BAI ĐI IN

Bao cao TH CNLM-MT 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

Nhom 2_08DSH2 GVHD: TS. Nguyn Hoai Hng

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Bao cao TH CNLM-MT Hinh 1.1.o OD canh trng bng spectrophotometerA)

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 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 = 0.540 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.


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Bao cao TH CNLM-MT

co c ng cong tng tng cn thc hin o OD nh bang sau: Thi im 08:15 ... 12:00 ... Thi gian (min) 0 ... 225 ... OD H s pha OD pha OD hc loang ... 0.055 .... 0,824 ... logODh.c -1.26 ... -0.08 ...

loang 0.055 n=1 ... ... n=4 ...

0.685 (1ml+3ml); 0.206 ... ...

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/maxNhom 2_08DSH2 GVHD: TS. Nguyn Hoai Hng trang 4

Bao cao TH CNLM-MT - 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 bao1.5.

ng chun t bao: tng quan tuyn tinh gia OD (OD50%) - D dang s dung1.11.2.

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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Bao cao TH CNLM-MT 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 chroococcumNhom 2_08DSH2 GVHD: TS. Nguyn Hoai Hng trang 8

Bao cao TH CNLM-MT 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 a Petri nhn ging mt lng Ashby (2)_50ml lc 150v/ph mt thch nghing Ashby (1) + gar -8ml/ng

2. Tin hnh th nghim:2.1.

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


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Bao cao TH CNLM-MT -

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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Bao cao TH CNLM-MT 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 agar (g/l) (1) 20 mannitol 0.2 0.2 0.1 0.2 5 15 7 300C 48 h Thch nghing Cy ria Mi trng nhn ging (g/l) (2) 10 sucrose 0.5 0.5 0.2 0.1 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 K2HP4trang 11


Bao cao TH CNLM-MT 0.16 g/l KH2PO4 0.2 g/l MgSO4.7H2O 0.134 g/l FeSO4 0.2 g/l NaCl 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

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


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Bao cao TH CNLM-MT 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 . HSPL OD hc log ODtrang 13


Bao cao TH CNLM-MT 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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Bao cao TH CNLM-MT 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.


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Bao cao TH CNLM-MT 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 lngNhom 2_08DSH2 GVHD: TS. Nguyn Hoai Hng trang 16

Bao cao TH CNLM-MT 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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Bao cao TH CNLM-MT Bng 2.4: Thit lp ng chun t bo Huyn ph t bo ml 4 3.5 3 2.5 2 1.5 1 0.5 nc mui sinh l 0 0.5 1 1.5 2 2.5 3 3.5 HSPL 10 10 10 10 10 10 10 10 OD 600 0.403 0.346 0.308 0.208 0.17 0.107 0.078 0.039 Cfu/ml 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 phmNhom 2_08DSH2 GVHD: TS. Nguyn Hoai Hng trang 18

Bao cao TH CNLM-MT 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 15000rpmNhom 2_08DSH2 GVHD: TS. Nguyn Hoai Hng trang 19

Bao cao TH CNLM-MT 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 IAAII.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 Gi tr xc nh, tnh ton 15 5trang 20


Bao cao TH CNLM-MT Dung dch Khong Ml 1000 Khoang vi lng: 8,842.10-3 Ging 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 Ml OD Cfu/ml Cfu/ml G G Cfu/g +/+/0,2955 1,07.10-10 9,64 30,9. 10-10 15 Khoang a lng: 1,554 15 ml

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.

o

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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Bao cao TH CNLM-MT 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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Bao cao TH CNLM-MT 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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Bao cao TH CNLM-MT nc + Bnh tam gic 100 ml, 500 ml + Becher 1000 ml.-

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

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

II.2.2 Nhn ging-

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 g/l (1) 30 5 5 20 7.0 300Ctrang 24

Mi trng nhn ging g/l (2) 30 5 5 7.0 300C


Bao cao TH CNLM-MT Thi gian Lc Th tch mi trng Th tch ging 24h Thc nghing 15-18h 150-200 v/min 50 ml 1 ml

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 Cao nm men 20 g/l Tinh bt tan 20 g/l NaCl 5 g/l MgCl2 0.2 g/l Dung dch khong MnCl2 0.2 g/l 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 gian 1 8 (t) 0 OD 600 0,144 H s pha long 20 2,88 OD hc Log OD hc 0,459trang 25

Soi knh

1/pht 0


Bao cao TH CNLM-MT 2 3 4 5 6 7 81.4 1.2 1 0.8 0.6 0.4 0.2 0 0 10 20 30 40 50 60 Series1

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

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 Hoai Hng trang 26

Bao cao TH CNLM-MT 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) 4Na2SO3 + I2 I2 + 2Na2S2O3 - Tin hnh Xc nh th tch dung dch iod cn thit cho th nghim-

Na2SO4

3Na2SO4 + 2NaI NaI + 2Na2S4O6

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

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,


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Bao cao TH CNLM-MT 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 a ml I2 Bng thiosulfat

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; Ch lc (ml) = 35,7 ml mg O2/l/pht 6,93 5,53 9 9,2 ODhctrang 28

TB (mg O2/l/pht)

(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 OD HSPL(ln)

9,1

ODTB


Bao cao TH CNLM-MT OD1 150 Vong/phut 250 Vong/phut 0,335 0,371 OD2 0,313 0,370 60 45 OD1 20,1 16,695 OD2 18,78 16,65 19,44 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 dngNhom 2_08DSH2 GVHD: TS. Nguyn Hoai Hng trang 29

Bao cao TH CNLM-MT 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 (khng b sung bo t) TN1 (b sung bo t ch TN2 (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 15 ml Dung dch dinh dng 50 ml Cm go 250 g/bao ( m 6%) Pepton 25 g/kg 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 mlNhom 2_08DSH2 GVHD: TS. Nguyn Hoai Hng trang 30

Bao cao TH CNLM-MT 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 tlag Tlog max

n v Pht Pht 1/pht Pht

Gi tr xc nh,tnh ton 120 (8:10h) (0 : 2h nui cy) 120 (10:12h) (2:4h nui cy) 0.2556 21.56

Td

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

Gi tr xc nh,tnh ton

B1: 0.660 kgtrang 31


Bao cao TH CNLM-MT 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


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Bao cao TH CNLM-MT 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.


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Bao cao TH CNLM-MT


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