Muaj yuav luag tag nrho cov tuam txhab uas muag covbromelain enzyme hmoovyog tov nrog papain.Nws nyuaj rau purify bromelain mus rau 5000GDU/g. tab sis nws yooj yim heev los ntxuav cov papain rau 1000,000GDU/g. txawm mus txog 2000,000GDU/g. tab sis cov haujlwm ntawm bromelain txawv ntawm papain. xim, ordour, dej solubility, thiab saj sib txawv nrog cov khoom cuav thiab cov khoom tseeb. Lawv yog qhov sib txawv ntawm txoj cai bromelain enzyme hmoov yog grey, tsis muaj ntxhiab, tsis qab thiab dej insoluble.
Tab sis qhov cuav bromelain muaj cov yam ntxwv hauv qab no:
● Nws yog lub teeb-daj, muaj ntxhiab tsw qab thiab me ntsis qab zib, thiab dej soluble. Nws yog tus yam ntxwv ntawm papain.
● Nws yog siv los txo qhov mob thiab txo qhov mob. tab sis yuav luag tsis muaj cov haujlwm no hauv papain.
● Nws yog siv los ua cov khoom noj khoom haus nkaus xwb, tsis yog khoom noj khoom haus ntxiv, tab sis qhov tseeb tuaj yeem yog qib pharma. Tab sis papain tsuas yog siv los ua nqaij tenderizer hmoov thiab tshuaj pleev ib ce.
Peb tau kuaj cov bromelain raws li SDS-PAGE gel thiab GELATIN DIGESTION UNIT ANALYTICAL thiab HPLC txoj kev, muaj tsawg lub hom phiaj molecules hauv SDS-PAGE gel txoj kev rau cov khoom cuav.
Hauv qab no yog SDS Page electrophoresis cov txiaj ntsig
Cov speckle nyob rau hauv cov saum toj no qhia tau hais tias tsuas yog cov khoom uas yog muaj tib speckle nyob rau hauv txoj cai qhov chaw nyob rau hauv daim duab, tab sis cov khoom cuav muaj me ntsis speckles rau nws, uas txhais tau hais tias qhov hnyav molecular yog NW nyuam qhuav pom.
Qhov hnyav molecular ntawm Bromelain yog txog 33000, tab sis qhov hnyav molecular ntawm papain yog 21000. Thiab HPLC Chromatogram:
Koj yuav pom tias tsuas yog txoj cai extract muaj qhov hnyav molecular ntawm bromelain thiab kuaj tau los ntawm HPLC.
Tab sis yog tias peb tsuas yog kuaj los ntawm Gelatin Digestion Unit Analytical Method (GDU) saib Annex II. Ob txoj cai thiab cov khoom cuav muaj cov txiaj ntsig zoo ib yam li hauv qab no:
Pineapple qia yog pheej yig heev, thiab tus nqi ntawm qia mus rau bromelain yog tsawg heev, muaj qee qhov ua paug hauv cov txheej txheem tsim khoom.
Yuav luag tag nrho cov bromelain enzyme hmoov Hoobkas nyob ze teb. Lawv siv cov qia tshiab. Nws yog tsis yooj yim sua kom xa cov qia nyob rau hauv ntev. Cov khoom siv raw yuav tsum tau ua kom sai li sai tau thaum nws tau sau rau hauv daim teb lossis nws yuav tsum muab khaws cia rau hauv qhov chaw qis qis (4-8 degree) kom tsis txhob muaj cov protein degradation. Hauv lub neej tiag tiag, cov chaw tsim khoom tsis tuaj yeem ua tau raws li ob qho ntawm cov xwm txheej saum toj no vim muaj ntau cov pineapples stems. Txoj kev tsim nyog yog qhov sib npaug ntawm kev tuav lub sijhawm thiab kev ua kom muaj protein ntau. Feem ntau, kev tsim cov khoom siv raw hauv lub caij nplooj ntoos hlav sau qoob loo, thiab khaws cov ntaub ntawv nyoos rau hauv qhov kub thiab txias (4-8 degree ).
Peb tab tom ua qee qhov kev ntsuam xyuas txog SDS Page electrophoresis, Hauv kev xav peb tuaj yeem tsa nws mus rau 10, 000 lossis 20, 000.tab sis nws yuav kim heev. 5000GDU / g thiab ua kom ruaj khov tej zaum ntawm malto kev txhawb nqa / microencapsulated ib lossis ib yam dab tsi ntxiv rau qib organic extraction / purification.
Annex I
● Txoj Kev 5 SDS polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE yog ib txoj kev denatured polyacrylamide gel electrophoresis. Lub hauv paus ntsiab lus ntawm kev sib cais ntawm cov protein nyob rau hauv txoj kev no yog ua raws li qhov tseeb tias feem ntau cov proteins tuaj yeem ua ke nrog anionic surfactant sodium dodecyl sulfate (SDS) hauv qhov hnyav piv los tsim ib qho nyuaj.
Tus nqi tsis zoo nqa los ntawm cov protein molecules deb tshaj li tus nqi ntawm cov protein molecules, tshem tawm cov nqi ntawm cov protein sib txawv thiab cais cov proteins raws li lawv cov molecular loj.
Txoj kev no yog siv rau kev txheeb xyuas qhov zoo, purity thiab impurity tswj, thiab txiav txim kom muaj nuj nqis ntawm cov protein.
1 Cov cuab yeej cuab tam
Tsis tu ncua voltage lossis tas li tam sim no fais fab mov, ntsug phaj electrophoresis tank, thiab nplaum ua pwm.
2. Reagents
(1) Dej.
(2) Sib cais gel tsis (4x, daws A) 1.5mol / L trihydroxymethyl aminomethane hydrochloric acid tsis. Qhov hnyav 18.15g ntawm trihydroxymethyl aminomethane thiab yaj nrog ib qho dej txaus. Kho tus nqi pH rau 88 nrog hydrochloric acid thiab dilute rau 100ml nrog dej.
(3) Nyhav 58. tsaus).
(4) Qhov hnyav 10g ntawm sodium dodecyl sulfate hauv 10 feem pua SDS tov (C tov), yaj hauv dej, thiab dilute rau 100ml.
(5) Kev lag luam reagent ntawm tetramethylethylenediamine tov (TEMED, D tov).
10% ammonium persulfate tov (E tov) Nyhav 10g ntawm ammonium persulfate, yaj nyob rau hauv dej, thiab dilute mus rau 100ml. Nws raug nquahu kom npaj nws ua ntej siv lossis khaws cia cais ntawm -20 degree rau 2 lub lis piam.
(7) Concentrated roj hmab buffer (4 ×, F tov) 0.5mol/L Trihydroxymethylaminomethane Hydrochloric acid buffer, hnyav 6.05g ntawm Trihydroxymethylaminomethane, ntxiv tus nqi tsim nyog ntawm cov dej kom yaj, kho tus nqi pH rau 6.8 nrog hydrochloric acid, thiab dilute nrog dej rau 100 ml.
(8) Electrode buffer (10 ×) Nyhav 30g ntawm trimethylaminomethane, 144g glycine, thiab 10g ntawm sodium dodecyl sulfate, yaj lawv hauv dej thiab dilute mus txog 800ml. Kho tus nqi pH rau 8.1-8.8 nrog hydrochloric acid, thiab dilute rau 1000ml nrog dej.
(9) Cov qauv tsis txo qis (4 ×) Qhov hnyav 303g ntawm trimethylaminomethane, 20mg ntawm bromophenol xiav, thiab 8.0g ntawm sodium dodecyl sulfate, ntsuas 40ml ntawm glycerol, yaj hauv dej thiab dilute mus txog 80ml. Kho cov pH rau 68 nrog hydrochloric acid, thiab dilute rau 100ml nrog dej.
(8) Electrode buffer (10 ×) Nyhav 30g ntawm trimethylaminomethane, 144g glycine, thiab 10g ntawm sodium dodecyl sulfate, yaj hauv dej thiab dilute mus txog 800ml. Kho tus nqi DH rau 81-88 nrog hydrochloric acid, thiab dilute rau 1000ml nrog dej
(9) Cov qauv tsis txo qis (4 ×) Qhov hnyav 3.03g ntawm trimethylaminomethane, 20mg ntawm bromophenol xiav, thiab 80g ntawm sodium dodecyl sulfate, ntsuas 40ml ntawm glycerol, yaj hauv dej, thiab dilute mus txog 80ml. Kho cov pH mus rau 6.8 nrog hydrochloric acid, thiab dilute rau 100ml nrog dej.
(10) Txo cov tshuaj tsis haum (4 ×) Nyhav 3.03g ntawm trimethylaminomethane, 20mg ntawm bromophenol xiav, thiab 80g ntawm sodium dodecyl sulfate, ntsuas 40ml ntawm glycerol, yaj thiab dilute nrog dej txog li 80ml, thiab ntxiv. - 20ml mercaptoethanol, kho pH rau 6.8 nrog hydrochloric acid, dilute nrog dej rau 100ml (los yog 3.03g ntawm trimethylaminomethane, 20mg ntawm bromophenol xiav, 8.0g ntawm sodium dodecyl sulfate, ntsuas 40ml ntawm glycerol, yaj nyob rau hauv dej thiab dilute. rau 80ml, kho pH rau 6.8 nrog hydrochloric acid, dilute nrog dej mus rau 100ml, thiab ua ntej siv, ntxiv dithiothreitol rau 100mmol / L).
● Annex II
Gelatin Digestion Unit Analytical Method (GDU)
A. Lub Hom Phiaj: Cov txheej txheem no yog siv los txiav txim siab txog kev ua haujlwm proteolytic ntawm Bromelain Enzyme Powder.
B. Khoom siv:
1. pH ntsuas
2. Qhov kub thiab txias dej da dej ntawm 45.0 degree ± 0.1 degree
3. Kev Ntsuas Ntsuas
4. Volumetric flasks
5. Volumetric pipettes
6. Timer
7. Digital Burette (Qhov tseeb ntawm 0.1 ml)
8. Fum hood
9. Tsis siv neeg Pipetters
C. Kev Tiv Thaiv Kev Nyab Xeeb:
Qhov kev sim no yuav tsum tau ua nyob rau hauv lub fume hood.
1. Siv cov txheej txheem kuaj kev nyab xeeb.
2. Formaldehyde: Npaj thiab khaws cia rau hauv lub fume hood txhua lub sijhawm. Paub carcinogen thiab teratogen.
3. Peroxide: Muaj zog oxidizer
D. Reagents thiab Reagent npaj:
1. Dej Distilled: kwv yees li 500 ml: kho rau pH ntawm 4.5 nrog 0.1 N HCI.
2. Gelatin substrate:
a. Dissolve 25 grams ntawm gelatin (Mikrobiologie. 1.04070) nyob rau hauv 375 ml dej kub, coj mus rau ib tug boil. Txias rau 45 degrees.
b. Kho cov pH rau 4.5 nrog 0.1 N HCI thiab dilute mus rau 500 ml pH 4.5 distilled dej.
c. Khaws cov gelatin substrate ntawm 45 degree.
3. Kev daws tsis haum:
a. Ntxiv 15 gm NaCl maj mam mus rau 40-50 ml dej hauv 150ml beaker thiab do kom yaj.
b. Ntxiv 0.570 ml acetic acid.
c. Kho cov pH rau 4.5 nrog 0.2N HCL (ntim yuav ntau dua 100ml.)
4.3% Hydrogen peroxide:
a. Pipette 2.5 ml ntawm 30 feem pua Hydrogen peroxide (Tshuag tov) rau hauv 25 ml volumetric hwj thiab dilute rau ntim nrog pH 4.5 distilled dej.
5. 37 feem pua Formaldehyde pH 9.0: a. Kho qhov ntim txaus (100} ml) ntawm formaldehyde rau pH 9.0 nrog 0.1 N NaOH (kwv yees li 20 ml ntawm formaldehyde ib tus qauv ua ntej hloov pH.)
Gelatin Digestion Unit Analytical Method (GDU) - cont.
6. 0.100 N NaOH: (Purchased standardized) a. Kev daws teeb meem: tus qauv ntawm 0.100 N
E. txheej txheem:
1. Kev npaj Enzyme:
a. Xam Kev Npaj Enzyme
Qhov hnyav {{0}}/Target kev ua (kwv yees li 0.05 g lossis 50 mg)
A. Ntsuas cov enzyme precisely rau hauv 50 ml volumetric hwj
B. Ntxiv 8.3 ml ntawm cov tshuaj tsis haum.
b. Cia sawv ntsug rau 30 feeb ntawm chav tsev kub.
C. Dilute rau 50 ml siv pH 4.5 distilled dej, ntxiv ib tug me me do bar thiab do rau ib tug ntxiv 10 mus rau 15 feeb.
2. Cov txheej txheem Enzyme:
a. Pipette 25 ml ntawm Gelatin substrate rau hauv ib qho ntawm ob 100 ml beakers uas muaj cov tuav tuav thiab muab tso rau hauv ib da dej ntawm 45 degree rau 5 feeb, ib qho rau Test Solution thiab lwm yam rau lub Blank Solution.
b. Test Solution:
1) Ntxiv 1.
2) Tom qab raws nraim 20 feeb ntawm incubation ntawm 45 degree, ntxiv 0.1 ml ntawm 3 feem pua hydrogen peroxide thiab swirl.
3) Incubate rau 5 feeb ntxiv.
4) Tshem tawm lub beaker los ntawm cov dej da dej thiab nrog kev nplawm tas li ntxig rau pH sojntsuam.
5) Sau cov pH tom qab 10 vib nas this (Pib pH)
6) Kho rau pH 6.0 nrog 0.1 N NaOH. (kwv yees li 2-4 ml)
* Ceebtoom: Thaum kho cov pH rau 6.0 ceev faj ntawm pH 5.8; pH nce qeeb thiab feeb ntxiv ntawm NaOH ntawm lub sijhawm no yuav ua rau pH nce ntxiv.
7) Txuas ntxiv stirring tas li, ntxiv 10 ml ntawm 37 feem pua formaldehyde pH 9.0.
8) Sau cov pH tom qab 10 vib nas this thiab 1 feeb.
9) Titrate rau pH 9.0 nrog 0.1 N NaOH.
10) Sau qhov ntim titration.
Qhov no yog lub xeem titer, T.c.
Blank Solution: Qhov Kev daws Blank yuav tsum tau khiav ib txhij nrog Kev Ntsuas Kev Ntsuas. Qhov no ua tiav los ntawm kev pib qhov kev txiav txim siab Blank Solution 12 feeb tom qab pib qhov kev daws teeb meem. Qhov ntawd muab sijhawm rau koj los ua kom tiav qhov kev ntsuam xyuas ntawm Kev Ntsuas Kev Ntsuas ua ntej yuav tsum tau mus nrog Blank Solution. 1) Ntxiv 0.1 ml ntawm 3 feem pua ntawm hydrogen peroxide rau lub beaker uas tau xaiv rau cov tshuaj dawb huv thiab yoj.
2) Tom qab raws nraim 20 feeb ntawm incubation ntawm 45 degree ntxiv 1.0 ml ntawm bromelain tov thiab swirl.
3) Incubate rau 5 feeb ntxiv.
GELATIN DIGESTION UNIT ANALYTICAL METHOD (GDU) - cont.
4) Tshem tawm lub beaker los ntawm cov dej da dej thiab nrog kev nplawm tas li ntxig rau pH sojntsuam.
5) Sau cov pH tom qab 10 vib nas this (Pib pH)
6) Kho rau pH 6.0 nrog 0.1 N NaOH. (kwv yees li 2-4 ml)*Saib Daim Ntawv Ceeb Toom saum toj no.
7) Txuas ntxiv stirring tas li, ntxiv 10 ml ntawm 37 feem pua formaldehyde pH 9.0.
8) Sau cov pH tom qab 10 vib nas this thiab 1 feeb.
9) Titrate rau pH 9.0 nrog 0.1 N NaOH.
10) Sau qhov ntim titration. Qhov no yog qhov dawb huv titer, B.
F. Kev suav:
1. Cov ntsiab lus: Ib Gelatin Digestion Unit yog cov enzyme uas yuav tso tawm, tom qab 20 feeb kev zom ntawm 45 degree, 1 mg ntawm amino nitrogen los ntawm tus qauv gelatin tov ntawm pH 4.5 lossis pH 5.5 (GDU pH 4.5 lossis pH 5.5).
GDU/g {{0}} (TB) x 14 x N x 50 Wt (g) Qhov twg: T=Test titer (ml 0.1 N NaOH) B=Blank titer (ml 0.1 N NaOH) N=Normality of standardized NaOH (ie 0.100) Wt (g)=Pib qhov hnyav ntawm enzyme
G. Kev Ntsuas Kev Ntsuas:
1. Enzyme npaj yog diluted mus rau ib qho concentration ntawm kwv yees li 0.001 g/ml (1 mg/ml) los yog rau bromelain enzyme hmoov concentrate kwv yees li ntawm 1-2 GDU/ml. Cov ntaub ntawv siv tau raug soj ntsuam nrog txhua qhov kev khiav kom paub meej cov txheej txheem raug.
Guanjie muab cov hmoov bromelain ntau, siv txoj kev xeem GDU.Peb cov txheej txheem ntau lawm ua haujlwm raws li CGMP tus qauv kev cob qhia, siv peb cov kab ntau lawm thoob plaws ob lub chaw tsim khoom, thiab ob lub chaw kuaj mob ywj pheej. Rau txhua qhov kev nug txog peb cov khoom, thov hu rau peb ntawminfo@gybiotech.com.
