CN1222613C - Producing process for extracting allinase from garlic - Google Patents
Producing process for extracting allinase from garlic Download PDFInfo
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- CN1222613C CN1222613C CN 03100419 CN03100419A CN1222613C CN 1222613 C CN1222613 C CN 1222613C CN 03100419 CN03100419 CN 03100419 CN 03100419 A CN03100419 A CN 03100419A CN 1222613 C CN1222613 C CN 1222613C
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 3
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
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- ADFCQWZHKCXPAJ-UHFFFAOYSA-N indofine Natural products C1=CC(O)=CC=C1C1CC2=CC=C(O)C=C2OC1 ADFCQWZHKCXPAJ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention relates to a producing process for extracting alliinase from garlic, which has the following steps: firstly, adding alliinase extraction liquid for pulping and separating clear liquid; secondly, depositing the clear liquid by steps to obtain coarse alliinase; thirdly, dialysing the coarse alliinase for removing salt, regulating isoelectric point, and cooling the coarse alliinase for drying to obtain alliinase. The present invention is suitable for producing medical alliinase in batches by using fresh garlic as raw materials. The alliinase products obtained by the present invention have high purity after the proof of SDS-gel electrophoresis, and the molecular weight of monomer of the alliinase products is 67000 Daltons by calculation according to the rf value of standard albumen (C: molecular weight is from 14400 to 97400), wherein the Michaelis constant K#-[m] is 3.6 mmol/l, and the average activity of the products is 765 U/g.
Description
Technical field
The present invention relates to extract from plant the method for allinase biotechnological formulation, is a kind of allinase production technique of extracting from bright garlic.
Background technology
Garlic (Allium sativum L.) is time-honored medicine food dual purpose plant.Garlic has another name called giant garlic, is the underground bulb of Liliaceae allium garlic (Allium sativum L.).Recent studies finds, that garlic has is anticancer, reducing blood-fat, kill effect such as fungi, becomes the focus that states such as moral, U.S., day are studied.Allinase (Alliinase, EC 4.4.1.4) has another name called alliin lyase, alkylthio halfcystine enzyme.Be present in the vacuole of cell.Be that six pyridoxal phosphates and two subunits form.280, there is maximum absorption at the 430nm place.Isoelectric pH: 4.9.Generally acknowledge at present allinase catalysis alliin (Alliin, 2-allyl cysteine sulfoxide) to produce serial volatility organic compounds containing sulfur such as allicin (Allicin, diallyl thiosulfinate), 4,5,9-trithiadodeca-1,6,11-triene 9-oxide (ajoenes) and bring into play multiple drug effect.
37 ℃ of best catalytic temperatures, pH:5.0-8.0 can be converted into 80% alliin allicin (Allicin, ally 2-propenethisulfinate) in 10 minutes.Allicin has another name called: diallyl thiosulfinate or allicin.
Allinase and alliin independently are present in the bulb of garlic respectively, smash these two kinds of materials of back to pieces and are in contact with one another, and decomposition reaction takes place rapidly.Therefore, it is very difficult extracting allinase from garlic.
Summary of the invention
The invention provides a kind of allinase production technique of from bright garlic, extracting.
Technical scheme of the present invention is achieved like this: a kind of allinase production technique of from bright garlic, extracting, carry out according to the following steps:
The first step garlic clove adds the making beating of allinase extracting solution and isolates clear liquid: earlier the garlic clove temperature is controlled in-5 ℃ to the 5 ℃ scopes, and controlled temperature is in-5 ℃ to 5 ℃ scopes, add the allinase extracting solution and wear into 100 to 200 purpose garlics slurry, the add-on of allinase extracting solution is 0.8 to 1.2 times of garlic clove weight, and further isolates first clear liquid;
The second step fractional precipitation obtains thick allinase: controlled temperature is in-5 ℃ to 5 ℃ scopes, under agitation ammonium sulfate is added in first clear liquid, make its whole dissolvings and control ammonium sulfate concentrations in 20% to 30% percent weight in volume concentration range, isolate second clear liquid then with specific gravity hydrometer; Under agitation ammonium sulfate is added in second clear liquid, make its whole dissolvings and control ammonium sulfate concentrations in 40% to 50% percent weight in volume concentration range, isolate the thick allinase of the first pasty state throw out then with specific gravity hydrometer;
The 3rd step dialysis desalting-adjusting iso-electric point-lyophilize obtains allinase: with the first pasty state throw out urethane bag of packing into, dialysis is 40 hours to 50 hours in the frozen water dialysis groove in mobile-5 ℃ to 5 ℃ of scopes, regulates with phosphoric acid then that to isolate the second pasty state throw out behind the isoelectric pH to 4.9 be the pasty state allinase.
Said extracted liquid contains the aqueous solution of complexing agent, oxidation inhibitor, buffer reagent and glycerol.
Above-mentioned complexing agent can be disodium ethylene diamine tetraacetate; Above-mentioned oxidation inhibitor can be mercaprol; Above-mentioned buffer reagent can be phosphoric acid salt.
The allinase extracting solution can be in Sodium phosphate dibasic-potassium phosphate buffer to add by the damping fluid volume and adds 0.1% to 0.3% Na2EDTA, 0.02% to 0.06% mercaptoethanol, 0.3% to 1.0% sodium-chlor, 5% to 15% glycerol.
In above-mentioned production technique, the second pasty state throw out can be put into freeze drier and carry out drying, make moisture content be lower than 2.0%, obtain the freeze-drying allinase, carry out airtight preservation with nitrogen or rare gas element then.
Above-mentioned separation equipment used can adopt freezing closed screenings self-separation paste roller mill or centrifugal dried machine or the low temperature tubular type separating centrifuge of falling.
It is the medicinal allinase of raw material mass production that the present invention is suitable for bright garlic.Gel electrophoresis proves that it has higher purity to gained allinase product of the present invention through SDS-.According to (C: molecular weight 14400～97400) to calculate its allinase product list polymer molecular amount be 67000 dalton to Rf value with standard protein.Michaelis-Menton constant K
mBe 3.6mmol/L.The average vigor of its product is: 765U/g.
Description of drawings
Accompanying drawing 1 is allinase (A, B), standard protein (C, 3mg/mL) electrophorogram;
Accompanying drawing 2 is substrate (alliin) and reaction product (pyruvic acid, allicin) efficient liquid phase chromatographic analysis (being HPLC) color atlas;
Accompanying drawing 3 is an allinase catalysis alliin polymerization kinetics curves.
Embodiment
The present invention is not subjected to the restriction of following embodiment, can determine concrete embodiment according to the technical scheme and the practical situation of the invention described above.
Embodiment, undertaken by following step:
The first step adds the making beating of allinase extracting solution and isolates clear liquid in garlic clove: earlier the garlic clove temperature is controlled in-5 ℃ to the 5 ℃ scopes, and controlled temperature is in-5 ℃ to 5 ℃ scopes, add the allinase extracting solution and wear into 100 to 200 purpose garlics slurry, the add-on of allinase extracting solution is 0.8 times or 1.2 times of garlic clove weight, and further isolates first clear liquid;
The second step fractional precipitation obtains thick allinase: controlled temperature is in-5 ℃ to 5 ℃ scopes, under agitation ammonium sulfate is added in first clear liquid, make its whole dissolvings and control ammonium sulfate concentrations in 20% to 30% percent weight in volume concentration range, isolate second clear liquid then with specific gravity hydrometer; Under agitation ammonium sulfate is added in second clear liquid, make its whole dissolvings and control ammonium sulfate concentrations in 40% to 50% percent weight in volume concentration range, isolate the thick allinase of the first pasty state throw out then with specific gravity hydrometer;
The 3rd step dialysis desalting-adjusting iso-electric point-lyophilize obtains allinase: with the first pasty state throw out urethane bag of packing into, dialysis is 40 hours to 50 hours in the frozen water dialysis groove in mobile-5 ℃ to 5 ℃ of scopes, regulates with phosphoric acid then that to isolate the second pasty state throw out behind the isoelectric pH to 4.9 be the pasty state allinase.
Preferably the second pasty state throw out is put into freeze drier and carry out drying, make moisture content be lower than 2.0%, obtain the freeze-drying allinase, carry out airtight preservation with nitrogen or rare gas element then, help prolonged preservation like this.
In the above-described embodiments: extracting solution is the aqueous solution that contains complexing agent, oxidation inhibitor, buffer reagent and glycerol; Complexing agent can be disodium ethylene diamine tetraacetate; Oxidation inhibitor can be mercaprol; Buffer reagent can be phosphoric acid salt; The allinase extracting solution is preferably in Sodium phosphate dibasic-potassium phosphate buffer to add by the damping fluid volume and adds 0.1% or 0.3% Na2EDTA, 0.02% or 0.06% mercaptoethanol, 0.3% or 1.0% sodium-chlor, 5% or 15% glycerol.
Separate equipment used and adopt freezing closed screenings self-separation paste roller mill or centrifugal dried machine or the low temperature tubular type separating centrifuge of falling.
Be to be the affirmation of allinase to the foregoing description products obtained therefrom below:
One, sds gel electrophoresis is measured allinase product purity and molecular weight:
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) condition: adopt 10% separation gel, 4.5% concentrates glue making sheet.Point sample amount 20 μ L.CC type vertical electrophoresis groove, glycerine-tri methylol amino methane-HCl-sodium laurylsulfonate damping fluid.Voltage 20mA electrophoresis launches, coomassie brilliant blue staining.The result shows a band.According to (C: molecular weight 14400～97400) Rf value gets regression equation: Y=-1.7297X+5.6399 (r=0.97) with standard protein.
Calculating the single polymer molecular amount of allinase sample (lot number: A:011105, B:020120) is 67000 dalton.As shown in Figure 1.
Two, the allinase vigor is tired:
Set up the method that HPLC measures substrate alliin, product allicin and pyruvic acid simultaneously.As shown in Figure 2.Allinase product 0.100g, purified water made dissolving in ultrasonic 30 seconds, made the 1.00mg/ml test liquid.With alliin (Indofine Chemical Company, Inc.USA content 99.5%.) make standard reference material.Hypersil-BDS-C
18Chromatographic column (4.6mm * 250mm, 5um), 35 ℃ of column temperatures, moving phase: methanol-water (3: 2), flow rate: 0.8ml/min, 214nm detects.Sample size 10 μ l measure.Vigor with adjacent alliin and pyruvic acid chromatographic peak counting yield (allinase) is tired.Five batches of allinase products of embodiment gained catalysis activity average out to: 765U/g.
The enzyme amount (Ω) that 1 allinase unit of activity (U)=per minute cracking 1 μ mol alliin (substrate) needs.Five batches of average allinase vigor of allinase product are: 765U/g.
In the formula: W ' is alliin reduction (μ g); 177.2 be the alliin molecular weight; 5 is reaction times (min); W is trial-product sampling amount (g); N is the trial-product extension rate.
Three, enzyme kinetics curve and parameter
The allinase product adds substrate (alliin) and is made into test solution respectively.Ultrasonic mixing 30 seconds, 35 ℃ of water bath heat preservations.Respectively at 5,15,25min, and 1,3,5,7,9,11, that 13hr draws reaction solution is an amount of, adds the methyl alcohol mixing.Cross 0.45 μ m filter membrane, HPLC sample introduction 10 μ l obtain color atlas.Calculate alliin, pyruvic acid and allicin content.Draw the enzymatic reaction curve, as shown in Figure 3.
The characteristic reactions curve that shows allinase: 5 minutes is that the enzymatic reaction initial velocity (presents first order reaction: S+E → P with in.Its rate equation is :-d[S]/dt=k
1[S]); Reach maximum reaction velocity in 25 minutes and (present mixed class reaction: E+S ES → E+P).Allicin concentration in time increases, and 15 minutes to its concentration of 13hr basicly stable (25 minutes-13hr is near zeroth order reaction).
Catalytic cracking reaction meets Michaelis-Menton equation: 1/V=0.129/[S]+0.0358.
Regression equation: Y=35.78+0.129X (r=0.996)
Maximum reaction velocity: V
Max=27.9molmin
-1Mg
-1
Michaelis-Menton constant: K
m=3.6mmol/L.
Claims (10)
1, a kind of allinase production technique of extracting from bright garlic is characterized in that carrying out according to the following steps:
The first step adds the making beating of allinase extracting solution and isolates clear liquid in garlic clove: earlier the garlic clove temperature is controlled in-5 ℃ to the 5 ℃ scopes, and controlled temperature adds the allinase extracting solution and wears into 100 to 200 purpose garlics slurry-5 ℃ to 5 ℃ scopes, the add-on of allinase extracting solution is 0.8 to 1.2 times of garlic clove weight, and further isolates first clear liquid;
The second step fractional precipitation obtains thick allinase: controlled temperature is in-5 ℃ to 5 ℃ scopes, under agitation ammonium sulfate is added in first clear liquid, make its whole dissolvings and control ammonium sulfate concentrations in 20% to 30% percent weight in volume concentration range, isolate second clear liquid then with specific gravity hydrometer; Under agitation ammonium sulfate is added in second clear liquid, make its whole dissolvings and control ammonium sulfate concentrations in 40% to 50% percent weight in volume concentration range, isolate the thick allinase of the first pasty state throw out then with specific gravity hydrometer;
The 3rd step dialysis desalting-adjusting iso-electric point-lyophilize obtains allinase: with the first pasty state throw out urethane bag of packing into, dialysis is 40 hours to 50 hours in the frozen water dialysis groove in mobile-5 ℃ to 5 ℃ of scopes, regulates with phosphoric acid then that to isolate the second pasty state throw out behind the isoelectric pH to 4.9 be the pasty state allinase.
2, the allinase production technique of extracting from bright garlic according to claim 1 is characterized in that the allinase extracting solution is the aqueous solution that contains complexing agent, oxidation inhibitor, buffer reagent and glycerol.
3, the allinase production technique of extracting from bright garlic according to claim 2 is characterized in that complexing agent is a disodium ethylene diamine tetraacetate; Oxidation inhibitor is mercaprol; Buffer reagent is a phosphoric acid salt.
4, according to claim 1 or the 2 or 3 described allinase production technique of from bright garlic, extracting, it is characterized in that the allinase extracting solution in Sodium phosphate dibasic-potassium phosphate buffer, adding 0.1% to 0.3% Na2EDTA, 0.02% to 0.06% mercaptoethanol, 0.3% to 1.0% sodium-chlor, 5% to 15% glycerol by the damping fluid volume.
5, according to claim 1 or the 2 or 3 described allinase production technique of from bright garlic, extracting, it is characterized in that the second pasty state throw out is put into freeze drier carries out drying, make moisture content be lower than 2.0%, obtain the freeze-drying allinase, carry out airtight preservation with nitrogen or rare gas element then.
6, the allinase production technique of from bright garlic, extracting according to claim 4, it is characterized in that the second pasty state throw out is put into freeze drier carries out drying, make moisture content be lower than 2.0%, obtain the freeze-drying allinase, carry out airtight preservation with nitrogen or rare gas element then.
7, according to claim 1 or the 2 or 3 described allinase production technique of from bright garlic, extracting, it is characterized in that separating equipment used and adopt freezing closed screenings self-separation paste roller mill or centrifugal dried machine or the low temperature tubular type separating centrifuge of falling.
8, the allinase production technique of extracting from bright garlic according to claim 4 is characterized in that separating equipment used and adopts freezing closed screenings self-separation paste roller mill or centrifugal dried machine or the low temperature tubular type separating centrifuge of falling.
9, the allinase production technique of extracting from bright garlic according to claim 5 is characterized in that separating equipment used and adopts freezing closed screenings self-separation paste roller mill or centrifugal dried machine or the low temperature tubular type separating centrifuge of falling.
10, the allinase production technique of extracting from bright garlic according to claim 6 is characterized in that separating equipment used and adopts freezing closed screenings self-separation paste roller mill or centrifugal dried machine or the low temperature tubular type separating centrifuge of falling.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03100419 CN1222613C (en) | 2003-01-13 | 2003-01-13 | Producing process for extracting allinase from garlic |
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|---|---|---|---|
| CN 03100419 CN1222613C (en) | 2003-01-13 | 2003-01-13 | Producing process for extracting allinase from garlic |
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| CN1424397A CN1424397A (en) | 2003-06-18 |
| CN1222613C true CN1222613C (en) | 2005-10-12 |
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| CN 03100419 Expired - Fee Related CN1222613C (en) | 2003-01-13 | 2003-01-13 | Producing process for extracting allinase from garlic |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9023623B2 (en) | 2010-06-22 | 2015-05-05 | Xinjiang Ailexin Pharmaceutical Co., Ltd. | Method for preparing of allicin injection and low-temperature continuous stirring ultrafiltration device thereof |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305482B (en) * | 2013-06-28 | 2016-01-27 | 南昌航空大学 | A kind of method of fast purifying CYP119 enzyme |
| KR20150041425A (en) * | 2013-10-08 | 2015-04-16 | (주)태영에프에이 | Method for preparing alliinase derived from garlic |
| CN106957883A (en) * | 2017-04-06 | 2017-07-18 | 山东晨隆晟世生物科技有限公司 | A kind of allicin synthesizes the process of 4,5,9-trithiadodeca-1,6,11-triene 9-oxide |
| CN108913678A (en) * | 2018-07-23 | 2018-11-30 | 江苏挺卫实业有限公司 | A kind of separating and extracting process of the garlic allinase for cereal beverage |
| CN113512543A (en) * | 2021-06-08 | 2021-10-19 | 新疆农业职业技术学院 | Ultrasonic-based garlic allinase rapid extraction process |
| CN113564134A (en) * | 2021-06-08 | 2021-10-29 | 新疆农业职业技术学院 | Process for extracting alliinase effective component from garlic |
| CN114304289B (en) * | 2022-01-24 | 2023-09-05 | 青岛博恩高科生物技术有限公司 | Extraction method for comprehensive utilization of fresh garlic as raw material |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9023623B2 (en) | 2010-06-22 | 2015-05-05 | Xinjiang Ailexin Pharmaceutical Co., Ltd. | Method for preparing of allicin injection and low-temperature continuous stirring ultrafiltration device thereof |
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