Nghiên cứu xử lý sinh khối Bacillus clausii để thu bào tử.pdf

B Y T

TRNG I HC DC H NI

V TH NGC BCH

NGHIN CU X L SINH KHI

Bacillus clausii THU BO T

KHA LUN TT NGHIP DC S

H NI - 2013

B Y T

TRNG I HC DC H NI

V TH NGC BCH

NGHIN CU X L SINH KHI Bacillus clausii THU BO T

KHA LUN TT NGHIP DC S

Ngi hng dn:

TS. m Thanh Xun

Ni thc hin:

D

H NI - 2013

LI CM N

Vi tt c s knh trng, ti xin gi li cm n chn thnh v su sc

ti c gio TS. m Thanh Xun Ging vin b mn Cng nghip Dc

Trng i hc Dc H Ni, ngi lun ng vin, tn tnh hng dn,

gip ti trong sut qu trnh thc hin ti.

Ti xin chn thnh cm n DS. L Ngc Khnh, cng ton th cc thy

c gio v cc anh ch k thut vin b mn Cng nghip Dc to iu

kin, gip ti trong sut thi gian nghin cu hon thnh kha lun.

Ti cng xin cm n Ban gim hiu, Phng o to, cc thy c gio

trng i hc Dc H Ni du dt ti trong sut qu trnh hc tp v rn

luyn ti trng.

Cui cng, ti xin by t lng bit n su sc ti gia nh, bn b

lun bn, ng vin v gip ti trong cuc sng v hc tp.

H Ni, ngy 20 thng 5 nm 2013

Sinh vin

V Th Ngc Bch

MC LC Li cm n Mc lc Cc ch vit tt Danh mc cc bng Danh mc cc hnh t vn .........1 Chng 1: TNG QUAN 1.1. i cng v probiotics 1.1.1. nh ngha .........2 1.1.2. Lch s pht trin ca probiotics ........2 1.1.3. Vai tr ca probiotics .........2 1.1.4. Cc ch phm probiotics trn th trng ........4 1.2. Bacillus clausii

1.2.1. Bacillus clausii .......5 1.2.2. Bo t Bacillus clausii ....7 1.3. S hnh thnh bo t v cc phng php thu bo t 1.3.1. S hnh thnh bo t .....10 1.3.2. Sc khng ca bo t ...11 1.3.3. Cc phng php gii phng ni bo t ...11 Chng 2: NGUYN LIU, HA CHT, THIT B PHNG PHP NGHIN CU 2.1. Nguyn liu, ha cht, thit b 2.1.1. Nguyn liu, ha cht ...........14 2.1.2. My mc, thit b ..........15 2.1.3. Mi trng s dng ..........16 2.2. Ni dung nghin cu 2.2.1. La chn cc phng php x l sinh khi Bacillus clausii to nguyn liu cha bo t .....16

qu trnh bo qun .........17 2.3. Phng php nghin cu 2.3.1. Hot ha ging t ch phm Enterogermina ........17 2.3.2. Gi ging trn thch nghing .......17 2.3.3. Chun b dch nhn ging .....17 2.3.4. Phng php thu bo t ....18 2.3.5. Phng php m s lng bo t cn sng trong mt lng sn phm thu c theo nguyn tc pha long lin tc ..........20 2.3.6. Kim tra kh nng bo t b ny mm tr li trong iu kin bo qun

.........................................................................................................................21

Chng 3: THC NGHIM KT QU V BN LUN 3.1. La chn cc phng php x l sinh khi Bacillus clausii to nguyn liu cha bo t 3.1.1. X l nhit sinh khi t bo to nguyn liu th...22 3.1.2. Gii phng ni bo t t nguyn liu th bng phng php vt l (phng php siu m) ...23 3.1.3. Gii phng ni bo t t nguyn liu th bng phng php ha hc .24 3.1.4. S dng tc nhn sinh hc (enzyme lysozyme) .......29

trong qu trnh bo qun

2SO4 10% (80C)..32 3.2.2. Kho st kh nng bo t b ny mm tr li trong qu trnh bo qun .........................................................................................................................34

KT LUN V KIN NGH.......36 Ti liu tham kho

DANH MC CC CH VIT TT

AAD Antibiotic Associated Diarrhea Bnh tiu chy lin quan

n khng sinh

ADN Acid Deoxyribo Nucleic

ATTC American Type Culture Bo tng ging vi sinh vt

M

B. clausii

Bacillus clausii

B. subtilis

Bacillus subtilis

B. coagulans

Bacillus coagulans

CFU

Colony Forming Units

S n v khun lc

E. coli Escherichia coli

EDTA

Ethylen Diamine Tetra Acetic acid

IBD

Inflammatory Bowel Diseases Bnh vim rut cp

LAB

Lactic Acid Bacteria Nhm vi khun lactic

SDS Sodium Dodecin Sulfate

DANH MC CC BNG

Bng 2.1: Cc ha cht s dng trong nghin cu.

Bng 2.2: Cc my mc thit b s dng trong nghin cu.

3.3: 2SO4

.

Bng 3.2: Kt qu m s lng bo t sng st sau qu trnh x l bng 2

tc nhn l H2SO4 10% (80C) v lysozyme.

DANH MC CC HNH V

Hnh 1.1: VSL#3 l hn hp gm 1 loi Streptococcus, 3 loi

Bifidobacterium v 4 loi Lactobacillus.

Hnh 1.2: Sn phm Yakult ni ting ca Yakult Nht Bn c cng b

c cha L. casei Shirota.

Hnh 1.3: Sn phm Biosubtyl-II ca cng ty vacxin v sinh phm s 2,

Nha Trang, Vit Nam c ng gi di dng gi bt ung cha bo t B.

subtilis.

Hnh 1.4: Bo t B. clausii.

Hnh 1.5: Bo t B. coagulans.

Hnh 1.6: Mt ct ngang ca 1 bo t Bacillus subtili.

Hnh 1.7: Ch phm Erceflora ca cng ty Sanofi Aventis c ng gi

2 t bo t trong 5ml. Sn phm c lu hnh Philippines.

Hnh 1.8: Ch phm Probacin ca cng ty INPHARM, s.r.o., R cha 5 t

bo t B. clausii / 10ml. Sn phm c lu hnh ti cng ha Sec.

Hnh 1.9: Ch phm Enterogermina ca cng ty Sanofi Aventis.

Hnh 1.10: S hnh thnh bo t.

Hnh 3.1: Hnh nh tiu bn ca nguyn liu th.

Hnh 3.2: Hnh nh sau x l bng NaOH 20% 80C trong 80 pht.

Hnh 3.3: Hnh nh tiu bn sau x l sinh khi vi H2SO4 10% 80C

trong 80 pht.

Hnh 3.4: Hnh nh tiu bn ca sn phm x l bng lysozyme t l 40ml

lysozyme (1mg/ml)/1g nguyn liu th trong 120 pht.

Hnh 3.5: Biu th hin s tng quan v % sinh khi sau x l so vi

nguyn liu ban u ca 3 phng php NaOH, H2SO4 10% (80C) v

lysozyme.

1

Bacillus

. Trong nhm

cc Bacillus c s dng lm probiotics, chi B. clausii

.

B. clausii

.

, v

.

B. clausii :

1. La chn cc phng php x l sinh khi Bacillus clausii to

nguyn liu cha bo t.

2. Kho st a bo t sau x l y mm trong

qu trnh bo qun.

2

CHNG 1: TNG QUAN

1.1. i cng v probiotics:

1.1.1. nh ngha:

Thut ng probiotics l mt thut ng c dng ch cc vi khun

c li cho con ngi v ng vt [32].

1.1.2. Lch s pht trin ca probiotics:

Cc vi khun c li cho c th con ngi ln lt c pht hin ra t

hn mt th k trc. Nhm cc vi khun lactic (LAB) c Elie

Metchnikoff (1845 1916) pht hin ra khi ng nghin cu mi lin quan

gia ch n c s dng sa ln men vi i sng khe mnh ca ngi

nng dn Bulgari. Cng thi gian , ln u tin Bifidobacterium c bc

s nhi khoa ngi Php Henry Tissier - phn lp t phn ca nhng a tr s

sinh c b m.

Chi Bacillus c s dng trong cc ch phm sinh hc trong 1 thi

gian di vi ch phm c bit n nhiu nht l Enterogermina (c ng

k ti Italy nm 1958) [30].

Hin nay, probiotics c a vo cc ch phm di nhiu dng:

thuc, thc phm, thc phm chc nng. Ngoi chi Lactobacillus v

Bifidobacterium l cc probiotics c s dng ph bin v lu i th

Enterococcus, Bacillus, Streptococcus v Pediococcus cng ang c ng

dng rng ri [11].

1.1.3. Vai tr ca probiotics:

iu tr tiu chy do cc nguyn nhn:

- Do s dng khng sinh (AAD): Mt nghin cu ti M nm 2006

ch ra rng: Saccharomyces boulardii, Lactobacillus rhamnosus GG, v hn

3

hp ca 2 loi trn lm gim ng k s pht trin ca bnh tiu chy lin

quan n khng sinh.

- Do tc nhn gy nhim trng: Phn ln cc trng hp tiu chy cp

tr em do rotavirus gy ra, v mt nghin cu nm 2000 cho thy rng

vic s dng Lactobacillus v / hoc Bifidobacterium lm gim thi gian tiu

chy tr em di 36 thng tui t 30 - 40% [28].

- Do x tr ung th: Tnh trng tiu chy do x tr bnh nhn ung th

c gim ng k sau khi s dng mt ch phm ng kh gm 1 loi

Streptococcus, 3 loi Bifidobacterium v 4 loi Lactobacillus. [15].

- Do khng dung np lactose: Nhng ngi khng dung np lactose

(do thiu enzym lactase), sau khi tiu th mt lng lactose ln (khong

18g) s gp tnh trng tiu chy, y hi, bun nn. Tuy nhin, tnh trng ny

s gim ng k nu nhng ngi ny s dng ch phm sa ln men, v

trong sa ln men, nng lactose gim i rt nhiu (trung bnh l 30%), v

trong sa ln men cn cha mt enzym lactase c ngun gc t cc vi sinh

vt ln men gip tiu ha lactose [16].

iu tr bnh vim rut (IBD):

IBD thng c bit n l bnh nhim trng ng rut mn tnh, c

cc t ti pht cp tnh. Cc nghin cu lm sng chng minh hiu qu ca

probiotics trong vic duy tr s thuyn gim ca IBD c cng b [12].

iu tr cc bnh ngoi ng rut:

- Phng nga v trong 1 s trng hp c th iu tr cc nhim khun

ng niu v nhim khun m o [18].

- Can thip tch cc vo cc bnh d ng, v d nh bnh chm d ng

ph n mang thai v tr s sinh [19].

4

- Lm gim mc cholesterol trong mu nhng ngi mc chng bo

ph, tiu ng, tim mch, tuy nhin mc gim ny khng nhiu nh cc ch

phm statin [21].

- Gim nguy c si thn do nh hng n s bi tit oxalate trong nc

tiu, do l gim s hnh thnh si [17].

1.1.4. Cc ch phm probiotics trn th trng:

C khong hn 30 loi vi sinh vt thuc 6 chi (Lactobacillus,

Bifidobacterium, Enterococcus, Streptococcus, Pediococcus, Bacillus) ang

c s dng lm ch phm probiotics dng n l hoc phi hp

Hnh 1.1: VSL#3 l hn hp gm 1 loi Streptococcus, 3 loi

Bifidobacterium v 4 loi Lactobacillus.

Cc ch phm probiotics cng c nhiu dng bo ch khc nhau. Hnh

thc ph bin nht l cc sn phm t sa v cc loi thc phm c b sung

cc vi sinh vt c li. Bn cnh l cc ch phm bo ch di dng vin

nang, gi bt ung, cm, hn dch ung... Trong cc dng bo ch ny vi sinh

vt c th c bao di cc dng khc nhau (bao tan trong rut, vi nang ha,

bao kp) trnh c tc ng bt li ca acid dch v v mui mt. Tuy

nhin nu s dng vi sinh vt di dng bo t th khng cn bao v bn thn

dng bo t c kh nng khng vi acid dch v v mui mt n rut

non v ny mm gn nh nguyn vn ti [4].

5

Hnh 1.2: Sn phm Yakult ni

ting ca Yakult Nht Bn c

cng b c cha L. casei Shirota.

Hnh 1.3: Sn phm Biosubtyl-II

ca cng ty vacxin v sinh phm s

2, Nha Trang, Vit Nam c ng

gi di dng gi bt ung cha

bo t B. subtilis.

Liu lng ca cc ch phm probiotics cng rt khc nhau ty thuc

vo tng chng vi sinh vt v tng ch phm, nhng hu ht cc ch phm

u cung cp mt liu lng vi sinh vt trong phm vi 1- 10 t CFU/liu,

nhng mt s sn phm chng minh c hiu qu liu thp hn, v d

nh Bifidobacterium infantis 35624 t hiu qu iu tr vi liu 100 triu

CFU/ngy, trong khi sn phm VSL#3 cha n 450 t CFU/liu [30].

Hin nay, trn th trng c dng ch phm synbiotics l s kt hp ca

probiotics v prebiotics. Prebiotics l nhng cht hu nh khng b tiu ha

bi h tiu ha ca vt ch, c tc dng h tr s pht trin ca probiotics. S

phi hp ny s to nn mt hiu ng tng hp trong vic iu tr [30].

1.2. Bacillus clausii:

1.2.1. Bacillus clausii:

c im phn loi:

Theo phn loi ca Bergey (1994) B. clausii thuc:

6

B: Eubacteriales

H: Bacillaceae

Chi: Bacillus

Loi: Bacillus clausii

c im phn b:

Bacillus clausii phn b nhiu ni nhng ch yu l trong t, c th l

t vn (B. clausii KSM- K16, B. clausii DSM 8716, ATCC 31084...), hoc

t st (B. clausii DSM 9784). Ngoi ra c th tm thy B.clausii trong

nc (B. clausii MB9 c phn lp t mu nc vng ven bin pha ng

n ), trong bn [24].

c im hnh thi

Bacillus clausii c hnh que ng n l hoc kt thnh chui, l vi khun

Gram dng, c kh nng di ng, hnh thnh bo t c hnh bu dc.

B. clausii c kch thc chiu rng 1-2m, chiu di 5m [34].

iu kin nui cy

- Nhu cu oxy: B. clausii l vi khun hiu kh do trong qu trnh nui

cy cn c s cp kh.

- V nhit : nhit nui cy thch hp l t 15 500C. Mi chng c

nhit ti u ring nh: chng B. clausii DSM 8716 c nhit ti u l

300C, chng B. clausii KSM-K16 c nhit ti u l 400C, B. clausii ATCC

31084 c nhit ti u l 370C[36].

- V pH: khong pH nui cy thch hp l t 7 10,5. Chng B. clausii

KSM-K16 c pH ti u l 9,0 [36].

- V dinh dng, B. clausii c th s dng nhiu ngun cacbohydrat

khc nhau nh: glucose, galactose, mannose, sorbitol, 2- ketogluconat...;

ngun nit, phospho, cc nguyn t vi lng.

7

c im sinh ha

Vi khun B. clausii c kh nng thy phn c casein, gelatin v tinh bt,

nhng khng thy phn c Tween 20, 40 hoc 60. Cho phn ng oxidase,

catalase dng tnh, kh nitrate thnh nitrit [25].

1.2.2. Bo t Bacillus clausii:

c im bo t:

Hnh 1.4: Bo t B. clausii [32]. Hnh 1.5: Bo t B. coagulans.

Bo t Bacillus clausii cng mang nhng c im chung ca bo t:

- Bo t l dng sng tim sinh ca vi khun. N c nhng c im c

bit gip vi khun tn ti trong nhng iu kin khc nghit v ny mm

tr li khi gp iu kin thun li. Mi vi khun ch c th hnh thnh mt

bo t [2].

- Cu to:[22]

o ADN nm trong li bo t.

o V bo v bao quanh li bo t.

o o bo t trong mng.

o o bo t ngoi dy.

8

Hnh 1.6: Mt ct ngang ca 1 bo t Bacillus subtilis [22].

u im ca bo t khi s dng lm probiotics:

Bo t c kh nng khng cao vi iu kin khc nghit ca mi

trng v s ny mm tr thnh dng hot ng khi iu kin mi trng

thun li nn s c mt s u im ni tri so vi dng khng simh bo t

nh Lactobacillus spp.:

- Trong qu trnh sn xut, cc iu kin nh sy, ng kh khng lm

nh hng ti s lng v cht lng ca bo t.

- Trong qu trnh bo qun, lu hnh, ch phm probiotic t bo t t chu

nh hng ca iu kin mi trng nn c n nh cao.

-

n rut non v ny mm ti

m bo hiu qu iu tr.

- Tm li, v nguyn tc, mt liu lng xc nh ca bo t c th c

bo qun v thi hn m khng cn lm lnh, v ton b liu vi khun vo

ng tiu ha s t mc nguyn vn rut non [13].

Ch phm trn th trng:

Trn th gii, bo t Bacillus clausii c ch bi nhng u im ca

n v c ng dng ch yu trong iu tr tiu chy tr em:

Lp o ngoi Lp o trong V bo t Li bo t ADN

9

Hnh 1.7: Ch phm Erceflora ca cng ty Sanofi Aventis c ng gi

2t bo t trong 5ml. Sn phm c lu hnh Philippines [37].

Hnh 1.8: Ch phm Probacin ca cng ty INPHARM, s.r.o., R cha 5 t

bo t B. clausii / 10ml. Sn phm c lu hnh ti cng ha Sec [35].

th trng Vit Nam, ch phm cha Bacillus clausii c nhc n

kh nhiu l Enterogermina. Enterogermina l mt ch phm probiotics c tc

dng hu hiu trong iu tr tiu chy tr em. Enterogermina cha bo t vi

khun B. clausii di 2 dng: dng huyn dch trong ng nha (2 t bo t

trong 5ml) v dng vin nang (2 t bo t trong 1 vin nang). Enterogermina

c ch nh trong cc trng hp:

- iu tr v phng nga ri lon khun ch ng rut v bnh l km

hp thu vitamin ni sinh.

- iu tr h tr phc hi h khun ch ng rut b nh hng khi

dng thuc khng sinh hoc ha tr liu.

10

- Ri lon tiu ha cp v mn tnh tr em do nhim c hoc ri lon

khun ch ng rut v km hp thu vitamin.

B. clausii c kh nng khng khng sinh, do khi s dng khng sinh

nn ung ch phm xen k vi khong thi gian dng khng sinh ngn

nga cc tc dng khng mong mun ca khng sinh (gy lon khun rut).

Ch phm Enterogermina cha 4 loi B. clausii, mi loi khng mt s

loi khng sinh nht nh: O/C khng chloramphenicol, N/R khng

novobiocin v rifampicin, T khng tetracycline, SIN khng streptomycin v

neomycin [38].

Hnh 1.9: Ch phm Enterogermina ca cng ty Sanofi Aventis.

1.3. S hnh thnh bo t v cc phng php thu bo t:

1.3.1. S hnh thnh bo t

Hnh 1.10: S hnh thnh bo t [2].

1 n

2 0

n 7

n 5

n

6

n

4

n 3

11

Pha 1: Trong t bo sinh dng, ADN c phn chia thnh chromosome

ring bit.

Pha 2: Mng t bo cht ln su vo phn chia t bo hnh thnh 2 phn

khng u nhau.

Pha 3: Phn nhn bo t mang ADN ln su vo t bo cht.

Pha 4: V bo t c hnh thnh.

Pha 5: o bo t c hnh thnh.

Pha 6: T bo m ly gii gii phng bo t.

Qu trnh hnh thnh bo t mt khong 6 - 8h, l qu trnh tng i

phc tp v khng th o ngc to ra c cu trc bn vng chu c

iu kin khc nghit .

Khi gp iu kin thun li v nhit , m, dinh dng, cp kh

bo t s ny mm tr thnh t bo sinh dng [2].

1.3.2. Sc khng ca bo t:

Bo t vi khun c sc khng cao i vi cc iu kin bt li:

- khng vi nhit cao: v nc trong bo t phn ln trng thi

lin kt, nn khng c kh nng gy bin tnh protein. Hn na, trong bo t,

protein tn ti di dng lin kt vi canxi dipicolinat thnh mt phc cht c

tnh n nh cao vi nhit .

- khng vi cc cht ha hc (acid, kim), cc cht st khun: v

cu trc v bo t gm nhiu lp, t tnh thm thu nn cc cht ha hc kh

c th tc ng n t bo [2].

1.3.3. Cc phng php gii phng ni bo t:

12

Trong khi quy trnh gii phng enzym ni bo hay vt cht di truyn

c nghin cu kh nhiu trong v ngoi nc th quy trnh gii phng v

thu ni bo t vn cn t c bit n.

Tuy nhin, vn c mt nguyn tc chung cho c ba vn ny l: mun

thu c cc phn t ni bo, vic u tin l phi ph v v t bo.

C ba phng php chnh gii phng phn t ni bo khi vi sinh

vt l phng php vt l, ha hc v enzym. Tuy nhin, khng phi tt c

cc k thut u thch hp trn quy m ln. quy m sn xut cng nghip,

ngi ta thng gp kh khn trong vic thit k cng sut cn thit cho th

tch ln v loi b nhit c sinh ra trong qu trnh ph v t bo.

:

- Ph v t bo bng phng php s dng sng siu m: Phng

php ny ch thch hp quy m phng thi nghim..

- Ph v t bo bng my ng ha cao p:

Loi my c s dng ph bin ph v t bo l Manton - Gaulin

APV. Dch huyn ph t bo c y vi p sut v vn tc cao qua 1 van

x c ming hp. Cc t bo s b v v gii phng cc cht bn trong khi lm

gim p sut sau van. Phng php ny thch hp ph v cc vi khun n

bo, khng thch hp vi cc loi sng thnh tp on dng si.

- Ph v t bo bng cch nghin hoc khuy vi cc bt, ht thy

tinh hoc thp:

Dch huyn ph t bo c lc cng vi ht thy tinh hoc thp nh (d

= 0,2- 1,0 mm) th t bo s b ph v bng lc ct do s va chm vi cc ht

ny.

- Ph v t bo bng phng php lnh ng:

13

Huyn ph t bo di dng bt nho ri em lm lnh -20C, sau

em nn di p sut cao qua cc l hp ca my nn Hughes. T bo s b

ph v do s thay i pha v thay i th tch cng nh lc ct ca cc tinh

th .

Phng php ha hc:

- X l kim:

Mi trng kim (pH = 11,6- 12,5) s lm thy phn mng t bo. X

l bng kim c s dng thnh cng trong tch chit quy m nh v

ln cc protein vi khun. V d, enzym tr liu, L-asparaginase, c th c

gii phng khi Erwinia chrysanthemi bng cch t bo pH =11,0-12,5

trong 20 pht.

- S dng cht ty ra:

Trong iu kin pH, lc ion v nhit nht nh, cc cht ty ra nh

natrilaurylsulfat, tween 20, triton s t hp vi lipoprotein mng to ra cc

mixen lm suy yu mng t bo.

Phng php enzym:

Lysozyme thy phn peptidoglucan c trn thnh t bo vi khun, lm

suy yu thnh t bo, t ph v t bo. Mc d qu trnh thao tc n gin

v nh nhng, nhng k thut ny t c s dng cho quy m cng nghip,

c l do gi thnh tng i cao ca lysozyme v kh nng a vo cc tc

nhn gy nhim bn [9], [10].

14

CHNG 2: NGUYN LIU, HA CHT, THIT B, NI

DUNG V PHNG PHP NGHIN CU

2.1. Nguyn liu, ha cht, thit b

2.1.1. Nguyn liu, ha cht

Cc ha cht s dng:

Bng 2.1: Cc ha cht s dng trong nghin cu

Ha cht Xut x

Ha cht s dng pha mi trng

Natri clorid Trung Quc

Pepton Trung Quc

Cao tht Trung Quc

Thch bt Vit Nam

Ha cht s dng thu bo t

Lysozyme Vin kim nghim

Na2EDTA Trung Quc

H2SO4 98% Trung Quc

NaOH Trung Quc

KH2PO4 Trung Quc

KCl Trung Quc

Natrilaurylsulphat (SDS) Trung Quc

Cc dung dch s dng trong ti:

- Dung dch m phosphate pH 7,6:

Pha dung dch KH2PO4 0,2M (500ml): 0,20,5136=13,6 g

Pha dung dch NaOH 0,2M (500ml): 0,20,540=4,0 g

15

Trn 50ml dung dch KH2PO4 0,2M vi 42,8ml dung dch NaOH 0,2M ri

thm nc va 200ml.

- Dung dch KCl 1M: Ha tan 7,45g KCl trong va 100ml nc.

- Dung dch NaCl 1M: Ha tan 5,84g NaCl trong va 100ml nc.

- Dung dch H2SO4 10%: 6ml H2SO4 c vo khong 50ml nc ,

ch tan ht ri thm nc va 100ml.

- Dung dch NaOH cc nng :

Dung dch NaOH 20%: Ha tan 20g NaOH trong va 100ml nc

ui CO2.

Dung dch NaOH 10%: Thm nc ct va 100ml vo 50ml dung dch

NaOH 20%.

Dung dch NaOH 0,4%: Thm nc va 100ml vo 4ml NaOH 10%.

- Dung dch SDS 3%: Ha tan 3g SDS trong va 100ml nc ct.

- Dung dch EDTA pH 8: Ha tan 18,61g Na2EDTA trong khong 80ml

nc ct, thm NaOH 10% vo iu chnh pH v 8,0. Thm nc ct va

100ml.

- Dung dch lysozym1mg/ml: ha tan 0,100g lysozymtrong va

100ml nc ct tit trng.

2.1.2. My mc, thit b

Bng 2.2: Cc my mc thit b s dng trong nghin cu

Thit b Xut x

T cy Bioair (Italy)

My ly tm Rotofix (c)

Ni hp ALP (Nht)

T lnh Toshiba (Nht)

16

T m Memmert (c)

T sy Memmert (c)

T lc Bioshake (c)

My vortex IKA (c)

L vi sng Daewoo (Hn Quc)

Ni cch thy Trung Quc

Branson (USA)

Knh hin vi Labomed M

Cn k thut Sartorius (c)

Cn phn tch Sartorius (c)

Bnh ht m Trung Quc

Pipet, ng nghim, u cn, bnh nn, cc c m, a thy tinh, ng ly

tm, giy lc...

2.1.3. Mi trng s dng

Mi trng canh thang (MT 1)

Natri clorid 0,5g

Cao tht 0,5g

Pepton 1g

Nc my v 100ml

Mi trng thch thng (MT 2) = MT 1 + 1,8g thch

Mi trng thch khng (MT 3)

Thch bt 2,0g

Nc ct v 100ml

17

2.2. Ni dung nghin cu

2.2.1. La chn cc phng php x l sinh khi Bacillus clausii to

nguyn liu cha bo t.

2.2.2. Kho st l y mm

trong qu trnh bo qun.

2.3. Phng php nghin cu

2.3.1. Hot ha ging t ch phm Enterogermina:

Pha 100 ml mi trng canh thang (MT1) trong bnh nn dung tch 250

ml, y bng nt bng khng thm nc, hp tit trng iu kin 1atm

trong 20 pht, ngui. Tin hnh cy ging B. clausii t ch phm vo bnh

nn trong t cy v trng. Nui cy trong my lc 37C, 110 vng/pht

trong 24h.

Sau 24h, vi khun pht trin lm c mi trng.

2.3.2. Gi ging trn thch nghing:

Pha mi trng canh thang thch (MT 2), un si cho ng nht cc

thnh phn trong mi trng, chia ra cc ng nghim, mi ng 6 ml, nt kn,

hp tit trng 1 atm trong 20 pht. ngui bt ri t nghing. Dng que

cy v trng, cy ging trong bnh hot ha ging ln thch nghing theo

hnh ziczac trong t cy v trng, trong t m 37C, trong khong 24h. Sau

khi khun lc mc th ct ging vo t lnh.

nh k 2 thng cy truyn ging nhm gi hot tnh vi khun.

2.3.3. Chun b dch nhn ging:

Chun b 100ml mi trng MT1 trong bnh nn dung tch 250ml. Hp

tit trng 1atm trong 20 pht. ngui, dng que cy v trng cy khun

lc trong ng nghim cha ging vo mi trng. trong my lc 110

vng/pht 370C trong 24h.

18

2.3.4. Phng php thu bo t:

a. To nguyn liu th cha bo t (c dng t do v dng cn nm

trong t bo) [3]:

Chun b 4 n 5 bnh nn c dung tch 250ml, mi bnh cha 100ml

mi trng MT1, hp tit trng 1atm trong 20 pht, ngui. Cy 10ml

dch nhn ging vo mi bnh, em lc 370C, 110 vng/pht trong my lc

trong 4 ngy. Tin hnh x l nhit dch nui cy bng cch un cch thy

100C trong 1 gi.

Ly tm dch sau x l nhit 4000 vng/pht trong 15 pht, thu cn.

Phn tn u cn trong nc ct tit trng, vortex k ra sch mi

trng nui cy, ri ly tm (4000 vng/pht trong 15 pht) thu c nguyn

liu th cha bo t.

b. Giai on gii phng ni bo t:

Nguyn liu th thu c sau s l nhit dch nui cy gm c t bo sinh

dng non cht, t bo cha ni bo t v bo t t do c gii phng

[3]. V vy, cn phi s dng cc tc nhn khc nhau ph v dng sinh

dng, gii phng ni bo t.

Gii phng ni bo t bng phng php ha hc:

Ly khong 0,5g nguyn liu th chun b mc a., phn tn u

trong 30ml dung dch ha cht:

- H2SO4 10% 2 iu kin: nhit phng (30C) v 80C trong 80

pht.

- NaOH 0,4%, 10%, 20% 80C trong 80 pht.

- SDS 3% [7] 80C trong 80 pht.

- EDTA pH8 [5] 2 iu kin: nhit phng (30C) v 80C trong

80 pht.

19

Sau thi gian , tn hnh x l loi xc t bo sinh dng, lm tiu bn

theo Ogieska [8] nh gi kh nng gii phng ni bo t ca cc phng

php ha hc cc iu kin khc nhau.

Gii phng ni bo t bng phng php vt l: (phng php siu

m)

Ly khong 0,5g nguyn liu th vo bnh nn 100ml, phn tn u

trong 30ml nc ct. em dch phn tn siu m b 40

KHz [10], sau cc khong thi gian khc nhau ly mu, lm tiu bn theo

Ogieska [8] nh gi kh nng gii phng ni bo t ca phng php siu

m.

Gii phng ni bo t bng phng php sinh hc: (s dng enzym

lysozyme)

Ly khong 0,5g nguyn liu th, phn tn trong 10ml dung dch

m phosphat pH 7,6, thm dung dch lysozym(1 mg/ml), 37C [6], sau

khong thi gian khc nhau, ly mu lm tiu bn theo Ogieska xc nh

thi im cc t bo sinh dng b ph v hon ton.

c. Thu bo t:

- Dch x l trn c em ly tm thu cn (4000 vng/pht trong 20

pht).

- Phn tn cn trong nc ct tit trng, vortex k ra sch ha

cht, ri ly tm thu cn (4000 vng/pht trong 20 pht).

Chun b cc cc dung dch KCl 1M, NaCl 1M, v nc ct trong cc bnh

nn to, hp tit trng 1 atm trong 20 pht, ngui.

- Ha cn trong 30ml dung dch KCl 1M loi v t bo, ly tm dch

thu cn (4000 vng/pht trong 10 - 15 pht).

- Tip tc ha cn trong 30ml dung dch NaCl 1M loi hon ton

v t bo, ly tm dch thu cn (4000 vng/pht trong 10- 15 pht).

20

- Thu cn, ra bng nc ct tit trng, lm 3 ln thu c bo t [3].

- Hiu qu x l c nh gi bng cch: lm tiu bn theo Ogieska

[8] ri soi di knh hin vi quang hc ( phng i 1000 ln, vt knh du),

phng php m s lng vi sinh vt theo nguyn tc pha long lin tc v

phng php cn tnh t l gia khi lng sinh khi sau x l so vi

lng nguyn liu th ban u. Cng thc tnh:

2.3.5. Phng php m s lng bo t cn sng trong mt lng sn

phm thu c theo nguyn tc pha long lin tc

Chun b bnh nn dung tch 250ml cha chnh xc 100ml nc ct.

Cc ng nghim mi ng cha chnh xc 9ml nc ct. Hai bnh nn cha

mi trng thch thng (MT2) v mi trng thch khng (MT3). Hp tit

trng 1 atm trong 20 pht.

mi trng thch thng chun b trn vo cc a petri ( tit

trng 1 atm trong 20 pht), dn u, ngui.

Cho khi lng sinh khi cn m s lng vi khun vo bnh nn

cha 100ml nc ct hp tit trng, ngui trn, lc cho phn tn u.

Ht chnh xc 1ml dch cha vi khun bnh nn pha long vo ng nghim

th nht, lc u, sau t ng nghim th nht li ht chnh xc 1ml cho

vo ng nghim th 2 Lm nh vy cho ti ng nghim cui cng cn pha

long.

Nh 1ml dch cha vi khun cn kho st a petri mi trng

thch thng trn, dng que trang dn u. mi trng thch khng

dinh dng ngui n khong 45C ln trn, nui cy trong t m 37C

trong 24 gi.

Cng thc tnh s lng vi khun

21

X= 10.a.k

Trong :

a: s lng lc khun trung bnh m c mi nng pha long.

k: pha long.

X: s lng vi khun cn sng trong lng sinh khi em m.

2.3.6. Kim tra kh nng bo t b ny mm tr li trong iu kin bo

qun

Bo t thu c sau qu trnh x l gii phng ni bo t c bo

qun 2 iu kin sau:

- iu kin bo qun trong mi trng kh: sy bo t 60C trong

24 gi ri bo qun trong ti polymer, gi trong bnh ht

(28- 30C).

- iu kin bo qun trong mi trng lng: gi bo t trong nc ct

2 ln, bo qun iu kin phng.

Ti cc thi im khc nhau (1, 3, 5 thng) ly mu lm tiu bn theo

Ogieska [8], kim tra xem bo t c b ny mm hay khng, nu trn tiu

bn c hnh nh ca t bo sinh dng bt mu xanh th bo t b ny mm,

ngc li th kt lun bo t khng b ny mm. ng thi ly mu nui cy

tr li vo mi trng dinh dng thch hp xem bo t cn kh nng pht

trin trong iu kin thun li khng.

22

Chng 3: THC NGHIM, KT QU V BN LUN

3.1. La chn cc phng php x l sinh khi Bacillus clausii to

nguyn liu cha bo t:

Nh c sc khng ca mnh, bo t vi khun probiotics c th i qua

c hng ro bo v l acid dch v v mui mt ny mm nguyn vn

rut non.

Bacillus claussi c kh nng sinh ni bo t, vic s dng cc phng

php khc nhau l nhm ph b v t bo, gii phng ni bo t. Hn na,

xc t bo sinh dng khi b ph v cng d dng c loi b hn trong qu

trnh ra. Cc th nghim sau y thc hin ph b v t bo vi cc tc nhn

khc nhau nhm tm ra c phng php x l hiu qu nht.

3.1.1. X l nhit sinh khi t bo to nguyn liu th [3]:

Mc tiu:

To ra c sinh khi cha ch yu l bo t.

Tin hnh:

Nui Bacillus clausii iu kin 37C, lc 110 vng/pht. Sau 4 ngy,

dch nui cy c un cch thy 100C trong 1 gi. Sau ly tm dch

nui cy thu cn. Ra li sinh khi theo quy trnh nu

mc 2.3.4 (c).

Kt qu:

Hnh nh trn tiu bn cho thy: trong sinh khi tn ti ng thi 3 dng l

t bo sinh dng bt mu xanh, bo t t do bt mu v bo t mu

nm trong t bo sinh dng (cha c gii phng). Trong

chim a phn l bo t t do v ni bo t cha c gii phng.

23

Hnh 3.1: Hnh nh tiu bn ca nguyn liu th.

Nhn xt, :

Kt qu thu c cho thy, sau 4 ngy nui cy v c x l nhit th

trong sinh khi cha ch yu l bo t. Kt qu ny ph hp vi kt qu

nghin cu ca tc gi Nguyn Th Hin [3]. Sau 4 ngy nui cy th cht

dinh dng trong dch ln men gim nhiu to iu kin thun li cho t

bo sinh dng chuyn thnh dng bo t. Di tc dng ca nhit cao

(100C trong 1 gi), t bo sinh dng cn non cht i

Bacillus clausii 55C [36]), t bo sinh dng gi chuyn thnh dng

bo t. V th, trong sinh khi thu c sau qu trnh ny c c 3 dng: t bo

sinh dng cht, bo t t do v ni bo t vn nm trong t bo sinh

dng.

thu c nguyn liu cha bo t tinh khit, cn phi ph v t bo

sinh dng nhm 2 mc ch l gii phng ni bo t ra khi t bo sinh

dng v loi c hon ton dng sinh dng ra khi sinh khi cha bo t

(v khi b ph v, cc t bo sinh dng d dng b loi b cng dch ni trong

qu trnh ra).

.

3.1.2. Gii phng ni bo t t nguyn liu th bng phng php vt l

(phng php siu m):

24

:

.

Tin hnh:

Phn tn 0,5g nguyn liu th trong 30ml nc ct tit trng, em siu m

40 KHz.

C 1 gi ly mu 1 ln, lm tiu bn theo phng php Ogieska kim

tra kh nng gii phng ni bo t ca phng php siu m.

Kt qu:

Kt qu s dng tc nhn l sng siu m gii phng ni bo t sau cc

khong thi gian t 1- 6 gi cho thy: ch n thi gian 5 gi th t bo sinh

dng mi bt u b ph v, th hin hnh nh trn tiu bn bt u thy

xut hin cc mnh bt mu xanh m, c th y l cc mnh v t bo, c

nhiu bo t t do bt mu , t l t bo sinh dng gim, bt mu km,

hnh nh nh (c th do tc dng ca vic siu m trong thi gian di).

Nhn xt, :

Vi thi gian siu m trong 5 gi l thi gian di,

hiu qu x l khng hon ton, qu trnh siu m cn

sinh nhit, m thanh nh hng n thnh gic ca con ngi

. iu ny

ph hp vi l thuyt v vic s dng phng php siu m trong vic ph v

v t bo: siu m l phng php ph v v t bo n gin, thun tin

nhng ch thch hp quy m phng th nghim, rt kh ng dng quy m

cng nghip [10].

3.1.3. Gii phng ni bo t t nguyn liu th bng phng php ha hc:

:

25

c tc nhn ha hc l SDS, EDTA, NaOH, H2SO4

Bacillus clausii .

a. S dng tc nhn l SDS 3%:

Trn c s kt qu nghin cu ca nhm tc gi ca vin Sinh hc nhit

i trong vic ph v t bo Lactobacillus spp. [7].

Tin hnh:

Ly 0,5 g nguyn liu th 2.3.4

(a), vi 30ml dung dch SDS 3% 80C trong 80 pht. Ra li

.

Kt qu:

Vic s dng SDS 3% 80C trong 80 pht x l sinh khi cha

bo t Bacillus clausii khng c hiu qu tch bo t khi v t bo, trn tiu

bn vn c c 3 dng tn ti ca B. clausii l dng t bo sinh dng, bo t

t do v ni bo t vn cn nm trong t bo sinh dng.

Nhn xt, :

Mc d cng l chng Gram (+) nhng c th bn cht thnh t bo 2 loi

Lactobacillus v Bacillus c th c s khc bit, dn n s khc bit v hiu

qu x l. , theo l thuyt, SDS ph v v t bo vi khun bng cch

ko cc phn t lipid ra khi cu trc thnh t bo, lm suy yu thnh t bo,

t gii phng cc thnh phn ni bo nn phng php ny s th hin

hiu qu trong vic ph v t bo vi khun Gram (-) hn l vi khun Gram (+)

[10].

b. S dng EDTA pH 8:

26

p dng phng php ph v t bo E. coli tch chit plasmid ca tc

gi Mai Phng Hoa [5].

Tin hnh:

Ly 0,5 g nguyn liu th 2.3.4

(a), vi 30ml dung dch EDTA pH 8 :

80C trong 80 pht.

theo quy trnh nu mc 2.3.4 (c).

Kt qu:

S dng EDTA pH 8 c iu kin khc nhau l nhit phng v

80C u khng cho hiu qu gii phng ni bo t. Trn tiu bn vn nhn r

3 dng tn ti ca B. clausii dng sinh dng bt mu xanh cn nhiu.

Nhn xt, n:

Kt qu ny khc so vi kt qu ph v t bo E. coli thu plasmid ca

tc gi hc bit c th do cu trc

khc nhau ca 2 i tng th nghim: E. coli l trc khun Gram (-) cn

Bacillus l trc khun Gram (+), thnh t bo ca chng khc nhau ch yu l

v thnh phn peptidoglycan v acid techoic: trong khi thnh vi khun Gram

(+) cha t l ln 2 thnh phn ny th thnh vi khun Gram (-) cha t

peptidoglycan hn v khng cha acid techoic [2].

c. S dng tc nhn l NaOH:

Da trn c s vic gii phng enzym ni bo L-asparaginase ra khi

Erwinia chrysanthemi bng cch t bo pH = 11,0-12,5 trong 20 pht

[10]. Th nghim c tin hnh vi NaOH cc nng 0,4%, 10%, 20%.

Tin hnh:

Ly 0,5 g nguyn liu th 2.3.4

(a), vi 30ml dung dch NaOH c nhau (0,4%, 10%, 20%)

27

80C trong 80 pht.

mc 2.3.4 (c) ri cn lng sinh khi sau x l.

Kt qu:

S dng NaOH cc nng khc nhau trong cng 1 iu kin th

nghim l trong 80 pht 80C u khng hiu qu trong vic gii phng

ni bo t.

NaOH 0,4% v 10% hu nh khng c tc dng ph v v t bo Bacillus

clausii gii phng ni bo t. Trn tiu bn vn nhn thy r 3 dng tn ti

ca B. clausii, dng sinh dng bt mu xanh cn nhiu.

NaOH 20% lm cc thnh phn c trong sinh khi t bo b kt dnh thnh

tng m bt mu xanh trn tiu bn.

Hnh 3.1: Hnh nh sau x l bng NaOH 20% 80C trong 80 pht.

Kt qu cn sinh khi sau x l:

: 0,50g.

l: 0,07g.

T l % gia sinh khi sau x l so vi sinh khi trc x l:

Nhn xt, :

28

Kt qu khc so vi kt qu th nghim ph v

t bo Erwinia chrysanthemi gii phng enzym ni bo L-asparaginase.

iu ny c th do s khc nhau gia cu trc thnh t bo ca 2 i tng

th nghim: Erwinia chrysanthemi l vi khun Gram (-) v Bacillus l vi

khun Gram (+) (nh nu c th mc 3.1.2 (b)). Hn na, khi tng nng

NaOH ln qu cao (20%) th cc thnh phn ca t bo c th b bin tnh

gy kt dnh cc thnh phn c trong sinh khi gy cn tr qu trnh thu bo

t.

d. S dng tc nhn l H2SO4 10%:

Tin hnh:

Ly 0,5 g nguyn liu th 2.3.4

(a), vi 30ml dung dch H2SO4 10% : (30C

80C trong 80 pht. Ra li sinh khi, theo

quy trnh nu mc 2.3.4 (c) ri cn lng sinh khi sau x l.

Kt qu:

Vi iu kin thng, H2SO4 10% khng th hin tc dng ph v v t

bo.

Vi iu kin un cch thy 80C trong 80 pht, trn tiu bn nhn r hnh

nh bo t bt mu , t bo sinh dng gim v bt mu m nht.

29

Hnh 3.3: Hnh nh tiu bn sau x l sinh khi vi H2SO4 10%

80C trong 80 pht.

Kt qu cn sinh khi sau x l:

Khi lng tru : 0,50g.

: 0,21g

T l % gia sinh khi sau x l so vi sinh khi trc x l:

Nh :

Vi vic x l sinh khi bng H2SO4 10%, un cch thy 80C trong 80

pht, hnh nh trn tiu bn cho thy hiu qu x l

(siu m, SDS, EDTA, NaOH). Tuy nhin, lng

sinh khi sau khi ra cn li rt t (42% so vi lng sinh khi ban u) m

vn cn ln dng sinh dng. Theo tc gi Nguyn Th Hin [3], iu kin

nui cy tng t, lng bo t chim n trn 80% so vi khi lng sinh

khi ban u, chng t mt lng kh ln bo t 50%) b mt i

trong qu trnh x l bng H2SO4 10% v un nng 80C trong 80 pht.

3.1.4. S dng tc nhn sinh hc (enzym lysozym):

a. S dng lysozym vi t l 40ml lysozym (1mg/ml)/1g nguyn liu th:

p dng t l c s dng trong KLTN ca tc gi Nguyn Th Hin [3]

l 20ml lysozym (1mg/ml) cho khong 0,5 g sinh khi.

Tin hnh:

Ly 0,5 g nguyn liu th 2.3.4

(a), vi 20ml lysozym

30

. Ra li sinh khi,

n theo quy trnh nu mc 2.3.4 (c) ri cn lng bo t thu c.

Kt qu:

Sau 120 pht, hnh nh trn tiu bn cho thy r c rt nhiu mnh v t

bo, khng pht hin cn dng t bo sinh dng v t bo sinh dng cha

ni bo t. Sau khi ra, thu c hnh nh tiu bn ch c bo t tinh khit.

Hnh 3.4: Hnh nh trn tiu bn ca sn phm x l bng lysozym t l

40ml lysozym (1mg/ml)/1g nguyn liu th trong 120 pht.

Kt qu cn sinh khi sau x l:

c x l: 0,50g.

: 0,40g.

T l % gia sinh khi sau x l so vi sinh khi trc x l:

Nhn xt, :

lysozym .

s ( )

31

[3] enzym

.

S 3 :

lysozym 2SO4 80C v x l bng NaOH 10%.

3 2SO4

10% lysozym.

Khi lng

(g)

Khi lng

(g) k.l

NaOH 0,5 0,07 14% (++)

H2SO4 0,5 0,21 42% (+)

Lysozym 0,5 0,40 80% (-)

Hnh 3.5: Biu th hin s tng quan v % sinh khi sau x l so vi

nguyn liu ban u ca 3 phng php NaOH 10%, H2SO4 10% (80C) v

lysozym.

32

3.1 v th 3.5

lysozym l cao nht (80%), H2SO4

(14%). P ng H2SO4

phng cho tt nht trong cc phng php ha

hc v vt l nhng lysozym

(

kh ). Nh v , trong cc phng php x l tin

hnh, phng php sinh hc l hiu qu nht, sn phm thu c sch dng t

bo sinh dng, sinh khi thu c t t l cao so vi nguyn liu ban u.

Tuy nhin, gi thnh ca lysozym rt cao, s y cao chi ph sn xut. V

vy, cn tm ra lng lysozym thp nht cn hiu qu x l sch dng sinh

dng trong nguyn liu th cha bo t tit kim chi ph sn xut, gim

gi thnh sn phm.

b. S dng lysozym vi cc t l khc nhau:

Tin hnh:

Ly 0,5 g nguyn liu th 2.3.4

(a), vi 30ml dung dch lysozym 37C

trong 120 pht.

mc 2.3.4 (c).

lysozym t

: 40, 20, 15, 10ml.

Kt qu:

Vi cc th tch lysozym (1mg/ml)/1g sinh khi ln lt l 40, 20, 15ml,

hnh nh trn tiu bn ch nhn thy bo t tinh khit, khng pht hin dng t

bo sinh dng v dng t bo sinh dng cha ni bo t.

33

Vi 10ml lysozym (1mg/ml) dng cho 1g nguyn liu th th trn tiu bn

sau x l bt u thy xut hin dng sinh dng bt mu xanh nm ln vi

cc bo t bt mu .

Nh :

Kt qu trn cho thy, lng lysozym thp nht cn t hiu qu x l

gii phng ni bo t l 15ml lysozym (1mg/ml)/1g sinh khi t. T l ny

thp hn 2,6 ln so vi t l c s dng trong nghin cu ca tc gi

Nguyn Th Hin (40ml lysozym (1mg/ml)/1g sinh khi) [3]. iu ny c

ngha trong vic tit kim chi ph sn xut, gim gi thnh sn phm. Nu tip

tc gim t l lysozym xung cn 10ml lysozym (1mg/ml)/1g sinh khi t

iu kin 37C trong 120 pht th vn cn t bo sinh dng cha b ph

v, v vi cng 1 lng sinh khi, th tch lysozym gim xung lm tng

nng c cht, trong khi nng enzym gim xung lm gim kh nng

tip xc enzym c cht, lm gim hiu qu x l gii phng ni bo t ca

lysozym.

Tm li, kt qu ca phn 3.1 cho thy trong

, enzym lysozym

lysozym 15ml lysozym

t, 37C trong 120 pht enzym

.

lysozym . Tc nhn H2SO4

(10%, 80C trong 80 pht) l tc nhn gii phng ni bo t tt nht trong

cc tc nhn vt l, ha hc (lm gim dng t bo sinh dng, t l sinh khi

cn li l 42%). Mt khc, H2SO4 li l 1 tc nhn d kim, r tin, cht lng

n nh. Cho nn, vic s dng phng php ny gii phng ni bo t

Bacillus cn c nghin cu thm.

34

3.2. Kho st ca bo t sau x l v ny mm

trong qu trnh bo qun:

S

, sau khi thu

c dng bo t ca B. clausii, cn

mc bo t b ny mm

.

3.2.1.

lysozym 2SO4 10% (80C):

:

Mt yu cu quan trng ca bo t thu c sau qu trnh x l l phi c

t l sng cao.

(sn phm ch cn bo

t, t l sn phm thu c so vi nguyn liu ban u cao (80%))

enzym

. Th nghim ny c thc hin so snh

ca bo t thu c sau qu trnh x l

nhn lysozym 2SO4 10% (80C).

Tin hnh:

lysozym 2SO4

2.3.5.

Kt qu:

:

35

Bng 3.2: Kt qu m s lng bo t sng st sau qu trnh x l

bng 2 tc nhn l H2SO4 10% (80C) v lysozym.

(g) (cfu)

S

(cfu)

H2SO4

10%

0,21 1201010

5711010

Lysozym

1mg/ml

0,40 > 3001013

> 751014

Nhn xt:

Kt qu bng 3.2 cho thy t l bo t sng/1g sinh khi sau x l bng

phng php lysozym nhiu hn rt nhiu so vi phng php H2SO4 10%

(gp trn 1000 ln), thm vo , bo t thu c phng php lysozym

tinh khit hn bo t thu c phng php H2SO4 10% (80C). Do ,

cht lng bo t thu c sau x l bng phng php lysozym cao hn

nhiu cht lng bo t thu c sau phng php x l bng H2SO4 10%

(80C). Mt khc, khi lng bo t thu c sau khi x l cng 1 lng sinh

khi ban u (0,5g) ca phng php s dng lysozym l 0,4g cao gp 2 ln

ca phng php s dng H2SO4 10% 80C (0,21g). Nh vy, phng php

s dng lysozym x l sinh khi Bacillus clausii to nguyn liu cha bo

t l hiu qu nht trong cc phng php tin hnh c v cht lng v s

lng bo t.

3.2.2. Kho st kh nng bo t b ny mm tr li trong qu trnh bo

qun:

36

Trn th trng hin nay, cc ch phm cha bo t c bo ch di 2

dng l hn dch ung (trong nc) v ch phm kh (gm c dng bt ung,

vin nn, vin nang). Trong iu kin bo qun bo t c th ny mm tr li

lm gim cht lng ca nguyn liu cng nh ca ch phm cha bo t.

Nguyn nhn c th do cc tp cht cha c loi sch trong qu trnh x l.

Cc th nghim di y nhm xc nh nguyn liu bo t to ra c b ny

mm trong 2 iu kin bo qun thng thng l bo qun kh v bo qun

trong nc hay khng. Bn cnh tin hnh kim tra xem trong qu trnh

bo qun, bo t c cn kh nng ny mm trong iu kin thun li khng.

Tin hnh:

Bo t thu th nghi bng lysozym c chia thnh 2

phn. Bo qun 2 iu kin:

- Mt phn em sy kh 60C ri ct trong ti polymer,

bo qun trong bnh ht m.

- Mt phn bo qun trong nc ct hp tit trng, yn iu kin

nhit phng.

Ti cc thi im 1, 3, 5 thng, theo di mu sc v mi ca mu lu v

ly mu lm tiu bn theo Ogieska xem bo t c b ny mm trong iu kin

bo qun hay khng.

ng thi dng que cy v trng cy bo t vo mi trng dinh dng

hp tit trng, em nui cy trong my lc n nhit iu kin 37C, tc

lc 110 vng/pht trong 24 gi. Sau 24 gi, thu mu lm tiu bn, c kt

qu.

Kt qu:

Sau 5 thng th kt qu theo di vn khng c g thay i:

Cm quan:

37

- Trong mi trng lng, bo t mu trng ng, lng y l, phn nc

trn trong sut, khng mu, khng mi.

- Trong iu kin bo qun kh, nguyn liu bo t c mu nu nht,

kh, khng mi.

Hnh nh tiu bn:

c 2 mu lu, trn tiu bn ch c bo t mu

Kt qu nui cy:

Vi c 2 mu lu, bo t u pht trin lm c mi trng dinh

dng. Hnh nh trn tiu b nui cy l hnh nh trc khun

Gram (+).

Nhn xt:

Trong iu kin thi gian theo di l 5 thng th nguyn liu cha bo

t c to ra bng quy trnh x l vi tc nhn lysozym vn m bo c 2

yu t l: khng ny mm trong iu kin bo qun v ny mm trong iu

kin thun li.

Tm li, kt qu ca mc 3.2 cho thy

lysozym

t

trn 751014

bo t/1g sinh khi sau x l. , m

enzym lysozym

enzym lysozym

,

.

38

KT LUN V KIN NGH

1. Kt lun:

Trn c s kt qu cc th nghim tin hnh, ti t c mt s kt

lun sau:

1.1. La chn cc phng php x l sinh khi Bacillus clausii to

nguyn liu cha bo t:

Bacillus clausii

(SDS, EDTA, NaOH, H2SO4

. Sau qu trnh x l bng

lysozyme thu c bo t tinh khit, t l khi lng bo t so vi sinh khi

ban u cao (80%). T l enzyme lysozyme thp nht t hiu qu x l l

15ml lysozyme (1mg/ml)/1g sinh khi t trong iu kin 37C trong 120

pht. Phng php s dng H2SO4 10% (80C, 80 pht) cng cho kt qu

sch hu ht dng sinh dng nhng t l sinh khi cn li qu thp (42%).

1.2. l m

trong qu trnh bo qun:

sau khi

lysozyme trn 251013

cfu/1g nguyn liu ,

2SO4 10% 80C (5711010

cfu/1g nguyn liu

) , ,

n ,

c: ng

.

39

2. Kin ngh:

Do hn ch v thi gian v ha cht nghin cu, ti hon thin hn cn

tip tc cc nghin cu sau:

- Ngh kho st cc yu t nh hng n hiu qu gii phng

ni bo t ca tc nhn H2SO4 (nng , nhit x l, thi gian x l, cht

xc tc) .

- Theo di n nh ca bo t thu c sau qu trnh x l

ca bo t ).

TI LIU THAM KHO

Ting Vit:

- B mn Vi sinh Sinh hc, Trng i Hc Dc H Ni

(2007), Vi sinh vt hc, H Ni, tr. 22 24.

- Nguyn Ln Dng (2012), Vi sinh vt hc, Nxb Khoa hc v k

thut, H Ni, tr. 31- 35.

- Nguyn Th Hin (2012), Kho st kh nng hnh thnh bo t

ca vi khun Bacillus clausii, Kha lun tt nghip Dc s,

trng i hc Dc H Ni.

- Nguyn Trng Hip, Bi Tng Hip, Nguyn Vn Vinh (2009),

Bn v kh nng sng st ca vi sinh vt trong cc sn phm

probiotics, Tp ch dc hc, 393, b Y t, tr. 4- 7.

- Mai Phng Hoa (2012), Thc hnh k thut di truyn, trng

i hc Nguyn Tt Thnh, thnh ph H Ch Minh, tr. 11- 12.

- Trn Thu Hoa (2002), Nghin cu kh nng dng bo t

Bacillus subtilis ti t hp lm nguyn liu thuc chng nga

qua nim mc, lun n tin s Dc hc, trng i hc Y Dc

thnh ph H Ch Minh, tr. 33- 35.

- Nguyn Duy Long, Phan T Nh, Nguyn Minh Nht, Hong

Quc Khnh (2007), Tuyn chn cc chng Lactobacillus sinh

tng hp polyhydroxybutyrate, bo co nghin cu khoa hc,

Phng vi sinh ng dng, Vin sinh hc nhit i.

- L Xun Phng (2008), Th nghim vi sinh vt hc, i hc

Nng, tr. 54 55.

- Trn Th Thanh (2003), Cng ngh vi sinh, Nxb Gio dc.

- ng Th Thu (2012), Cng ngh Enzyme, Nxb Khoa hc v k

thut, H Ni, tr. 25- 29.

Ting Anh:

- Adam JK, Bharti O, Naidu KSB (2012), Probiotics: Recent

understandings and biomedical applications, Curr Trends in

Biotechnol.Pharma., 6, p. 1-14.

- A.L Hart, A.J. Stagg, M.A. Kamm (2003), Journal of Clinical

Gastroenterology, 36, p. 111- 119.

- Barbosa TM, Serra CR, La Ragione RM, Woodward MJ,

Henriques AO (2005), Screening for Bacillus isolates in the

broiler gastrointestinal tract, Appl Environ Microbio, 71, p. 968-

978.

- C. Ganesh Kumar, Han-Seung Joo, Yoon-Mo Koo, Seung R.

Paik and Chung-Soon Chang (2004), Thermostable alkaline

protease from a novel marine haloalkalophilic Bacillus clausii

isolate, World Journal of Microbiology & Biotechnology, 20, p.

351357.

- Delia, P., Sansotta, G., Donato, V., Frosina, P., Messina, G., De

Renzis, C., Famularo, G. (2007), Use of probiotics for

prevention of radiation-induced diarrhea, World Journal of

Gastroenterology, 13 (6), p. 912-915.

- Fooks, L.J., Fuller, R., Gibson, G.R. (1999), Prebiotics,

probiotics and human gut microbiology, International Dairy

Journal, 9(1), p. 53-61.

- J.C. Lieske, D.S. Goldfarb, C. De Simone, C. Regnier (2005),

Use of a Probioitic to Decrease Enteric Hyperoxaluria, Kidney

International, 68(3), p. 12441249.

- K.C. Anukam, E. Osazuwa, G.I Osemene, F. Ehigiagbe, A.W.

Bruce, G. Reid (2006), Azole vaginal gel to treat symptomatic

bacterial vaginosis, Microbes and Infection, 12, p. 2772.

- K. Kukkonen, E. Savilahti, T. Haahtela, K. Juntunen-Backman,

R. Korpela, T. Poussa, T. Tuure, M. Kuitunen (2007),

Probiotics and prebiotic galacto-oligosaccharides in the

prevention of allergic diseases: A randomized, double-blind,

placebo-controlled trial, Journal of Allergy and Clinical

Immunology, 119(1), p. 192- 198.

- K Suresh Babu Naidu, Jamila K Adam, Patrick Govender

(2012), The use of probiotics and safety concerns, African

Journal of Microbiology Research, 6(41), p. 6871-6877.

- L.A. Simons, S.G. Amansec, P. Conway (2006), Nutrition

Metabolism and Cardiovascular Diseases, 16(531).

- L. Nicholson, Nobuo Munakata, Gerda Horneck, Henry J.

Melosh and Peter SetlowWayne (2000), Resistance of Bacillus

Endospores to Extreme Terrestrial and Extraterrestrial

Environments, Microbiology and Molecular Biology Reviews,

64(3), p. 548- 572.

- Nicholson, W.L. and P. Setlow (1990), Sporulation,

germination, and outgrowth, pp. 391 450.

- N. K. Asha Devi1, K. Balakrishnan, R. Gopal and S.

Padmavathy (2008), Bacillus clausii MB9 from the east coast

regions of India: Isolation, biochemical characterization and

antimicrobial potentials, Current science, 95, p. 627- 636.

- Preben Nielsen, Dagmar Fritze and Fergus G. Priest (1995),

Phenetic diversity of alkaliphilic Bacillus strains: proposal for

nine new species, Microbiology, 141, p. 1745-1761.

- Rosa Lippolis, Antonio Gnoni, Anna Abbrescia, Damiano

Panelli, Stefania Maiorano, Maria Stefania Paternoster, Anna

Maria Sardanelli, Sergio Papa, Antonio Gaballo (2011),

Comparative proteomic analysis of four Bacillus clausii strains:

Proteomic expression signature distinguishes protein profile of

the strains, Journal of proteomics, p. 1 - 10.

- Saavedra, J. (2000), Probiotics and infectious diarrhea, The

American Journal of Gastroenterology, 95 (1), p. 16-18.

- Spinosa MR, Braccini T, Ricca E, De Felice M, Morelli L, Pozzi

G, Oggioni MR (2000), On the fate of ingested Bacillus

spores, Res Microbiol, 151, p. 361-368.

- Tran C Dong, Pham H Van, Simon M Cutting (2009), Bacillus

Probiotics, School of Biological Sciences, Royal Holloway

University of London, Egham, Surrey, TW20 0EX, UK.

- World Gastroenterology Organisation (2011), Global

Guidelines: Probiotics and prebiotics.

- World Health Organization, Food and Agriculture Organization

of the United Nations (2011), Health and Nutritional Properties

of Probiotics in Food including Powder Milk with Live Lactic

Acid Bacteria.

Mt s trang web:

- http://www.anabio.com.vn/vn/san-pham-va-dich-vu/bacillus-

clausii.

- http://www.atcc.org.

- http://eurekamag.com/keyphrase/b/001/bacillus-clausii.php.

- http://www.lekynainternetu.cz/p/probacin/.

- http://microbewiki.kenyon.edu/index.php/Bacillus_clausii.

- http://www.mims.com/Philippines/drug/info/Erceflora.

- http://www.sporegen.com/bacilluspro.html.