B GIO DC V O TO

B Y T

TRNG I HC DC H NI

NINH TH KIM THU

NGHIN CU VAI TR CA ALGINAT

TRONG QU TRNH TO NGUYN
LIU PROBIOTICS CHA
Lactobacillus acidophilus

LUN VN THC S DC HC

H NI 2013

B GIO DC V O TO

B Y T

TRNG I HC DC H NI

NINH TH KIM THU

NGHIN CU VAI TR CA ALGINAT

TRONG QU TRNH TO NGUYN
LIU PROBIOTICS CHA
Lactobacillus acidophilus

CHUYN NGNH CNG NGH DC PHM V BO CH

M S 60720402

Ngi hng dn khoa hc:

1.

TS. m Thanh Xun

2.

ThS. Nguyn Th Trinh Lan

H NI 2013

MC LC
DANH MC

Trang

T VN

Chng 1: TNG QUAN

1.1. i cng v probiotics

1.1.1. Khi nim

1.1.2. Cc vi sinh vt c s dng trong ch phm probiotics

1.1.3. C ch tc dng

1.1.3.1. C ch tc dng

1.1.3.2. ng dng ca probiotics

1.2. Tnh hnh nghin cu v s dng probiotics trn th gii v Vit

Nam
1.2.1. Tnh hnh nghin cu sn xut v s dng cc ch phm
probiotics trn th gii
1.2.2. Tnh hnh nghin cu sn xut v s dng cc ch phm probiotics
Vit Nam
1.3. Lactobacillus acidophilus

10

1.3.1. c im hnh thi, sinh l

10

1.3.2. Kh nng chuyn ha carbohydrat

11

1.3.3. Cc dng ch phm probiotics cha L. acidophilus

11

1.4.

12

Alginat

7
7

1.4.1. Tnh cht

12

1.4.2. ng dng

13

1.4.3. Mt s nghin cu s dng alginate trong bo ch probiotics

13

1.5.

Phng php ng kh

15

1.5.1. u nhc im ca phng php ng kh

16

1.5.2. ng dng

16

1.5.3. Cc t dc bo v thng dng trong ng kh vi sinh vt

16

1.6.

19

Nang cng

1.6.1. c im dng thuc nang cng

19

1.6.2. nh gi cht lng nang cng

20

Chng 2. NGUYN LIU, THIT B V PHNG PHP NGHIN

CU
2.1. Nguyn vt liu v thit b

21

2.1.1. Nguyn vt liu s dng

21

2.1.2. Mi trng s dng trong nghin cu

22

2.1.3. Cc dung dch v hn dch s dng trong nghin cu

22

2.1.4. Thit b

22

21

Phng php nghin cu

23

2.2.1. Phng php nhn ging

23

2.2.2. Phng php nui cy thu sinh khi

23

2.2.3. Phng php ng kh

24

2.2.4. Phng php tit khun Tyndall

25

2.2.5. Phng php to thuc bt cha L. acidophilus

25

2.2.6. Phng php to nang cng cha L. acidophilus

25

2.2.7. Phng php xc nh hm m

25

2.2.

2.2.8. Phng php xc nh s lng VSV theo nguyn tc pha long

lin tc

26

2.2.9. Phng php nh gi nh hng ca alginate n kh nng sng

27

st ca Lactobacillus acidophilus trong qu trnh ng thuc bt

2.2.10. Phng php nh gi nh hng ca alginate n kh nng

28

sng st ca Lactobacillus acidophilus trong qu trnh ng thuc nang

cng
Chng 3. THC NGHIM, KT QU V BN LUN

30

3.1.

Nghin cu nh hng ca alginate n kh nng sng st ca vi

khun Lactobacillus acidophilus trong qu trnh ng kh

30

3.1.1. nh gi th cht ca cc nguyn liu cha Lactobacillus

acidophilus to thnh sau khi ng kh

30

3.1.2. nh gi m v tc ht m ca cc mu ng kh vi sinh
vt vi t dc bo v l sa gy v alginate

33

3.1.3. Kho st s lng vi khun Lactobacillus acidophilus sng st

sau ng kh

36

3.1.4. So snh kh nng sng st ca vi sinh vt trong nguyn liu ng

kh c v khng c kt hp alginate v sa gy

39

3.2.

Nghin cu nh hng ca alginate n kh nng sng st ca vi

khun Lactobacillus acidophilus trong qu trnh ng thuc bt v ng
nang cng

42

3.2.1. Nghin cu nh hng ca alginate n kh nng sng st ca vi

khun L. acidophilus trong qu trnh ng thuc bt

43

3.2.2. Nghin cu nh hng ca alginate n kh nng sng st ca vi

khun L. acidophilus trong qu trnh to nang cng

45

Chng 4: BN LUN

48

KT LUN V XUT

55

DANH MC CC CH VIT TT

B. subtilis

: Bacillus subtilis

Cfu (Colony- Forming Units)

: S n v khun lc

FAO (Food and Agriculture Organization)

: T chc nng lng th gii

Glass : mt trng thi nhit ng qu bo ha khng bn vi nht cao

HDL (High density lipoprotein)

: Lipoprotein t trng cao

HDSK

: Hn dch sinh khi

IDF (Internation Dairy Federation)

: Lin on b sa th gii

Kl/kl

: Khi lng/khi lng

Kl/tt

: Khi lng/th tch

L. acidophilus

: Lactobacillus acidophilus

LAB (Lactic acid bacteria)

: Nhm vi khun lactic

LDL (Low density lipoprotein)

: Lipoprotein t trng thp

MRS (de Man, Rogosa, Sharpe)

: Mi trng nui cy vi khun

MT

: Mi trng

PPI (Proton Pump Inhibitor)

: c ch bm proton

S. boulardii

: Saccharomyces boulardii

Tt/kl

: Th tch/khi lng

VSV

: Vi sinh vt

WHO (World Health Organization)

: T chc Y t th gii

DANH MC CC BNG

Tn bng

STT

Trang

Bng 1.1 Cc th h bo ch ca ch phm cha probiotics

Bng 1.2 Mt s ch phm probiotics trn th trng Vit Nam

Bng 1.3 C ch bo v ca t dc trong qu trnh ng kh

17

Bng 2.1 Cc ha cht dng trong nghin cu

21

Bng 2.2 Cc thit b dng trong nghin cu

22

Bng 3.1 Th cht ca cc mu ng kh L. acidophilus vi cc t

31

dc bo v ti thi im ngay sau khi ng kh

Bng 3.2

Bin thin hm m (%) ca mt s mu ng kh L.

34

acidophilus vi t dc bo v khc nhau theo thi gian

Bng 3.3 S lng vi khun sng st trong 4 mu sau ng kh

37

Bng 3.4 T l VSV sng st sau ng kh so vi mu ban u

37

Bng 3.5 Hm m ca cc mu ng kh vi t dc bo v l hn

40

hp sa gy vi alginate cc nng
Bng 3.6 S lng vi sinh vt sng st tnh trn 1g bt sau ng kh

41

trong cc mu ng kh s dng kt hp alginate v sa

gy.
Bng 3.7 S lng vi sinh vt sng st tnh trn 1g bt ng kh

44

Bng 3.8 S lng vi sinh vt sng st trong 1 vin nang trong mi

45

trng acid HCl pH 1,2 vi tc khuy 50 v/pht

Bng 3.9 T l vi sinh vt sng st ca mu alginate so vi mu tinh
bt trong mi trng pH 1,2

46

DANH MC CC HNH V BIU

Tn bng

STT

Trang

Hnh 1.1

Hnh nh L. acidophilus trn knh hin vi in t.

11

Hnh 1.2

Cng thc cu to ca acid - L- guluronic v acid - D-

12

mannuronic
Hnh 3.1

Hnh nh mu ng kh vi nc ct.

32

Hnh 3.2

th biu th hm m trung bnh ca cc mu ngay sau

34

ng kh v trong thi gian bo qun.

Hnh 3.3

Hnh nh mu ng kh vi alginate ngay sau khi tho khi

35

my v sau 3 pht ngoi mi trng.

Hnh 3.4

th biu din s lng vi khun L. acidophilus sng st

38

trong cc mu sau ng kh
Hnh 3.5

Hm m ca cc mu ng kh vi t dc bo v l hn

40

hp sa gy vi alginate cc nng
Hnh 3.6

S lng vi sinh vt sng st tnh trn 1g bt sau ng kh

42

trong cc mu ng kh s dng kt hp alginate v sa gy.

Hnh 3.7

Biu th hin s lng vi sinh vt sng st sau khi th

44

trong mi trng acid HCl pH 1,2

Hnh 3.8

S lng vi sinh vt sng st trong 1 vin nang trong mi

trng acid HCl pH 1,2 vi tc khuy 50 v/pht

46

LI CM N
Vi s knh trng v long bit n su sc ti xin g i l i c
Thanh Xun v ThS. Nguyn Th Trinh Lan, nh ng ng
v ch b o cho ti t nh ng b c

i th

n n TS. m
t n t nh h ng

u ti n cho n khi ti hon thi n lu n vn n

ng th i, ti xin g i l i c

n ti Ds. L Ngc Khnh cng cc th

cc anh ch k thu t vi n trong b n Cng Nghip Dc

hn p n ti c ng xin g i l i c
c gio tr

ng i hc

ti trong th i gian ti hc t p ti tr
V cui c ng l l i c

n n an gi

v to

c gio,

to i iu ki n gip

ti trong sut qu tr nh thc hi n ti, ch b o ti trong th i gian l thc nghi

th

hi u c ng ton th cc
i iu ki n thu n l i cho

ng

n ti g i ti M ca ti, gia

nh, ng

i thn v bn b

ng vi n, gip ti trong sut qu tr nh hc t p v hon thnh lu n vn tt nghi p

o th i gian l thc nghi
n c n c nhiu thiu s t
kh a lu n

i r t

c ng nh kin th c ca b n thn c hn, kh a lu n

ong nh n

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ca cc th

c hon thi n hn

i xin chn thnh c

i, ng 30, thng , n
c vin
s

inh h Kim Thu

c, bn b

T VN
Probiotics thuc nhm cc vi sinh vt sng khng th thiu i vi sc
khe con ngi. Nhng vi sinh vt ny a vo c th khi cn sng vi mt
lng y s c li cho sc khe ca vt ch. Chng thng c b sung
km vi ch n hoc qua cc ch phm cha probiotics.
Vi sinh vt hay c s dng nht trong cc ch phm probiotics l
nhm vi sinh vt sinh acid lactic, m trong in hnh l Lactobacillus
acidophilus.
Tuy nhin, probiotics km n nh do bt li ca iu kin bo qun
cng nh hng ro sinh hc ca vt ch. Do vy, cc nghin cu trong nc
cng nh trn th gii hin nay u c thc hin vi mc tiu gip n nh
vi sinh vt. Trong , alginat l t dc c nghin cu nhiu nht trn th
gii. Nhng trong nc cc nghin cu v t dc ny cn rt hn ch.
Xut pht t thc t trn, chng ti thc hin ti:
N
p ob o

a alginat
s

Lactobacillus acidophilus.

Vi cc mc tiu:
alginat
.
alginat

C
1.1.

1. TNG QUAN

probiotics

1.1.1. Khi nim

Probiotics bt ngun t ting Hy Lp c ngha l s sng. Khi nim
probiotics u tin xut pht t nh khoa hc Eli Metchnikoff, trong cun
sch Ko di s sng ca ng a ra 1908. ng cho rng nhng ngi nng
dn Bulgary sng lu l v h thng xuyn s dng sa chua c cha vi
khun lactic, cc vi khun ny c li cho vi sinh vt ng rut. [55]
Thut ng probiotics c gii thiu ln u tin vo nm 1953 bi
Kollath. Probiotics c nh ngha l cc yu t c ngun gc t vi khun,
kch thch s pht trin ca cc vi khun khc. [55]
Nm 1974, Parker pht trin nh ngha ny, ng cho rng probiotics
l nhng vi sinh vt v nhng c cht gip cn bng h vi khun ng
rut. Sau n nm 1989, Fuller thay i nh ngha ny, ng cho rng
probiotics l thc phm b sung mt s vi sinh vt sng c ch cho vt ch
nhm cn bng h vi sinh vt ng rut ca n. nh ngha ny nhn mnh
vai tr kh nng sng ca vi sinh vt. Nm 1998, Salminen nh ngha
probiotics l nhng thc phm cha vi khun sng nh hng c li cho sc
kho. Nm 2002 Marteau nh ngha probiotics hon chnh hn
probiotics l nhng ch phm cha t bo vi sinh vt hay nhng thnh phn
ca t bo vi sinh vt m nh hng c li cho sc kho. [23], [55]
Nm 2002, T chc Y t th gii (WHO) v T chc Lng thc th
gii (FAO) a ra nh ngha probiotics c coi l hon chnh nht hin nay:
Probiotics l nhng vi sinh vt sng m khi s dng vi lng ph hp s
em li li ch cho sc khe ca sinh vt ch. [23], [55]
1.1.2. Cc vi sinh v

c s dng trong ch ph m probiotics

Probiotics c chia lm 3 nhm chnh sau [6]:

2

- Nhm vi khun lactic (chim phn ln): gm 2 chi ph bin l chi

Lactobacillus v Bifidobacterium.
- Nhm khng phi vi khun lactic:
+ Chi Propionibacterium: P. freudenreichii, P. cyclohexanicum,...
+ Chi Bacillus: B. subtilis, B. clausii,...
+ Chi Brevibacillus: B. laterosporus,
+ Chi Sporolactobacillus: S. laevolacticus,
+ Chi Escherichia: E. coli,...
- Nhm nm men: ch yu l chi Saccharomyces cerevisiae.
Vic la chn cc vi sinh vt trn dng lm nguyn liu probiotics u
phi da vo cc tiu ch sau:
C kh nng bm dnh vo nim mc ng tiu ha ca vt ch
Chu c pH thp d dy v acid mt rut non
Khng sinh c t v khng gy bnh cho vt ch
C kh nng sinh enzym, cht khng sinh hoc cc sn phm cui cng
m vt ch c th s dng c
C kh nng sng v c tr trong rut
D nui cy v c kh nng tn ti c lp trong mt thi gian di
L. acidophilus l trc khun chim t l ch yu trong s cc vi sinh vt c
ch c tr on trn ca ng tiu ha. N c kh nng lm gim s lng
cc vi sinh vt hoc nm c hi rut non; c kh nng sinh lactase - mt
enzym quan trng trong chuyn ha sa; c lin quan n qu trnh sn xut
mt s vitamin nhm B nh: niacin, acid folic, pyridoxin; tng cng chc
nng ca h thng min dch; gim s tiu dit vi sinh vt c ch do s dng
khng sinh di ngy.[6]
Vi cc c im trn, L. acidophilus l vi sinh vt c la chn u
tin sn xut cc ch phm probiotics.
3

1.1.3. C

tc dng c a probiotics

1.1.3.1. C

tc dng [3],[4],[6],[7]

- Cnh tranh nng lng, v tr bm: Probiotics cnh tranh cht dinh
dng v nng lng vi cc vi khun gy bnh duy tr v pht trin. Bng
cch chim lnh v bm cht vo thnh rut, probiotics ngn nga cc vi
khun c hi tn cng v pht trin.
- Tng cng chc nng chng ca nim mc rut: Probiotics sng
ti ng rut cng ng thi tng cng chc nng chng ca nim mc
rut v gim thiu s di chuyn ca cc vi khun v khng nguyn t rut vo
mch mu. Chc nng ny gip gim s nhim khun v d ng i vi cc
khng nguyn c trong thc phm.
- Sn sinh ra cc cht c ch: Mt s probiotics sn sinh ra cc khng
sinh nh bacteriocins gip ngn nga v tiu dit mm bnh. Trong qu trnh
ln men ng, cc thnh phn khc nhau ca probiotics sn sinh ra acid
lactic gip lm gim pH ca rut v ngn cn s pht trin ca cc vi khun
khng cn thit. Mt s sn phm sinh ra trong qu trnh trao i cht, qu
trnh ln men chuyn ha carbohydrat nh butyrat v acid butyric c kh nng
chng ung th.
- Kch thch p ng min dch: Probiotics c kh nng lm tng c
phn ng min dch c hiu v khng c hiu bng cch kch thch cc t
bo phn ng min dch (macrophases, lymphocytes) v tng cng s sn
xut ra cc thnh phn min nhim (cytolines, immunoglobulins, interferon).
- Tng cng chc nng tiu ha: Probiotics trong rut non gip gim
lng mt trong c th v gim cholesterol. Probiotics sn sinh ra cc enzym
khc phc qu trnh tiu hy carbohydrate v lm thun tin hn qu trnh
rut hp th nng lng t cc cht dinh dng. Probiotics cng ng thi ln

men nhng carbohydrat khng tiu ha c trong rut non v sn xut ra

vitamin B, K.
1.1.3.2. ng dng c a probiotics:
Probiotics c ng dng:
Trong cc bnh tiu ha [16], [20], [27]
- Tng kh nng tiu ha lactose v hot ng ca cc enzym khc
- H tr trong iu tr tiu chy do s dng khng sinh
- Tc dng ln Helicobacter pylori
Tng cng min dnh [22], [39]
- Kch thch min dch nim mc
- Tng cng min dch, gim phn ng d ng
- Gim nguy c nhim vi khun, nm
- Gim nguy c mc ung th
Chng tng huyt p, gim cholesterol mu [14], [26]
ng dng trong nui trng thy sn
1.2. Tnh hnh nghin c u v s dng probiotics trn th gii v Vit Nam
1.2.1. Tnh hnh nghin c u s n xut v s dng cc ch ph m probiotics
trn th gii
Vic s dng thc phm c probiotics (nh 1 thnh phn t nhin ca
thc phm hoc thc phm ln men) c bit n t lu, nhng vic
nghin cu h vi sinh vt ng rut v s dng probiotics mi thc s pht
trin t nhng nm 80 ca th k 20. Nhng nghin cu v c im phn
loi v qun th vi sinh vt ng rut ngi v ng vt c tin hnh
bi Apajalahti v cs (1998) [13]; Vander Wielen v cng s (2000) [49]
cho thy nu nh trong rut non ca ngi Bacteroides v Bifidobacterium
chim u th th g l Ruminococcus v Streptococcus. Bng k thut gen,
cc nh nghin cu ch ra rng ch c khong 20 n 50% s loi vi sinh
5

vt ng rut ng vt c phn lp, nui cy nh ngun probiotics.

Netherwood v cs (1999) [38]; Gong v cs (2002) [23]; Zhu v cs (2002) [54]
s dng k thut gen nghin cu s thay i cu trc qun th v c
im sinh hc ca h vi sinh vt ng rut ng vt di tc ng ca
probiotics. Tuy nhin, cho n nay nhng nhn t no gp phn to nn mt
h vi sinh vt cn bng hoc lm ri lon s cn bng ca h vi sinh vt
ng rut cng cha c hiu bit y . c rt nhiu nghin cu v
vai tr ca probiotics i vi i sng ng vt nh tc ng ca probiotics
i vi h thng min dch nim mc rut (Schat v Mayer, 1991)[46];
(Hersbberg v Mayer, 2000) [25] i vi s thay i ca nim mc rut non
vt nui (McCracken v Lorenz, 2001). [37]
Nhng nh hng c li ca probiotics th hin nhiu kha cnh khc
nhau nhng nhng hiu bit ca con ngi v c ch tc ng ca probiotics
cn rt hn ch. C mt s tc gi cho rng hiu qu ca probiotics trong vic
c ch s pht trin ca cc vi khun gy bnh trong ng tiu ha ca ng
vt c ngha rt quan trng. S km hm c thc hin theo nhng cch
sau: cnh tranh cht dinh dng, sn xut c t v cc sn phm trao i
(cc acid bo bay hi, cc cht ging khng sinh...), cnh tranh v tr bm dnh
nim mc rut v kch thch h thng min dch rut. [22], [40]
a phn cc sn phm probiotics s dng theo ng ung, chng phi
chu tc dng ca dch v, cng nh ca acid mt v th vi sinh vt b cht rt
nhiu v khng n rut c hoc n rut vi s lng rt t khng gy
tc dng. Vic m bo kh nng sng st ca vi sinh vt probiotics trong sn
xut, bo qun, lu hnh, v t l sng st cao khi n rut khng n gin, do
vy c nhiu hng nghin cu khc nhau nh ci tin phng php ng
kh to bt [32], [33], p dng phng php ln men 2 bc, phng php vi
nang ha [15], [21], phng php s dng kt hp vi t dc bo v [41],
6

[42]. Vic bo v cc vi sinh vt probiotics chng sng st v pht huy tc

dng khi n rut l mt vn quan trng v ang c gii quyt.
1.2

am

nc ta, cc ch phm probiotics chim lnh th trng hn 10

nm; tuy nhin vic nghin cu sn xut probiotics phc v cho i sng cn
rt mi m v bt u c quan tm trong khong mt thp k gn y. n
nay, trn th trng nc ta c mt cc sn phm probiotics ca bn th h
bo ch nh sau: [6]
B
T

1.1: C

bo

m VSV

T 1 VSV sng (trong

(Non-

sa chua, phomat,

coated)

kim chi, )
Bo t VSV.

p m

p ob o

N m

Gi r s dng

VSV gn nh khng

nhiu.

sng st khi qua d dy

v dch mt.

Thun li cho nui

Mt nhiu thi gian

cy, sn xut, lu

pht trin thnh VSV

hnh.

chm tc dng, hn ch
kh nng cnh tranh vi
VSV gy bnh.

Probiotics ng

T l sng nhit

T l VSV b cht vn

kh.

thng cao hn

cao.

T 2 VSV a vo di Bo v VSV khi i

Mt thi gian mng bao

Enteric-

dng vin nn,

qua d dy v dch

tan r gii phng

coated

vin nang c lp

mt.

VSV.

bao ngoi tan

trong rut.
7

T 3 Dng bao vi nang.

Hon thin tng Cha khc phc c

Micro-

ca th h 2.

nhc im ca th h

encapsul

2: kh nng phng thch

ated

ti rut cha tt, tan

chm trong rut.

T 4 Lp bn trong l

VSV c t l sng

i hi cng ngh cao,

Dual

protein, lp ngoi

cao. Chu c

k thut bo ch hin

coated

l polysaccharid.

acid dch v v

i, trang thit b t

mui mt. Lp bao

tin.

bn trong c tnh

Gi thnh cao.

thc y phng
thch VSV ti ch.
Mt s c s c sn xut nguyn liu probiotics nc ta l Cng ty
TNHH mt thnh vin Pasteur Lt, Cng ty TNHH mt thnh vin vaccin
v sinh phm Nha Trang, vi mt s ch phm quen thuc trn th trng nh
Enzym biosub, Biosubtyl DL, Viabiovit, Vivac bio, Healthy liver. Tuy nhin,
nguyn liu ch yu trong cc ch phm probiotics ca Cng ty TNHH mt
thnh vin Pasteur Lt cng nh cng ty TNHH mt thnh vin vaccin v
sinh phm Nha Trang u l chng vi sinh vt Bacillus subtilis. V mi y
xut hin thm cng ty ANABIO R$D, c nh my sn xut nguyn liu
probiotics hin i khng km so vi cc nc trn th gii. Ngoi vic sn
xut nguyn liu probiotics c in trong nc nh nguyn liu cha chng vi
sinh vt Bacillus subtilis, ANABIO cn c thm nguyn liu cha cc chng
vi sinh vt lactic. Do vy, th trng ch phm probiotics khng ch ln v s
lng m cn a dng v chng loi vi sinh vt, ng thi gi thnh sn phm
cc ch phm probiotics c h nhiu ln, ngi s dng c th s dng
8

sn phm cht lng cao m gi thnh li r. Tuy nhin, sn phm y vn

ch yu thuc th h 1 nn kh nng bo v VSV vn cn rt thp (Bng 1.2.)
B ng 1.2: Mt s ch phm probiotics trn th trng Vit Nam
STT T B d

bo

N s x

L-Bio-3D

300mg vi khun: L. acidophilus, Me-Auspharm

B. longum, L. rhamnosus

Antibio

Vit Nam

Gi bt ung 1g cha 108 cfu L. Organon M

acidophilus

Biosubtyl

Gi 1g cha 107 - 108 vi khun B. Vin vaccin

LD

subtilis v L. acidophilus

Lt Vit
Nam

Biolacto

Vin nang cha t nht 108 vi Union M

khun L. acidophilus v L. bul
garicus

Lactomed

Vin nang 320 mg cha:

II Dong - Hn

L. bifidus 2 106 vi khun

Quc

L. acidophilus 2 106 vi khun

Streptococcus faecalis 2 106 vi
khun
6

Ultraflore

Vin nang 0,2 g cha 2 109 nm Sobio Php

men Saccharomyces cerevisiae

Antibio

Vin nang cha 2 108 2 109 Lyocentre

-philus

vi khun L. acidophilus

Probio

Gi bt ng kh cha 109 L. Imexpharm

Php

acidophilus
9

Abiiogran

Vit Nam

Thuc cm, 1g cha 108 cfu L. Dae Han New

acidophilus

Pharm Hn
Quc

10

Lactomin Hn hp Probiotics bao vi nang

cha L. acidophilus, B. longum, L.

Austrapharm

rhamnosus 300 mg

Vit Nam

Laclean

Bt cm pha hn dch cha Lactic

Cell-biotech

gold

Acid Bacteria 1,25 x 109,

Hn Quc

plus

11

Mebipharm-

L.acidophilus (100 t cfu/g); L.

rhamnosus (100 t cfu/g); B.
longum (50 t cfu/g); B. bifidum
(50 t cfu/g); Enterococcus
faecium (100 t cfu/g)
12

Kidlac

Bt cm pha hn dch ung:

Cell-biotech

Lactic Acid Bacteria 0,5 x 109, L.

Hn Quc

acidophilus (100 t cfu/g), B.

longum (50 t cfu/g), B. breve (50
t cfu/g), Enterococcus faecium
(100 t cfu/g)

1.3.

Lactobacillus acidophilus

1.3.1.

m hnh thi, sinh l

L. acidophilus thuc nhm vi sinh vt sinh acid lactic, l trc khun

Gram dng, kch thc t 0,6 0,9 1,5 6,0 m, mc n hoc i hoc
to chui ngn, khng c bo t, khng di ng, k kh khng bt buc, phn
10

ng catalase m tnh, pht trin ti u 370C, khng pht trin 20 220C

hoc nhit cao hn 480C [4], [12], [44].

Hnh 1.1: Hnh nh L. acidophilus trn knh hin vi in t.

1.3.2. Kh

yn ha carbohydrat

L. acidophilus chuyn ha glucose, lactose, sucrose sinh acid lactic,

nhng khng sinh kh; khng chuyn ha glycerol, erythritol, D-arabinose, Larabinose, D-ribose, D-xylose. [6], [12]
1.3.3. Cc dng ch ph m probiotics ch a L. acidophilus
Trn th trng dc phm hin nay, cc ch phm cha L. acidophilus
ch yu l dc phm v thc phm chc nng bo ch di dng bt, cm,
nang cng.
Khc vi ch phm khc, ch phm probiotics phi duy tr s lng VSV
sng st nht nh trong thi hn s dng cng nh trong h thng tiu ha
ca ngi s dng. Do , phng php sn xut v thnh phn ch phm
probiotics c ci bin khc so vi ch phm thng thng.
V thnh phn, ch phm probiotics thng c thm cc t dc bo v
nh cht x, ng,
V phng php sn xut nguyn liu, hin nay c rt nhiu phng php
sn xut nhm tng kh nng bo v VSV. in hnh l phng php by
bng cch to vi nang [21], phng php phun sy [15],Trong , mt
trong nhng phng php t ra hiu qu v c s dng nhiu l phng
11

php ng kh. Phng php ng kh gip cho VSV n nh v gip VSV

c t l sng st cao trong thi gian bo qun. [17]
1.4.

Alginate

1.4.1. Tnh cht

Alginate c ngun gc ch yu l t to nu, rong m. Alginate l tn
ch chung acid alginic v 1 s mui ca n nh natri alginate, kali alginate.
Acid alginic l mt heteropolyme saccharid mch thng cu to t hai
gc uronic l acid - L- guluronic (G) v acid - D- mannuronic (M).[43]

Acid L Guluronic

Acid D Mannuronic

Hnh 1.2: Cng thc cu to ca acid - L- guluronic v acid - Dmannuronic

Ty theo ngun gc ca alginate m di trung bnh ca mch phn
t, di ca mi block, t l v trnh t kt hp ca chng vi nhau c khc
nhau. iu ny lm cho tnh cht ca alginate bin i trong mt di rng.
Mui natri ca acid alginic d ht m, tuy nhin kh n nh nu c
bo qun trong iu kin mt m v m tng i thp. Dung dch Natri
alginate n nh nht pH 410, pH < 3 acid alginic b kt ta li, hp tit
khun c th lm gim nht ca dung dch [24], [43], [50].

12

Thc t natri alginate khng tan trong ethanol (95%), ether,

chloroform, v hn hp ethanol nc c cha hn 30% ethanol. Khng tan
trong cc dung mi hu c khc v trong dung dch acid c pH<3. Tan chm
trong nc, to dung dch keo nht [43], [18].
Ty theo ngun gc ca phng php sn xut m natri alginate c
nht khc nhau, dao ng t 10 3000.u. Thng thng nht ca dung
dch natri alginate 1% (kl/tt) c nht khong 20-400 mPas. nht ca
natri alginate thay i theo nng , nhit , pH v s c mt ca cc ion
kim loi [43]. Dung dch natri alginate 1% vi dung mi nc c pH t 7,2
[43].
1.4.2.

Trong cng ngh bo ch dc phm, alginate c s dng lm t

dc r, t dc dnh trong vin nn, t dc kim sot gii phng trong bo
ch vin gii phng ko di. Natri alginate cn c s dng lm t dc n
trong dng vin nang, t dc tng nht, n nh th cht trong cc ch
phm dng kem, gel, bt nho dng ti ch, cht nh ha gip n nh th
cht nh tng, hn dch. Trong thi gian gn y natri alginate c s dng
bo ch dng vi nang v siu vi nang cha dc cht, hay c s dng
trong mt s dng ch phm tc dng ti ch khc nh thuc nh mt, nh
mi [43]. Trong cng ngh vi sinh, alginate c s dng trong k thut bt
ng t bo v bt ng enzym, ngoi ra hin nay n ang c nghin cu
ng dng to vi nang cha vi sinh vt tng kh nng sng ca chng
trong cc iu kin bt li [21], [31], [41].
Trong ngnh y t, loi vi si sn xut t natri v calci alginate c s
dng bng vt thng, cm mu v lm vt thng mau lnh hn. Ngoi
ra, do tnh trng n trong nc v kh nng bm dnh ca dng gel trong

13

nc, natri alginate c s dng trong mt s thuc dng ngn cn tro

ngc d dy- thc qun, gim chng hi [43].
1.4.3. M

alginate

Trong nhng nm gn y vic nghin cu tm ra phng php v t

dc thch hp nhm ci thin kh nng sng st ca vi khun probiotics
ngy cng c quan tm. Mt s nghin cu gn y v vi nang ha vi sinh
vt bng vic kt hp dung dch alginate vi dung dch CaCl2 lm gia tng
s lng VSV sng st trong cc iu kin bt li nh: nhit cao, acid thp
d dy, mui mt... [21], [31], [43]. Latha Sabikhi thc hin nghin cu
nm 2010 v nh gi kh nng bo v L. acidophilus trong ch phm
probiotics bng phng php to vi nang vi alginate. Vi nguyn liu ban
u cha khong 109 cfu/g, mu khng c bo v u c s lng sng st
sau 1h, 2h trong iu kin pH 1 gim xung cn khong 105 106 cfu/g (gim
khong 1000 ln), cn vi phng php to vi nang th sau 1h trong pH 1 s
lng Lactobacillus acidophilus LA1 gim t 107 cfu/g lc ban u xung
cn khong 106 cfu/g (ngha l gim khong 10 ln), sau 2h trong iu kin
acid pH 1, s lng VSV ca mu bao vi nang cn 105 cfu/g (gim 100 ln so
vi ban u). Cn trong th nghim c s dng alginate lm t dc n th
sau 1h trong mi trng acid HCl pH 1,2 s lng vi sinh vt gim t 1,7 x
109 cfu/g xung cn khong 1,9 x 107 cfu/g (tng ng khong xp x 100
ln), cn ti thi im sau 2h lng vi sinh vt cn li l khong 1,1 x 107
cfu/g (gim khong 155 ln so vi ban u) [31].
Mt s nghin cu khc li cho thy vic to nang cng vi cc mui
alginate ca cc kim loi ha tr mt (mui natri alginate hoc kali alginate)
cng cho tc dng bo v vi sinh vt. Randolph Stanley Porubcan nghin cu
to nang cng probiotics cha natri alginate vi hm lng bin thin t 10%
n 99%. Kt qu ca nghin cu cho thy t l phi hp ti u ny l 20
14

60%. Cng theo nghin cu ca Randolph Stanley Porubcan, s lng VSV

b cht trong mi trng acid pH 1,6 khng nhiu, c th trc khi th trong
mi trng acid pH 1,6 th s lng cfu/ nang l 9,0x109, sau khi th trong
mi trng acid pH 1,6 trong 90 pht s lng cfu/ nang khng cha alginate
nh hn 104, cn s lng cfu/ nang cha alginate l 4x108. Kt qu nghin
cu cng cho thy khi s dng alginate lm t dc n trong nang cng
lm gia tng t l VSV sng st trong mi trng acid pH thp [42].
1.5. P

p p

ng kh c nh ngha nh l mt quy trnh to s n nh m trong

vt cn ng kh c lm ng lnh trc, tip theo lng dung mi c
trong vt (dung mi thng l nc) c loi i nh qu trnh thng hoa, sau
l qu trnh kh m lin kt cn li n gi tr m s khng th khin VSV
pht trin hoc xc tc cc phn ng ha hc [17], [32].
ng kh gm c 3 giai on chnh: tin ng, thng hoa v lm kh
G

Tin ng l giai on u tin ca qu trnh ng kh, thng c tin

hnh nhit -700C n -800C. giai on ny phn ln nc c tch
ra khi dc cht v t dc, h tch thnh nhiu pha. Kt thc giai on
ny, c th c trng thi kt tinh, v nh hnh hoc kt tinh kt hp vi v
nh hnh. [17], [11]
G

giai on ny vt c t trong bung sy, tin hnh ht chn khng.

Nc c chuyn trc tip t trng thi rn sang trng thi hi m khng
qua giai on lng. Trong giai on ny nhit v p sut bn trong bung
sy c duy tr mc di nhit ng bng. [17], [11]
Giai o lm k

15

 giai on ny nhit tng nhanh, m trong vt l m lin kt v

trng thi lng. Qu trnh sy trong giai on ny ging nh qu trnh sy
trong cc thit b sy chn khng bnh thng. Nhit mi cht trong
bung sy lc ny cng cao hn nhit giai on thng hoa. [11]
1.5.1.

ng kh c rt nhiu u im so vi cc phng php sy khc nh:

Gi nguyn mu sc, cu trc, hng v; Gi c hot tnh sinh hc; Bo v
nguyn vn cc vitamin nh lc cn ti, khng lm bin cht albumin Gi
nguyn c th tch ban u ca vt sy nhng c cu trc xp nn d hp
th nc tr li trng thi ban u. Tuy nhin, phng php ng kh cng
c nhng nhc im ring khin n cha c s dng rng ri: gi thnh
thit b cao; vn hnh phc tp, i hi ngi thao tc phi c trnh k
thut cao; tiu hao in nng ln lm gi thnh sn phm tng cao [11].
1.5.2.

Do nhng u nhc im trn nn phng php ng kh ch c p

dng trn cc sn phm c yu cu cht lng cao, ng thi km bn vi
nhit cao. Trong ngnh Dc c ng dng sn xut cc bt ng kh
pha tim (v d: khng sinh penicillin, cephalexin), lm kh cc ch
phm sinh hc nh albumin, vi sinh vt. Ngoi ra ng kh cng c s
dng nh mt phng php bo qun trong ngnh cng nghip thc phm
bo qun 1 s sn phm khc.
1.5.3. C

Qu trnh ng kh vi sinh vt tuy c nhiu u im nhng n cng l

1 trong nhng nguyn nhn lm gim s lng vi sinh vt sng st trong qu
trnh to nguyn liu v ch phm probiotics. c bit giai on tin ng l
nguyn nhn gy ln p lc cho thnh t bo vi khun. ng lnh lm cho lp
lipid mng t bo d b tn thng. Mt nghin cu khc li cho rng s thay
16

i hnh thi t nhin ca lipid mng v s thay i cu trc ca cc protein

nhy cm l nguyn nhn chnh gy ra s suy gim s lng t bo trong qu
trnh ng kh [29]. Mt s ti liu li cho rng chnh vic hnh thnh tinh th
ni phn t c th l nguyn nhn gy ph v mng t bo. Cc t dc
bo v c thm vo trong thnh phn nguyn liu em ng kh nhm: bo
v t bo trong qu trnh ng kh v m bo sc chu ng ca vi sinh vt
trong qu trnh lm kh [32].
Mt trong nhng phng php c s dng gia tng s sng st
ca vi sinh vt trong qu trnh ny l b sung vo nguyn liu ng kh cc
tc nhn bo v. Cc tc nhn ny nhm bo v t bo vi sinh vt trong qu
trnh ng kh. Cc t dc chia thnh cc nhm chnh sau: nhm
carbohydrate (surcrose, lactose, trehalose, maltose, glucose, galactose,
raffinose), nhm polymer (gelatin, xanthan gum, maltodextrans, dextran),
nhm protein (sa gy, trypsin, pepton, trytone, cao tht,), polyols
(mannitol, glycerol, sorbitol, butanol,), nhm amino acid (Na glutamate,
proline, glutamate,), nhm chng oxy ha (acid ascorbic, Na thiosulfate,)
v mt s t dc bo v khc nh mt ong, Na acetate, Ca chloride,[17].
C ch bo v ca cc nhm t dc ny trong qu trnh ng kh c tm
tt trong bng 1.3 [17]:
B

3: C ch bo v ca t dc trong qu trnh ng kh

STT Nhm

Thay th lin kt hydro trong lp mng t bo

Carbohydrate

bo

lipid kp
2

Polymer

Lm gim t l lin kt vi nc

Protein

Cung cp hm lng acid amin, enzym phn gii

17

protein thnh acid amin, vitamin, lm tng kh

nng m
4

Polyols

Thay th nc bay hi t mng t bo lipid kp

Amino acid

n nh protein

Chng oxy ha

Dn gc t do

Mt s t dc bo Tng hot tnh enzym, thay th lin kt hydro

v khc

trong lp mng lipid kp,

i vi qu trnh ng kh t bo vi sinh vt, nhm carbohydrate,

nhm protein, nhm amino acid v nhm polymer l cc nhm hay c s
dng nht bo v vi sinh vt trong qu trnh ng kh. Trong khng th
khng k n nhm t dc bo v rt hay c s dng l protein vi t
dc in hnh: sa gy.
S

Sa gy hay cn gi l skim milk l cc sn phm sa m trong

thnh phn c cha khng qu 0,5 % cht bo (trong khi sa nguyn cht
cha hm lng cht bo khong 3,5%, sa tch b-butter milk cha khong
1% cht bo).
Sa gy c trong cc ch phm ng kh probiotic v n c tc dng
bo v tt probiotic trong qu trnh ng kh. Theo nhiu tng kt nhiu
nghin cu trong v ngoi nc t trc ti nay th sa gy s dng nng
khong 10% cho t l sng st cao nht. Tc dng bo v ny c th do n
cha t l dinh protein v carbohydrat- nhng cht m c kh nng bo v t
bo. Thnh phn dinh dng chnh trong sa gy gm 32- 35,7 % protein v
48,4- 54,1 % lactose.
Lactose c trong sa gy cng nh mt s disaccharid khc c th thm
qua thnh t bo, sau chng hnh thnh lin kt hydro gia ng- protein.
18

Cc lin kt ny gip duy tr cu trc ca protein mng khi nc b loi i.

Protein gip hnh thnh trng thi glass xung quanh v bn trong t bo to ra
mt mi trng nht bn trong v ngoi t bo gip ngn cn s bin i
ca t bo mc thp nht [17].
Theo mt nghin cu khc th protein trong sa gy c th to thnh
lp o bo v trn thnh t bo, ng thi sa gy cn cung cp nhng cht
m tan trong nc (mui phosphat, mui citrat) gip n nh pH trong qu
trnh ng kh [32].
Nguyn liu ng kh vi sa gy va to cu trc xp gip qu trnh
b nc d dng, nhng ng thi cng lm cho nguyn liu ng kh ht m
kh nhanh nu khng c bo qun tt.
1.6.

Nang c ng

1.6.1.

m dng thu c nang c ng

Nang cng gelatin l mt dng thuc phn liu gm mt v rng

ng thuc (bng gelatin), gn lin vi thuc v a vo c th cng vi
thuc. Sau khi tan r gii phng thuc, v ng c tiu ha trong c th
[2]. Hin nay, nang cng l dng bo ch ph bin nht vi cc ch phm
probiotics sau dng tinh bt.
i vi ch phm probiotics, vic to dng thuc nang cng gip bo
v vi sinh vt trnh mt phn tc ng bt li ca ngoi mi nh m, nh sng
do vy lm tng t l sng st ca vi sinh vt. Thnh phn t dc ch yu
trong nang cng l t dc n, vi in hnh l tinh bt, cc loi ng n,
ng i nh saccarose, lactose, glucose,... Ngoi ra, c thm mt s thnh
phn khc nh t dc trn, t dc mu, t dc iu hng iu v,
i vi nang cng cha probiotics, thnh phn cn c thm cc t dc
gip bo v vi sinh vt trong iu kin bo qun, iu kin ngoi mi, nh
sng, m, hay bo v vi sinh vt bn vng qua acid d dy. in hnh l
19

nhm cht x Fructooligosaccharide, Vegetable cream, inulin, l nhm t

dc hay c s dng nht.
1.6.2

ng nang c ng

Mun hon thin cng thc v quy trnh sn sut nang cng, ng thi
m bo thu c cc nang cng c cht lng ng nht qua cc l m khc
nhau cn phi nh gi cht lng nang cng da trn cc ch tiu cht lng.
Mt s ch tiu cht lng ca nang cng cn nh gi l: [2]
ng u v hm lng
ng u v khi lng
r: nang cng phi r trong vng 30 pht
Trc nghim ha tan: Th ha tan gip nh gi tc gii phng
dc cht, gip cho cc nh sn sut la chn, sng lc cng thc v cc
thng s k thut trong qu trnh sn xut, ng thi y cng l mt ch tiu
nh gi cht lng sn phm.
Cc th nghim khc: Xc nh hm lng dc cht, mc ng
nht v hm lng dc cht, tin hnh theo nh ch dn trong cc chuyn
lun c th ca Dc in i vi tng loi ch phm c th.

20

2. NGUYN LIU, THIT B

V PHNG PHP NGHIN CU

2.1. N

l
y

2.1.1.

Chng vi sinh vt: Lactobacillus acidophilus ATCC 4653

Nguyn liu :
- Alginat:

S dng dng mui Natri alginat.

Ngun gc: to nu
- V nang gelatin:

C nang: c s 1

Mu sc: mu xanh
- Di y l bng cc t dc c s dng trong nghin cu
B

2.1: C

o
T

Glucose

Trung Quc

TC NSX

Pepton

Merk-c; n

TC NSX

Cao tht

Merk- c

TC NSX

Cao nm men

Merk- c

TC NSX

K2HPO4

Trung Quc

TC NSX

MgSO4.7H2O

Trung Quc

TC NSX

Calci clorid

Trung Quc

TC NSX

Sa gy

Trung Quc

TC NSX

Alginate

Trung Quc

TC NSX

Triamoni citrat

Trung Quc

TC NSX

21

Acetat natri

Trung Quc

TC NSX

MnSO4.4H2O

Trung Quc

TC NSX

HCl

Trung Quc

TC NSX

Tinh bt

Vit Nam

TC NSX

V nang gelatin

Vit Nam

TC NSX

2.1.2. M

MRS lng (MT1).

Glucose

20g

Acetat natri

5g

Pepton

10g

K2HPO4

2g

Cao tht

10g

MgSO4.7H2O

0,2g

Cao nm men

5g

MnSO4.4H2O

0,05g

Triamoni citrat

2g

Nc my v 1000ml

pH

6,8 7,0

Mi trng MRS thch (MT2).

MT2= MT1 + thch agar (22g/1000ml mi trng).
2.1.3. C

Hn dch sa gy 10%, 20%

Dung dch alginate 1%, 2%, 5%
Dung dch HCl pH 1,2 (pha theo DVN4)
2.1.4. T

b d

T cy v trng (Sanyo)

o
N

Nht
22

T m CO2 (Sanyo)

Nht

T lnh su

T lnh LG

Hn Quc

Thit b ng kh

My li tm

Ni hp tit khun

ALP - Nht

Cn phn tch (Satorious)

Cn k thut (Satorious)

My o hm m Pressica

Thy in

My khuy t Wisd

Hn Quc

My lc (Vortex)

Trung Quc

H thng th ha tan ERWEKA DT 60

a petri, ng nghim, u cn,

2.2.

2.2.1. P

p p

Cn, ong cc thnh phn trong cng thc mi trng MRS lng (cng
thc nu trong mc 2.1.2). Ha tan ht cc thnh phn. Phn chia mi trng
vo cc ng nghim sch bng phu thy tinh, mi ng nghim 10ml MRS
lng. y kn bng nt bng. Hp tit khun mi trng 115oC / 20 ph
trong ni hp tit khun. Sau khi tit khun ngui xung khong 37
40oC. Cy ging vo ng nghim trn (thao tc cy phi c thc hin trong
t cy v trng). cc ng nghim c cy ging VSV trong t m 5%
CO2 trong 24h 37 1oC [9].

23

2.2.2. P

Cn, ong cc thnh phn trong cng thc mi trng MRS lng (theo
cng thc nu trong mc 2.1.2). Ha tan cc thnh phn trong cng thc trong
cc c m. Chuyn 100ml mi trng MRS lng vo bnh nn dung tch thch
hp. y kn ming bnh bng nt bng. Hp tit khun 115oC /20ph trong
ni hp t ng.Ly ra, ngui xung khong 37 40 oC. Cy ging vo cc
bnh nn trn trong t cy v trng, mi bnh 1 ng ging 10ml. trong t
m CO2 5% trong 24h nhit 371oC.
2.2.3. P

Nui cy VSV trong cc bnh nn cha mi trng MRS iu kin

37oC trong 24h trong t m CO2 5% (nh phng php nu trong mc 2.3.3).
ng nht dch nui cy bng cch lc bnh cha dch nui cy v khuy t
trong 10 pht, ong vo cc ng li tm mi ng 10ml hn dch sinh khi
(HDSK) trn, li tm cc ng ny vi tc 4000 vng/ph. Ly cc ng li tm
khi my, loi b phn dch trong, gi li phn cn sinh khi pha di, cho t
dc n chun b sn v c hp tit khun, ngui, lc trn my lc
(Vortex) cho sinh khi thu c phn tn u trong dung dch t dc n.
hn dch cc mu ny ra a petri c lm sch v hp tit khun t
trc, y kn bng giy nhm, trong t lnh su -80oC cho ng rn hon
ton, sau tin hnh giai on lm kh trong my ng kh. To cc mu
ng kh vi thnh phn nh sau:
Mu 1: SK + 10 ml nc ct.
Mu 2: SK + 10 ml dch li tm
Mu 3: SK + 10 ml sa gy 10%.
Mu 4: SK + 10 ml alginate 2%.

Tin ng: Cc mu c lm ng lnh trong t lnh su -80oC cho

ti khi mu ng rn hon ton. Tin ng trong thi gian 24h.

24

Lm kh: Cc mu c lm kh trong khng gian lnh ca my ng

kh nhit khong -54oC, 0,055mbar. Lm kh trong thi gian 24h.

Kt thc qu trnh lm kh, ly mu ra khi thit b chuyn vo bao
polyme c kha, xc nh s vi sinh vt sng st hoc y kn ri bo qun
trong t lnh nhit 4oC.
2.2.4. P

p p k

Ty d ll

Cho nguyn liu cn tit khun vo trong cc c m, y kn. cc

70 80oC trong 1h. Ly ra, cc ng nguyn liu vo t m 37oC trong
24h. Sau 24h, ly cc ng nguyn liu ra, lp li nh trn 3 ln.
2.2.5. P

p p o

L. acidophilus

Chun b t dc: Tinh bt, Natri alginate c tit khun theo phng

php Tyndall

To thuc bt cha L. acidophilus: Tin hnh trong t cy v trng,

trn t dc v bt ng kh theo t l 1:1 to thnh khi bt ng nht,

thu c bt thuc cha tinh bt v bt thuc cha Natri alginate.
2.2.6. P

p p o nang c ng ch a L. acidophilus

Chun b cc t dc: Tinh bt, Natri alginat c tit khun theo

phng php Tyndall

To nang cng cha L. acidophilus: Sau khi c hp tit trng, t

dc v bt ng kh trn ng lng to thnh khi ng nht. Sau em

ng thnh nang cng bng my to nang th cng.

Bo qun mu trong l nt kn nhit phng.

2.2.7. P

p p x

m m

Tin hnh o trn thit b o hm m Prescisa.

Lm nh mu cn o hm m. Cho ln mt a ca thit b khong 1g
mu, dn u mu ra mt a, y np, chy my. Ch my gia nhit trong

25

khong 5 pht hoc n khi my hin kt qu. c, ghi li s liu hin trn
mn hnh (a1). Hm m ca mu l hiu s ca (100- a1) %.
2.2.8. P

p p x

s l

VSV

eo

c pha long

lin
Cn, ong chnh xc cc thnh phn theo cng thc mi trng MRS
c (nu trong mc 2.1.2). Ha tan cc thnh phn tan trong nc, phn tn
thch vo dung dch ng trong bnh nn thch hp. y kn bng nt bng.
Hp tit khun 115oC/ 20 pht trong ni hp tit khun. Ly bnh ng mi
trng ra khi ni hp, phn phi mi trng ln cc a petri c ra
sch, hp tit khun v sy kh (b dy lp thch trn cc a khong 2mm)
sao cho mt thch nhn, phng. Ch cho thch ngui v ng rn li, y kn.
Chun b cc ng nghim sch mi ng cha 9ml nc ct, y kn,
hp tit khun 115oC / 20 pht, ngui xung nhit khong 37 40oC.
Xc nh s lng VSV trong 1ml dch nui cy ban u.
Ht chnh xc 1ml hn dch sinh khi pha vo ng nghim th nht, lc
u. Ht chnh xc 1ml dch c ng nht trong ng nghim th nht
pha long sang ng nghim th 2 (cha 9ml nc ct c hp tit khun
115oC / 20 pht), lc u. C tip tc pha long nh vy cho ti nng
pha long cui cng (trong th nghim ny l 10-7, 10-8, 10-9). Cy tri ln mi
a petri 0,5ml dch trong cc ng pha long trn (lm trn 3 nng cui,
mi nng cy trn 2 a).
Mu xc nh s lng VSV cn sng sau ng kh
Lm nh nguyn liu, cn chnh xc khi lng ca mu, sau pha
vo bnh nn cha 100ml nc ct c hp tit khun 115 oC / 20 pht.
Phn tn u mu trong H2O bng khuy t, sau ht chnh xc 1ml dch t
bnh nn pha vo ng nghim cha 9ml nc ct, ri tip tc pha long, cy
nh i vi mu l cht lng nh trn.
26

 Mu xc nh s lng VSV sng st trong iu kin mi trng pH thp:

+ M

b : Cn chnh xc khong 1,0g mu cn th, cho vo bnh nn

cha 100ml acid HCl pH 1,2 (pha theo Ph lc 2) c hp tit khun

115oC / 20 pht. Khuy t vi tc 50 vng/ph trong 1h, 2h. Ly mu ti
cc thi im 0h, 1h, 2h, tin hnh pha long lin tc nh cc bc nu
trn n (nng pha long 10-4, 10-5; 10-6; 10-7), cy tri dch trong 3 nng
pha long cui cng ln mt thch MRS, mi a 0,5ml, dn u dch trn
b mt thch bng que chang c tit khun.
+ M

: Ti cc thi im 15ph, 30ph, 1h, 2h, ht ly 1 ml dch

mu th, pha long lin tc n nng 10-3, 10-4, sau cy tri dch trong 3
nng pha long cui cng ln mt thch MRS, mi a 0,5 ml, dn u
dch ln trn b mt thch bng que chang c tit khun
cc a ny trong t CO2 5%, nhit 37oC.
Tin hnh xc nh s lng VSV trn cc mu:
- 1ml dch nui cy sau 24h.
- Mu to thnh sau ng kh.
- Mu sau khi trong dch acid pH 1,2.
Sau 48h c kt qu
S lng VSV c trong 1g nguyn liu hoc 1 ml dch nui cy ban u
c tnh theo cng thc:

Trong :
m: khi lng mu em tin hnh xc nh s lng.
X: s VSV c trong 1 g hoc 1 ml mu.
An: S khun lc mc trn cc a petri c cy cc nng pha long n.

27

2.2.9. P

p p

st c a L. acidophilus

ng c a alginate n kh

sng

thuc bt

Nui cy vi sinh vt trong bnh nn cha 200ml mi trng MRS lng

trong 24h (theo phng php nu trong mc 2.2.2).
Thu gn b mt phn dch trong, gi li khong 60ml hn dch sinh
khi pha y, to mu tin ng gm: hn dch sinh khi trn ng lng
vi hn dch sa gy 20%.
ng kh cc mu (theo phng php nu trong mc 2.2.3)
m s lng vi sinh vt c trong 1g nguyn liu bt ng kh theo
phng php pha long nu trong mc 2.2.8
Cho tng mu vi khi lng 1g vo bnh nn cha 100ml dung dch
HCl pH 1,2 . Khuy t vi tc 50 vng/ pht. Cc mu c thnh phn nh
sau:
Mu a: Bt ng kh L. acidophilus vi hn dch sa gy10% (Bt
ng kh).
Mu b: Bt ng kh + tinh bt.
Mu c: Bt ng kh + alginate.
Trong cc mu bt ng kh phi t tiu chun trn v hm m,
th cht v s lng VSV.
Sau 1h, ng nht ha mu trong bnh. Nhanh chng ht chnh xc 1 ml
dch trong bnh tin hnh pha long lin tc theo mc 2.2.8.
trong t CO2 5%, sau 48h tin hnh m s lng khun lc. Tnh s
lng vi sinh vt cn sng st (theo cng thc nu trong phn 2.2.8.)
2.2.10. P

p p

ng c a alginate n kh

sng st c a L. acidophilus trong qu trnh

c ng

S dng my th ha tan ERWEKA DT60 nh gi s lng L.

acidophilus cn sng sau khi th trong iu kin mi trng acid pH 1,2
28

iu kin th:
- Thit b: gi quay.
- Tc quay: 50 1 vng/pht.
- Mi trng th: 200 ml mi trng pH 1,2.
- Nhit mi trng th: 37 0,5oC.
- Thi gian ly mu: 15 ph, 30ph, 1h, 2h.
Tin hnh:
To bt ng kh t tiu chun v hm m, th cht v s lng VSV.
Phn tn nh bt ng kh. em bt ng kh ng nang vi t dc
alginate v tinh bt vi t l 1:1. Cc mu c thnh phn nh sau:
Mu 1: Bt ng kh + tinh bt.
Mu 2: Bt ng kh + alginate.
em mi nang cho vo trong mt cc th ha tan n mi trng
acid pH 1,2 t 370C. Sau tin hnh khuy vi tc khuy 50 vng/pht.
Ti cc thi im 15 pht, 30 pht, 1h, 2h, ht 1 ml dch v tin hnh m
VSV theo phng php pha long lin tc mc 2.2.8.

29

C
3.1.

3. THC NGHIM, KT QU V BN LUN

Nghin c u

khun L. acidophilus

ng c a alginate n kh
o

sng st c a vi

Trong nhng nm gn y alginate c nghin cu s dng kh nhiu

trong cc ch phm probiotics v cho thy kh nng bo v vi sinh vt khi
nhiu iu kin bt li [15], [41], [42]. Cc nghin cu di y c tin
hnh nhm nh gi tc dng ca alginat trong qu trnh ng kh vi sinh vt
v so snh tc dng ca n vi mt nguyn liu c dng ph bin trong
ng kh l sa gy.
Trong sn xut, bt c nguyn liu no cng cn phi c tiu chun
lm mc nh gi cht lng, gip sn phm cht lng tt. i vi nguyn
liu probiotics, theo tiu chun ca IDF, s lng VSV trong 1 gam ch phm
t 108-109 cfu/g. Mt khc, i vi nguyn liu ng kh, hm m l mt
tiu chun quan trng, v hm m cao lm cho nguyn liu d hp th nc
tr li [11], kh m bo t l VSV sng cao. Mt tiu ch na cn quan tm
l ti xp ca nguyn liu, ti xp ln s gip nguyn liu d phn tn,
ng u hm lng s tt hn. Do vy, nguyn liu sau khi ng kh cn
t c tiu chun nu ln di y:
Th cht ca nguyn liu to thnh sau khi ng kh: ti xp
Hm m sau ng kh vi natri alginate: <5%
S lng vi sinh vt sng st sau khi ng kh vi cht bo v l natri
alginate: >108 cfu/g

30

Cc nghin cu di y nhm nh gi tc dng ca alginate trong

qu trnh ng kh v so snh vi alginate nhng u da trn mc tiu t
c tiu chun trn.
3.1.1.

Mt trong nhng vai tr quan trng ca t dc s dng trong qu

trnh ng kh l gi cho ch phm c th cht n nh, trnh b ht m trong
qu trnh bo qun. Mt khc, y l ch phm probiotics nn t dc cn c
thm mt vai tr quan trng na l bo v VSV, gi cho VSV c t l sng
st cao sau khi ng kh. Theo cc nghin cu cng b th sa gy 10%
va bo v VSV tt, va gi cho ch phm c th cht n nh [32], [33]. Do
vy, trong th nghim ny, mt trong nhng t dc dng so snh vi
alginate l sa gy 10%, v ch tiu so snh l s n nh ca th cht mu
sau khi ng kh.
Trong cc nghin cu to ch phm vi nang hay bt phun sy cha
probiotics, t dc natri alginate u c s dng vi nng 2% [15], [21].
Do vy, t dc alginate c kho st trong nghin cu ny cng c s
dng vi nng 2%.
Mc tiu:
Kho st th cht ca bt ng kh cha VSV vi t dc alginate
nng 2%, ng thi so snh vi sa gy 10% v dch ly tm.
Kt qu:
Th cht ca cc mu thu c c th hin trong bng 3.1.
B

3 : Th cht ca cc mu ng kh L. acidophilus vi cc t dc bo
v ti thi im ngay sau khi ng kh
T d

T
31

bo
Nc ct

Sn phm thu c l 1 lp rt mng, dnh a. Rt kh

ly ra ht khi dng c cha.

Dch li

Sn phm thu c l 1 lp rt mng, dnh a. Rt kh

tm

ly ra ht khi dng c cha

Sa gy

Th cht ng nht, kh, xp, d lm ti, d dng ly ra khi

10%

dng c cha

Alginate

Th cht ng nht, nh, xp, kt thnh mng, rt kh lm nh.

2%

D ly ra khi dng c cha nu thc hin thao tc ny trc

khi mu b ht m

Nhn xt:
Khi khng dng t dc n, cc mu ng kh L. acidophilus c
to thnh l mt lp mng bm vo dng c cha, kh ly ra, ng thi cc
mu ng kh ny cng nhanh chng ht m tr li.

Hnh 3.1: Hnh nh mu ng kh vi nc ct.

32

Cc mu ng kh L. acidophilus vi t dc l hn dch sa gy10%

v dung dch alginate 2% u cho th cht c xp ln. Tuy nhin, mu
ng kh vi alginate c th cht xp hn nhng li b kt thnh mng, nhanh
b ht m v kh phn tn nh.
Tm li, s dng dung dch alginate lm t dc trong ng kh gip
thu c mt mu nguyn liu c th cht xp, d dng ly ra khi dng c
cha hn so vi cc mu khng s dng t dc. Tuy nhin s dng alginate
lm t dc trong ng kh s to ra nguyn liu nhanh b ht m, kh phn
tn nh thnh dng bt mn, vic ny dn n kh ng nht gia s lng
VSV probiotics cc l m trong qu trnh bo ch to sn phm tip theo.
3.1.2.

y alginate

Hm m l mt ch s gy nh hng ln n kh nng sng st ca vi

sinh vt trong qu trnh bo qun. Hm m ln l nguyn nhn gy gim s
lng vi sinh vt sng st. Trong phng php ng kh, tiu chun hm m
ca cc mu ngay sau ng kh l khng nh hn 5%.V vy, cc th nghim
di y c thc hin nhm nh gi hm m ca cc mu cc thi im
ngay sau ng kh, v trong qu trnh bo qun trong ti polymer kn nhit
4oC.
Tuy nhin, khi thc hin nh gi th cht v t l VSV sng st cc
mu mc 3.1.1 nhn thy: th cht ca cc mu s dng nc (mu 1) v
dch ln men (mu 2) khng thch hp to nguyn liu nn vic theo di
hm m ca mu 1 v mu 2 theo thi gian khng cn nhiu ngha. Do
chng ti thc hin th nghim nh gi hm m v tc ht m theo thi
gian ca 2 mu c th cht t ti xp, l mu ng kh vi sa gy 10%
v mu ng kh vi alginate 2%.

33

 Mc tiu: Xc nh hm m ca cc mu ngay sau khi ng kh v tc

ht m ca cc mu trong thi gian bo qun.
Kt qu: Hm m ca cc mu c th hin trong bng 3.2.

3 . Bin thin hm m (%) ca mt s mu ng kh L. acidophilus

vi t dc bo v khc nhau theo thi gian
Hm m (%)
T

bo q

10%

natri alginate 2%

2,8 + 0,3

6,5 + 1,6

Sau 2 ngy

3,4 + 0,7

10,8 + 2,9

3,5 + 0,7

12,5 + 3,3

3,5 + 0,6

13,3 + 3,0

ys

T o

Mu cha sa gy 10%: mu 3 trong mc 3.1.1

34

Mu cha alginate 2%: mu 4 trong mc 3.1.1

Hnh 3.2: th biu th hm m trung bnh ca cc mu ngay sau ng kh

v trong thi gian bo qun.
Nhn xt:
Kt qu a ra bng 3.2 cho thy hm m ca mu ng kh vi sa
gy 10% ngay sau ng kh l 2,8% t tiu chun hm m ca cc ch phm
thuc bt (nh hn 5%). Sau 2 tun bo qun, hm m ca mu ng kh vi
sa gy thay i khng ng k v kt qu o hm m ca mu vn t gi tr
nh hn 5%. Trong khi , mu ng kh L. acidophilus vi alginate 2% li
c hm m kh cao v bin i kh ln gia cc mu khc nhau khi ng kh
trong cng mt iu kin, cng thi gian nh nhau, hm m thu c ca mu
4 l 6,5% ngay sau ng kh.
ng thi, tc ht m ca mu ng kh vi sa gy cng chm
hn so vi mu ng kh vi alginate. Sau 2 tun bo qun, hm m ca mu
ng kh s dng alginate 2% c hm m kh cao, t 13,3 + 3,0, trong khi
mu ng kh vi sa gy vn t gi tr nh hn 5%.

Hnh 3.3. Hnh nh mu ng kh vi alginate ngay sau khi tho khi my

v sau 3 pht ngoi mi trng.

35

Trong qu trnh tin hnh th nghim, khi tho sn phm ra khi my

ng kh nu khng nhanh chng bo qun trong bao b kn th nguyn liu
ng kh vi alginate s ht m rt nhanh v d b bt dnh vo dng c cha
hoc chy nc.
Tm li, ngay sau ng kh v trong qu trnh bo qun, mu ng kh
vi Natri alginate 2% u c hm m v tc ht m cao hn so vi mu
ng kh vi sa gy 10%. Khi s dng t dc bo v l sa gy, hm m
ca mu ng kh kh n nh theo thi gian, trong khi hm m ca mu
ng kh vi alginate li thay i kh nhiu, mi ch sau 2 tun bo qun
trong bao polymer kn hm m ca mu ln ti khong 13%. Nh vy mu
ng kh vi alginate 2% kh c th m bo yu cu v hm m.
3.1.3. K

nh gi tnh kh thi ca vic s dng ca bt k t dc no lm t

dc bo v trong ng kh Lactobacillus acidophilus l nh gi s lng
v t l vi sinh vt sng st sau khi ng kh. c coi l c tc dng bo
v trong ng kh th trc tin t dc phi cho thy kh nng ci thin
c s lng cng nh t l vi sinh vt sng st trong qu trnh ng kh, t
nht l so vi cc mu ng kh khng s dng t dc bo v. V vy, th
nghim tip theo c tin hnh vi tiu ch nhm xc nh s lng v t l
vi sinh vt sng st ca cc mu s dng t dc alginate sau qu trnh ng
kh.
Mc ch
nh gi nh hng ca dung dch alginate 2% ln s lng v t l
sng st ca vi sinh vt trong qu trnh ng kh, ng thi so snh vi mu
ng kh khng s dng t dc v mu ng kh s dng hn dch sa
gy10%.
36

 Kt qu v nhn xt
S lng vi sinh vt Lactobacillus acidophilus c trong 1ml dch nui
cy trc ng kh l khong 1,09 x 109 VSV/ 1ml.

3 3 S lng vi khun sng st trong 4 mu sau ng kh

(Tnh theo khi lng tng ng 1ml dch nui cy ban u)

S l

M 1

M 2

( )

(d h ln men)

Ln 1

2,9 x 106

7,9 x 105

5,1 x 108

1,7 x 107

Ln 2

2,5 x 106

8,5 x 105

7,1 x 108

2,2 x 107

Ln 3

2,3 x 106

7,7 x 105

4,6 x 108

2,3 x 107

2,57 x 106

8,03 x 105

5,6 x 108

2,07 x 107

0,31 x 106

0,42 x 105

1,32 x 108

0,32 x 107

Trung
bnh
RSD

( )

sng st

(s

y 10%)

M 4
(alginate 2%)

3 4: T l VSV sng st sau ng kh so vi mu ban u

M 1

T l VSV

M 3

0,24%

M 2
(d

me )

0,07%

37

M 3
(s

y 10%)

51,38%

M 4
(alginate 2%)

1,9%

Trong :

Mu 1: ng kh L. acidophilus vi dch li tm.

Mu 2: ng kh L. acidophilus vi nc ct.
Mu 3: ng kh L. acidophilus vi sa gy 10%.
Mu 4: ng kh L. acidophilus vi alginate 2%.

Hnh 3.4. th biu din s lng vi khun L. acidophilus sng st

trong cc mu sau ng kh
Kt qu trong bng 3.3 cho thy ng kh vi t dc l dung dch
alginate 2% cho t l vi sinh vt sng st thp hn nhiu so vi t dc dng
ph bin l sa gy 10%. Tuy vy, s liu cng cho thy alginate 2% c tc
dng bo v khi s dng lm t dc ng kh vi t l sng st ca mu ny
cao gp 27,1 ln so vi mu ng kh khng c t dc bo v.
Nh vy, alginate c tc dng bo v Lactobacillus acidophilus trong
qu trnh ng kh. Khi ng kh vi t dc l alginate 2% cho t l sng st
ca L. acidophilus l 1,90% cao gp 27,1 ln so vi mu chng ng kh vi
nc ct, v cao gp 7,91 ln so vi mu ng kh vi dch li tm. Tuy nhin

38

t l sng ca L. acidophilus trong mu ng kh vi alginate cn km sa

gy nhng vn t s lng vi sinh vt l 107.
3.1.4. S

alginate

T kt qu th nghim 3.1.3 cho thy alginate c tc dng bo v vi

sinh vt trong qu trnh ng kh, tuy nhin nu s dng mt mnh lm t
dc ng kh th tc ht m ca nguyn liu to thnh ln lm cho
nguyn liu kh m bo yu cu v hm m. Mt khc nguyn liu to thnh
sau khi ng kh L. acidophilus vi alginate 2% cho t l vi sinh vt sng st
thp hn so vi khi ng kh vi sa gy 10%. M tiu chun s lng VSV
sng st l tiu chun quan trng nht ca ch phm probiotics nn trong qu
trnh ng kh phi kt hp vi sa gy 10% t c tiu chun trn. Do
chng ti thit k th nghim tip theo: ng kh vi sinh vt s dng kt
hp t dc l dung dch alginate cc nng 1%, 2%, 5% v sa gy 10%
nhm ci thin th cht cng nh t l sng st VSV ca mu ng kh vi
alginate.
Mc tiu:
S b so snh t l sng st ca vi sinh vt ca mu s dng t dc
n l sa gy 10% v mu s dng sa gy c kt hp thm dung dch
alginate.
Kt qu v nhn xt
V th cht
C 4 mu u to ra bt nguyn liu c th cht ng nht, ti xp,
kh, mu m dn theo nng alginate c trong mu. Nh vy vic thay
i nng alginate t 1 5% khng lm nh hng n th cht ca cc
mu.
Hm m
39

3 5: Hm m ca cc mu ng kh vi t dc bo v l hn hp sa
gy vi alginate cc nng
Hm m (%)
T

ian

0% alginate 1% alginate 2% alginate 5% alginate

0h

2,8 + 0,3

3,1 + 0,4

3,6 + 0,5

3,7 + 0,5

Sau 2 ngy

3,4 + 0,7

3,4 + 0,5

4,0 + 0,6

4,1 + 0,6

Sau 1 tun

3,5 + 0,7

3,7 + 0,6

4,3 + 0,6

4,6 + 0,6

Sau 2 tun

3,6 + 0,7

4,0 + 0,7

4,4 + 0,6

4,7 + 0,7

Hnh 3.5: Hm m ca cc mu ng kh vi t dc bo v l hn hp sa
gy vi alginate cc nng
Hm m ca cc mu ngay sau ng kh c kt hp sa gy 20% v
alginate 1%, 2%, 5% t t: 2,8% 3,7%, gi tr ny gn tng ng vi
hm m ca mu ng kh vi sinh vt vi t dc l sa gy 10% (hm m
2,8%), v t tiu chun hm m ca dng thuc bt [2]. Hm m sau thi
40

gian bo qun trong ti polyme kn ca cc mu ny vo khong 3,4 4,7%

trong khi mu ng kh vi sa gy 10% c hm m 3,4 3,6%. Nh vy xt
v th cht v hm m th khi kt hp alginate v sa gy 10% th nguyn liu
to thnh c th cht v hm m tng ng vi mu ng kh ch s dng
sa gy, nhng t l alginate cng cao th ch phm c mu cng m v hm
m cng ln hn so vi mu c hm lng alginate thp (tuy nhin s thay
i v hm m gia 3 mu ny l khng ng k).
V s lng vi sinh vt Lactobacillus acidophilus sng st sau ng kh
S lng vi sinh vt sng st c nu trong bng 3.6 cho thy tuy mu
kt hp alginate 2% vi hn dch sa gy 10% cho t l sng cao nht 26,1 x
108 cfu/g so vi 20,2 x 108 cfu/g ca mu ng kh vi sa gy v 21,2 x
108cfu/g; 24,3 x 108cfu/g ca 2 mu cha 1% v 5% alginate. Tuy nhin s
lng sng st trong mu ng kh khng c s khc nhau nhiu (c 4 mu
u cha khong 109 VSV/1g).
S lng VSV sng st c th hin trong bng sau:
B

3 6. S lng vi sinh vt sng st tnh trn 1g bt sau ng kh trong

cc mu ng kh s dng kt hp alginate v sa gy.
S l

( x108 )

0%

1%

2%

5%

alginate

alginate

alginate

alginate

Ln 1

8,51

14,50

29,40

23,92

Ln 2

21,89

9,05

34,52

27,84

Ln 3

20,42

29,91

4,90

1,29

20,2

21,2

26,1

24,3

Trung
bnh

41

Hnh 3.6: S lng vi sinh vt sng st tnh trn 1g bt sau ng kh trong

cc mu ng kh s dng kt hp alginate v sa gy.
Nh vy khi kt hp vi sa gy 10% ng kh, natri alginate th
hin rt t kh nng gia tng t l sng st trn Lactobacillus acidophilus.
Nguyn nhn c th do t l natri alginate s dng kh thp, hn na nh th
nghim 3.1.3 ta thy tc dng bo v vi sinh vt ca alginate nh hn kh
nhiu so vi sa gy (khong 27 ln). Do vy khi kt hp alginate vi sa gy
to hn hp t dc khi ng kh ta s khng thy c s thay i ln trn
t l sng st. Mt khc, s kt hp t dc s lm cho qu trnh bo ch tr
ln phc tp hn, nguyn liu to thnh vi cc mu c nng alginate cao
c mu sc khng p, ht m cng cao hn tuy khng nhiu.
Tuy nhin v c th cht, hm m, cng nh s lng vi sinh vt sng
st trong mu phi hp alginate vi sa gy u t gi tr tt hn so vi mu
ng kh s dng ring alginate.
3.2.

Nghin c u

ng c a alginate n kh

khun Lactobacillus acidophilus

nang c ng
42

sng st c a vi
thuc b

Cc kt qu nghin cu nu trong mc 3.1.2 cho thy alginate c tc

dng lm gia tng t l sng st ca vi sinh vt trong qu trnh ng kh. Tuy
nhin vic s dng n nh mt t dc bo v trong ng kh vi sinh vt cn
nhiu hn ch v sn phm to thnh c th cht v hm m khng thch hp.
Do vy cc th nghim tip theo c thit k nhm kho st kh nng bo v
ca natri alginate khi s dng lm t dc n trong cc ch phm cha vi
sinh vt. Mt trong nhng phng php th tc dng bo v ph bin nht
hin nay l th tc dng bo v ca cc t dc s dng ln t l sng st ca
vi sinh vt khi tip xc vi iu kin acid d dy. Th nghim ny tin hnh
th nghim trn cc mu: nguyn liu ng kh vi sa gy 10% v khi c
tin hnh trn ng lng bt ng kh ny vi t dc n l natri alginate,
tinh bt - mt t dc n c dng ph bin hin nay c s dng i
chng.
3.2.1. N
k

L. acidophilus

alginate k
q

Mc tiu:
Nghin cu nh hng ca alginate n kh nng sng st ca vi khun
L. acidophilus c s dng lm nguyn liu thuc bt
Kt qu, nhn xt
Mu s dng alginate lm t dc n cho t l vi sinh vt sng st sau
1h v 2h cao hn khong 37 ln so vi khi s dng t dc n l tinh bt v
mu khng s dng t dc l mu bt ng kh. Mu s dng alginate cho
s lng VSV sng st vo khong 107, trong khi mu s dng tinh bt v
mu khng s dng t dc (mu bt ng kh) cho s lng VSV sng st
khong 105 n 106.

43

3 7. S lng vi sinh vt sng st tnh trn 1g bt ng kh

trong mi trng acid HCl pH 1,2
S l

Sau 1h
M

M b

Sau 2h
M

M b

L 1 1,0 x 109 9,8 x 106 6,6 x 105

6,3x 105

4,1x 106

2,0x 105

1,9 x 105

L 2 1,9 x 109 2,2 x 107 3,1 x 105

2,9x 105

1,3x 107

2,6x 105

2,6 x 105

L 3 2,2 x 109 2,4 x 107 4,6 x 105

4,7x 105

1,6x 107

4,0x 105

4,3 x 105

SLTB 1,7 x 109 1,9 x 107 4,8 x 105

4,6x 105

1,1x 107

2,9x 105

2,9 x 105

0,6 x 109 5,6 x 107 1,7 x 105 1,7 x 105

2,2 x 107

1,0 x 105

1,2 x 105

SD

Trong : Mu a: Bt ng kh L. acidophilus vi hn dch sa gy10%.

Mu b: Bt ng kh + tinh bt.
Mu c: Bt ng kh + alginate.

Hnh 3.7. Biu th hin s lng vi sinh vt sng st sau khi th trong mi
trng acid HCl pH 1,2

44

Hai mu b v a cho trong cc ln cng th nghim th cho s lng

VSV sng st ngang nhau, t 4,8x105 cfu/g sau 1h v 2,9x105 sau 2h. iu
ny chng t vic b sung thm t dc n l tinh bt khng h gip ci
thin s lng VSV sng st trong iu kin acid d dy.
Tm li, kt qu th nghim cho thy mu nguyn liu s dng Natri
alginate lm t dc n cho t l sng st trong mi trng acid pH 1,2 cao
hn so vi mu th vi bt ng kh v mu bt ng kh c s dng thm
t dc n l tinh bt. T c th s b kt lun natri alginate khi s dng
lm t dc n trong ch phm vi sinh vi t l cao (trong th nghim ny l
50%) cho hiu qu lm gia tng t l sng st ca L. acidophilus ATCC 4653.
3.2.2. N
k

L. acidophilus

alginate k
q

Mc tiu: Nghin cu nh hng ca alginate n kh nng sng st

ca vi khun L. acidophilus trong qu trnh to nang cng
Kt qu:
B

3.8: S lng vi sinh vt sng st trong 1 vin nang trong mi trng

acid HCl pH 1,2 vi tc khuy 50 v/pht
S l

Sau 15 pht

Sau 30 pht

M 1

M 2

M 1

M 2

Sau 1h
M 1

M 2

Sau 2h
M 1 M 2

L 1

1,5x108

1,2x104

1,5x106

1,0x103

1,1x105 1,0x102 3,0x104

L 2

2,7x108

1,6x104

1,9x106

1,3x103

1,5x105 1,5x102 3,9x104

L 3

3,3x108

1,8x104

2,1x106

1,6x103

1,8x105 1,9x102 4,1x104

SLTB

2,5x108

1,5x104

1,8x106

1,3x103

1,5x105 1,5x102 3,7x104

SD

0,9x108

0,3x104

0,3x106

0,3x103

0,4x105 0,5x102 0,6x104

Ch thch: (-):khng thy xut hin khun lc

45

Bng 3.9: T l vi sinh vt sng st ca mu alginate

so vi mu tinh bt trong mi trng pH 1,2
M 1

M 2

T l VSV sng sau 15 pht

120

T l VSV sng sau 30 pht

115

T l VSV sng sau 1h

240

T l VSV sng sau 2h

Hnh 3.8: S lng vi sinh vt sng st trong 1 vin nang trong mi

trng acid HCl pH 1,2 vi tc khuy 50 v/pht

Nhn xt:
Kt qu bng cho thy mu nang c s dng alginate lm t dc n
cho t l sng st VSV trong mi trng acid pH 1,2 ln hn so vi mu
nang c s dng tinh bt lm t dc n. Trong th nghim ny, t l VSV
sng st ca mu s dng alginate cao gp hn 100 ln so vi mu s dng
tinh bt. Ngay sau 15 pht, s lng VSV sng st ca mu nang cha tinh
46

bt gim mnh xung 1,5x104 cfu/nang, trong khi mu nang cha

alginate vn gi c 1,8x106 cfu/nang. Sau 1,5 gi, s lng VSV sng st
trong mu nang alginate gim xung cn 3,7x104, trong khi mu nang tinh
bt s lng VSV l 1,5x102. Nh vy, i vi mu nang tinh bt, lng
VSV sng st b gim mnh ngay trong 15 pht u, cn nang alginate lng
VSV sng st b gim mnh sau 1,5 gi.
Tuy nhin, sau 2 gi trong mi trng acid pH 1,2 hu nh khng cn
VSV sng st. Do vy, cn tm thm cc bin php ci thin khc, lm gia
tng t l sng st ca VSV trong mi trng acid thp.

47

4.1.

Nghin c u

ng c a alginate n kh
o

khun L. acidophilus
4.1.1.

4: BN LUN

c ng y

sng st c a vi

Khi nh gi th cht ca nguyn liu to thnh sau khi ng kh, mu

ng kh vi dch sa gy 10% v mu ng kh vi dung dch alginate 2%
c th cht xp hn so vi mu ng kh vi nc ct v dch ly tm. Tuy
nhin, mu ng kh vi alginate li b kt thnh mng, kh lm nh. Vic
mu ng kh vi dung dch natri alginate to thnh dng nguyn liu kh
nghin nh thnh dng bt mn c th c gii thch nh sau: alginate thc
cht l mt heteropolyme saccharid mch thng cu to t hai gc uronic l
acid - L- guluronic (G) v acid - D- mannuronic (M) [43]. Chnh cu trc
chui polymer ca alginate lm cho nguyn liu to thnh sau ng kh c
cu trc dng cc mng. Trong qu trnh hp tit khun do tc ng ca nhit
cao (> 70oC) nn cc chui polymer ny mt phn b polymer ha lm
gim nht ca dung dch polymer trc khi kt hp vi sinh khi v cng
l l do cc mng nguyn liu ng kh to thnh khi ng kh vi alginate
khng c cu trc mng di m ch l cc cu trc dng mng xp, ngn (vi
mm). V chnh cu trc dng chui polymer ca alginate khin cho nguyn
liu to thnh kh c th c lm nh thnh dng bt mn.
Khc vi vic s dng alginate trong ng kh, khi s dng alginate
lm t dc vi nang ha, sn phm probiotics c to ra c th cht tt
hn. C th, trong th nghim ca Asma Sohail v cng s [15], vi nang cha
probiotics c to thnh bi alginate 2% v CaCl2 bng phng php phun
sy c kch thc phn b ng u t 10 40 m v hnh dng trn u p.
48

Tm li, s dng dung dch alginate lm t dc trong ng kh gip

thu c mt mu nguyn liu c th cht xp, d dng ly ra khi dng c
cha hn so vi cc mu khng s dng t dc. Tuy nhin s dng alginate
lm t dc trong ng kh s to ra nguyn liu nhanh b ht m, kh phn
tn nh thnh dng bt mn, vic ny dn n kh ng nht gia s lng
VSV probiotics cc l m trong qu trnh bo ch to sn phm tip theo.
4.1.2.

y alginate

Hm m v tc ht m ca mu ng kh vi alginate 2% ngay sau

ng kh v trong thi gian bo qun 2 tun u ln hn so vi mu ng
kh vi sa gy 10%, c bit ch trong 2 tun bo qun hm m ca mu
ng kh vi alginate 2% ln ti khong 13%, trong khi hm m ca
mu ng kh vi sa gy 10% vn t tiu chun nh hn 5%. Trong hnh
3.3 cho thy mu ng vi alginate ht m rt nhanh, ngay sau 3 pht
chuyn t dng ti xp sang dng do. iu ny c th gii thch l do natri
alginate l mui ca acid alginic, alginic l acid c cu to bi cc gc
uronic. Gc uronic cha nhiu nhm phn cc, a nc [43], do vy tc
ht m ca alginate ngoi mi trng rt nhanh. Hm m 13% sau thi gian
bo qun 2 tun l qu ln, khng m bo c tiu chun ca nguyn liu,
do vy khng s dng ring alginate 2% lm t dc trong qu trnh ng kh
c, c th kt hp b sung vi sa gy hoc cc t dc khc.
V tiu ch hm m nguyn liu, sa gy vn t. Do vy, sa gy l
mt trong nhng la chn u tin lm t dc bo v VSV trong khi ng
kh. Cn theo nghin cu ca Lim Chi Ming, sa gy c tc dng bo v
VSV trong qu trnh ng kh l do protein trong sa gy to thnh lp o
bo v trn thnh t bo v cung cp cht m gip n nh pH trong sut qu
trnh ng kh [32]. Trn thc t, trong cc ch phm ng kh, sa gy
49

thng c s dng km thm vi cc nhm t dc khc nh carbohydrat,

polymer, polyols, aminoacid, tng cng tc dng bo v VSV [17].
4.1.3. K

Kt qu kho st t l sng st ca L. acidophilus sau ng kh cho thy

khi s dng cht bo v l hn dch sa gy10% cho s lng vi sinh vt sng
st cao nht (khong 108), tip theo l mu ng kh vi dung dch alginate
(khong 107), mu ng kh vi dch ln men (106), mu ng kh vi nc
ct (105). Kt qu ny tng t vi kt qu ca Lim Chi Minh v cng s
thc hin nm 2008 [32]. Theo kt qu cng b ca Lim Chi Minh, t l
sng st ca VSV sau ng kh ca sa gy (13,03%) cng cao hn so vi
nc ct (0,07%). c bit, theo Lim Chi Minh, t l sng st ca VSV sau
khi ng kh vi nc (0,07%) tng ng vi kt qu nghin cu trn
chng vi sinh vt khc cng thuc chi Lactobacillus l Lactobacillus
salivarius vi nc (0,08%).
Tc dng bo v vi sinh vt trong qu trnh ng kh c th c gii
thch nh sau:
Natri alginate c cu trc polyme nn s trng n trong nc, to ra
c xung quanh v bn trong t bo VSV mt mi trng c nht tng
t gip gim thiu bin i cu trc phn t mc thp nht [32].
Vi sa gy c thnh phn ch yu gm lactose v protein chnh l
nhng yu t quyt nh kh nng bo v ca chng. Protein c trong sa to
ra mi trng c nht cao gip bo v cu trc t bo vi sinh vt, trong khi
thnh phn lactose s to lin kt hydro gia ng - protein, lin kt ny
gip n nh protein t bo, gip duy tr cu trc ca protein mng khi nc
b loi i. S n nh mng t bo v protein cu trc bng lin kt hydro ca
cc disaccharid cn c gi l l thuyt thay th nc [17]. Cn theo
nghin cu khc th protein trong sa gy c th to nn mt lp o bo v
50

trn thnh t bo. Trong sa gy cn cha nhiu cht tan nh mui phosphat,
citrat c kh nng m gip n nh pH mu khi m dung mi mt dn trong
qu trnh ng kh [32].
Nh vy, alginate c tc dng bo v Lactobacillus acidophilus trong
qu trnh ng kh. Khi ng kh vi t dc l alginate 2% cho t l sng st
ca L. acidophilus l 1,90% cao gp 27,1 ln so vi mu chng ng kh vi
nc ct, v cao gp 7,91 ln so vi mu ng kh vi dch li tm. Tuy nhin
t l sng ca L. acidophilus trong mu ng kh vi alginate cn km sa
gy nhng vn t s lng vi sinh vt l 107.
4.1.4. S

alginate

Khi kt hp sa gy vi alginate vi t l tng dn n 5%, khng thy

c s gia tng v t l sng st ca VSV. Mt khc, vic kt hp ny lm cho
qu trnh bo ch phc tp hn nn vic kt hp vi alginate vi t l nh
trong th nghim ny l khng kh thi.
Dianawati Dianawati v cng s cng thc hin nghin cu nm 2012
[19] v phi hp cc t dc bo v trong qu trnh ng kh, l nh gi
tc dng bo v ca vic phi hp 5 loi protein v 3 loi ng i vi B.
longum trong qu trnh ng kh. 5 loi protein c nghin cu l natri
caseinate 12%, whey protein 12%, natri caseinate:whey protein 6%:6%, sa
gy 12%, du u nnh 12% c phi hp ln lt vi 3 loi ng l
glycerol (3% kl/tt), mannitol (3% kl/tt), maltodextrin (3% kl/tt) to ra 15 dch
nh tng. Lng B. longum v nh tng c a vo khi ng kh theo
t l 1:4. Kt qu cho thy nh tng phi hp gia whey protein natri
casenate v Glycerol gip VSV c t l sng st cao sau ng kh, t 99,2%.
Cn i vi nh tng phi hp gia natri casemate v manitol, t l VSV
sng st sau ng kh, sau qu trnh tip xc vi acid v sau qu trnh tip
51

xc vi mui mt ln lt l 97,4%, 81,6% v 99,3%. Kt qu cui cng cho

thy sa gy c tc dng bo v vt tri so vi du u nnh v loi ng
c nhiu nhm OH bo v tt hn maltodextrin [19]. iu ny li khng nh
mt ln na tc dng bo v VSV vt tri ca sa gy trong qu trnh ng
kh.
4.2.

Nghin c u

ng c a alginate n kh

khun L. acidophilus trong qu trnh

4.2.1. N
k

L. acidophilus

thuc b

alginate k
q

sng st c a vi
c ng
s

Theo kt qu nghin cu cho thy alginate c tc dng bo v VSV tt

hn so vi tinh bt trong mi trng pH 1,2. Tc dng bo v ca alginate
trn cc vi sinh vt trong iu kin acid pH thp ca d dy c th gii thch
nh sau: khi tip xc vi dung dch acid pH thp mui natri alginate chuyn
thnh dng acid alginic khng tan. Acid alginic trng n to ra mt lp
mng bao quanh bt ng kh cha vi sinh vt, cch li vi sinh vt vi iu
kin bt li ca mi trng xung quanh [30].
So snh kt qu th nghim ny vi kt qu trong nghin cu ca Latha
Sabikhi nm 2010 khi nh gi kh nng bo v L. acidophilus trong ch
phm probiotics bng phng php to vi nang vi alginate. Vi nguyn liu
ban u cha khong 109 cfu/g, mu khng c bo v u c s lng sng
st sau 1h, 2h trong iu kin pH 1 gim xung cn khong 105 106 cfu/g
(gim khong 1000 ln) [31]. nghin cu ca Latha Sabikhi vi phng
php to vi nang bo v th sau 1h trong pH 1 s lng Lactobacillus
acidophilus LA1 gim t 107 cfu/g lc ban u xung cn khong 106 cfu/g
(ngha l gim khong 10 ln), sau 2h trong iu kin acid pH 1, s lng
VSV ca mu bao vi nang cn 105 cfu/g (gim 100 ln so vi ban u). Cn
trong th nghim c s dng alginate lm t dc n th sau 1h trong mi
52

trng acid HCl pH 1,2 s lng vi sinh vt gim t 1,7 x 109 cfu/g xung
cn khong 1,9 x 107 cfu/g (tng ng khong xp x 100 ln), cn ti thi
im sau 2h lng vi sinh vt cn li l khong 1,1 x 107 cfu/g (gim khong
155 ln so vi ban u) [31].
Tm li, kt qu th nghim cho thy mu nguyn liu s dng Natri
alginate lm t dc n cho t l sng st trong mi trng acid pH 1,2 cao
hn so vi mu th vi bt ng kh v mu bt ng kh c s dng thm
t dc n l tinh bt. T c th s b kt lun natri alginate khi s dng
lm t dc n trong ch phm vi sinh vi t l cao (trong th nghim ny l
50%) cho hiu qu lm gia tng t l sng st ca L. acidophilus ATCC 4653.
4.2.2. N
k

L. acidophilus

alginate k
q

Kt qu th nghim cho thy t l VSV sng st trong mi trng acid

pH 1,2 ca mu nang cng cha alginate gp 40 ln so vi mu nang cng
cha tinh bt. iu ny c th gii thch nh sau: khi tip xc vi dung dch
acid pH thp mui natri alginate chuyn thnh dng acid alginic khng tan.
Acid alginic trng n to ra mt lp mng bao quanh bt ng kh cha vi
sinh vt, cch li vi sinh vt vi iu kin bt li ca mi trng xung quanh
[30]. Do vy, t l VSV sng st ca mu s dng alginate ln hn so vi
mu s dng tinh bt.
Mt khc, t l sng st gia 2 mu alginate v tinh bt trong mi
trng acid pH 1,2 nang cng ln hn so vi thuc bt. C th, i vi
thuc bt, t l ny l 40, trong khi i vi thuc nang cng t l ny l
120. Nguyn nhn l do acid alginic trng n to lp kt dnh vi v gelatin,
trong khi mu thuc bt khng c s kt dnh ny nn v nang nhanh b v
hn so vi mu alginate. S kt hp ny lm cho t l sng st VSV trong
mu alginate cao hn nhiu ln so vi mu tinh bt ch phm nang cng.
53

Kt qu ny cng tng t nh kt qu ca Randolph Stanley Porubcan

thc hin nm 2006 [42]. Trong nghin cu ca Randolph Stanley Porubcan,
ng cng to nang cng probiotics cha Natri alginate vi hm lng t 10%
n 99%. Kt qu ca nghin cu cho thy s lng VSV b cht trong mi
trng acid pH 1,6 khng nhiu, c th trc khi th trong mi trng acid
pH 1,6 th s lng cfu/ nang l 9,0x109, sau khi th trong mi trng acid
pH 1,6 trong 90 pht s lng cfu/ nang khng cha alginate nh hn 104,
cn s lng cfu/ nang cha alginate l 4x108 [42]. Kt qu nghin cu cng
cho thy khi s dng alginate lm t dc n trong nang cng lm gia
tng t l VSV sng st trong mi trng acid pH thp.
Tuy nhin, sau 2 gi trong mi trng acid pH 1,2 cho kt qu hu nh
khng cn VSV sng st. Do vy, cn phi c bin php phi hp vi cc t
dc bo v khc hoc thay i dng bo ch tng t l VSV sng st.

54

KT LUN V XUT
KT LUN

I.

ti thu c cc kt qu sau:
1.
k

L. acidophilus

alginate k

k :

Khi s dng dung dch natri alginate 2% lm t dc bo v trong qu

trnh ng kh lm tng t l sng st ca L. acidophilus so vi mu ng

kh vi nc ct (khong 27 ln).

Th cht ca sn phm thu c sau ng kh vi alginate 2%: kh

lm nh v nhanh b ht m tr li.

Khi kt hp sa gy vi natri alginate cc nng 1%, 2%, 5% to ra

c nguyn liu c th cht tt tng ng vi dng ring t dc l sa

gy. Nhng t l sng st li khng c thy s khc bit ln gia cc mu, t
khong 109cfu/g.
2. nghin c u
khun L. acidophilus

ng c a alginate n kh
o

sng st c a vi

thuc bt v thuc nang c ng

nh gi c tc dng bo v vi sinh vt ca alginate khi s dng

lm t dc n cho dng thuc bt probiotic trong iu kin tip xc vi mi

trng acid HCl pH 1,2 cho s lng vi sinh vt sng st khong (10 6 107
cfu/g) cao hn khong 37 ln so vi cc mu khng s dng thm t dc
n v mu bt ng kh c kt hp t dc n thng dng l tinh bt.

nh gi c tc dng bo v vi sinh vt ca alginate khi s dng

lm t dc n cho dng thuc nang cng: trong iu kin tip xc vi mi

trng acid pH 1,2 vi tc khuy 50 v/pht, mu nang cng vi alginate
lm t dc n cho t l VSV sng st cao hn 100 ln so vi mu nang
cng vi tinh bt lm t dc n, v t l ny cao hn so vi mu thuc bt.

55

II.

XUT
Do thi gian c hn nn ti cha cp c ht cc vn c lin

quan, chng ti xut mt s hng nghin cu nh sau:

Nghin cu v n nh ca nang v thuc bt vi thi gian di ti 1

nm.

Nghin cu cc bin php kt hp t dc alginat vi cc t dc bo

v khc trong qu trnh to thuc bt v ng nang cng lm gia tng s

lng VSV sng st trong mi trng acid pH thp

Nghin cu mt s bin php bo ch khc ca t dc alginat vi

VSV nh phng php vi nang ha, phng php bao pellet,

56

TI LIU THAM KHO

Ti liu ting Vit
1. B mn Cng nghip Dc, Trng i hc Dc H Ni (2009), K thut sn
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