nghiên cứu vai trò của alginat trong quá trình tạo nguyên liệu probiotics chứa...
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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 NGUYNLIU PROBIOTICS CHALactobacil lus acidophi lus
LUN VN THC S DC HC
H NI 2013
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B GIO DC V O TO B Y T
TRNG I HC DC H NI
NINH TH KIM THU
NGHIN CU VAI TR CA ALGINATTRONG QU TRNH TO NGUYN
LIU PROBIOTICS CHALactobacil lus acidophi lus
CHUYN NGNH CNG NGH DC PHM V BO CHM S60720402
Ngi hng dn khoa hc:
1. TS. m Thanh Xun
2. ThS. Nguyn Th Trinh Lan
H NI 2013
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MC LC
DANH MC Trang
T VN 1
Chng 1: TNG QUAN 2
1.1. i cng v probiotics 2
1.1.1. Khi nim 2
1.1.2. Cc vi sinh vt c s dng trong ch phm probiotics 2
1.1.3. C ch tc dng 4
1.1.3.1. C chtc dng 5
1.1.3.2. ng dng caprobiotics 5
1.2. Tnh hnh nghin cu v sdng probiotics trn thgii v Vit
Nam
5
1.2.1. Tnh hnh nghin cu sn xut v s dng cc ch phm
probiotics trn thgii
7
1.2.2. Tnh hnh nghin cu sn xut v s dng cc ch phm probiotics
Vit Nam
7
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.
Alginat 12
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
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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.
Nang cng 19
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
21
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 cu22
2.1.3. Cc dung dch v hn dch s dng trong nghin cu22
2.1.4. Thit b22
2.2. Phng php nghin cu 23
2.2.1. Phng php nhn ging 232.2.2.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
2.2.8.Phng php xc nh s lng VSV theo nguyn tc pha long
in tc
26
2.2.9.Phng php nh gi nh hng ca alginate n khnng sng 27
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st ca Lactobacillus acidophilus trong qu trnh ng thuc bt
2.2.10.Phng php nh gi nh hng ca alginate n kh nng
sng st ca Lactobacillus acidophilus trong qu trnh ng thuc nang
cng
28
Chng 3. THC NGHIM, KT QU V BN LUN 30
3.1. Nghin cu nh hng ca alginate n khnng 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 khnng 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
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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 khunMT : 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
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DANH MC CC BNG
STT Tn bng Trang
Bng 1.1 Cc th h bo ch ca ch phm cha probiotics 7
Bng 1.2 Mt s ch phm probiotics trn th trng Vit Nam 9
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
dc bo v ti thi im ngay sau khi ng kh
31
Bng 3.2 Bin thin hm m (%) ca mt s mu ng kh L.
acidophilus vi t dc bo v khc nhau theo thi gian
34
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
hp sa gy vi alginate cc nng
40
Bng 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.
41
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
trng acid HCl pH 1,2 vi tc khuy 50 v/pht
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
46
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DANH MC CC HNH V BIU
STT Tn bng 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-
mannuronic
12
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
ng kh v trong thi gian bo qun.
34
Hnh 3.3 Hnh nh mu ng kh vi alginate ngay sau khi tho khi
my v sau 3 pht ngoi mi trng.
35
Hnh 3.4 th biu din s lng vi khun L. acidophilus sng st
trong cc mu sau ng kh
38
Hnh 3.5 Hm m ca cc mu ng kh vi t dc bo v l hn
hp sa gy vi alginate cc nng
40
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.
42
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
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
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LI CM N
Vi s knh trng v long bit n su sc ti xingi li c n n TS. m
Thanh Xunv ThS. Nguyn Th Trinh Lan,nhng ngi th tn tnh hng n
v ch bo cho ti t nhng bc u tin cho n khi ti hon thin lunvn n
ng thi, ti xin gi li c n tiDs. L Ngc Khnhcng cc th c gio,
cc anh ch k thut vin trong b n Cng Nghip Dc to i iu kin gip
ti trong sut qu trnh thc hin ti, ch bo ti trong thi gian l thc nghi.
hn p n ti cng xin gi li c n n an gi hiu cng ton th cc
th c gio trng i hc c i v to i iu kin thun li cho
ti trong thi gian ti hc tp ti trng
V cui cng l li c n ti gi ti M ca ti, gia nh, ngi thn v bn b
ng vin, gip ti trong sut qu trnh hc tp v hon thnh lunvntt nghip
o thi gian l thc nghi cng nh kin thc ca bn thn c hn, kha lun
n cn c nhiu thiu st i rt ong nhn c s gp ca cc th c, bn b
kha lun c hon thin hn
i xin chn thnh c n
i, ng 30, thng , n
cvin
s inh hKim Thu
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T VN
Probiotics thuc nhm cc vi sinh vt sng khng th thiu i vi sckhe con ngi.Nhng vi sinhvt nya vo c th khi cn sng vimt
lng y s c li cho sc khe ca vtch.Chng thngc 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 dobt 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 hinvi 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 a alginat o q o y l
pobos Lactobacil lus acidophil us.
Vi cc mc tiu:
alginat
.
alginat
http://vi.wikipedia.org/w/index.php?title=K%C3%BD_ch%E1%BB%A7&action=edit&redlink=1http://vi.wikipedia.org/w/index.php?title=K%C3%BD_ch%E1%BB%A7&action=edit&redlink=1http://vi.wikipedia.org/w/index.php?title=K%C3%BD_ch%E1%BB%A7&action=edit&redlink=1http://vi.wikipedia.org/w/index.php?title=K%C3%BD_ch%E1%BB%A7&action=edit&redlink=1http://vi.wikipedia.org/w/index.php?title=K%C3%BD_ch%E1%BB%A7&action=edit&redlink=1 -
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C 1. TNG QUAN
1.1. probiotics
1.1.1.
Khi nim
Probiotics bt ngun tting Hy Lp c ngha l ssng.Khi nim
probiotics u tin xut pht t nh khoa hc Eli Metchnikoff, trong cun
sch Ko di ssng 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 ngprobiotics c gii thiu ln u tin vo nm 1953 biKollath. Probiotics c nh ngha l cc yu tc ngun gc tvi khun,
kch thch spht 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 bsung mt svi sinh vt sng c ch cho vt chnhm cn bng hvi 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 chphm cha tbo vi sinh vt hay nhng thnh phn
ca tbo vi sinh vt m nh hng c li cho sc kho. [23], [55]
Nm 2002, Tchc Y t thgii (WHO) v Tchc 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 sdng 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 sdng trong chphm probiotics
Probiotics c chia lm 3 nhm chnh sau [6]:
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- Nhm vi khun lactic (chim phn ln): gm 2 chi ph bin l chi
Lactobacillus vBifidobacterium.
- Nhm khng phi vi khun lactic:+ ChiPropionibacterium: P. freudenreichii, P. cyclohexanicum,...
+ ChiBacillus: B. subtilis, B. clausii,...
+ ChiBrevibacillus: B. laterosporus,
+ Chi Sporolactobacillus: S. laevolacticus,
+ ChiEscherichia: E. coli,...
- Nhm nm men: chyu 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 khnng bm dnh vo nim mc ng tiu ha ca vt ch
Chu c pH thp ddy v acid mt rut non
Khng sinh c tv khng gy bnh cho vt ch
C khnng sinh enzym, cht khng sinh hoc cc sn phm cui cng
m vt chc thsdng c
C khnng sng v c tr trong rut
Dnui cy v c khnng tn ti c lp trong mt thi gian di
L. acidophilusl trc khun chim tlchyu trong scc vi sinh vt c
ch c tr on trn ca ng tiu ha. N c khnng lm gim s lng
cc vi sinh vt hoc nm c hi rut non; c khnng 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 hthng min dch; gim stiu dit vi sinh vt c ch do sdng
khng sinh di ngy.[6]
Vi cc c im trn,L. acidophilusl vi sinh vt c la chn u
tin sn xut cc chphm probiotics.
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1.1.3.C tc dng ca probiotics
1.1.3.1. C tc dng [3],[4],[6],[7]
- Cnh tranh nng lng, v tr bm:Probiotics cnh tranh cht dinhdng 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 sdi chuyn ca cc vi khun v khng nguyn trut vo
mch mu. Chc nng ny gip gim snhim khun v dng i vi cc
khng nguyn c trong thc phm.
- Sn sinh ra cc cht c ch:Mt sprobiotics 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 spht 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 nhbutyrat v acid butyric c khnng
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 thv gim cholesterol. Probiotics sn sinh ra cc enzym
khc phc qu trnh tiu hy carbohydrate v lm thun tin hn qu trnh
rut hp thnng lng tcc cht dinh dng. Probiotics cng ng thi ln
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men nhng carbohydrat khng tiu ha c trong rut non v sn xut ra
vitamin B, K.
1.1.3.2. ng dng ca probiotics:Probiotics c ng dng:
Trong cc bnh tiu ha [16], [20], [27]
- Tng khnng tiu ha lactose v hot ng ca cc enzym khc
- Htrtrong iu trtiu chy do sdng khng sinh
- Tc dng lnHelicobacterpylori
Tng cng min dnh [22], [39]- Kch thch min dch nim mc
- Tng cng min dch, gim phn ng dng
- 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 cu v sdng probiotics trn thgii v Vit Nam
1.2.1. Tnh hnh nghin cu sn xut vsdng cc chphm probioti cs
trn thgii
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 phttrin 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 Ruminococcusv Streptococcus. Bng k thut gen,
cc nh nghin cu ch ra rng ch c khong 20 n 50% s loi vi sinh
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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 cim 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 cngcha 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 nrut 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],
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[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 T x m V 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 thh
bo ch nh sau: [6]
B 1.1: C bo pm pobos
T m VSV m N m
T 1
(Non-
coated)
VSV sng (trong
sa chua, phomat,
kim chi, )
Gi r s dng
nhiu.
VSV gn nh khng
sng st khi qua d dy
v dch mt.
Bo t VSV. Thun li cho nui
cy, sn xut, lu
hnh.
Mt nhiu thi gian
pht trin thnh VSV
chm tc dng, hn ch
kh nng cnh tranh vi
VSV gy bnh.
Probiotics ng
kh.
T l sng nhit
thng cao hn
T l VSV b cht vn
cao.
T 2
Enteric-
coated
VSV a vo di
dng vin nn,
vin nang c lp
bao ngoi tan
trong rut.
Bo v VSV khi i
qua d dy v dch
mt.
Mt thi gian mng bao
tan r gii phng
VSV.
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T 3
Micro-
encapsulated
Dng bao vi nang. Hon thin tng
ca th h 2.
Cha khc phc c
nhc im ca th h
2: kh nng phng thchti rut cha tt, tan
chm trong rut.
T 4
Dual
coated
Lp bn trong l
protein, lp ngoi
l polysaccharid.
VSV c t l sng
cao. Chu c
acid dch v v
mui mt. Lp bao
bn trong c tnh
thc y phng
thch VSV ti ch.
i hi cng ngh cao,
k thut bo ch hin
i, trang thit b t
tin.
Gi thnh cao.
Mt sc sc 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 schphm quen thuc trn thtrng nh
Enzym biosub,Biosubtyl DL, Viabiovit, Vivac bio, Healthy liver. Tuy nhin,
nguyn liu chyu trong cc chphm 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 vtBacillus 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 thgii. Ngoi vic sn
xut nguyn liu probiotics cin trong nc nh nguyn liu cha chng vi
sinh vtBacillus subtilis, ANABIO cn c thm nguyn liu cha cc chng
vi sinh vt lactic. Do vy, thtrng chphm probiotics khng chln vs
lng m cn a dng vchng loi vi sinh vt, ng thi gi thnh sn phm
cc chphm probiotics c hnhiu ln, ngi sdng c thsdng
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sn phm cht lng cao m gi thnh li r. Tuy nhin, sn phm y vn
chyu thuc thh1 nn khnng bo vVSV vn cn rt thp (Bng 1.2.)
Bng 1.2:Mt schphm probiotics trn thtrngVit Nam
STT T
B d T p d bo N s x
1 1 L-Bio-3D 300mg vi khun: L. acidophilus,
B. longum, L. rhamnosus
Me-Auspharm
Vit Nam
2 1 Antibio Gi bt ung 1g cha 108 cfu L.
acidophilus
OrganonM
3 1 Biosubtyl
LD
Gi 1g cha 107 - 108vi khunB.
subtilisvL. acidophilus
Vin vaccin
LtVit
Nam
4 1 Biolacto Vin nang cha t nht 10 vi
khunL. acidophilusvL. bul
garicus
UnionM
5 1 Lactomed Vin nang 320 mg cha:
L. bifidus2 106vi khun
L. acidophilus2 106vi khun
Streptococcus faecalis2 106vi
khun
II Dong - Hn
Quc
6 1 Ultraflore Vin nang 0,2 g cha 2 109nm
men Saccharomyces cerevisiae
SobioPhp
7 1 Antibio
-philus
Vin nang cha 2 1082 109
vi khunL. acidophilus
Lyocentre
Php
8 1 Probio Gi bt ng kh cha 109 L. Imexpharm
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acidophilus Vit Nam
9 2 Abiiogran Thuc cm, 1g cha 10 cfu L.
acidophilus
Dae Han New
PharmHnQuc
10 3 Lactomin
plus
Hn hp Probiotics bao vi nang
chaL. acidophilus, B. longum, L.
rhamnosus 300 mg
Mebipharm-
Austrapharm
Vit Nam
11 4 Laclean
gold
Bt cm pha hndchchaLactic
cid Bacteria1,25 x 109,
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
aecium(100 t cfu/g)
Cell-biotech
Hn Quc
12 4 Kidlac Bt cm pha hndch ung:
Lactic Acid Bacteria 0,5 x 109,L.
acidophilus (100 t cfu/g),B.
longum (50 t cfu/g),B. breve (50
t cfu/g), Enterococcus faecium
(100 t cfu/g)
Cell-biotech
Hn Quc
1.3. Lactobaci ll us acidophi lus
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 t0,60,9 1,56,0 m, mc n hoc i hoc
to chui ngn, khng c bo t, khng di ng, kkh khng bt buc, phn
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ng catalase m tnh, pht trin ti u 370C, khng pht trin 20220C
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, L-
arabinose, D-ribose, D-xylose. [6], [12]
1.3.3. Cc dng chphm probiotics cha L. acidophil us
Trn th trng dc phm hin nay, cc chphm cha L. acidophilus
chyu l dc phm v thc phm chc nngbo chdi dng bt, cm,
nang cng.
Khc vi chphm khc, chphm probiotics phi duy tr slng VSV
sng st nht nh trong thi hn sdng 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 chphm thng thng.
V thnh phn, chphm probiotics thng c thm cc t dc bo v
nh cht x, ng,
Vphng 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 quv c s dng nhiu l phng
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php ng kh.Phng php ng kh gip cho VSV n nh v gip VSV
c tlsng st cao trong thi gian bo qun. [17]
1.4.
Alginate1.4.1.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 LGuluronic Acid DMannuronic
Hnh 1.2:Cng thc cu to ca acid - L-guluronic v acid - D-
mannuronic
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 alginatebin i trong mt di rng.
Mui natri ca acid alginic dht 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].
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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
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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 sTrong 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 snghin cu gny v vinang 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 109cfu/g, mu khng c bo v u c s lng sngst
sau 1h, 2h trong iu kin pH 1 gim xung cn khong 105 106 cfu/g (gim
khong 1000 ln), cn viphng 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 106cfu/g (ngha l gim khong 10 ln), sau 2h trong iu kin
acid pH 1, s lng VSV ca mu bao vi nang cn 105cfu/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 vtgim t 1,7 x
109cfu/g xung cn khong 1,9 x 107cfu/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 cukhc licho thy victo nang cng vicc 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
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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 9,0x10
9
, sau khi th trongmi 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 pp k
ngkh cnh 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 o
Tin ngl giai on u tin ca qu trnh ng kh, thng c tin
hnh nhit -700C n -800C. giai on ny phnln 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 o o
giai on ny vt c t trong bung sy, tin hnh ht chn khng.
Nc cchuyn trc tip t trng thi rn sang trng thihi m khng
qua giai on lng. Trong giai on ny nhit v psutbn trong bung
sy c duy tr mcdi nhit ng bng. [17], [11]
Giai o lm k
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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 trongbung sy lc ny cng cao hn nhit giai on thng hoa. [11]
1.5.1. m
ngkh c rt nhiuu 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 phpng 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 nnphng phpng 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
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i hnh thi t nhin ca lipid mng v s thay i cu trcca cc protein
nhy cm l nguyn nhn chnh gy ra s suy gim s lng tbo 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 trongqu 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,), nhmchng 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 ngkh c tm
tt trong bng 1.3[17]:
B 3:C ch bo v ca t dc trong qu trnh ng kh
STT Nhm C bo
1 Carbohydrate Thay th lin kt hydro trong lp mng t bo
lipid kp
2 Polymer Lm gim t l lin kt vi nc
3 Protein Cung cp hm lng acid amin, enzym phn gii
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protein thnh acid amin, vitamin, lm tng kh
nng m
4 Polyols Thay th nc bay hi t mng t bo lipid kp5 Amino acid n nh protein
6 Chng oxy ha Dn gc t do
7 Mt s t dc bo
v khc
Tng hot tnh enzym, thay th lin kt hydro
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 lprotein vi t
dc in hnh: sa gy.
S y
Sa gy hay cn gi l skim milk l cc sn phm sa m trongthnh 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% chot 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 phndinh 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.
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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 ica 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 cng
1.6.1. m dng thuc nang cng
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 trnhmt phn tc ng bt li ca ngoi mi nh m, nh sng
do vy lm tng t lsng st ca vi sinh vt. Thnh phn t dc chyu
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 sthnh
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 vvi sinh vt bn vng qua acid ddy. in hnh l
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nhm cht x Fructooligosaccharide, Vegetable cream, inulin,l nhm t
dc hay c sdng nht.
1.6.2 ng nang cngMun 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 mkhc
nhau cn phi nh gi cht lng nang cng da trn cc chtiu cht lng.
Mt schtiu cht lng ca nang cng cn nh gi l: [2]
ng u vhm lng
ng u vkhi lng
r: nang cng phi r trong vng 30 pht
Trc nghim ha tan: Thha tan gip nh gi tc gii phng
dc cht, gip cho cc nh sn sut la chn, sng lc cng thc v cc
thng skthut trong qu trnh sn xut, ng thi y cng l mt chtiu
nh gi cht lng sn phm.
Cc th nghim khc: Xc nh hm lng dc cht, mc ng
nht vhm lng dc cht, tin hnh theo nh chdn trong cc chuyn
lun cthca Dc in i vi tng loi chphm cth.
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C 2. NGUYN LIU, THIT B
V PHNG PHP NGHIN CU
2.1. Ny l b2.1.1.y
Chng vi sinh vt:Lactobacillus acidophilusATCC 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 d o
T N 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
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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 C b d o
T b N
T cy v trng(Sanyo) Nht
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T m CO2 (Sanyo) Nht
T lnh su c
T lnh LG Hn Quc
Thit b ng kh c
My li tm c
Ni hp tit khun ALP -Nht
Cn phn tch (Satorious) c
Cn k thut(Satorious) c
My o hm mPressica Thy in
My khuy tWisd Hn Quc
My lc (Vortex) Trung Quc
Hthng thha tan ERWEKA DT 60 c
a petri, ng nghim, u cn,
2.2. P pp
2.2.1.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 115
o
C / 20 phtrong 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].
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2.2.2.P y
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 trongcc 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 CO25% 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 CO25% (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
tikhi mu ng rn hon ton.Tin ng trong thi gian 24h.
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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 baopolyme 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 pp k Tydll
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 pp o b 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 pp o nang cng cha 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 khtrn ng lng to thnh khi ng nht. Sau em
ng thnhnang cngbng my to nang th cng.
Bo qun mu trong l nt kn nhit phng.
2.2.7.P pp 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
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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 pp x s l VSV eo y c pha longlin
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 phalong 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.
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Mu xc nhs lngVSVsng st trong iu kin mi trng pHthp:
+ 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.
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2.2.9.P pp ng ca alginate n kh sng
st ca L . acidophiluso q thuc bt
Nui cy vi sinh vt trong bnh nn cha 200ml mi trng MRS lngtrong 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 trnng lng
vi hn dchsa 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 liubt ng kh theophng 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 CO25%, 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 pp ng ca alginate n kh
sng st ca L. acidophilustrong qu trnh cng
Sdng my thha tan ERWEKA DT60 nh gi slngL.
acidophiluscn sng sau khi thtrong iu kin mi trng acid pH 1,2
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iu kin th:
- Thit b: giquay.
-Tc quay: 501 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. embt ng khng 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.
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C 3. THC NGHIM, KT QUV BN LUN
3.1. Nghin cu ng ca alginate n kh sng st ca vi
khun L. acidophiluso q k
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 tinhnh nhmnh 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 phmt 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 chna 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: 108cfu/g
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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. y
Mt trong nhng vai tr quan trng ca t dc s dng trong qu
trnh ng kh l gi cho ch phm cth cht n nh, trnh b ht mtrong
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 alginateu c s dng vi nng 2%[15], [21].
Do vy, t dc alginate c kho st trong nghin cu nycng c s
dngvi 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 chtca 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
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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
tm
Sn phm thu c l 1 lp rt mng, dnh a. Rt kh
ly ra ht khi dng ccha
Sa gy
10%
Th cht ng nht, kh, xp, d lm ti, d dng ly ra khi
dng c cha
Alginate
2%
Th cht ng nht, nh, xp, kt thnh mng, rt kh lm nh.
D ly ra khi dng c cha nu thc hin thao tc ny trckhi mu b ht m
Nhn xt:
Khi khng dng t dc n, cc mu ng kh L. acidophilusc
to thnh l mtlp 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.
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Cc mu ng khL. acidophilusvi 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 chtxp hn nhng li bkt thnh mng, nhanhb 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 gias lng
VSV probiotics cc l m trong qu trnh bo ch to sn phm tip theo.
3.1.2. m m m
y alginate
Hm m l mt ch sgy 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 chunhm 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 quntrong 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 ca2 mu c th cht t ti xp, lmu ng kh vi sa gy10%
v mu ng kh vi alginate 2%.
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Mc tiu:Xc nh hm m ca cc mu ngay sau khi ng khv tc
ht m ca cc mu trongthi gian bo qun.
Kt qu: Hm m ca cc mu c th hin trong bng 3.2.
B 3.Bin thinhm m (%) ca mt s mu ng kh L. acidophilus
vi t dc bo v khc nhau theo thi gian
T bo qHm m (%)
M s y
10%
M
natri alginate 2%
Ny s k 2,8 + 0,3 6,5 + 1,6
Sau 2 ngy 3,4 + 0,7 10,8 + 2,9
S 1 3,5 + 0,7 12,5 + 3,3
S 2 3,5 + 0,6 13,3 + 3,0
To : Mu cha sa gy 10%: mu 3 trong mc 3.1.1
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Mu cha alginate2%: mu 4 trong mc 3.1.1
Hnh 3.2: th biu th hm m trung bnh cacc mu ngay sau ng kh
v trong thi gian bo qun.
Nhn xt:
Kt qu a ra bng 3.2cho 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 gythay i khng ng k v kt qu o hm m ca mu vn t gi tr
nh hn 5%. Trong khi , mu ng khL. acidophilusvi 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.
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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 chahoc chy nc.
Tm li, ngay sau ng kh v trong qu trnhbo qun, mu ng kh
viNatri 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 bov 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 trongng 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 sovi 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 mus dng t dc alginate sau qu trnh ng
kh.
Mc ch
nh gi nh hng cadung dch alginate 2% ln s lng v t l
sng st ca vi sinh vt trong qu trnh ng kh, ng thiso snh vi mu
ng kh khng s dng t dc v mu ng kh s dng hn dch sa
gy10%.
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Kt qu v nhn xt
S lng vi sinh vt Lactobacillus acidophilusc trong 1ml dch nui
cy trc ng kh l khong 1,09 x 10
9
VSV/ 1ml.
B 33S 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 s M 1
()
M 2
(dh ln men)
M 3
(s y 10%)
M 4
(alginate 2%)
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
Trung
bnh2,57 x 106 8,03 x 105 5,6 x 108 2,07 x 107
RSD 0,31 x 106 0,42 x 105 1,32 x 108 0,32 x 107
B 34:T l VSV sng st sau ng kh so vi mu ban u
M 1
()
M 2
(d l me)
M 3
(s y 10%)
M 4
(alginate 2%)
T l VSV
sng st0,24% 0,07% 51,38% 1,9%
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Trong : Mu 1: ng khL. acidophilusvi dch li tm.
Mu 2: ng khL. acidophilusvi nc ct.
Mu 3: ng khL. acidophilusvi sa gy10%.
Mu 4: ng khL. acidophilusvi alginate 2%.
Hnh 3.4. th biu din s lng vi khun L. acidophilus sngst
trong cc mu sau ng kh
Kt qu trongbng 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 dcdng
ph bin l sa gy10%. Tuy vy, s liu cng cho thyalginate 2% c tc
dng bo v khi s dng lm t dc ng kh vi t l sng st ca mu ny
cao gp 27,1ln 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 ngkh vi t dc l alginate 2% cho t l sng st
caL. 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
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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 y alginate y
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 khL. 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 kth 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 cavi sinh vt ca mu s dng tdc
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
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B 35:Hm m ca cc mu ng kh vi t dc bo v l hn hp sa
gy vi alginate cc nng
T ianHm m (%)
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
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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 liuto 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 acidophilussng stsau 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 gy10% 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 mung kh khng c s khc nhau nhiu (c 4 mu
u cha khong 109VSV/1g).
S lng VSV sng st c th hin trong bng sau:
B 36. 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 s ( x108 )
0%
alginate
1%
alginate
2%
alginate
5%
alginate
Ln 1 8,51
14,50 29,40 23,92Ln 2 21,89 9,05 34,52 27,84
Ln 3 20,42 29,91 4,90 1,29
Trung
bnh20,2 21,2 26,1 24,3
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Hnh 3.6:Slng 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 thnghim 3.1.3 ta thy tc dng bo v vi sinh vt ca alginate nh hn kh
nhiu so vi sa gy (khong 27ln). Do vy khi kt hp alginate vi sa gy
to hn hp t dc khi ng kh ta s khng thy c s thayi 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 cu ng ca alginate n kh sng st ca vi
khun Lactobacil lus acidophil uso q thuc b
nang cng
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Cc kt qu nghin cu nu trong mc 3.1.2cho 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 cnnhiu hn ch v sn phm to thnh c th cht v hm m khng thch hp.
Do vy ccth nghimtip theo c thit knhm 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 alginate k s s
kL. acidophiluso q b
Mc tiu:
Nghin cu nh hng caalginate n kh nng sng st ca vikhun
L. acidophilusc 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 37ln 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 btv
mukhng s dng t dc (mubt ng kh)cho s lng VSV sng st
khong 105 n 106.
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B 37. S lng vi sinh vt sng st tnh trn 1g bt ng kh
trong mi trng acid HCl pH 1,2
S l s
B Sau 1h Sau 2h
M M b M M M b M
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
SD 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
Trong : Mu a: Bt ng khL. acidophilusvi 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
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Hai mu b v a cho trong cc ln cng th nghim th cho s lng
VSV sng st ngang nhau, t 4,8x105cfu/g sau 1h v 2,9x105sau 2h. iu
ny chng t vic b sung thm t dc n l tinh bt khng h gip cithin 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 caL. acidophilus ATCC 4653.
3.2.2.N alginate k s s
kL. acidophiluso q o
Mc tiu:Nghin cu nh hng ca alginate n kh nng sng st
ca vi khunL. acidophilus trong qu trnh tonang 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 s
B Sau 15 pht Sau 30 pht Sau 1h Sau 2h
M 1 M 2 M 1 M 2 M 1 M 2 M 1 M
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 hinkhun lc
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Bng 3.9:Tlvi 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 1 120
T l VSV sng sau 30 pht 1 115
T l VSV sng sau 1h 1 240
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 bngcho 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 nghimny, t l VSV
sng st ca mu s dng alginate cao gp hn100 lnso vi mu s dng
tinh bt.Ngay sau 15 pht, s lng VSV sng st ca mu nang cha tinh
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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 3,7x10
4
, trong khi mu nang tinhbt 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 phpci thin khc, lm gia
tng t l sng st ca VSV trong mi trng acid thp.
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C 4: BN LUN
4.1.
Nghin cu ng ca alginate n kh sng st ca vikhun L. acidophiluso q k
4.1.1. c ngy
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 trnhhp 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 CaCl2bngphng php phun
sy c kch thc phn b ng u t 10 40 m v hnh dng trn u p.
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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 alginatelm 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 gias lng
VSV probiotics cc l m trong qu trnh bo ch to sn phm tip theo.
4.1.2. m m m
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 sungvi 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
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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 caL. acidophilussau ng kh cho thy
khi s dng cht bo v lhn 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 Minhv 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 stca 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 trcpolyme nn s trng n trong nc, to ra
c xung quanh v bn trong t boVSV 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 trongsa gy c th to nn mt lp o bo v
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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
caL. 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 nh in
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 y
alginate y
Khi kt hp sa gyvi 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 scng thc hin nghin cunm 2012
[19] v phi hp cc t dc bo v trong qu trnh ng kh, lnh gi
tc dng bo vca 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. LngB. 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
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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 nhmt ln na tc dng bo v VSV vt tri ca sa gy trong qu trnh ng
kh.
4.2. Nghin cu ng ca alginate n kh sng st ca vi
khun L. acidophilustrong qu trnh thuc b cng
4.2.1.N alginate k s s
k L. acidophiluso q o b
Theo kt qu nghin cu cho thy alginate c tc dng bo v VSV tt
hn so vi tinh bt trong mi trngpH 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 probioticsbng phng php to vi nang vi alginate. Vi nguyn liu
ban u cha khong 109cfu/g, mu khng c bo v u c s lng sng
st sau 1h, 2h trong iu kin pH 1 gim xung cn khong 10 5 106 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
acidophilusLA1 gim t 107 cfu/g lc ban u xung cn khong 106cfu/g
(ngha l gim khong 10 ln), sau 2h trong iu kin acid pH 1, s lng
VSV ca mu bao vi nang cn 105cfu/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
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trng acid HCl pH 1,2 s lng vi sinh vt gim t 1,7 x 109cfu/g xung
cn khong 1,9 x 107cfu/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 10
7
cfu/g (gim khong155 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 chot 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 caL. acidophilus ATCC 4653.
4.2.2.N alginate k s s
k L. acidophiluso q o
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.
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Kt quny 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.
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KT LUN V XUT
I. KT LUN
ti thu c cc kt qu sau:1. alginate k s s
k L. acidophiluso q k:
Khi s dngdung dch natri alginate 2% lm t dc bo v trong qu
trnh ng kh lm tng t lsng st caL. acidophilus so vi mu ng
kh vi nc ct (khong 27ln).
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 lsa
gy. Nhng t l sng st li khng c thy s khc bit ln gia cc mu , t
khong 109cfu/g.
2. nghin cu ng ca alginate n kh sng st ca vi
khun L. acidophiluso q thuc bt v thuc nang cng
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.
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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 vn 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,
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