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MT S PHNG PHP TA PROTEIN
I. Phng php ta bng cc tc nhn gy ta l dung mi hu c (Ethanol 80%,
Aceton) ha tan ca protein trong dung dch ph thuc vo nhiu yu t, mt trong s l hng s in mi ca dung dch. Nhn chung, nhng phn t dung mi c hng s in mi ln (nh nc, dimethylsulphoxid) c th n nh tng tc gia chng vi cc phn t protein v to iu kin thun li cho s ha tan ca protein trong dung dch. Ngc li, cc dung mi vi hng s in mi nh (aceton, ethanol) ngn cn s phn tn ca cc phn t protein trong mi trng. Do , ha tan ca nhng phn t protein gim v xy ra kt ta do s lm gim hng s in mi hin hu ca mi trng. iu ny c c bng cch thm mt dung mi ha tan trong nc nh aceton vo dung dch cha protein. S ta bng aceton hoc ethanol 80% c nhiu thun li hn v n tng i r, c sn dng tinh khit vi t cht tp nhim gy c hay c ch i vi enzym, do nhit bay hi ca dung mi thp nn d tch b dung mi khi ch phm enzym bng phng php sy nh bng qut gi. II. Phng php ta bng mui Ngi ta c th dng mui (NH4)2SO4, NaCl ta protein v cc mui ny va lm trung ha in (do cc ion tc ng tng h vi cc nhm tch in tri du), va loi b lp v hydrat ca phn t protein. Cc protein khc nhau c th b kt ta vi nng mui khc nhau, v vy c th dng mui tch ring cc protein ra khi hn hp ca chng. Phn th nghim 1. Dng c v ha cht Dng c Ha cht Pipet 5ml, 10ml Nguyn liu thm (da) Becher 50ml, 100ml, 250ml Etanol 96% Bnh tia Mui (NH4)2SO4 My ly tm 2. Thc hnh Cn khong 80g n 100g thm, gi nt vt ly nc ri ly tm 6000 vng/pht trong 15 pht ri ong xem th tch ca nc thm l bao nhiu, ghi nhn th tch V. Sau chia i th tch V nc thm va thu c thnh hai phn bng nhau ri tin hnh ta protein bng hai tc nhn ta khc nhau: Ethanol 80% v mui (NH4)2SO4 Quy trnh kt ta enzym Bromelin bng cn 80% Chi ngn, v qu
Lm sch
Ta cn 4oC / 2 gi
Thu ta
Sy kh
Hn hp dch da, nhit phng 30 pht Ly tm 6000 v/p, trong 15 pht Thu ta B dch trong
Sy kh Bromelin Lu : c mui(NH4)2SO4 bo ha 70% ta phi trn vi t l 47,2g mui trong 100ml nc thm. 3. Kt qu: Hy tnh hm lng protein c trong 100g nguyn liu ban u.
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Dung dch Tyrosin chun (ml) 0 0,2 0,4 0,6 Lng Tyrosin tng ng (M) 0 0,2 0,4 0,6 Dung dch HCl 0,2N (ml) 5,0 4,8 4,6 4,4 Dung dch NaOH 0,5N (ml) 10 10 10 10 Thuc th Folin (ml) 3 3 3 3 Lc mnh, sau 10 pht o OD bc sng 660nm
ng s 1 l ng th khng (TK), cc ng cn li l ng th nghim (TN). V ng chun Tyrosin tng quan gia lng Tyrosin (M) v OD (OD = ODTN ODTK).
Vi: H Protease : hot enzym protease (UI/g) V : tng th tch hn hp trong ng th tht hoc ng th khng (ml) v : th tch dch lc em phn tch (ml) t : thi gian thy phn (pht) m : khi lng mu enzym em i xc nh hot tnh (g) L: pha long enzym M Tyrosin: lng M Tyrosin trong v (ml) suy ra t ng chun
thy gii trong 30 pht nhit phng. Tip , em tt c cc ng un cch thy trong 2 pht, lm lnh di vi nc. Ht 5 ml ca hn hp cho vo mt erlen 250ml v nh phn ng glucose nh sau: cho thm vo bnh 10ml dung dch Iod N/10 v 15 ml dung dch NaOH N/10. Thi gian nh NaOH phi ko di khong 2 pht (nh tng git). yn trong bng ti 15 -20 pht. Ly ra thm vo 2ml dung dch H2SO4 N. nh phn lng iod thm cn tha bng dung dch Na2S2O3 N/20 (dng h tinh bt cho thm vo lc cui phn ng lm cht ch th mu).
IV. Kt qu Hot tnh ca invertase c biu th bng s mol saccharose thy gii bi lng enzym invertase c trong 1g men bia trong 1 pht nhit phng th nghim.
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a: th tch ly ra trong mi ng nh ng glucose (ml) A: th tch tng cng trong mi ng (ml) b: th tch dung dch enzym cho vo mi ng (ml) B: tng th tch dung dch enzym c c t mg men (ml) t: thi gian thy gii tnh bng pht.
XC NH HOT TNH ENZYM ALPHA AMYLASE (THEO PHNG PHP SMITH & ROE)
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Nguyn tc: S v c ha cht m l s bin i tt c cc cht m d nm di dng no (hu c, v c, protein) thnh hp cht v c l amonium sulfat (NH4)2SO4 bng cch un si vi H2SO4 m c v cht xc tc.
H2SO4 m c Cht m Xc tc, t0 (NH4)2SO4
Thc hnh: ht chnh xc 1ml nu l nguyn liu lng hay cn chnh xc 1g nu l nguyn liu kh nghin nhuyn cho ng nht, cho vo mt bnh c cao c dung tch t 60ml n 100ml (bnh kjeldahl). Thm vo 5ml H2SO4 m c v khong 0,5g cht xc tc. un si hn hp trong t ht kh c t 2 n 3 gi cho n khi dung dch tr thnh trong sut v c mu xanh do tri nht. ngui v pha long thnh 100ml vi nc. Cht xc tc dng y l hn hp xay nhuyn ca: K2SO4 v CuSO4 c t l theo trng lng l 9: 1 Thc hin 3 s th tht (c cht m) v 3 s th khng c cht m (thay cht m bng nc ct) II. Phng php kjeldahl:
Nguyn tc: Cht m c v c ha nm di dng Amonium sulfat em cho tc dng vi cht kim mnh nh NaOH s phng thch ra amoniac
(NH4)2SO4 + 2NaOH 2NH4OH + Na2SO4
Sau lng amoniac c hi nc ni cun bng mt dng c my Parnas Wargner v c dn n mt bnh tam gic c cha mt lng tha H 2SO4. T y cho php chng ta xc nh c lng amoniac phng thch ra, c ngha l xc nh c lng m c trong mu nguyn liu.
Bnh xt nc ct Becher 250 ml, 100ml, 50ml Erlen 250ml Bnh nh mc 100ml, 50ml Buret 25ml Pipet 1ml, 5ml, 10ml, 20ml. ng ong 25ml
Dung dch H2SO4 N/100 Dung dch NaOH N/100 (dung dch ny sau khi pha xong dng ngy th ng nng , nhng lu s b carbonat ha nn nng khng cn ng nh ban u, v th phi xc nh li h s chnh l x bng cch nh phn n vi dung dch acid chun (acid oxalic, acid phtalic) Dung dch metil 0,5% pha trong ru etylic.
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Cch thc hnh: un si bnh nc my Parnas Wargner. t erlen c cha 25ml H2SO4N/100 v vi git metil vo phn ui ca my sao cho phn nhn nhng chm vo dung dch. Ht 10ml dung dch v c ha pha long thnh 100ml, em vo my Parnas Wargner; thm vo 10ml NaOH m c ( ghi nh l khi cho mu cng nh NaOH m c vo my, phi cho xung t t bng kp Mohr, nu cho chy xung qu nhanh th dung dch s b my ht ra ngoi). Tip tc un si bnh nc v cho hi nc li cun amoniac sang erlen trong 3 pht, h erlen xung v tip tc un thm 2 pht na, trng vi ca my bng mt tia nc ct, tt c u cho chy vo erlen ng H2SO4 N/100. Ly erlen ra v nh phn H2SO4 tha bng dung dch NaOH c chun l x N/100 cho u. Khi dung dch i mu (t mu hng nht sang mu da cam): thc hin 3 s th thut v 3 s th khng ly tr s trung bnh. Cch tnh: Nu nguyn liu l cht lng, thng thng lng m c tnh bng s gam m trong 1 lt, nu nguyn liu l cht rn, th trc ht phi tnh m, sau lng m c tnh bng phn trm (s gam m c trong 100g nguyn cht liu) theo kh tuyt i) Gi s nguyn liu l cht lng. Ly 1ml nguyn liu em v c ha v pha long thnh 100ml, sau ht 10ml em chng ct bng my Parnas Wargner. Gi V0 l th tch dung dch NaOH x N/100 (tr s trung bnh ca 3 ln th khng). Gi Vt th tch dng dch NaOH x N/100 (tr s trung bnh ca 3 ln th tht). Vy:
V = V0 Vt
l lng NaOH tng ng vi lng amoniac phng thch bi 10ml dung dch v c ha pha long.
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S gam m c trong 10ml dung dch v c ha pha long l: S gam m c trong 1000gl dung dch v c ha pha long hay trong 1ml nguyn liu
14.Vx.10 5.100 = 14.Vx10 4 g 10
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KHO ST LIPID
I. Kho st tnh ha tan ca lipid
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NH LNG VITAMIN C
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Ha cht
Erlen 100ml - Dung dch HCl 5% - Pipet 5ml, 10ml - Dung dch Na2S2O3 0,01N - Buret - Dung dch tinh bt 1% - Bnh nh mc 50ml - Dung dch iod 0,01N - Becher - Ci chy s - Phu thy tinh 3. Cch thc hnh a. Chun b mu vt: Cn N (g) mu vt v nghin nhanh chng trong ci s vi 5ml HCl 5%. Vitamin C rt d b oxi ha trong khng kh nht l khi c s hin din ca cc ion kim loi (Fe, Cu). V vy trong khi chun b mu phi ct hoc nghin bng dao khng r v lm nhanh. Dch chit thu c cho vo bnh nh mc 50ml v thm nc ct (ra ci chy v b vi ln bng nc ct ri vo bnh), khuy u ri lc. b. nh phn: - nh chun li dung dch iod N/100 vi dung dch Na2S2O3 0,01N tnh h s hiu chnh T ca dung dch iod. - Chun b hai erlen. Ht vo mi bnh 20ml dch chit c cha vitamin C, 5 -10 git dung dch h tinh bt 1% v chun ngay bng dung dch iod 0,01N n khi c mu xanh
4. Kt qu
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a: th tch (ml) dung dch iod 0,01N dng khi chun . T: h s hiu chnh dung dch iod 0,01N vi dung dch Na2S2O3 0,01N V: th tch chung ca dch chit (50ml) v: th tch dch chit ly chun (20ml) N: trng lng mu phn tch 0,00088: s gram vitamin C tng ng vi 1ml dung dch iod 0,01N
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