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CHNG 2: VT LIU PHNG PHP

2.1. VT LIU: 2.1.1. Chng vi sinh vt dng trong nghin cu
Chng nm mc Aspergillus niger, Aspergillus awamori v Rhizopus sp nhn t phng th nghim B mn Sinh ho trng i hc KHTN Tp HCM. Ging nm men Saccharomyces cerevisiae nhn t phng th nghim B mn Sinh ho trng i hc KHTN Tp HCM.

2.1.2. Nguyn liu.

Cm go. B khoai m. Tru. Khong dinh dng.

2.1.3. Mi trng nui cy nm mc

2.1.3.1. Mi trng gi ging [30], [9] Mi trng gi ging PGA (Potato Glucose Agar) - Khoai ty - Glucose - Agar - Nc ct C cht 70g Tru to xp 20g Thnh phn khong theo mi trng Czapeck: - NaNO3 - KH2PO4 - MgSO4 - FeSO4 3g/l 1g/l 0.5g/l 0.01g/l 200g 20g 20g 1000ml

2.1.3.2. Mi trng bn rn nui nm mc thu enzyme

Nc ct + thnh phn khong Czapeck to m 50%.

-33-

2.1.4. Mi trng nui nm men [32]

2.1.4.1. Mi trng gi ging Mi trng Hansen - Glucose - KH2PO4 - MgSO4.7H2O - Agar - Nc ct - ng kh - DAP - Urea - pH - Mt t bo 50g 3g 2-5g 20g 1000ml

2.1.4.2. Mi trng nui nm men thu sinh khi 4% 0.15% 0.15% 5.0 ~ 2*105 tb/ml

2.2. PHNG PHP NGHIN CU 2.2.1. Phng php xc nh pH [11]

Tin hnh: Cn chnh xc 10g nguyn liu ha tan trong 100ml nc, dng que thy tinh khuy u. lng sau 1 gi, ly dch trong em o vi my o pH.

2.2.2. Phng php xc nh m [5]

Nguyn tc: Dng sc nng lm bay hi nc trong nguyn liu. Cn trng lng mu trc v sau khi sy kh tuyt i. Trng lng mu gim khi sy kh tuyt i chnh l lng nc c trong mu. Tin hnh: Cn trng lng b (sau khi sy kh tuyt i). Cn m(g) mu. Cho mu vo b ri em sy n trng lng khng i, a vo bnh ht m lm ngui n nhit phng. Cn li c mu + b bng cn phn tch chnh xc n 0.0002g.

-34 m ca mu c tnh theo cng thc sau: * 100% Trong : - m1: khi lng mu + b trc sy - m2: khi lng mu + b sau sy - m: khi lng mu ban u

2.2.3. Phng php xc nh lng ng kh theo Miller [17], [29]

2.2.3.1. Nguyn tc Phng php ny da trn c s phn ng to mu gia ng kh vi thuc th 3,5- dinitrosalisylic (DNS). Cng mu ca hn hp phn ng t l thun vi nng ng kh. Da theo th chun ca glucose tinh khit vi thuc th acid dinitrosalisylic s tnh c hm lng ng kh ca mu nghin cu. Kh ha

Mu vng

Mu cam

Hnh 2.1: C s ha hc ca phn ng nh lng ng kh 2.2.3.2. Ho cht: Thuc th acid dinitrosalisylic (DNS): cn 1g DNS pha trong 20ml dung dch NaOH 2N, thm vo 50ml nc ct v 30g mui sodium potassium tartrate. un nh cho tan ri nh mc ti 100ml. Dung dch glucose mu (1mg/ml): cn chnh xc 100mg D-glucose ha tan trong 100ml nc ct. 2.2.3.3. Cch tin hnh Dng ng chun glucose

-35T dung dch glucose chun, pha cc dung dch glucose chun c nng t 0 1000 (mg/ml). ng nghim 0 1 2 3 4 5 6 7 8 9 10 Nng glucose 0 (mg/ml) Dung dch glucose 0 chun (ml) Nc ct (ml) 10 9 8 7 6 5 4 3 2 1 0 Ht 1 ml glucose vi cc nng khc nhau cho vo cc ng nghim sch theo th t t 0-10 DNS 1% (ml) 1 1 1 1 1 1 1 1 1 1 1 Lc u, em un cch thy trong 10 pht. Lm ngui v o mt quang bc sng 540nm V ng chun glucose vi trc tung l mt quang, trc honh l nng glucose. Tin hnh th nghim: phn ng ca dung dch ng cn phn tch vi thuc th DNS c tin hnh nh trong phn xy dng th chun. Nng ng kh trong mu c xc nh nh vo ng chun glucose dng. 1 2 3 4 5 6 7 8 9 10

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

2.2.4. nh lng tinh bt theo phng php thy phn bng acid [2], [7]
Nguyn tc: Di tc dng ca HCl tinh bt b thu gii thnh glucose theo phng trnh: (C6H12O5)n +n H2O nC6H12O6 Xc nh hm lng glucose to thnh, nhn vi h s chuyn i tinh bt thnh ng l 0.9 ta suy ra hm lng tinh bt c trong nguyn liu. Tin hnh:

-36Cn 1g nguyn liu c cha tinh bt cho vo cc, thm 50 ml nc ct lc u, yn trong vng khong 30 - 45 pht lc qua giy lc ra cn bng nc ct khong 5- 6 ln, chc thng giy lc chuyn cn vo bnh cu (bnh hon lu) bi 25ml dung dch HCl 5%. Lp bnh cu vo h thng hon lu trong vng 3 gi ly h thng ra ngui sau trung ho bng NaOH 5% n pH = 5-6. Chuyn hn hp vo bnh nh mc 100ml thm nc cho n vch, lc u v lc qua giy lc. Ly 1 ml dch lc em i xc nh hm lng ng glucose to thnh theo phng php DNS, t suy ra hm lng tinh bt tng ng. Cch tnh Hm lng tinh bt ca nguyn liu

A(%) =
A: hm lng tinh bt.

a * V * n * 0.9 * 100 m

a : nng glucose (mg/ml) n: s ln pha long (ln) m: khi lng mu (g) 0.9: h s chuyn i glucose thnh tinh bt

2.2.5. Xc nh Nit tng s theo phng php Kjeldahl [2], [7], [26]
2.2.5.1. Nguyn tc Di tc dng ca H2SO4 c, nhit cao, cc hp cht c cha nit b phn hu v b oxy ho n CO2 v H2O. Cn nit chuyn thnh amoniac (NH3) v tip tc kt hp vi H2SO4 to thnh mui amoni sunfat. Khi cho tc dng vi cht kim mnh nh NaOH s phng thch ra NH3 (NH4)2SO4 + 2NaOH = Na2SO4 + H2O + NH3 - Lng NH3 phng thch ra c hi nc li cun bng mt dng c l my Parnas-wargner v c dn n bnh hng c cha H3BO3

-372NH4OH + 4H3BO3 = (NH4)2B4O7 + 7H2O - nh lng amoni tetraborat to thnh bng dung dch H2SO4 0,1N. T tnh c lng N tng trong mu. 2.2.5.2. Tin hnh V c ho nguyn liu Cn 1g mu ti hoc 0,1 n 0,3g mu kh nghin nh. Cho mu vo bnh Kjeldahl. Nu mu lng th dng pipet, ht 2 n 5ml cho vo bnh Kjeldahl thm 5 ml H2SO4 m c v 0,5 xc tc (hn hp 9K2SO4:1CuSO4). nghing bnh trn bp t trong t hoffer un khong 2-3 gi cho n khi dung dch tr nn trong sut v c mu xanh da tri nht, ngui. Chuyn dch sang bnh nh mc 50ml trong bnh Kjeldahl nhiu ln bng nc ct v m dn nc ct n vch nh mc. Ct m t bnh hng (c cha 20ml H3PO4 3% thm vi git ch th Tasiro) vo vi ng sinh hn. Ht 10ml dung dch v c ho, cho vo phu ca my ct m v m kho cho vo bnh phn ng, trng phu 3 ln bng nc ct. Cho 10ml dung dch NaOH m c cho xung bnh t t, trng mt t nc ct. my li cun trong 5 pht ri h bnh tam gic xung thm 2 pht na. nh lng (NH4)2B4O7 to thnh bng dung dch H2SO4 0,1N, dung dch chuyn t mu xanh lc sang mu tm . (NH4)2B4O7 + H2SO4 + 5H2O 2.2.5.3. Tnh kt qu Hm lng Nit tng s c trong mu c tnh theo cng thc sau: Nmg% = V*1,42*100 *(50/10) m*(100-w) Trong : - V: Dung dch H2SO4 0,1N dng chun (ml) - 1,42: S mg nit ng vi 1ml H2SO4 0,1N (NH4)2SO4 + 4H3BO3

-38- m: Trng lng mu phn tch ( mg). - w: m mu phn tch (%) (w/w) - 100: H s chuyn thnh (%)

2.2.6. Phng php xc nh s lng vi sinh vt

2.2.6.1. nh lng mt t bo trc tip bng bung m hng cu [9], [10], [16], [30] Nguyn tc Bung m Goriaep l mt phin knh dy hnh ch nht, gia l phn lm phng, ti y c k mt li gm 4, 5 hnh vung c din tch tng cng l 1mm2 v c chia thnh 25 vung ln c din tch mi l 1/25mm2. B dy ca khong trng bung m l 1/10mm. Mi nh c din tch 1/400mm2. Do vy, mi ln c th tch l 1/250mm3 hay 1/250 x 103cm3 = 4 x 10-6ml. Tin hnh Lc mnh dch huyn ph, nh mt git bng pipet ln b mt bung m k vi cnh ca l knh. t bung m ln bn knh hin vi. Bt u m t bo sau khi nh git dch t 3 n 5 pht. m t nht 5 ln, ly tr s trung bnh. m bo mt t bo khng qu ln (ln hn 200 t bo trong mt ln). Cch tnh kt qu S t bo trn mt ml mu phn tch (N) c tnh nh sau N (t bo/ml) = (a x K) / v Trong a: S t bo trung bnh c trong mt ln. v: Th tch mt ln = 4 x 10 -6ml K: H s pha long mu. Vy N (t bo/ml) = (a x K)/ (4 x 10 -6) = 0,25 x a x K x 10 6

-392.2.6.2. nh lng mt t bo bng phng php o mt quang [9], [15] Nguyn tc T bo vi sinh vt l mt thc th nn khi hin din trong mi trng cn nh sng, lm phn tn chm nh sng ti cng lm mi trng tr nn c. c ca huyn ph t l vi mt t bo. Do vy c th nh lng mt t bo mt cch gin tip thng qua o c bng my so mu bc sng 610nm. Tin hnh Xy dng ng tng quan tuyn tnh gia c v mt t bo. Pha long mt huyn ph nm men cn kim nghim c mt t bo bt k thnh cc huyn ph khc nhau c c o OD 610 nm t cc gi tr ln cn 0,1; 0,2; 0,3; 0,4; 0,5. o OD 610nm ca cc huyn ph va c pha, ghi nhn cc s o thc t. Dng phng php m trc tip di knh hin vi (dng bung m hng cu) xc nh mt t bo (N/ml) ca cc huyn ph ny. Tnh gi tr log (N/ml) cho mi gi tr mt N/ml tng ng vi c. V ng biu din ca log (N/ml) theo OD 610nm. Xc nh mt t bo theo c: - o c ca mt huyn ph t bo cn xc nh mt . - T gi tr OD 610nm o c, suy ra s lg (N/ml) v tr s mt N/ml t ng chun. V th ng chun biu din tng quan gia gia gi tr OD v lgx.

2.2.7. Phng php nui vi sinh vt

2.2.7.1. Phng php nui cy nm mc thu enzyme [9] Chun b mi trng bn rn c thnh phn theo mc 2.1.3.2. Cho 20g mi trng vo erlen 250, em hp kh trng 1210C, 1atm trong 30 pht. Thm vo ng ging cc chng nm mc 10ml nc ct v trng 0,05% agar, lc u. Xc nh lng t bo nm mc theo mc 2.2.6.

-40Thi gian nui: ty thuc vo chng nm s c cc thi gian ti u khc nhau. Nhit nui nm mc l nhit phng. Tin hnh nui ng thi nm mc vi c cht c t l khc nhau v kho st thi gian ti u cho glucoamylase c hot cao nht. 2.2.7.2. Phng php nui cy nm men thu sinh khi [5] Chun b mi trng lng nui cy nm men theo mc 2.1.4.2. Cho 100ml mi trng vo erlen 250, em hp kh trng 1210C, 1atm trong 30 pht. Thm vo ng ging Saccharomyces cerevisea 10ml nc ct v trng 0,05% agar, lc u. Xc nh lng t bo nm men theo mc 2.2.6. Ht 1ml ging cy vo mi erlen (vi lng t bo khong 2*105). Sau 72h nui cy lc, ly dch nui ly tm thu sinh khi nm men dng paste.

-41-

2.2.8. Phng php xc nh thi gian thch hp thu nhn enzyme glucoamylase [9]
Nguyn tc: Mi loi sinh vt c mt ng cong tng trng khc nhau. Ti mi thi im khc nhau ca qu trnh sinh trng, chng s dng cc ngun c cht khc nhau trong mi trng nui cy bng cch tit ra h enzyme thy phn c hiu. Kho st kh nng sinh tng hp enzyme glucoamylase theo thi gian nui cy ca nm mc nhm xc nh thi gian ti u thu nhn c enzyme c hot cao nht. Tin hnh: Chun b mi trng bn rn c thnh phn theo mc 2.1.3.2. Cho 20g mi trng vo erlen 250, em hp kh trng 1210C, 1atm trong 30 pht. Thm vo ng ging cc chng nm mc 10ml nc ct v trng 0,05% agar, lc u, cy vo mi erlen 1ml dch huyn ph. Nui mc iu kin nhit phng th nghim, trung bnh 300C. Ti cc thi im 24h, 30h, 36h, 42h, 48h, 54h, 60h, 66h v 72h tin hnh kho st hot enzyme glucoamylase. Biu din s bin i hot enzyme glucoamylase theo thi gian sinh trng khc nhau ca nm mc chn ra thi im nm mc cho hot glucoamylase cao nht Chn thi im m nm mc cho hot glucoamylase cao nht thc hin nui cy thu ch phm enzyme.

2.2.9. Phng php tch chit v thu nhn dung dch enzyme
2.2.9.1. Phng php tch chit v thu nhn dch enzyme th [9] Nguyn tc: enzyme glucoamylase l enzyme ngoi bo nn vic tch chit kh n gin. Nm mc tit enzyme glucoamylase ra ngoi v ngm vo mi trng bn rn nui nm mc, nn chit enzyme ch cn nghin nh

-42v ha tan canh trng vo trong nc ct hoc dung dch m c pH thch hp, chit ly dch lng. Dch enzyme thu c l dung dch enzyme dng th. Tin hnh Cn 20g canh trng nui nm mc nghin nhuyn cho vo erlen, thm 100ml nc ct hay dung dch m. Lc 30 pht, lc qua vi, ly tm dch lc 4000 vng/pht trong 10 pht. Thu ly dch ly tm, nu thy dch cha trong th lc qua giy lc ri nh mc cho 100ml. Dch enzyme thu c l dung dich enzyme glucoamylase th. 2.2.9.2. Phng php thu nhn ch phm enzyme glucoamylase t dch enzyme bi tc nhn ta cn [8], [14] Nguyn tc Nguyn tc kt ta protein l da vo ho tan ca chng. ho tan ca protein trong dung dch ph thuc vo nhiu yu t v mt trong s l hng s in mi ca dung dch. Cc dung mi vi hng s in mi nh (aceton, ethanol. . .) ngn cn s phn tn ca cc phn t protein gim v xy ra s kt ta do vic lm gim hng s in mi hin hu ca mi trng. iu ny c c bng cch thm mt dung mi ho tan trong nc (aceton, ethanol) vo dung dch cha protein. Tin hnh Cho t t cn 96% lnh (tng git bng buret v khuy u) vo dung dch protein (enzym th) trong iu kin lnh nhit t 4-80C, vi t l Vcn/V dd enzyme = 3/1. yn trong t lnh 15 pht. Ly tm thu kt ta vi tc 5000 vng/pht, thi gian 10 pht. Kt ta c sy kh nhit nh hn 400C. Nghin nh thnh dng bt. 2.2.9.3. Phng php tnh hiu sut thu nhn ch phm enzyme Thu ch phm enzyme nh mc 2.2.9.2 v tnh hiu sut thu nhn ch phm enzyme theo cng thc:

-43Trong : - %H : hiu sut thu nhn CPE - m(CPE): khi lng ch phm enzyme (g) - m(CT): khi lng canh trng (g)

2.2.10. Phng php xc nh hot glucoamylase [17], [22]

nh ngha n v hot [17] n v hot ca enzyme c tnh theo n v quc t UI (International Unit). Mi UI l hot ng ca enzyme xc tc ch th s chuyn ha 1 mmol c cht sau 1 pht iu kin thch hp nht. Hot ca enzyme glucoamylase Mt n v hot (UI) ca glucoamylase l lng enzyme xc tc thy phn tinh bt gii phng ra 1mg glucose trong 1 pht iu kin nhit v pH thch hp. Xc nh hot enzyme glucoamylase theo phng php so mu vi DNS. Cch tin hnh: Mu th khng: Ht 0,5ml ch phm enzyme glucoamylase pha long nng thch hp cho vo mt ng nghim sch v kh. Thm 0,5ml h tinh bt 2%. Thm 1ml thuc th DNS, un si 15 pht, lm ngui ri em xc nh mt quang. Mu th tht: Ht 0,5ml ch phm enzyme glucoamylase pha long nng thch hp cho vo mt ng nghim sch v kh. Thm 0,5ml h tinh bt 2%, thc hin phn ng 450C, pH 5.0 trong 10 pht. Thm 1ml thuc th DNS, un si 15 pht, lm ngui ri em xc nh mt quang. Cc dung dch sau phn ng ny c xc nh mt quang bc sng 540nm. Da vo ng chun glucose xc nh c lng ng kh to thnh sau khi thy phn bi enzyme glucoamylase. Hot glucoamylase tnh trn 1ml dch lc enzyme

-44-

Trong : - X: lng glucose gii phng c xc nh da vo ng chun (mg/ml) - n: h s pha long - t: thi gian phn ng - a: lng enzyme tham gia phn ng Hot glucoamylase tnh trn 1g canh trng

Trong : - H(UI/ml): n v hot enzyme trong 1ml dch lc - V: th tch dch enzyme thu c sau khi lc (ml) - m: khi lng canh trng ban u em trch ly (g)

2.2.11. Phng php xc nh hm lng protein theo Bradford [17], [8], [2]
Nguyn tc: Cc protein khi phn ng vi xanh Coomassie (Coomassive Brilliant Blue CBB) s hnh thnh hp cht mu c kh nng hp thu nh sng bc sng 595nm, cng mu t l vi nng protein trong dung dch. Ha cht: Thuc th Bradford: - Coomassie Brilliant Blue (CBB) - Ethanol 96o 4,7g 8.5g - Acid phosphoric 85% 10mg

-45CBB c lm ha tan trong ethanol 96o, sau b sung acid phosphoric 85%, nh mc vi nc ct n 1000ml. Dung dch Albumin 100mg/ml: cn chnh xc 10mg Albumin, ha vi nc ct thnh 100ml. Tin hnh: Dng ng chun Albumin ng nghim Dung dch Albumin 0.01% (ml) Nc ct (ml) Nng protein (mg/ml) 1 0 10 0 2 1 9 10 3 2 8 20 4 3 7 30 5 4 6 40 6 5 5 50

Ht 1ml dung dch Albumin c nng va pha trn vo cc ng nghim sch khc nhau theo th t tng ng t 1 n 6 Thuc th Bradford (ml) 5 5 5 5 5 5 Lc u, yn trong 10 pht. Sau em o mt quang bc sng 595nm V th biu din s bin thin mt quang (DOD) theo nng protein chun (mg/ml). Xc nh hm lng protein trong mu: ht 1ml dung dch protein cn phn tch, thm 5ml thuc th Bradford. Lc u, yn trong 10 pht, em o mt quang bc sng 595nm. T th ng chun suy ra hm lng protein c trong mu.

10-3 (mg/gCPE)

Trong : - HLpr: hm lng protein (mg/gCPE)

-46- x: hm lng protein suy ra t ng chun (mg/ml) - n: h s pha long - V: th tch dung dch enzyme - m: khi lng CPE

2.2.12. Phng php xc nh hot ring ca ch phm enzyme

i vi cc ch phm enzyme, ngoi vic xc nh mc hot ng cn phi nh gi mc tinh sch. i lng c trng cho mc tinh sch ca ch phm l hot ring. Hot ring ca enzym l s n v hot enzyme c tnh trn 1mg protein ca CPE.

Trong : - HR: hot ring ca ch phm enzyme - HC: tng hot enzyme UI/g CPE hay ml enzyme - HLpr: hm lng protein trong 1g CPE (mg)

2.2.13. Phng php kho st c tnh enzyme glucoamylase Nguyn tc: hu ht cc enzyme hot ng xc tc trong iu kin
nh nhng. Tuy nhin tng hiu qu xc tc th cc enzyme thng c gi trong nhng iu kin phn ng ti u v nhit , pH. Kho st s nh hng ca nhit ln hot ca enzyme

glucoamylase
Cn 0.1g CPE ho trong 50ml nc ct, thu c th tch dung dch enzyme glucoamylase. Pha long dch enzyme vi nng thch hp. Tin hnh xc nh hot chung ca enzyme vi cc nhit cn kho st: 30; 40; 50; 60; 70 (0C).

-47V ng biu din bin thin hot ca glucoamylase (trc tung) i vi nhit (trc honh). T suy ra nhit ti u cho ch phm enzyme glucoamylase.

Kho st s nh hng ca pH ln hot ca enzyme glucoamylase Pha m Na-acetate c cc gi tr pH 3.0; 3.5; 4.0; 4.5; 5.0; 5.5;
6.0; 6.5; 7.0. Tinh bt 2% pha trong m vi cc gi tr pH khc nhau. Cn 0.1g CPE ha tan trong 50ml dung dch m Na-acetate vi cc gi tr pH khc nhau, thu c th tch dung dch enzyme glucoamylase. Pha long dch enzyme vi nng thch hp. Tin hnh xc nh hot chung ca enzyme vi cc gi tr pH cn kho st. V ng biu din bin thin hot ca glucoamylase (trc tung) i vi pH (trc honh). T suy ra pH ti u cho ch phm enzyme glucoamylase.

2.2.14. Phng php ng dng ch phm enzyme glucoamylase thy phn cc loi tinh bt [11]
Tin hnh kho st s thy phn cc loi c cht khc nhau ca ch phm enzyme glucoamylase trong iu kin pH v nhit thch hp nht. Chun b - Dung dch enzyme vi cc nng 0,1UI/ml dd E; 0,2UI/ml dd E; 0,3UI/ml dd E; 0,4UI/ml dd E; 0,5UI/ml dd E: cn chnh xc lng CPE pha trong 100ml dung dch m Na-acetate vi pH thch hp. - Dung dch c cht 10%: cn chnh xc 10g c cht ha tan trong 100ml dung dch m Na-acetate vi pH thch hp. Tin hnh Cho 40ml dung dch c cht 2% vo bnh tam gic 250ml t ln bp n nhit vi nhit ti u cho phn ng xc tc ca enzyme. Thm vo 10ml dung dch CPE, lc u thc hin phn ng thy phn c cht ca enzyme glucoamylase. Ti cc thi im cch nhau 30 pht, ht 1ml dung

-48dch phn ng em xc nh lng ng kh theo phng php Miller (mc 2.2.3). Kt qu Tnh DOD theo tng thi im thy phn, tng ng l lng ng kh to thnh cc thi im khc nhau. V th biu din s bin thin ca lng ng kh to thnh theo tng thi gian thy phn. 2.2.15. Phng php s dng phn mm Microsoft Excel trong x l tnh ton kt qu V cc bng, th, biu bng table, chart wizard c sn trong Excel. S dng cc hm Average, Stdev tnh, trung bnh, lch chun ca cc s liu.