bung m hng cu.

3.4.2.4. B tr th nghim
Th nghim b tr 1 yu t, lp li 2 ln vi 4 nghim thc.
Ch Men
Mu phm bnh m u Nho
Ln 1
Ln 2
 24

3.4.3. Th nghim 2: Kho st nh hng ca mi trng nui cy v thi gian

thu hoch ln s sinh trng ca nm men Saccharomyces
T kt qu th nghim 1, chng ti chn 2 chng c kh nng sinh trng tt
nht lm i tng nghin cu cho th nghim 2.

3.4.3.1. Mc ch
Tm ra mi trng nui cy v thi gian thu hoch thch hp cho s lng t
bo nm men ln nht.

3.4.3.2. Thng s c nh
Th tch mi trng nui cy: 300ml.
Nhit nui cy: nhit phng.
S lng mu ban u.
C sc kh.

3.4.3.3. Ch tiu theo di

S lng t bo nm men trong 1 ml bng phng php m trc tip trn
bung m hng cu, n v tnh tb/ml.
S lng t bo nm men sng trong 1g ch phm sau 22 ngy bo qun
nhit phng bng phng php m khun lc trn mi trng thch Sabouraud,
n v tnh l cfu/g.
Phng php m t bo trc tip trn bung m hng cu ch cho bit tng
s t bo trong dch nui cy (bao gm c s t bo sng v t bo cht). Vi mc
ch nui cy nm men dng lm ch phm sinh hc nn yu cu s lng t bo
sng trong ch phm phi cao. Phng php m khun lc trn a cho php xc
nh s lng t bo nm men sng trong ch phm, t ta c th nh gi cc
yu t nh hng n sc sng ca nm men trong ch phm.
 25

3.4.3.4. B tr th nghim
Th nghim b tr gm 2 yu t mi trng nui cy v thi gian thu hoch,
lp li 3 ln.
Yu t mi trng nui cy c 2 mc :
T1:200 ml mi trng r ng 60B + 100 ml nc chit gi u.
T2: 200 ml mi trng cm go + 100 ml nc chit gi u.
Yu t thi gian thu hoch c 3 mc : 36 gi, 48 gi v 60 gi.

3.4.4. Th nghim 3: Kho st nh hng ca acid ascorbic (vitamin C) v

cht nn ln thi gian sng ca nm men trong ch phm
Sau khi tin hnh xong th nghim 2,chng ti thu hoch sinh khi t bo
nm men v sn xut thnh ch phm. Th nghim trn sinh khi nm men vi cht
nn l cm go v bt m, trong chia thnh 3 phn:
Phn 1: khng b sung vitamin C.
Phn 2: b sung 50/000 vitamin C.
Phn 3: b sung 10/00 vitamin C.
Sau 22 ngy bo qun nhit phng, tin hnh m s lng t bo nm
men cn sng trong ch phm bng phng php khun lc trn mi trng thch
Sabouraud.

3.4.4.1. Mc ch
Xc nh c thi gian thu hoch v iu kin bo qun thch hp nm
men c kh nng sng lu nht trong ch phm.

3.4.4.2. Thng s c nh
Thi gian bo qun: 22 ngy.
Nhit bo qun: nhit phng.
S lng t bo nm men mi mu: 109 tb/g.
 26

3.4.4.3. Ch tiu theo di

S lng t bo nm men sng trong 1 g ch phm bng phng php m
khun lc trn a, n v tnh l cfu/g.

3.4.5. Th nghim 4: Kho st nh hng ca mi trng nui cy, cht nn v

thi gian thu hoch ln sc sng ca nm men trong ch phm
Cc mu ly t th nghim 3 v ch tiu theo di cng l s lng t bo sng
trong 1 g ch phm (n v tnh l cfu/g), nhng lc ny khng xt n nh hng
ca acid ascorbic (vitamin C).

3.4.6. Th nghim 5: Kho st nh hng ca vi khun Bacillus subtilis ln s

sinh trng ca nm men Saccharomyces cerevisiae trong iu kin nui cy
chung
S. cerevisiae t ng ging c tng sinh trn mi trng Sabouraud lng
trong 24 gi nhit phng, B. subtilis t ng ging c tng sinh trn mi
trng TSB (Tryptone Soya Broth) trong 24 gi 370C ri cy vo cc bnh nui
cy theo t l sau:
Bnh 1: 1 ml dch S. cerevisiae + 0 ml dch B. subtilis (k hiu 1S+0B).
Bnh 2: 1 ml dch S. cerevisiae + 1 ml dch B. subtilis (k hiu 1S+1B).
Bnh 3: 1 ml dch S. cerevisiae + 0,1 ml dch B. subtilis (k hiu 1S+0,1B).
Bnh 4: 1 ml dch S. cerevisiae + 0,01 ml dch B. subtilis (k hiu
1S+0,01B).

3.4.6.1. Mc ch
Tm hiu nh hng ca B. subtilis ln s pht trin ca S. cerevisiae.

3.4.6.2. Thng s c nh
Th tch mi trng nui cy: 200 ml.
Nhit nui cy: nhit phng.
Thi gian nui cy: 48 gi.
 27

S lng mu ban u.
C sc kh.

3.4.6.3. Ch tiu theo di

S lng t bo nm men trong 1 ml dch nui cy bng phng php m
trc tip trn bung m hng cu. n v tnh: tb/ml.
S lng t bo nm men sng trong 1 g ch phm sau 22 ngy bo qun
nhit phng bng phng php m khun lc trn mi trng thch Sabouraud.
n v tnh: cfu/g.

3.4.6.4. B tr th nghim
Th nghim b tr gm 2 yu t, lp li 2 ln.
Yu t mi trng nui cy c 2 mc :
Mi trng r ng: 150 ml mi trng r ng 60B + 50 ml nc chit
gi u.
Mi trng cm go: 150 ml mi trng cm go + 50 ml nc chit gi
u.
Yu t nng B. subtilis c 4 mc : 0, 100, 10-1, 10-2.

3.4.7. Phng php m s t bo nm men bng bung m hng cu

Ly 1ml dch nm men nui cy pha long vi 9ml nc mui sinh l 90/00,
ta c nng 10-1, ri pha tip n 10-2, 10-3 chn nng thch hp cho vic
m c d dng v c chnh xc cao. Sau nh ln bung m hng cu v
m di knh hin vi vt knh 40X. Kt qu c tnh theo cng thc:
S t bo nm men/1 ml = a*4000*1000*H
Trong :
A: s t bo trong 1 nh
H: h s pha long = 1/ pha long
4000 = h s chuyn thnh 1 mm3 = 1/th tch nh = 1/4000 mm
1000 = h s chuyn mm3 thnh ml (1 ml = 1000 m3)
 28

3.4.8. Phng php m s t bo sng ( s khun lc trn a thch)

T bo vi sinh vt sng l t bo c kh nng sinh trng to thnh mt
qun th. Trn b mt mi trng c, qun th ny to nhng m c hnh dng,
mu sc ring bit c gi l khun lc. T s khun lc mc trn a thch suy ra
s t bo sng c trong mu cy trn mt thch. y n v tnh l cfu/ml,
ngha l s n v hnh thnh khun lc trong 1 ml n v th tch.
Cch tin hnh
Pha long mu: ly 1 g ch phm pha long vi 9 ml nc mui sinh l 90/00
ta c nng pha long l 10-1, tip tc ht 1 ml dch th trn pha vi 9 ml nc
mui sinh l 90/00 ta c nng pha long 10-2 v lm nh th khi c c nng
pha long thch hp. tch ri cc t bo, cn pha long mu km theo lc
mnh dch pha long.
Cy tri dch pha long trn a thch Sabouraud: ta s cy mi mu 3
nng pha long v ng vi mi pha long chun b 2 a thch. Sau khi lc li
dch pha long, dng pipette v trng ht 0,1ml dch mu nh ln gia a thch.
Dng que cy trang v trng gt git dch tri u khp cho ti khi mt thch kh
ro.
Sau em nhit phng trong 24 48 gi v m s khun lc mc
trn a. Ch cn phn bit cc khun lc l hnh thnh do tp nhim v khng
tnh chng.
Tnh kt qu
cfu/ml(g) = a*1/K*1/V
A: s khun lc trung bnh xut hin trn cc a cy c cng pha long.
V: th tch dch pha long c cy trn mt a thch.
K: pha long ca dch cy.
Lu : xem xt s khun lc 3 nng pha long k tip nhau(v d 10-5,
10-6, 10-7). Khi nng pha long gim 10 ln, s khun lc trn a cng gim xp
x 10 ln v trn 2 a c cng nng pha long ch chnh lch nhau khong 10%.
 29

iu chng t vic tch ri cc t bo, pha long cng nh dn u chng trn

mt thch din ra hon ho v s liu thu c l ng tin cy.
 30

Chng 4
KT QU V THO LUN
4.1. Th nghim 1: Kho st s sinh trng ca cc chng phn lp trong
mi trng r ng 60B
Chng ti em 4 chng nm men phn lp c t 4 ngun mu khc nhau
nui trong mi trng r ng 60B vi thi gian nui cy l 36 gi nhm tm
chng c kh nng pht trin tt nht tin hnh cc th nghim tip theo c
thun li hn. S liu thu c ban u c n v tnh l tb/ml (ph lc Bng 7.1)
s chuyn i v gi tr logarit x l thng k.
Bng 4.1: S lng t bo nm men trong 1 ml dch nui cy theo
phng php m trc tip trn bung m hng cu (qui v gi tr logarit)
Ch Men
Mu phm bnh m u Nho
Ln 1 8,450 8,204 8,459 8,566
Ln 2 8,332 8,061 8,130 8,415
Trung bnh 8,391 8,133 8,295 8,491

Qua Bng 4.1 cho thy s lng t bo nm men thuc cc chng phn lp
gim dn theo th t sau:

Nho: 8,491
Ch phm: 8,391
u : 8,295
Men bnh m: 8,133
S khc bit ny c ngha v thng k vi P<0,05 (ph lc Bng ANOVA
7.1).
Cc loi nm men cng mt ging khng phi bao gi cng ng ha vt
cht nh nhau. C th ni, cc loi khc nhau (d l mt ging) ng ha cc ngun
 31

dinh dng l khc nhau nn tc sinh trng ca chng trn cng mt iu kin
nui cy l khc nhau. Do , tuy cc chng phn lp t 4 ngun mu trn u
thuc ging Saccharomyces nhng khi nui cy trong cng iu kin mi trng r
ng 60B th s lng t bo ca chng khc nhau l hp l.
Da theo s liu Bng 4.1, chng ti chn 2 chng c phn lp t nho
v ch phm lm i tng tin hnh cc th nghim tip theo. Sau khi nhum
xem hnh thi v th nghim cc phn ng ln men ng, chng ti xc nh c
chng nm men phn lp t dch qu nho thuc loi S. cerevisiae, chng phn lp t
ch phm sinh hc (tn thng mi l Ultra Levure) thuc loi S. boulardii.

Hnh 4.1: Phn lp nm men t Hnh 4.2: Phn lp nm men t

ch phm sinh hc Ultra Levure dch qu nho
 32

Hnh 4.3: Hnh thi S. boulardii

Hnh 4.4: Hnh thi S. cerevisiae

 33

4.2. Th nghim 2: Kho st nh hng ca mi trng nui cy v thi gian

thu hoch ln s sinh trng ca nm men Saccharomyces
Sau khi kho st nui cy 2 loi S. boulardii v S. cerevisiae trn mi trng
r ng 60B, mi trng cm go 3 mc thi gian 36 gi, 48 gi, 60 gi,
chng ti thu nhn s liu v s lng t bo nm men trong mi nghim thc (ph
lc Bng 7.2, 7.3, 7.4 v 7.5). Tip theo, chng ti x l thng k trn s liu
logarit c chuyn i t dng s liu nguyn ban u (n v tnh l tb/ml, cfu/g).

4.2.1. Saccharomyces boulardii

Bng 4.2a: S lng t bo S. boulardii trong 1 ml dch nui cy theo
phng php m trc tip trn bung m hng cu (qui v gi tr logarit).
Mi trng R ng Cm go
Thi gian 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 8,427 8,505 8,394 8,453 8,536 8,385
Ln 2 8,443 8,491 8,533 8,362 8,398 8,293
Ln 3 8,282 8,411 8,445 8,394 8,447 8,470
Bng 4.2b: gi tr trung bnh ca bng 4.2a
36 gi 48 gi 60 gi Chung
R ng 8,384 8,469 8,457 8,437
Cm go 8,403 8,460 8,383 8,415
Chung 8,394 8,465 8,420
Qua Bng 4.2 cho thy tng s t bo S. boulardii trong dch nui cy cc
nghim thc u cao. Xt v mi trng nui cy, s lng t bo S. boulardii trn
mi trng r ng cao hn mi trng cm go (8,437>8,415). V thi gian thu
hoch, s lng t bo S. boulardii thi im 48 gi cao hn 60 gi v 36 gi
(8,465>8,420>8,394). Th nhng, s khc bit ny khng c ngha v mt thng
k vi P>0,05.
Nhn chung, mi tng quan c hai yu t mi trng nui cy v thi gian
thu hoch nh hng ln s lng t bo nm men khng c ngha thng k vi
P>0,05 (ph lc Bng ANOVA 7.2).
 34

Bng 4.3: S lng t bo S. boulardii trong 1 g ch phm theo phng

php m s khun lc trn a (qui v gi tr logarit)
Mi
trng R ng Cm go
Thi gian 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 7,537 8,546 8,834 8,175 9,041 8,537
Ln 2 8,361 8,189 8,474 9,442 8,525 8,516
Ln 3 8,975 8,406 8,569 9,389 8,331 9,316
Bng 4.3b: gi tr trung bnh ca bng 4.3a
36 gi 48 gi 60 gi Chung
R ng 8,291 8,380 8,626 8,432
Cm go 9,002 8,632 8,790 8,808
Chung 8,647 8,506 8,708
Qua Bng 4.3b cho thy s lng t bo S. boulardii sng trong ch phm
cc nghim thc u cao. Xt v mi trng nui cy, s lng t bo S. boulardii
sng trn mi trng cm go cao hn mi trng r ng (8,808>8,432). S khc
bit ny c ngha v thng k vi P<0,05. V thi gian thu hoch, s lng t bo
S. boulardii sng thi im 60 gi cao hn 36 gi v 48 gi (8,708>8,647>8,506).
Th nhng, s khc bit ny khng c ngha v mt thng k vi P>0,05.
Nhn chung, mi tng quan gia yu t mi trng nui cy v thi gian
thu hoch nh hng ln s lng t bo S. boulardii sng trong ch phm khng
c ngha thng k vi P>0,05 (ph lc Bng ANOVA 7.3).
Vi phng php m khun lc trn a, chng ti khng tin hnh m
lin ngay khi thu hoch m ch phm c bo qun sau 22 ngy mi m. Qua
Bng 4.3, chng ti nhn thy s lng t bo cn sng trong ch phm khi nui
cy trn mi trng cm go cao hn trn mi trng r ng. C kh nng l mi
trng cm go c thnh phn dinh dng y hn mi trng r ng, c bit
c cha nhiu vitamin B, p ng c nhu cu hot ng sinh l ca t bo nm
men v lm tng sc sng ca chng trong ch phm.
Chng ti nghin cu nui cy nm men vi mc ch dng lm ch phm
sinh hc nn yu cu s lng t bo cn sng trong ch phm phi cao. Do , c
 35

th kt lun mi trng cm go l mi trng thch hp nui cy S. boulardii

hn mi trng r ng.

4.2.2. Saccharomyces cerevisiae

Bng 4.4a: S lng t bo S. cerevisiae trong 1 ml dch nui cy theo
phng php m trc tip trn bung m hng cu (qui v gi tr logarit)
Mi
trng R ng Cm go
Thi gian 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 8,359 8,617 8,486 8,543 8,539 8,606
Ln 2 8,459 8,453 8,486 8,539 8,558 8,468
Ln 3 8,287 8,509 8,517 8,380 8,519 8,350
Bng 4.4b: gi tr trung bnh ca bng 4.4a
36 gi 48 gi 60 gi Chung
R ng 8,368 8,526 8,496 8,463
Cm go 8,487 8,539 8,475 8,500
Chung 8,428 8,533 8,486
Qua Bng 4.4b cho thy tng s t bo S. cerevisiae trong dch nui cy
cc nghim thc u cao. Xt v mi trng nui cy, s lng t bo S. cerevisiae
trn mi trng cm go cao hn mi trng r ng (8,5>8,463). Th nhng, s
khc bit ny khng c ngha v mt thng k vi P>0,05. V thi gian thu hoch,
s lng t bo S. cerevisiae thi im 48 gi cao hn 60 gi v 36 gi
(8,533>8,486>8,428). S khc bit ny c ngha v thng k vi P<0,05.
Nhn chung, s khc bit v mi tng quan gia mi trng v thi gian
thu hoch nh hng ln tng s t bo S. cerevisiae trong dch nui cy khng c
ngha thng k vi P>0,05 (ph lc Bng ANOVA 7.4).
T kt qu s lng t bo S. cerevisiae thu hoch vo thi im 48 gi cao
hn thi im 60 gi v 36 gi Bng 4.4 cho php ta c th kt lun thi gian
thch hp nht thu hoch sinh khi t bo nm men l 48 gi sau khi nui cy.
 36

iu ny cng ph hp vi kt qu th nghim ca nhiu tc gi trc (Nguyn

Khc Tun, 1996 v Lng Th Phng Tho, 2005).
S lng t bo nm men tng gim theo qui lut, ph hp vi cc giai on
sinh trng ca chng. Trc thi im 36 gi nui cy, nm men trong giai on
thch nghi vi mi trng nn s lng t bo tng khng ng k. T 36 48 gi
l giai on logarit, thi im s lng t bo nm men tng theo cp s nhn v t
gi tr cc i khong 48 gi. Trong khong thi gian s t bo pht trin o t,
khi cc cht dinh dng trong mi trng khng phi l v tn, m ngc li
ngy mt gim i, hn na trong mi trng xut hin v tch t cc sn phm trao
i cht khng cn thit i vi t bo. Do , lm mt i iu kin thun li cho
sinh trng ca t bo, chng s gi ci, thoi ha, cui cng xy ra hin tng t
phn nn s lng t bo s gim.
Bng 4.5a: S lng t bo S. cerevisiae trong 1 g ch phm theo phng
php m s khun lc trn a (qui v gi tr logarit)
Mi
trng R ng Cm go
Thi gian 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 8,686 8,567 9,475 7,808 7,665 8,929
Ln 2 8,737 8,625 8,290 9,006 8,125 8,130
Ln 3 10,012 8,829 9,620 8,581 8,438 8,818
Bng 4.5b: gi tr trung bnh ca bng 4.5a
36 gi 48 gi 60 gi Chung
R ng 9,145 8,674 9,128 8,982
Cm go 8,465 8,076 8,626 8,389
Chung 8,805 8,375 8,877
Qua Bng 4.5b cho thy s lng t bo S. cerevisiae sng trong ch phm
cc nghim thc u cao. Xt v mi trng nui cy, s lng t bo S. cerevisiae
sng trn mi trng r ng cao hn mi trng cm go (8,982>8,389). S khc
bit ny c ngha v thng k vi P<0,05. V thi gian thu hoch, s lng t bo
S. cerevisiae sng thi im 60 gi cao hn 36 gi v 48 gi
(8,877>8,805>8,375). Th nhng, s khc bit ny khng c ngha v mt thng
k vi P>0,05.
 37

Nhn chung, s khc bit v mi tng quan gia yu t mi trng nui cy

v thi gian thu hoch ln s lng t bo S. cerevisiae sng trong ch phm khng
c ngha thng k vi P>0,05 (ph lc Bng ANOVA 7.5).
Chng nm men S. cerevisiae m chng ti em kho st c phn lp t
dch qu nho nn c th n thch nghi v sinh trng trn mi trng r ng tt
hn trn mi trng cm go, mi trng r ng c hm lng ng cao hn
mi trng cm go. Do , chng ti c th kt lun nui cy S. cerevisiae trn
mi trng r ng thch hp hn mi trng cm go.

4.3. Th nghim 3: Kho st nh hng ca acid ascorbic (vitamin C) v cht

nn ln sc sng ca nm men trong ch phm
Vic thu hoch v bo qun ch phm nh hng nhiu n sc sng ca
nm men trong ch phm. n thi im thu hoch sinh khi t bo nm men,
chng ti tin hnh ly tm dch nui cy ly sinh khi v em trn sinh khi vi
2 loi cht nn l cm go v bt m. Cht nn c x l trc bng vic sy kh
1100C/45 pht, sau chia lm 3 phn:
Phn 1: nguyn, khng b sung vitamin C.
Phn 2: b sung vitamin C vi hm lng l 50/000.
Phn 3: b sung vitamin C vi hm lng l 10/00.
Sau khi trn sinh khi nm men vi cht nn, chng ti lm kh ch phm
bng cch sy 380C/10 gi. ng gi v nhit phng trong 22 ngy ri
tin hnh kim tra s lng t bo nm men sng theo phng php m s khun
lc trn a. S liu thu c ban u tnh bng n v cfu/g (ph lc Bng 7.6, 7.7)
s c qui v gi tr logarit x l thng k.
 38

4.3.1. Saccharomyces boulardii

Bng 4.6: S lng t bo S. boulardii trong 1 g ch phm theo phng
php m s khun lc trn a (qui v gi tr logarit)
Cht nn Cm M
Hm lng vitamin 0 50/000 10/00 0 50/000 10/00
Ln 1 8,081 8,212 8,311 8,885 8,338 8,716
Ln 2 8,503 8,388 8,420 9,022 8,339 8,853
Ln 3 8,535 8,610 8,350 9,245 9,141 9,812
Bng 4.6b: gi tr trung bnh ca bng 4.6a
0 50/000 10/00 Chung
Cm go 8,373 8,403 8,360 8,379
Bt m 9,051 8,606 9,127 8,928
Chung 8,712 8,505 8,744
Qua Bng 4.6b cho thy s lng t bo S. boulardii sng trong ch phm
cc nghim thc u cao. Xt v cht nn, s lng t bo S. boulardii sng trong
bt m nhiu hn trong cm (8,928>8,379). V hm lng vitamin C, s lng t
bo S. boulardii sng trong cht nn b sung vitamin C vi hm lng 10/00 nhiu
hn so vi hm lng 50/000 v khng b sung vitamin C (8,744>8,712>8,505). Th
nhng, s khc bit ny khng c ngha v mt thng k vi P>0,05 (ph lc
Bng ANOVA 7.6).

4.3.2. Saccharomyces cerevisiae

Bng 4.7: S lng t bo S. cerevisiae trong 1 g ch phm theo phng
php m s khun lc trn a (qui v gi tr logarit)
Cht nn Cm M
Hm lng vitamin 0 50/000 10/00 0 0
5 /000 10/00
Ln 1 8,269 8,684 8,359 9,150 8,423 8,558
Ln 2 8,352 9,999 9,019 8,771 8,635 8,370
Ln 3 9,572 8,919 9,325 9,242 9,719 10,155
Bng 4.7b: gi tr trung bnh ca bng 4.7a
0 50/000 10/00 Chung
Cm go 8,731 9,201 8,901 8,944
Bt m 9,054 8,926 9,028 9,003
Chung 8,893 9,064 8,965
 39

Qua Bng 4.7b cho thy s lng t bo S. cerevisiae sng trong ch phm
cc nghim thc u cao. Xt v cht nn, s lng t bo S. cerevisiae sng trong
bt m nhiu hn trong cm (9,003>8,944). V hm lng vitamin C, s lng t
bo S. cerevisiae sng trong cht nn c b sung vitamin C vi hm lng 50/000
nhiu hn so vi hm lng 10/00 v khng b sung vitamin C
(9,064>8,965>8,893). Th nhng, s khc bit ny khng c ngha v mt thng
k vi P>0,05 (ph lc Bng ANOVA 7.7).
V c s l thuyt, vic b sung vitamin C vo cht nn c nh hng ln
sc sng ca t bo nm men trong ch phm hay khng th vn cha c nghin
cu r. C kh nng do thi gian kho st cha lu (sau 22 ngy bo qun) nn
kt qu trn cng cha phn nh r bn cht vn .

4.4. Th nghim 4: Kho st nh hng ca mi trng nui cy, cht nn v

thi gian thu hoch ln sc sng ca nm men trong ch phm

4.4.1. Saccharomyces boulardii

Bng 4.8b: gi tr trung bnh ca bng 4.8a
36 gi 48 gi 60 gi Chung
ng-cm 8,264 8,096 8,341 8,234
Cm-cm 8,407 8,341 8,500 8,416
ng-m 7,897 8,206 8,761 8,288
Cm-m 9,345 8,653 9,020 9,006
Chung 8,478 8,324 8,656
Qua Bng 4.8b cho thy s lng t bo S. boulardii sng trong ch phm
cc nghim thc u cao. Xt v mi trng nui cy v cht nn, s lng t bo
S. boulardii sng gim dn theo th t sau:
Mi trng cm go v cht nn bt m: 9,006
Mi trng cm go v cht nn cm go: 8,416
Mi trng r ng v cht nn bt m: 8,288
Mi trng r ng v cht nn cm go: 8,234
Xt v thi gian thu hoch, s lng t bo S. boulardii sng thu hoch
thi im 60 gi cao hn 36 gi v 48 gi (8,656>8,478>8,324).
 40

Xt v mt thng k, s khc bit ca 2 yu t mi trng nui cy - cht

nn v thi gian thu hoch u c ngha vi P<0,05.
Nhn chung, mi tng quan gia 2 yu t ny ln s lng t bo
S. boulardii sng trong ch phm th s khc bit l c ngha v mt thng k vi
P<0,05 (ph lc Bng ANOVA 7.8).
V mi trng nui cy, th nghim ny cho kt qu ph hp vi th nghim
2, mi trng cm go thch hp nui cy S. boulardii hn mi trng r ng.
V thi gian thu hoch, qua Bng 4.8 chng ti c th kt lun thu hoch
S. boulardii vo thi im 60 gi sau khi nui cy s cho kt qu l s lng t bo
cn sng trong ch phm nhiu hn 36 gi v 48 gi.
 41

Bng 4.8: S lng t bo S. boulardii trong 1 g ch phm theo phng php m s khun lc trn a
(qui v gi tr logarit)

MT CN ng Cm Cm Cm ng M Cm M
Thi gian 36 Gi 48 Gi 60 Gi 36 Gi 48 gi 60 gi 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 7,619 7,403 8,209 8,302 7,834 8,681 6,380 8,380 8,887 8,491 8,920 8,932
Ln 2 8,412 8,397 8,328 8,505 8,567 8,381 8,380 7,674 8,048 9,445 8,508 8,660
Ln 3 8,762 8,488 8,485 8,414 8,623 8,437 8,930 8,564 9,349 10,098 8,532 9,468
Trung bnh 8,264 8,096 8,341 8,407 8,341 8,500 7,897 8,206 8,761 9,345 8,653 9,020

Bng 4.9: S lng t bo S. cerevisiae trong 1 g ch phm theo phng php m s khun lc trn a
(qui v gi tr logarit)

MT CN ng Cm Cm Cm ng M Cm M
Thi gian 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 8,902 8,097 8,868 7,732 7,487 7,689 8,606 8,628 9,302 7,626 8,242 9,014
Ln 2 9,538 8,720 8,470 10,255 7,993 7,989 8,903 8,508 8,447 8,850 7,486 8,569
Ln 3 10,014 8,909 9,120 8,605 8,393 8,408 10,340 9,729 9,929 9,785 8,428 9,362
Trung bnh 9,485 8,575 8,819 8,864 7,958 8,029 9,283 8,955 9,226 8,754 8,052 8,982
 42

4.4.2. Saccharomyces cerevisiae

Bng 4.9b: gi tr trung bnh ca bng 4.9a
36 gi 48 gi 60 gi Chung
ng-cm 9,485 8,575 8,819 8,960
Cm-cm 8,864 7,958 8,029 8,284
ng-m 9,283 8,955 9,226 9,155
Cm-m 8,754 8,052 8,982 8,596
Chung 9,097 8,385 8,764
Qua Bng 4.9b cho thy s lng t bo S. cerevisiae sng trong ch phm
cc nghim thc u cao. Xt v mi trng nui cy v cht nn, s lng t bo
S. cerevisiae sng gim dn theo th t sau:
Mi trng r ng v cht nn bt m: 9,155
Mi trng r ng v cht nn cm go: 8,960
Mi trng cm go v cht nn bt m: 8,596
Mi trng cm go v cht nn cm go: 8,284
Xt v thi gian, s lng t bo S. cerevisiae sng thu hoch thi im 36
gi cao hn 60 gi v 48 gi (9,097>8,764>8,385).
Xt v mt thng k, s khc bit ca tng yu t mi trng nui cy - cht
nn v thi gian thu hoch ln s lng t bo S. cerevisiae u c ngha vi
P<0,05.
Nhn chung, s khc bit v mi tng quan gia mi trng nui cy, cht
nn v thi gian ln sc sng ca t bo nm men khng c ngha v mt thng k
vi P>0,05 (ph lc Bng ANOVA 7.9).
th nghim 2, theo phng php m s t bo trc tip trn bung m
hng cu chng ti nhn c kt qu vo thi im 48 gi sau khi nui cy s
lng t bo S. cerevisiae cao hn 60 gi v 36 gi. Nhng th nghim ny, chng
ti kim tra s lng t bo sng trong ch phm (sau 22 ngy bo qun) theo
phng php m s khun lc trn a cho thy s lng t bo S. cerevisiae thu
hoch vo thi im 36 gi sau khi nui cy nhiu hn 60 gi v 48 gi.
 43

Nh chng ti ni yu cu t ra khi nui cy nm men lm ch phm

sinh hc l s t bo sng trong ch phm phi cao nn chng ti c th kt lun
thi im thu hoch S. cerevisiae thch hp nht l 36 gi sau khi nui cy.
V mi trng nui cy, kt qu th nghim ny ph hp vi th nghim 2,
mi trng r ng thch hp nui cy S. cerevisiae hn mi trng cm go.

4.5. Th nghim 5: Kho st nh hng ca vi khun Bacillus subtilis ln s

sinh trng ca nm men Saccharomyces cerevisiae trong iu kin nui cy
chung
Theo thng tin chng ti c bit, khi sn xut ch phm sinh hc ngi ta
thng nui cy ring tng loi vi sinh vt, sau mi phi trn cho ra thnh
phm. Vic mt kh nhiu cng sc, thi gian, chi ph cng cao nn chng ti
quyt nh kho st vic nui cy chung vi khun Bacillus subtilis v nm men
Saccharomyces cerevisiae trong mi trng r ng 60B v mi trng cm go.
Th nghim b tr gm 2 yu t (mi trng nui cy v nng vi khun
B. subtilis), thi gian nui cy l 48 gi. S liu ban u thu c tnh theo n v
tb/ml, cfu/g (ph lc Bng 7.8 v 7.9) s chuyn i theo gi tr logarit x l
thng k.
Bng 4.10a: S lng t bo S. cerevisiae trong 1 ml dch nui cy theo
phng php m trc tip trn bung m hng cu (qui v gi tr logarit)
Mi
R ng Cm go
trng
Mu 1S+0B 1S+1B 1S+0,1B 1S+0,01B 1S+0B 1S+1B 1S+0,1B 1S+0,01B
Ln 1 8,583 8,439 8,599 8,667 8,435 8,011 8,342 8,470
Ln 2 8,466 8,550 8,376 8,322 8,357 8,366 8,477 8,455
Bng 4.10b: gi tr trung bnh ca bng 4.10a
1S+0B 1S+1B 1S+0,1B 1S+0,01B Chung
R ng 8,525 8,495 8,488 8,495 8,493
Cm go 8,396 8,189 8,410 8,463 8,354
Chung 8,461 8,342 8,449 8,479
Qua Bng 4.10b cho thy tng s t bo S. cerevisiae trong dch nui cy
cc nghim thc u cao. Xt v mi trng nui cy, s lng t bo S. cerevisiae
 44

trong mi trng r ng cao hn mi trng cm go (8,493>8,354). S khc bit

ny c ngha v thng k vi P<0,05. V nng vi khun B. subtilis, s lng t
bo S. cerevisiae trong dch nui cy c b sung B. subtilis gim dn theo th t
sau:
Nng 10-2 dch B.subtilis nui 370C/24 gi: 8,479.
Nng 10-1 dch B.subtilis nui 370C/24 gi: 8,449.
Nng 100 dch B.subtilis nui 370C/24 gi: 8,342.
Th nhng, s khc bit ny khng c ngha v mt thng k vi P>0,05.
Nhn chung, s khc bit v mi tng quan gia mi trng v nng
B. subtilis ln tng s t bo S. cerevisiae trong dch nui cy khng c ngha v
mt thng k vi P>0,05 (ph lc Bng ANOVA 7.10).
Bng 4.11: S lng t bo S. cerevisiae trong 1 g ch phm theo phng
php m s khun lc trn a (qui v gi tr logarit)
Mi
R ng Cm go
trng
Mu 1S+0B 1S+1B 1S+0,1B 1S+0,01B 1S+0B 1S+1B 1S+0,1B 1S+0,01B
Ln 1 8,942 9,705 9,027 9,108 8,081 8,883 8,685 8,723
Ln 2 9,224 9,820 9,151 8,932 8,633 9,219 8,552 8,874
Bng 4.11b: gi tr trung bnh ca bng 4.11a
1S+0B 1S+1B 1S+0,1B 1S+0,01B Chung
R ng 9,083 9,763 9,089 9,020 9,291
Cm go 8,357 9,051 8,619 8,799 8,823
Chung 8,720 9,407 8,854 8,910
Qua Bng 4.11b cho thy s lng t bo S. cerevisiae sng trong ch phm
cc nghim thc u cao. Xt v mi trng, s lng t bo S. cerevisiae sng
trong mi trng r ng cao hn mi trng cm go (9,291>8,823). S khc bit
ny c ngha v thng k vi P<0,05. V nng vi khun B. subtilis, s lng t
bo S. cerevisiae sng trong ch phm c b sung B. subtilis gim dn theo th t
sau:
Nng 100 dch B.subtilis nui 370C/24 gi: 9,407
Nng 10-2 dch B.subtilis nui 370C/24 gi: 8,910
Nng 10-1 dch B.subtilis nui 370C/24 gi: 8,854
 45

Th nhng, s khc bit ny khng c ngha v mt thng k vi P>0,05.

Nhn chung, s khc bit v mi tng quan gia mi trng v nng
B. subtilis ln s lng t bo S. cerevisiae sng trong ch phm khng c ngha
v mt thng k vi P>0,05 (ph lc Bng ANOVA 7.11).
Yu t mi trng nui cy nh hng ln s lng t bo S. cerevisiae
c gii thch th nghim 2 v trong th nghim ny cng cho kt qu tng t.
C th l s lng t bo S. cerevisiae trn mi trng r ng cao hn mi trng
cm go.
Bng 4.10, s chnh lch s lng t bo S. cerevisiae gia 2 trng hp
1S+0B (1ml dch S. cerevisiae + 0 ml dch B. subtilis) v 1S+1B (1 ml dch
S. cerevisiae + 1 ml dch B. subtilis) trong mi trng r ng l 0,18%, trong mi
trng cm go l 1,25%. C th ni, trong mi trng r ng s c mt ca
B. subtilis nh hng khng ng k n s lng S. cerevisiae. Ngc li, trong
mi trng cm go (mi trng thng c s dng nui cy sinh khi
B. subtilis) s c mt ca B. subtilis nh hng ng k n s lng S. cerevisiae.
Chng ti c th kt lun khi nui cy chung B. subtilis v S. cerevisiae vi t l 1:1
trong mi trng cm go th s lng t bo S. cerevisiae gim ng k.
Mt im ng lu khc, tuy v mt thng k hc th khi nui cy chung
nng ca B. subtilis khng nh hng n s lng t bo S. cerevisiae nhng
theo chng ti nhn thy th vn c s nh hng v mt sinh hc. Da vo mt s
l do sau:
Khi thu hoch t bo S. cerevisiae, bng phng php m s t bo trc tip
trn bung m hng cu chng ti thu c kt qu s lng t bo S. cerevisiae
nui cy ring cao hn khi nui cy chung vi B. subtilis (8,461>8,423) (Bng
4.10).
Sau 22 ngy bo qun ch phm, chng ti kim tra s t bo sng trong ch
phm bng phng php m s khun lc trn a th nhn thy s lng t bo
S. cerevisiae nui cy chung vi B. subtilis cao hn khi nui cy ring
(9,057>8,720), (Bng 4.11).
 46

Ta nhn thy ban u s lng t bo S. cerevisiae nui cy ring cao hn

khi nui cy chung vi B. subtilis (8,461>8,423), nhng sau 22 ngy bo qun s
lng t bo S. cerevisiae khi nui cy ring cht nhiu hn khi nui cy chung
(8,720<9,057). Ngha l, khi nui cy chung B. subtilis c nh hng n
S. cerevisiae theo hng c li lm tng sc sng ca S. cerevisiae trong ch phm.
 47

Chng 5
KT LUN V NGH
5.1. Kt lun
Chng ti phn lp c t dch qu nho chng nm men thuc loi
S. cerevisiae v t ch phm sinh hc chng nm men thuc loi S. boulardii. C 2
chng u c kh nng sinh trng tt trn mi trng r ng 60B v mi trng
cm go.
Mi trng cm go thch hp cho s sinh trng ca loi S. boulardii hn
mi trng r ng 60B. Ngc li, loi S. cerevisiae sinh trng trn mi trng
r ng 60B mnh hn trn mi trng cm go.
Thi im thu hoch sinh khi t bo nm men thch hp nht l sau 48 gi
nui cy.
Vitamin C v cht nn (cm go, bt m) khng nh hng n sc sng ca
nm men trong ch phm (sau 22 ngy bo qun).
Thu hoch S. boulardii vo thi im 60 gi sau khi nui cy s cho kt qu
s lng t bo cn sng trong ch phm nhiu hn 36 gi v 48 gi. Cn thi
im thu hoch S. cerevisiae thch hp nht l 36 gi sau khi nui cy.
Trong nui cy chung, nng vi khun B. subtilis khng nh hng n s
lng t bo nm men S. cerevisiae.

5.2. ngh
Tin hnh th nghim kho st nh hng ca vitamin C ln sc sng t bo
nm men trong ch phm vi thi gian bo qun di hn 22 ngy.
m ng thi s lng t bo B. subtilis v S. cerevisiae trong th nghim
nui cy chung 2 loi.
 48

Kho st nui cy chung vi khun lactic thuc loi Lactobacillus acidophilus

vi nm men Saccharomyces cerevisiae trong mi trng r ng.
 49

Chng 6
TI LIU THAM KHO
1. Ban Nng Lm Nghip, U ban Khoa Hc - K Thut Nh Nc, 1970.
Nm men dng trong chn nui ln. Nh xut bn Khoa Hc - K Thut H Ni.

2. Lao Th Nga, 1987. K thut sn xut nm men bnh m v mt s loi nm

n. Nh xut bn TP.HCM.

3. Lm Thanh Hin, 1996. Vi sinh vt i cng. T sch i hc Nng Lm

TP.HCM.

4. Lng c Phm, 2006. Nm men cng nghip. Nh xut bn Khoa Hc -

K Thut.
5. Lng Th Phng Tho, 2005. Kho st s pht trin ca nm men
Saccharomyces sp. trn hai loi mi trng r ng v nc chit gi u. Kha
lun tt nghip Bc S Th Y, i hc Nng Lm TP.HCM.

6. L Nguyn Bo Trn, 2005. Kho st nui cy nm men Saccharomyces

dng lm ch phm b sung selen. Kha lun tt nghip C nhn Cng ngh sinh
hc, i hc M - Bn Cng TP.HCM

7. L Thanh Lm, 1996. Nghin cu di truyn hc mt s tnh trng c ngha

kinh t ca Saccharomyces spp. Lun n Ph Tin S Khoa Hc Sinh Hc, i hc
Quc Gia H Ni Khoa Hc T Nhin.

8. Nguyn c Lng, 2002. Cng ngh vi sinh tp 2.

9. Nguyn Khc Tun, 1996. Tuyn chn mt s chng nm men t bnh men
ru c truyn sn xut mt s ch phm sinh hc s dng trong chn nui ln.
Lun n Ph Tin S Khoa Hc Nng Nghip, i hc Nng Nghip I H Ni.

10. Nguyn Th Hng Phng, 2006. Kho st s pht trin ca nm men

Saccharomyces cerevisiae trn mi trng r ng cc iu kin khc nhau.
Kha lun tt nghip Bc S Th Y, i hc Nng Lm TP.HCM.
 50

11. Nguyn Vn Hng, 1990. Nghin cu c im di truyn hc ca mt s tnh

trng chng chu v lai to cc ni Saccharomyces c ngha kinh t. Lun n Ph
Tin S Sinh Vt Hc, i hc Tng Hp H Ni.

12. Phm Th Thm, 2004. Phn lp v nh danh mt s loi nm men thuc

ging Saccharomyces trong sn phm probiotic. Kha lun tt nghip C nhn
Cng ngh sinh hc, i hc M - Bn Cng TP.HCM.

13. V Th Minh c. Thc tp vi sinh vt hc. Nh xut bn i hc Quc Gia

H Ni.
 51

Chng 7
PH LC

Hnh 7.1: Bnh nui cy nm men

Hnh 7.2: Khun lc Saccharomyces Hnh 7.3: Ch phm sinh hc cha

sp. trn mi trng thch Sabouraud nm men Saccharomyces sp.
 52

7.1. Cc bng s liu th v s lng t bo nm men

Bng 7.1: S lng t bo nm men trong 1 ml dch nui cy theo phng
php m trc tip trn bung m hng cu (*108 tb/ml).
Ch Men
Mu phm bnh m u Nho
Ln 1 2,816 1,600 2,880 3,680
Ln 2 2,150 1,150 1,350 2,600

Bng 7.2: S lng t bo S. boulardii trong 1 ml dch nui cy theo

phng php m trc tip trn bung m hng cu (*108 tb/ml)
Mi
trng R ng Cm go
Thi gian 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 2,675 3,200 2,475 2,838 3,438 2,425
Ln 2 2,775 3,100 3,413 2,300 2,500 1,963
Ln 3 1,913 2,575 2,788 2,475 2,800 2,950

Bng 7.3: S lng t bo S. boulardii trong 1 g ch phm theo phng

php m s khun lc trn a (*108 cfu/g)
Mi
trng R ng Cm go
Thi gian 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 0,344 3,517 6,820 1,496 10,999 3,443
Ln 2 2,297 1,544 2,979 27,68 3,353 3,283
Ln 3 9,447 2,544 3,704 24,463 2,144 20,722
 53

Bng 7.4: S lng t bo S. cerevisiae trong 1 ml dch nui cy theo

phng php m trc tip trn bung m hng cu (*108 tb/ml)
Mi
trng R ng Cm go
Thi gian 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 2,288 4,138 3,063 3,488 3,463 4,038
Ln 2 2,875 2,838 3,063 3,463 3,613 2,938
Ln 3 1,938 3,225 3,288 2,400 3,300 2,238

Bng 7.5: S lng t bo S. cerevisiae trong 1 g ch phm theo phng

php m s khun lc trn a (*108 cfu/g )
Mi
trng R ng Cm go
Thi gian 36 gi 48 gi 60 gi 36 gi 48 gi 60 gi
Ln 1 4,849 3,689 29,857 0,643 0,462 8,497
Ln 2 5,457 4,219 1,949 10,129 1,335 1,350
Ln 3 102,835 6,738 41,710 3,812 2,743 6,576
 54

Bng 7.6: S lng t bo S. boulardii trong 1 g ch phm (*108 cfu/g)

36 gi 48 gi 60 gi
mu
ln 1 ln 2 ln 3 ln 1 ln 2 ln 3 ln 1 ln 2 ln 3
ng 0 cm 0,665 4,443 4,715 0,209 2,590 2,560 2,050 3,683 2,810
ng 5 cm 0,168 0,701 6,008 0,311 4,323 3,303 0,955 0,642 2,833
ng 1 cm 0,414 2,610 6,603 0,240 0,577 3,368 1,845 2,065 3,520
cm 0 cm 2,680 4,560 2,300 0,647 3,363 2,953 0,975 0,468 5,218
cm 5 cm 2,233 2,430 3,750 0,745 4,080 6,120 5,370 2,473 2,435
cm 1 cm 1,105 2,615 1,725 0,655 3,618 3,530 8,030 4,280 0,552
ng 0 m 0,022 0,151 14,178 6,825 0,498 2,528 11,590 2,275 4,598
ng 5 m 0,022 6,868 4,648 0,205 0,322 2,475 4,845 0,437 57,150
ng 1 m 0,027 0,178 6,695 0,162 0,596 5,998 6,695 0,638 5,280
cm 0 m 0,311 50,800 46,625 21,350 3,343 1,335 5,910 6,098 36,225
cm 5 m 1,745 0,542 10,458 3,180 2,978 1,988 3,070 1,948 6,283
cm 1 m 7,233 32,325 318,775 0,417 3,348 6,885 16,660 5,673 45,650
Bng 7.7: S lng t bo S. cerevisiae trong 1 g ch phm (*108 cfu/g)
36 gi 48 gi 60 gi
mu
ln 1 ln 2 ln 3 ln 1 ln 2 ln 3 ln 1 ln 2 ln 3
ng 0 cm 6,118 3,945 198,500 0,503 5,928 6,353 3,213 0,612 8,745
ng 5 cm 14,810 47,900 12,473 2,040 6,115 7,283 10,528 4,553 21,350
ng 1 cm 3,020 51,725 98,500 1,205 3,695 10,708 8,415 3,685 9,435
cm 0 cm 0,562 0,230 3,918 0,401 2,228 2,580 0,348 0,545 3,863
cm 5 cm 0,580 538,500 4,848 0,228 0,435 2,258 0,815 0,646 1,585
cm 1 cm 0,475 1,545 3,303 0,292 0,287 2,578 0,305 1,735 2,228
ng 0 m 3,580 6,968 7,170 6,875 2,510 7,123 56,500 3,285 74,675
ng 5 m 4,373 9,380 9,583 3,255 4,650 1,800 3,085 2,613 125,250
ng 1 m 4,148 7,628 640 2,613 2,503 151,750 0,489 2,503 55,025
cm 0 m 0,724 20,028 3,705 0,522 0,441 2,905 16,645 2,155 9,288
cm 5 m 0,192 0,621 118,225 0,426 0,192 4,398 4,550 8,423 55,025
cm 1 m 0,353 0,604 6,030 4,290 0,286 0,730 9,783 0,537 4,723
 55

Bng 7.8: S lng t bo trong S. cerevisiae trong 1 ml dch nui cy

theo phng php m trc tip trn bung m hng cu (*108 tb/ml)
Mi trng R ng Cm go
Mu 1S+0B 1S+1B 1S+0,1B 1S+0,01B 1S+0B 1S+1B 1S+0,1B 1S+0,01B
Ln 1 8,583 8,439 8,599 8,667 8,435 8,011 8,342 8,470
Ln 2 8,466 8,550 8,376 8,322 8,357 8,366 8,477 8,455
Trung bnh 8,525 8,495 8,488 8,495 8,396 8,189 8,410 8,463

Bng 7.9: S lng t bo S. cerevisiae trong 1 g ch phm theo phng

php m s khun lc trn a (*108 cfu/g)
Mi
R ng Cm go
trng
Mu 1S+0B 1S+1B 1S+0,1B 1S+0,01B 1S+0B 1S+1B 1S+0,1B 1S+0,01B
Ln 1 8,942 9,705 9,027 9,108 8,081 8,883 8,685 8,723
Ln 2 9,224 9,820 9,151 8,932 8,633 9,219 8,552 8,874
Trung bnh 9,083 9,763 9,089 9,020 8,357 9,051 8,619 8,799

7.2. X l thng k

7.2.1. Th nghim 1:Kho st s pht trin ca cc chng phn lp c trong

mi trng r ng 60B
Bng ANOVA 7.1
Analysis of Variance for SL
Source DF SS MS F P
CHUNG 3 0.2745 0.0915 5.34 0.014
Error 12 0.2055 0.0171
Total 15 0.4800

7.2.2. Th nghim 2: nh hng ca mi trng nui cy v thi gian thu

hoch n s lng t bo nm men

7.2.2.1. S lng t bo S. boulardii

Bng ANOVA 7.2
Analysis of Variance for SL, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

MT 1 0.00845 0.00845 0.00845 0.81 0.371
TG 2 0.05401 0.05401 0.02700 2.59 0.082
MT*TG 2 0.02871 0.02871 0.01436 1.38 0.259
Error 66 0.68721 0.68721 0.01041
Total 71 0.77838
 56

Bng ANOVA 7.3

Analysis of Variance for SL, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

MT 1 2.2476 2.2476 2.2476 4.57 0.036
TG 2 0.9455 0.9455 0.4727 0.96 0.387
MT*TG 2 2.2789 2.2789 1.1394 2.32 0.106
Error 66 32.4310 32.4310 0.4914
Total 71 37.9029

7.2.2.2. S lng t bo S. cerevisiae

Bng ANOVA 7.4
Analysis of Variance for SL, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

MT 1 0.02202 0.02202 0.02202 2.12 0.150
TG 2 0.13097 0.13097 0.06549 6.30 0.003
MT*TG 2 0.05414 0.05414 0.02707 2.61 0.081
Error 66 0.68556 0.68556 0.01039
Total 71 0.89269
Bng ANOVA 7.5
Analysis of Variance for SL, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

MT 1 8.0829 8.0829 8.0829 16.79 0.000
TG 2 2.1961 2.1961 1.0981 2.28 0.110
MT*TG 2 0.1044 0.1044 0.0522 0.11 0.897
Error 66 31.7654 31.7654 0.4813
Total 71 42.1488

7.2.3. Th nghim 3: nh hng ca acid ascorbic (vitamin C) v cht nn ln

sc sng ca nm men trong ch phm

7.2.3.1. S lng t bo S. boulardii

Bng ANOVA 7.6
Analysis of Variance for SL, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

CN 1 1.3000 1.3000 1.3000 2.49 0.116
VIT 2 0.5586 0.5586 0.2793 0.54 0.586
CN*VIT 2 0.4381 0.4381 0.2191 0.42 0.657
Error 210 109.4760 109.4760 0.5213
Total 215 111.7727

7.2.3.2. S lng t bo S. cerevisiae

Bng ANOVA 7.7
Analysis of Variance for SL, using Adjusted SS for Tests
Source DF Seq SS Adj SS Adj MS F P
CN 1 1.4787 1.4787 1.4787 2.30 0.130
VIT 2 0.7640 0.7640 0.3820 0.60 0.552
CN*VIT 2 1.5843 1.5843 0.7921 1.23 0.293
Error 210 134.7244 134.7244 0.6415
Total 215 138.5515
 57

7.2.4. Th nghim 4: nh hng ca mi trng nui cy, cht nn v thi

gian thu hoch ln sc sng ca nm men trong ch phm

7.2.4.1. S lng t bo S. boulardii

Bng ANOVA 7.8
Analysis of Variance for SL, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

MT-CN 3 15.2168 15.2168 5.0723 12.26 0.000
TG 2 4.3018 4.3018 2.1509 5.20 0.006
MT-CN*TG 6 7.8697 7.8697 1.3116 3.17 0.005
Error 204 84.3844 84.3844 0.4136
Total 215 111.7727

7.2.4.2. S lng t bo S. cerevisiae

Bng ANOVA 7.9
Analysis of Variance for SL, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

MT-CN 3 27.5532 27.5532 9.1844 19.64 0.000
TG 2 9.8236 9.8236 4.9118 10.50 0.000
MT-CN*TG 6 5.7882 5.7882 0.9647 2.06 0.059
Error 204 95.3864 95.3864 0.4676
Total 215 138.5515

7.2.5. Th nghim 5: nh hng ca vi khun Bacillus subtilis ln s sinh

trng ca nm men Saccharomyces cerevisiae trong iu kin nui cy chung

7.2.5.1. Theo phng php m trc tip trn bung m hng cu

Bng ANOVA 7.10
Analysis of Variance for SL, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

MT 1 0.10600 0.10600 0.10600 4.60 0.046
BA 2 0.07939 0.07939 0.03970 1.72 0.207
MT*BA 2 0.09024 0.09024 0.04512 1.96 0.170
Error 18 0.41482 0.41482 0.02305
Total 23 0.69045

7.2.5.2. Theo phng php m s khun lc trn a

Bng ANOVA 7.11
Analysis of Variance for SL, using Adjusted SS for Tests

Source DF Seq SS Adj SS Adj MS F P

MT 1 1.9471 1.9471 1.9471 10.55 0.004
BA 2 0.9100 0.9100 0.4550 2.47 0.113
MT*BA 2 0.4997 0.4997 0.2498 1.35 0.283
Error 18 3.3221 3.3221 0.1846
Total 23 6.6789
 58

7.3. Thnh phn cc loi mi trng

7.3.1. Mi trng Sabouraud

Glucose 40 g
Pepton 20 g
Agar 20 g
Nc ct 1000 g
Ph = 5,6

7.3.2. Mi trng r ng ma 60B

KH2PO4 20 g
MgSO4 5g
K2HPO4 20 g
(NH4)2SO4 40 g
R ng ma 60B 1000 ml
pH = 5 6.

7.3.3. Mi trng cm go
100 g cm ho trong 500 ml nc, un si, lc qua vi mn, pha thm nc
cho 1000 ml, b sung thm 5 g MgSO4, 3g KH2PO4.

7.3.4. Mi trng nc chit gi u

un si hn hp 200 g gi vi 1000 ml nc ri p ly nc.
Tt c cc loi mi trng u c hp kh trng bng autoclave 1210C/10
pht.

7.4. Phng php nhum n

Bc 1: lm vt bi
t 1 git nc v trng ln phin knh. Dng que cy ly 1 t nm men t
khun lc trn mi trng thch, ho u vo git nc v tri mng trn mt lame.
kh.
 59

Bc 2: c nh mu
H nh pha di phin knh qua ngn la n cn 3 4 ln hoc nh vi
git cn ngay vt tri v t. Mc ch: git cht t bo sng, gn cht t bo sng
vo phin knh khi ra khng b tri v lm cho t bo d bt mu.
Bc 3: nhum mu
Nh vi git thuc nhum n xanh methylen 0,1%.
Ra nc v lm kh.
Bc 4: quan st
Quan st hnh thi nm men bng knh hin vi di vt knh du 100X.