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Bai 1: Ly thuyt chung
NG HOC LN MEN THU SINH KHI VA QUY TRINH CHUNG SAN
XUT CH PHM SINH KHI.
1. ng hoc ln men thu sinh khi:
1.1.

Phng phap nui cy san xut sinh khi

- Nui cy theo me
- Nui cy lin tuc
- Nui cy theo me tip liu lin tuc
1.2.

Phng trinh ng hoc nui cy theo me

X= x0et ; N= N0 et ; OD= OD0 et

- Xac inh x bng phng phap khi lng

-

Xac inh N bng phng phap /trai ia nm khun lac

- Xac inh OD bng phng phap o uc

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Hinh 1.1.o OD canh trng bng spectrophotometer
A)

Mu trng: mi trng trong sut, cng anh sang n c t bao

quang in c coi la cng ln nht va OD c chinh v 0
bc song o (thng 600 610 nm).

B) Khi trong cuvette co vi khun, mt phn anh sang bi phn tan va khng
n c t bao quang in, OD>0.
Chu y: Chi o OD trc tip nu mi trng dinh dng khng mau hoc
mau nhat. Khi mi trng co mau nu phai ly tm mu, s dung mi
trng trong sut hoc nc mui sinh ly a v th tich ban u va lam
mu sang.
i vi mt s VSV, OD chi tuyn tinh vi nng t bao khi nng
nay thp vi vy chi nn nhn gia tri khi OD <=0.4 . Vi mu co OD>0.4
thi nn pha loang 2 ln.

Hinh 1.2. Tng tac gia gia tri OD va nng t bao.

Vi du: OD <0,4 ;OD = 0,270
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Pha loang mu 2 ln (1 ml mu + 1ml mi trng tit trung): OD = 0,135
OD hc (OD hiu chinh) = OD * n = 0.135*2= 0.270
OD>0,4 ; OD = 0.540
Pha loang mu 2 ln(1 ml mu + 1ml mi trng tit trung): OD = 0.288
OD hc (OD hiu chinh) = OD * n = 0.288*2 = 0.576
1.3.

ng cong tng trng vi sinh vt

Ve ng cong tng trng nhm xac inh cac thng s ng hoc nui cy:
ng hoc tng trng (x(t), N(t) hoc OD(t) ng cong (ham mu e)
Tuyn tinh hoa bng cach ly logx, logN hoc logOD.

Hinh 1.3. Tuyn tinh hoa ng cong tng trng bng cach ve thi logx,
logN, logOD theo t.

Nhom
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co c ng cong tng tng cn thc hin o OD nh bang sau:

Thi

Thi gian

OD

im
08:15

(min)
0

...
12:00
...

OD pha OD hc

logODh.c

loang
0.055 n=1

loang
-

0.055

-1.26

...

...

...

....

...

225

0.685 (1ml+3ml); 0.206

0,824

-0.08

...

...

...

...

H s pha

...
n=4
...

...

Hinh 1.4. ng cong tng trng VSV trong nui cy theo me (logOD theo t).
1.4.
-

Cac thng s t ng cong tng trng

tlag, tlog

- = [ln(OD/ODo)]/(t-to)=log(OD/ODo)/[loge*(t-to)]
=(logOD-logODo)/0.434(t-to)= 2.303(logOD-logODo)/(t-to)
- max
- td = thi gian th h = 2.303*log2/max = 0.69/max
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- xac inh nng sut sinh khi:
Nng sut R = (Xf-Xo)/(tf-to)
Cn xac inh Xf, Xo hoc Nf, N0
Mun vy cn thit lp ng chun t bao
1.5.

ng chun t bao: tng quan tuyn tinh gia OD (OD<=0.4) va

nng t bao.

Nng t bao xac inh bng phng phap khi lng, m trc tip hoc m
khun lac.
1.6.

Hiu sut

xac inh h s kinh t (hiu sut s dung c cht tng hp sinh khi) YX/S
Y = x/s = (Xf-Xo)/(So-S)

Nhom
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Quy trinh chung san xut sinh khi
Hinh 1.5. Quy trinh chung san xut sinh khi

ia Petri

Chun bi mi trng ln men

chinh

n lacn
trng
trn
1.7.(khu
Chu
bicmi
tr
ngmi
(mi trng ln men chinh, cach pha
trng gi ging)
mi
tr
g) ging thach
Chu y trong nhiu trng hp co s khac bit gia mi
tr
ngngi
nghing, mi trng nhn ging va mi trng ln men chinh.
Trong Tha
qua tricnhh nghing
pha tranh hin tng tao kt tua khi tit trung hoc phan ng
(mim
tr
ngtr
gi ngi
ng)p nn tit trung ring cac mt s thanh phn mi
Maillard,
t s
g h

Tit trung

trng ri trn chung sau khi a tit trung.

1.8.

(ch

Cy ging

tit trung)

Nhn ging

Ti l cy
% theo
(mi
trgi
ngngnhn
gith
ng)tich phai thoa iu kin v mt cy ging.
Xac inh Xo
1.9.

LnC
men
thunsinh
y gi
g khi

Ln men thu sinh khi

(t,oxy, ly mu, thi gian)

(tiTun
l %thu
thca
tich,thng
nngsqua
-Xotrinh: nhit , oxy.
Thi gian len men: xac inh da vao sinh ly sinh khi (sinh dng, trang
thai nghi, ken, bao t): kt hp o thng s ng hoc va soi kinh (nhum
hoc soi ung).

Downstream processing

1.10. Downstream processing

San

phm

Trong a s trng hp sinh khi phai c tacTa

h rao kho
chi sinh
phm
trng bng ly
tm hay loc tranh anh hng cua cac hp cht trao i cht bt li i vi
t bao. Mt s dang san phm, thu sinh khi di dang t bao nghi, ken hoc
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bao t, cac qua trnh Downstream Processing c giam thiu (han ch nng
lng), ch phm c tao trc tip t canh trng bng cach trn canh
trng vi cht mang. o la trng hp ch phm phn bon vi sinh va ch
phm x ly mi trng.
1.11. Tao ch phm
1.11.1.

Ch phm trn c s cht mang (carrier based products)

Tiu chun cht mang:

- Re
- Sn co trn thi trng
- Cha ham lng cht hu c cao
- Khng cha cht c
- Co kha nng gi nc tt (>50%)
- D dang s dung
1.11.2.

Ch phm long (liquid products)

Sinh khi thng c a v dang trang thai ngu, tt nht trong trng hp vi
sinh vt co ken, bao t

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BI 2: SN XUT CH PHM AZOTOBACTER SPP. (PHN BN C
NH M)
I.1.

L thuyt:

C nh N l kh nng ng ha N phn t ca mt s vsv v dung lm

ngun kim to t bo. Cc vsv c nh N sng t do c nhiu trong t, hay
sng cng sinh trong nt sn cy h u. Ngun N c th l nit phn t, cng
c th l mui amon, nitrate, nitrite, amoni acid. Ty thuc nng cc hp
cht c nit ny trong mi trng m qu trnh c nh nit phn t s b c ch
nhiu hay t. ng thi chng c nhu cu ln i vi P v Ca, cng nh c
nh nit mnh m chng cn Mo v B. Phn ln cc loi Azotobacter ch pht
trin pH ln hn 6 v m cao.
I.2.

Tao ch phm: Cng ngh san xut phn bon trn c s cht mang.

Cht mang phai ap ng nhng nhu cu sau:

- Re
- Sn co trn thi trng
- Cha ham lng cht hu c cao
- Khng cha cht c
- Co kha nng gi nc tt (>50%)
- D dang s dung
II. Thc hnh:
1. Vt liu:
VSV: Azotobacter chroococcum
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Ha cht: mi trng dinh dng nui cy vsv thng thng
- ng: mannitol, glucose, sucrose
- Ngun N: cao nm men
- Bao nilon
Dng c:
- Dng c thy tinh nui cy vsv: a Petri, ng nghim, bng khng thm nc
- Bnh tam gic
- Becher
Thit b:
- My lc
- My o quang ph
- Knh hin vi
gi ging
Cy truyn t

mt thch nghing Ashby (1) + gar -8ml/ng

a Petri
nhn ging

mt lng Ashby (2)_50ml lc 150v/ph

2. Tin hnh th nghim:

2.1.

Gi ging ( cy truyn t ia Petri vao thach nghing)

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-

Chun bi 10 ng thach nghing cha Ashby (1) 8ml/ng. Chon khun lac c
trng t ia petri cy vao ng thach nghing.

U 300 C, trong 48 gi.

2.2.

Nhn ging:

- Chun bi 4 binh tam giac mi trng long Ashby (2) 50ml/1binh : lc 150
vong/phut.
-

Kim tra OD sau 36 gi

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Bng 2.1: Thnh phn mi trng gi ging, nhn ging
Thnh phn
Ngun C
KH2PO4
K2HPO4
MgSO4.7H2O
K2SO4
NaCl
CaCO3
Yeast extract (cao nm men)
Agar
pH
T nui cy
Thi gian
Lc
Th tch mi trng
Th tch ging

Mi trng Ashby

Mi trng nhn

agar (g/l) (1)

20 mannitol

ging (g/l) (2)

10 sucrose
0.5
0.5
0.2

0.2
0.2
0.1
0.2
5

0.1

15
7
300C
48 h
Thch nghing
Cy ria

10
7
300C
36 h
140v/ph
50 ml
5 ml

2.3. Xc nh thng s ng hc:

- Chun bi mi trng ln men chinh (mi trng 3) theo doi thng s ng
hoc trong 48 gi.
- C cht gi hn l ng kh phn tch theo phng php DNS.

Bng 2.2: Thnh phn mi trng thu sinh khi (3)

Cao nm men 5g/l
Dung dch khong a lng

Glucose 15g/l
0.66 g/l K2HP4

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0.16 g/l KH2PO4
0.2 g/l MgSO4.7H2O
0.134 g/l FeSO4
0.2 g/l NaCl

Dung dch khong vi lng

(pha dung dch m nng
x10 sau s dng 10ml dung
dch m cho 1lit mi trng)

0.2 g/l CaCl2

0.0008 g/l CuSO4.5H2O
0.00024 g/l ZnSO4.7H2O
0.0028 g/l H2BO3
0.002 g/l Na2MoO4.4H2O
0.003 g/l MnSO4.H2O

Nhom
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Bng 2.3: Th nghim theo di cc thng s ng hc
TG

OD

Hinh

Binh Gi (t)
OD1 OD2 TB
HSPL OD hc log OD
1 8h
0 0.266 0.325 0.2955
1 0.2955 -0.52944
2 10h
2 0.28 0.33 0.305
1 0.305
-0.5157
3 12h
4 0.286 0.34 0.313
10 3.13
0.495544
2.7
4 14h
6 0.146 0.134
0.14
15 2.1
0.322219
5 16h
8 0.13 0.122 0.126
20 2.52
0.401401
6 18h
10 0.23 0.23
0.23
20 4.6
0.662758
7 20h
12 0.235 0.261 0.248
20 4.96
0.695482
8 8h
24
0.4 0.372 0.386
20 7.72
0.887617
9 12h
28 0.302 0.247 0.2745
20 5.49
0.739572
10 16h
32 0.218 0.209 0.2135
25 5.3375 0.727338
11 20h
36 0.202 0.392 0.297
15 4.455
0.648848
2.8
T bng th nghim trn ta v c ng cong tng trng ca Azotobacter spp

Hinh 2.1. thi ng cng tng trng cua Azotobacter spp

T ng cong tng trng trng trn ta thy im th 2 v im th 3 c h s
gc l ln nht. Nn ta chn 2 im tm
ng

Gi

TG

OD1

OD2

OD

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.
HSPL OD hc log OD
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2 10h
3 12h

2
4

0.28
0.286

TB
0.33 0.305
0.34 0.313

1
10

0.305
-0.5157
3.13 0.495544

(1)
Thi gian th h ca aztobacter l:
(2)
Ngoi ra trong giai on ny ta cn xc nh lng ng m azotobacter
s dng:

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Dng ng chun ng glucose:
ng
i chng
1
2
3
4
5

Nc ct
10
9.8
9.6
9.4
9.2
9

Glucose
0
0.2
0.4
0.6
0.8
1

OD 540
0
0.104
0.501
0.577
0.954
1.397

Hinh 2.3. thi ng chun glucose

T ng chun trn ta c c phng trnh ng chun: y = 1.373x 0.0977.

Nhom
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Suy ra lng ng tiu tn trong tng gi.
ng
1
2
3
4
5
6
7
8
9
10
11

Gi
8h
10h
12h
14h
16h
18h
20h
8h
12h
16h
20h

OD 540
0.904
0.885
0.869
0.846
0.76
0.768
0.697
0.157
0.3745
0.5245
0.362

HSPL
20
20
20
20
20
19
19
25
20
20
18

OD HC
18.08
17.7
17.38
16.92
15.2
14.592
13.243
3.925
7.49
10.49
6.516

x = (OD+0.0977)/1.373
13.2
12.96
12.72
12.39
11.14
10.69
9.71
2.92
5.52
7.71
4.81

Lng ng tiu thu theo thi gian

Hinh 2.4 thi th hin lng ng tiu thu theo thi gian cua Azotobacter spp
Kt lun:
-

T bng trn ta thy 3 ng nghim 9,10,11 l sai v : lng ng l ngun

c cht cho azotobacter tiu th. Tng ng vi thi gian cng lu th lng
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ng cng gim dn. Nhng 3 ng nghim trn th lng ng li tng ln.
-

Tom lai: Nhin vao thi trn thi ta thy lng ng giam dn theo thi

gian. u o chng to la s lng t bao tng dn theo theo thi gian

2.4.

Sn xut sinh khi:

Chun b mi trng ln men chnh (3) 238ml + cy 12ml/mu = 250ml.

1 binh lc 150v/ph, khong 48 gi va 1 binh lc 250v/ph
2.5.

Thit lp ng chun t bo:

T 1 huyn ph t bo, tin hnh pha long theo dy thp phn (106, 107, 108, 109)
v tin hnh tri a (mt Ashby agar), 300C 24 gi m khun lc.

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Bng 2.4: Thit lp ng chun t bo
Huyn ph

nc mui sinh

t bo ml
4
3.5
3
2.5
2
1.5
1
0.5

l
0
0.5
1
1.5
2
2.5
3
3.5

HSPL

OD 600

Cfu/ml

10
10
10
10
10
10
10
10

0.403
0.346
0.308
0.208
0.17
0.107
0.078
0.039

1.4.1010
1.225.1010
1.05.1010
0.875.1010
0.7.1010
0.525.1010
0.35.1010
0.175.1010

Phng trnh ng chun tnh s lng t bo:

Hinh 2.5. thi ng chun t bao.

T phng trinh ng chun t bao cua Azotobacter spp ta co th xac inh c
s lng t bao khi bit c OD cua no.

2.2.7. Phn tch ch phm

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-

Sinh khi: pha long, tri a , m khun lc, phi t 108cfu/g

Kh nng c nh m: chuyn mt khun lc vo mi trng Ashby lng

(8ml), nhit phng 2-4ngy, th NH4+ to thnh bng thuc th Nessler. Cc
bc tin hnh nh sau:
+ Chun b bnh 1 ( giai on ngh) v bnh 2 ( trn k lc ) v ging trn
mi trng thch ( mi trng asbly ) v 1 i chng m v 1 i chng dng
ly

Bnh 2
i chng m

i chng dng

Bnh 1

Ging trn mi
trng thch

Hinh 2.5. Th kha nng c inh dam cua Azotobacter spp

Kh nng sinh Auxin (IAA): Salkowskys technique
a 2ml canh ttrng vo tube Epp. Ly tm 10min trong 15000rpm
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Sau 1ml dung dch trn vi 2 ml thuc kh salkowsky: kt qu cho ra
khng mu. Vy trong mi trng trn khng c sinh IAA
II.4.

Kt qu tnh ton.

S lng t bao luc u va luc cui c tinh bng cach ta th vao phng trinh
n chun t bao: y=3.1914x +0.1257

Cfu/ml
Cfu/ml
Bng tm tt thng s ng hc
Thng s
tlag
tlog
td
X0 , N0
X,N
S0
S
Rmax= (X-X0)/(48)
Yx/s= (X-X0)/(S0-S)

n v
Min
Min
1/min
Min
Cfu/ml
Cfu/ml
mg/ml
mg/ml
Cfu/(ml*min)
Cfu/ml

Gi tr xc nh, tnh ton

120 ( 0 2gi)
120 (2 4gi)
0.019(1/ min) (1)
36.3 (2)
1,07.10-10
14,34.10-10
13,2
4,81
=(14,34.10-10 1,07.10-10 )/48 =0,276.10-10
=(14,34.10-10 1,07.10-10 )/ (13,2 - 4,81)=
1.58.10-10

Bng tm sn xut sinh khi/m:

Cfu/ml
Thng s
Glucose
Cao nm men

n v
G
G

Nhom
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Gi tr xc nh, tnh ton

15
5
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Dung dch
Khong

Ml

1000
Khoang vi lng:
8,842.10-3

Ging

Ml

Khoang a lng: 1,554

15 ml

OD
OD0
X0
OD
X
S0
S
M cht mang
M sn phm
Mt t bo trong sn phm
Kh nng c nh m
Kh nng sinh IAA

Cfu/ml
Cfu/ml
G
G
Cfu/g
+/+/-

0,2955
1,07.10-10
9,64
30,9. 10-10
15

C
Khng

Cu 3:S khc bit ca gia cc mi trng nui cy Azotobacter:

-

Mi trng Asbly gm c 2 mi trng: mi trng 1 v mi trng 2:

o

Mi trng 1: l mi trng thch v c cha agar v mi

trng ny khng cha yeast extract. Mi trng ny l mi
trng gi ging v khng cha nit.

Mi trng 2: khc vi mi trng 1 v n c cha cao nm

men. Cao nm men l thnh phn c9ha nit. y chnh la
mi trng nhn ging.

- Mi trng thu sinh khi: mi trng ny khc vi 2 mi trng trn l s

dng ng glucose.

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Hinh2.6.

Hinh
thai
khun
lac
Azotobacter: c dng nhy, n hi, kh
li, mt s dng nhn nheo, mu trng
trong.

Hinh 2.7. Hinh thai t bao Azotobacter spp:

khi nhun gram(m) thi co mau hng, hinh
que, hinh cu.
Hinh 2.8. Hinh thai tao ken

Azotobacter spp: Hinh giot nc co

cac t bao ken mau trng nu
bong,cac ken nm tp hp thanh tng
cum.

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Bi 3 . SAN XUT CH PHM BACILLUS SPP. H TR X LY NC
THAI GIAU PROTEIN VA TINH BT SAN XUT CH PHM BAO T
BACILLUS SUBTILIS
I .l thuyt
I.1 Vi khun Bacilluas subtilis
C nhiu hot tnh sinh hc :
_ Tit enzyme ngoi bo ( amylase, protease, cellulase, chitinase ) x l nc
thi, ging sn xut enzyme.
I.2 Sn xut sinh khi
Sn xut sinh khi di dng bo t ln men chm hoc ln men th rn.
I.3 Downstream processing
Ln men chm : Ly tm thu bo t, to ch phm
Ln men th rn : mi trng ng thi ng vai tr cht mang, sy.
I.4 To ch phm
Ch phm kh: trn cht mang sy
Ch phm lng: cht lng hn ch ny mm, nhim. V d nc + acetic acid
II. Chun b nguyn liu, dng c, tin hnh th nghim:
II.1 Chun b
-

VSV : Bcillus subtilis

Vt liu/ Ha cht: mi trng dinh dng nui cy VSV thng thng :

+ Tinh bt, cm go
+ Peptone, cao tht, cao nm men, (NH4)2SO4
+ Cc mui NaCl, MgCl2, MnCl2, FeSO2, ZnSO4
+ Cao
+ Gim n ( AcOH ln men)
+ Ha cht nhum Gram, nhum bo t.

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-

Dng c:
+ Dng c thy tinh nui cy VSV: a petri, ng nghim, bng khng thm

nc
+ Bnh tam gic 100 ml, 500 ml
+ Becher 1000 ml.
-

Thit b:
+ My lc
+ My spectrophotometer
+ Knh hin vi quang hc x 1000

II.2 Tin hnh th nghim

II.2.1 Cy truyn t a petri vo thch nghing:
-

Chun b cha thch nghing cha mi trng gi ging ( nutrient

agar ) : 2 ng/nhm. Chn khun lc c trng t a petri cy vo ng

thch nghing.
-

300C, 48 gi

II.2.2 Nhn ging

-

Chun b mi trng lng ( mt nhn ging) 50 ml : lc 150 vng/

pht. Kim tra OD sau 24 gi. Tnh ton lng ging cho ln men chm
v ln men b mt.
Bng 3.1 Thnh phn mi trng gi ging
Thnh phn
Tinh bt tan
Peptone
Cao nm men
Agar
PH
T nui cy

Mi trng gi ging

Mi trng nhn ging

g/l (1)

g/l (2)

30
5
5
20
7.0
300C

30
5
5

Nhom
2_08DSH2 GVHD: TS. Nguyn
Hoai Hng

7.0
300C
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TH CNLM-MT
Thi gian
Lc
Th tch mi trng
Th tch ging

24h

15-18h
150-200 v/min
50 ml
1 ml

Thc nghing

II.2.3 Xc nh cc thng s ng hc
-

Chun b mi trng ln men chnh (mt thu sinh khi) theo di

thng s ng hc trong 72 gi.Kt qu ghi chp vo bng 3.3

Bng 3.2 a Thnh phn mi trng thu sinh khi bo t (len men th rn)
Peptone 20 g/l

Tinh bt tan 20 g/l

Cao nm men 20 g/l

NaCl 5 g/l
MgCl2 0.2 g/l
MnCl2 0.2 g/l

Dung dch khong

CaCl2 0.2 g/l

FeSO4 0.2 g/l
ZnSO4 0.2 g/l

Tit trng mi trng : 1210C, 30 pht, cy ging 1%, 30-350C, lc, 250 vng/
pht 24-72h

Bng 3.3 Th nghim theo di cc thng s ng hc nui cy chm

ng

Gi

Thi

OD

H s

gian

600

pha

0,144

long
20

(t)
0

OD hc

Log OD

hc

1/pht

0,459

2,88

Nhom
2_08DSH2 GVHD: TS. Nguyn
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Soi knh

Bao
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TH CNLM-MT
2
3
4
5
6
7
8

10
12
14
8
12
16
20

2
4
6
24
28
32
36

0,130
0,217
0,236
0,248
0,309
,300
0,359

20
20
20
40
40
40
40

2,60
4,34
4,72
9,92
12,36
12
14,36

0,415
0,637
0,673
0,997
1,092
1,079
1,157

-0.0507
0.2556
0.0414
0.0414
0.0546
-0.007
0.0449

Hinh 3.2

1.4
1.2
1
0.8
Series1

0.6
0.4
0.2
0
0

10

20

30

40

50

60

Hinh 3.1 thi theo doi thng s ng hoc nui cy chim cua Bacilluas
subtilis.
II.2.4 Sn xut sinh khi bng phng php ln men chm
- Chun b mi trng ln men chnh ,238 ml/nhm, cy 12ml/mu.Lc
150,250 vng/pht, khong 48-72 gi.
- Kho st nh hng ca oxy ln s tng hp sinh khi ( cooper et al, 1944)
- Ha cht:
+Dung dch thiosulfat 0.1 N (Na2S2O3) hiu chnh nng
+Dung dch tinh bt 1%
+Dung dch iod 0.1 N mi pha ch
+Dung dch sulfit (Na2SO3) 0.8 N cha CuSO4 (0.001 M)
- Nguyn tc:
Nhom
2_08DSH2 GVHD: TS. Nguyn
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Mc thng kh c nh gi mt cch gin tip bng phng php
sulfit.Phng php da trn c s ca vic xc nh tc oxy ha sulfit thnh
sulfat khi c mt ca cht xc tc l ng. Lng sulfit d s phn ng vi iod.
Lng iod d s c chun bng thiosulfat.
Na2SO3 + O2
( CuSO4)

Na2SO4

4Na2SO3 + I2

3Na2SO4 + 2NaI

I2 + 2Na2S2O3

NaI + 2Na2S4O6

Nh vy oxy ha tan nhiu th tng ng th tch thiosulfat chun s ln.

- Tin hnh
Xc nh th tch dung dch iod cn thit cho th nghim
Cho dung dch iod 0.1N t burette chy dn vo bnh tam gic 250 ml cha

5 ml dd Na2SO3 mi pha ch cho n khi xut hin mu nu sng,ghi nhn th

tch iod a.
- Thm 0.5 ml dung dch tinh bt 1% vo v chun bng thiosulfat cho n
khi mt mu xanh lam(1-3 ml thiosulfat 0.1 N)
Cho ml dung dch I2 0.1N vo bnh tam gic 250 ml, chun bng

thiosulfat nh trn ( ch th tinh bt vo giai on cui ) n khi mt mu- V

thiosulfat khong 30-40 ml l c .
Xc nh tr s sulfit trc v sau khi thng kh
-5

ml sulfit cho vo bnh tam gic 250 ml cha sn a ml iod. y bnh bng np

knh vo ch ti 5-10 pht, sau dng nc ct trng knh y, thu ht

nc ra vo bnh tam gic trn, chun dch cha bng thiosulfat 0.1N. Th
tch thiosulfat chun l PN ml.
-

Bnh tam gic th tch nh bnh nui cy, thay mi trng bng dung dch
sulfit cng th tch, c t vo my lc trong mt thi gian xc nh
( thi gian chnh xc n hng pht ).Trc khi dng my lc 15-20 pht,

Nhom
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rt ml dung dch iod vo bnh 250 ml. Lc dng my lc, ly 5 ml dung
dch sulfit t cc bnh lc cho ngay vo bnh cha sn ml dd I2. Tip
tc lc bnh nui cy.Trong khi , gi cc bnh ly mu trong ti v em
chun bng thiosulfat 0.1 N nh trn,ghi nhn Px1 l th tch thiosulfat
chun .
- Sau 20 pht lc, lp li bc ly mu v chun nh trn, ghi nhn
Px2 l th tch thiosulfat chun .
5 ml Sulfit
Chun
Bng thiosulfat

a ml I2

hoa tan oxy trong dung dich sulfit tinh theo cng thc :
mg O2/l/pht = (PX-PN)/T *0.8*
Tnh Ton tr s sulfit
Bang 3.4. Lng oxy xp nht vao dung dich sulfit
PN = 1 ml;

= 35,7 ml

Ch lc

(0,05 N)
(0,1N)
= 7,2
= 3,6
150 vong/phut
= 10,3
= 5,15
= 11
= 5,5
250 vong/phut
= 11,25
= 5,6
Bang 3.5. Lng sinh khi thu c cua 2 ch lc
Ch lc

mg

TB (mg

O2/l/pht

O2/l/pht)

(ml)

OD

HSPL(ln)

Nhom
2_08DSH2 GVHD: TS. Nguyn
Hoai Hng

6,93
5,53
9
9,2
ODhc

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9,1

ODTB

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150
Vong/phut
250
Vong/phut

OD1

OD2

0,335

0,313

0,371

0,370

OD1

OD2

60

20,1

18,78

19,44

45

16,695

16,65

16,67

S liu t hai bang cho ta thy rng ch lc 150 vong/phut vi lng oxy
xm nhp 6,23 mg/l/phut cho lng sinh khi (ODTB = 22,23) cao hn ch lc
250 vong/phut, khng co s thay i v tui cua t bao.
Vy chon ch lc 150 vong/phut cho qua trinh ln men thu sinh khi
Bacilluas subtilis dang long (ln men chim).
II.2.6 Downstream processing
- Ly tm 4000 vng/pht x 15 pht ,ra sinh khi bng nc v trng 3 ln,
ha bo t vo nc mui sinh l v trng n th tch bng 1/10 th tch
trc ly tm.
II.2.7 To ch phm
- S dng gim n ( acid acetic ) 20% a PH v 3.8 4.2
- Bo qun nhit thng.
II.2.8 Phn tch ch phm
-

Pha long,trung ha bng NaOH 1M, a trn mi trng kim nh,phi

t 109 1010 cfu/ml

- Hot tnh enzyme: trn mi trng a sau khi m khun lc, trng a
bng dung dch Lugol, quan st vng trng xung quanh khun lc, nu kt
qu (+); c kh nng phn hy tinh bt; ngc li: khng c kh nng.
II.2.9 ng dung cua ch phm
Nguyn tc:
Ch phm lng : trung ha bng NaOH trc khi s dng
Nhom
2_08DSH2 GVHD: TS. Nguyn
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Lng bo t trong ch phm lng bng trong ch phm rn ( ln men b mt ) khi
cho vo nc thi.
Nc thi a v PH=7.0
Lc trong cng mt iu kin 150 vng/pht.
o COD ghi bng 3.6
Bng 3.6 COD mu th nghim ng dng
i chng

TN1

TN2

(khng b sung bo t)

(b sung bo t ch

(b sung bo t ch

phm lng)
phm rn)
II.2.10 Sn phm sinh khi bng phng php ln men b mt
Bng 3.7 Thnh phn mi trng thu sinh khi bo t bng ln men th
rn(mt len men chnh)
Th tch cy ging

Cm go 250 g/bao ( m 6%)

15 ml
Dung dch dinh dng

Pepton 25 g/kg

50 ml

Cao tht 10 g/kg

KH2PO4 10 g/kg

m 65%

CaO 5 g/kg
Lng nc b sung

Chun b 4 bich nilon mi trng nh bng 3.5. Tnh lng nc b sung sao cho
m mi trng 65%. Hp 1210C trong 30 pht. Cy 15 ml ging.Lc u.
nhit phng hoc 320C, 48 gi. Sy 40-450C.
Lng nc cn thm vao at m 65% la:
= 0,65 x = 1514 ml nc.
Lng nc thm vao mi bich la: 1514/4 = 379 ml
Nhom
2_08DSH2 GVHD: TS. Nguyn
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Vy lng nc cn thm vao mi bich at m 65% la 379 ml nc.
Tin hnh phn tch th nghim
II.3 Kt qu,tnh ton
Bng 3.8 Tm tt cc thng s ng hc.
Thng s

n v

Gi tr xc nh,tnh

Pht

ton
120 (8:10h) (0 : 2h nui

Pht

cy)
120 (10:12h) (2:4h nui

max

1/pht

cy)
0.2556

Td

Pht

21.56

tlag
Tlog

ODo
2.88
OD
Cfu/ml
nh hng oxy ha tan n sinh khi nui cy chm
Thng s
Lc 150 vng/pht
Oxy mg/l
6.9 (60 pht)
ODo
19,44
OD 24h
OD 48h
OD 72h
Soi knh 24h
Soi knh 48h
Soi knh 72h
Th tch sn phm
Nng bo t
Hiu qu x l
Bng 3.9 Thng s ln men b mt
Thng s
Khi lng mu
trc ln men
Khi lng mu

n v
g

Lc 250 vng/pht
9.1 (80 pht)
16,67

Gi tr xc nh,tnh ton

Nhom
2_08DSH2 GVHD: TS. Nguyn
Hoai Hng

B1: 0.660 kg
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B2: 0.657 kg
sau ln men
Mt bo t
Hot tnh enzyme
Hiu qu x l

B3: 0.655 kg
B4: 0.656 kg
6.35.1011
C/khng

Nhom
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II.3 Kt lun:
-

Trong san xut ch phm long 2 ch lc thi khng co s khac bit

v tui cua t bao, ch lc 150 vong/phut cho mt t bao (o
bng OD) cao hn ch lc 250 vong/phut.

- Qua trinh thc hanh do thi gian khng u nn chi tao c ch phm
rn.
c im hinh thai cua Bacillus subtilis: T bo vi khun B.subtilis c hnh

que, di, bo t hnh ovan nm xa tm hay gn tm c khuynh hng

phnh ra mt u, bt mau tim khi nhum Gram B.subtilis la vi khun
Gram dng.

Hinh 3.2. hinh thai t bao bacillus Soi kinh 24h: t

bao gram m co mau xanh tim
-

Qua trinh nhun bao t t bao bt mau o t bao bacillus subtilis bt mau
xanh.

Nhom
2_08DSH2 GVHD: TS. Nguyn
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Nhom
2_08DSH2 GVHD: TS. Nguyn
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