HC09BSH LOGO

Nui cy Aspergillus Oryzae thu nhn Lactase - ng dng

GVHD: PGS.TS Nguyn Thy Hng Nhm thc hin: on Ngc Mai Sng Phm Th Thanh Thy Nguyn Dng Ngc Lin Trn Trung Hiu

NI DUNG
1. Lactose v enzyme Lactase
2. Gii thiu v Aspergillus Oryzae 3. Nui cy A.oryzae thu nhn Lactase 4. ng dng ca Lactase

LACTOSE
C IM
Cng thc phn t C12H22O11

Phn t khi
Hnh dng Khi lng ring Nhit nng chy

342.30 g/mol
Tinh th mu trng 1.525 g/cm3 202.8 oC 668.9 oC 21.6 g/100 ml
3

Lactose Nhit si
tan/ nc

ENZYME LACTASE

b galactosidase (cn gi l Lactase) l enzyme thy phn ng lactose to thnh ng glucose v ng galactose.

ENZYME LACTASE

NI DUNG

NI DUNG
1. Lactose v enzyme Lactase
2. Gii thiu v Aspergillus Oryzae 3. Nui cy A.oryzae thu nhn Lactase 4. ng dng ca Lactase

Chng Aspergillus oryzae

Aspergillus oryzae l 1 dng nm si
Chi Aspergillus H Trichocomaceae B Plectascales Lp Ascomycetes.

A. oryzae tit ra mi trng cc enzyme thy phn nh pectinase, xylanase, cellulase, b galactosidase,khi sng trn mi trng c ngun c cht tng ng: pectin, xylan, cellulose, lactose,...

A. oryzae c ng dng rng ri trong: Trong cng ngh sn xut thc phm: nc tng, miso, ru sake. Trong cng ngh sn xut enzyme: cellulase, amylase, protease, hemicellulase v b galactosidase.

Cc loi thc phm

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Cc loi enzyme sn xut t A.oryzae

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Nm 2001, b gene di truyn ca A. oryzae c phn tch v gii m. H gene gm 8 nhim sc th vi 12 ngn gene v 37 triu cp base
(Galagan et al., 2005, Machida et al., 2005)

NI DUNG

NI DUNG
1. Lactose v enzyme Lactase
2. Gii thiu v Aspergillus Oryzae 3. Nui cy A.oryzae thu nhn Lactase 4. ng dng ca Lactase

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SN XUT CH PHM ENZYME VSV ging Nhn ging

Nui cy
[Chm] [B mt]

Ch phm enzyme th Tinh ch

Enzyme tinh khit

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Phng php nghin cu

Tuyn chn s b 3 chng sinh tng hp Lactase tt
MT thch lactose vi ch th X-gal

Nui cy Sinh Tng hp Lactase

Ln men chm My lc trn 200v/ph - pH 7 - 30oC - sau 48h

nh lng hot Lactase Tt nht A.oryzae 3 : enz ni bo 307,5 mU/g

Thy phn oNPG Galactose + oNP (vng)
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NGUYN VT LIU
MI TRNG GI GING PDA Glucose 20g Agar 20g Dch chit khoai ty 1 lit pH 7 MI TRNG HOT HA Lactose 20g Peptone 3g KH2PO4 1g NaNO3 0,5g KCl 0,5g H2O 1 lit
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TUYN CHN S B
Hot b - galactosidase Chng Vi sinh vt Ngoi bo (mU/ml) Ni bo (mU/g)

A.oryzae 1

8,96

70,3

Chng A. oryzae 3 c la chn nghin cu A.oryzae 2 9,84 81,87 thu nhn enzyme ni bo
A.oryzae 3 21,24 307,5

Hot b galactosidase ca 3 chng A.oryzae sau 48 gi nui cy 23

PHNG PHP NUI CY CHM

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LA CHN MI TRNG LN MEN

Mi trng Thnh phn Lactose(g) Peptone(g) Cao nm men(g) (NH4)2SO4(g) KH2PO4(g) K2HPO4(g) MgSO4(g)

MT1
10 10 10 5 3 1 0.5

MT2
40 0 1 5 3 1 0.5

MT3
5 0 5 3 5 0 0.4

MT4

Mi trng Lactose c bn

CaCO3(g)
H2O(ml) pH

0
1000 7

0.2
1000 6

0
1000 7
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KHO ST KH NNG SINH TNG HP LACTASE

Thi gian (gi) Mi trng

pH dch nui cy v hot b - galactosidase (mU/g)

24 36 48 60 72

pH
6,76

mU/g
0

pH
7,62

mU/g
111,61

pH
8,22

mU/g
367,01

pH
8,20

mU/g
529,47

pH
8,45

mU/g
128,58

MT1

MT2

6,15

Chn6,01 120,88 6,17 194,44 5,72 nui cy MT4 lm mi trng 285,34 5,98 0
0 6,88 45,32 7,05 165,00 6,76 180,01 6,88

169,02

MT3

6,79

277,4

MT4

6,20

43,57

6,53

264,13

6,79

398,61

6,78

507,00

6,78

738,80
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NH HNG CA NGUN CARBON

Thi gian (gi) Ngun Carbon (g/l)

Hot galactosidase ( mU/g)

24

36

48

60

72

Lactose
Saccarose

45.16
0

257.00
0

406.51
2.01

592.96
35.47

729.34
33.48

Maltose
Glucose Galactose

0
0 16.87

34.26
26.64 136.69

37.45
31.37 254.69

22.59
50.55 36.84

23.19
16.23 10.52

Hot b galactosidase khi nui cy A.oryzae 3 trn mi trng c ngun 27 Carbon khc nhau

Nng Lactose
Thi gian (gi) N. Lactose (g/l)

Hot galactosidase ( mU/g)

24 44.36 56.72 96.14 89.71
36 48

60 456.34 558.41 686.04 691.05

72 612.54 797.07 829.43 976.74

15 20 25 30

248.52 210.57 293.19 354.89

398.35 379.24 411.39 517.13

35

77.00

226.31

417.88

518.16

787.75

nh hng ca nng lactose n kh nng sinh tng hp galactosidase

nh hng ngun Nit

Thi gian (gi)
Ngun Nit (g/l) 24 Hot galactosidase ( mU/g)

36

48

60

72

2g peptone + 1g (NH4)2SO4

111.81

225.48

311.73

643.00

931.41

2g peptone + 1g NH4NO3
3g cao nm men 3g peptone

113.33
56.28 65.82 34.02

346.62
175.84 177.48 61.53

529.63
220.81 231.87 82.50

727.11
351.33 561.87 108.75

1017.60
537.00 815.00 165.82 29

Hot galactosidase khi nui cy A.oryzae 3 trn mi trng c ngun nit khc nhau

3g NH4NO3

nh hng nng Nit

Thi gian (gi) Ngun Nit (g/l) 0.5g peptone + 0g NH4NO3 24 Hot galactosidase ( mU/g) 36 48 60 72

67.02

136.73

333.01

451.74

674.61

1g peptone + 0.5g NH4NO3

2g peptone + 1g NH4NO3

182.02
119.22

481.26
328.70

627.96
548.10

994.83
781.26

1311.80
1008.71

3g peptone + 1.5g 103.33 304.66 490.68 692.08 969.25 NH4NO3 Chn ngun v nng Nit: 1g peptone + 0,5g NH4NO3 4g peptone + 2g NH4NO3

90.62

176.24

390.42

683.93

828.38
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MI TRNG 4

Thnh phn
Lactose Peptone NH4NO3 KH2PO4 MgSO4 KOH H2O 30g 1g 0,5g 1g 0,5g 0,5g 1000ml
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nh hng pH u
Thi gian (gi) pH u

Hot galactosidase ( mU/g)

24 89.80 79.99 206.71 221.69
36 48

60 589.71 643.13 958.38 1011.45

72 695.52 781.41 1237.40 1325.16

6.0 6.5 7.0 7.5

261.57 253.72 438.36 541.26

404.65 415.40 786.33 814.31

8.0

140.57

267.58

475.44

653.01

723.73

pH [7-7,5] thch hp. Nghin cu ng dng Chn pH 7

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NG THI QU TRNH LN MEN MT4, 200 v/ph 300C pH 7 Sau mi 12h thu 100ml dch nm mc

Xc nh sinh khi Hot tnh enzyme

o pH
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ng thi qu trnh ln men

KT LUN
Tuyn chn 3 chng c kh nng sinh tng hp b- galactosidase. cao nht l A. oryzae 3 Xc nh mi trng ph hp MT4 iu kin ti u [C] Lactose 30g/l [N] Peptone 1g/l + NH4NO3 0,5 g/l pH u 7 Thi gian ln men 72 h
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PHNG PHP LN MEN B MT

Solid-state fermentation(SSF)
D thc hin Quy trnh Cng ngh khng phc tp Lng enzyme to thnh ln Ch phm enzyme th d sy kh v bo qun D x l nhim Din tch b mt ln, kh c gii ha
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Ging VSV

A. oryzae phn lp t t nhim cht thi ca nh my sa Gi ging [PDA] Nui cy bo qun 40C

Chun b mi trng

Cm m + Tru [1:1] 12% NaNO3 + 1% Lactose + Hu c # m- Thanh trng- pH 5

K thut nui cy

10ml dch 1x10-7 (bo t/ml) 300C 7 ngy

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Ln men b mt
Trn 60g cm la m v v tru (t l 1:1) vo bnh thu tinh borosil 3000 ml Lm m vi nc ct v trng cha [ Lactose 12% w/w - NaNO3 1% w/w ] [ pH u 5.0 ] Tit trng mi trng cy chuyn 10ml huyn ph cha bo t nm si [ 30oC trong 6 ngy ]

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nh lng hot bng oNPG (Sigma Chemical USA) 1 n v hot = lng enzyme gii phng

1 mmol oNPG / 1 pht, iu kin th nghim

Xc nh hm lng protein tng vi Phng php Lowry
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SO SNH CC NGUN CARBON CARBON NH HNG NGUN

NH HNG NGUN NIT

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NH HNG CA M

pH

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Ti u ha iu kin
Ngun C Glucose cho hoat tnh enzyme mnh nht 386.6 mmol oNP/ml/pht
NaNO3> peptone> (NH4)2SO4 > urea >NH4NO3>Corn steep liquor m 90%

Ngun N

pH v nhit

Chn pH 5 v nhit 300C

Tho lun
Hot tnh enzyme lactase t cc vi sinh vt khc So snh phng php ln men & hiu qu enzyme c nh v t do iu kin nui cy nh hng ln ti hot tnh enzyme ca vi sinh vt iu kin ti u nui cy VSV thu enzyme c th khng ging vi iu kin hot ng ti u ca enzyme kt lun
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SO SNH 2 PHNG PHP LN MEN

YU T Ging VSV LN MEN CHM Phn lp t t nhim cht thi ca nh my sa MT lactose c bn Lactose Peptone + NH4NO3 7 300C 200 v/ph 72 h (3 ngy) 168 h (7 ngy)
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LN MEN B MT PTN Ha sinh- H Bch Khoa H Ni

Nguyn liu- C cht Ngun C Ngun N pH u Nhit Tc lc Thi gian ln men

Cm m+Tru m 90% Glucose NaNO3 5 300C

Thu enzyme Ln men chm

[C c CK]
hoc

Ly tm
Tch VSV Tp ko tan

[Trao i ion Phn hp th]

Thu Enzyme

Tinh sch
Enzyme tinh khit

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SN XUT ENZYME TINH KHIT

Nghin mn
Ph v thnh TB: enz tot ra

Trch ly

Dng nc dung mi ha tan

Lc

Ly phn dch

Kt ta Sy

Sy: d bo qun

Tinh ch kt ta

Cn enz khc & pro l

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SN XUT ENZYME

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Enzymes t nm si
iu kin ti u: pH [ 3 5 ] - nhit [ 55 60C ] Khng tinh khit. Do , ng dng ca lactase t nm si gii hn cc sn phm c acid cao cng nh cc dc phm. Enzyme b- galactosidase thng mi A. oryzae: Enzeco Fungul Lactase

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Hng ng dng Lactase

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NI DUNG

NI DUNG
1. Lactose v enzyme Lactase
2. Gii thiu v Aspergillus Oryzae 3. Nui cy A.oryzae thu nhn Lactase 4. ng dng ca Lactase

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Enzyme thng mi ch yu chit xut t Nm men: Kluyveromyces lactis, K. fragilis, K. marxianus, Candida kefyr Nm si: Aspergillus niger , A. oryzae Lactase l mt trong s t cc enzyme c sn xut quy m ln vi c 2 dng c nh v t do.

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NG DNG LACTASE
1981 Kligerman: Cho enzyme Lactase vo ly sa,

qua m trong t lnh: gim 70% lng lactose

Sa ngho lactose

1991: Nhiu thc phm ct gim lactose c y

mnh nghn cu v sn xut

Sa, ph mai, kem

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ng dng sn xut GOS

GOS l mt loi prebiotic, ng dng trong thc phm Cht ngt b sung Thay th saccharose Bnh tiu ng hay bo ph.

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Tc dng ca GOS
Tng sc khng vi vi khun gy bnh ng rut Kch thch min dch ng tiu ha Tng hp thu Canxi Qu trnh ln men sinh ra kh hi v nc phn mm v xp phng chng to bn

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Whey - casein

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Whey - casein
Ti sao sa cn c m Whey v Casein??

Whey

Casein

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ng dng trong sa ngho lactose

Hm lng lactose c trong cc loi sa

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ng dng trong sa ngho lactose

Thiu lactase tng i

Nguyn nhn

Tn thng rut non lp nim mc rut lactase khng sn sinh

Bm sinh
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ng dng trong sa ngho lactose

D ng sa b

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Xt nghim chn on

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Gii php

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ng dng trong sa ngho lactose

Sa u nnh c lng protein cao gn bng sa b, nhng t canxi hn sa b. Sa u nnh khng c lactose.

Sa go khng c gluten Lng asen trong sa go khuyn co khng nn cho tr di 4,5 tui ung sa 63 go.

Lactose free milk

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Kt lun
b galactosidase m ra nhiu hng nghin cu mi. Nhng nghin cu ny c tim nng ng dng vo nhiu lnh vc khc nhau: cng ngh thc phm y hc Cng ngh c nh enzyme ng mt vai tr ln trong ch bin sa v whey. Vic s dng -galactosidase c nh trong thy phn lactose l mt ch khoa hc v cng ngh ng quan tm
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CM N S LNG NGHE CA C V CC BN

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