Combined preparation method of garlic extract
阅读说明：本技术 一种大蒜提取物的联合制备方法 (Combined preparation method of garlic extract ) 是由 朱颖秋 王鹏 钟斌 王庚申 于 2020-12-25 设计创作，主要内容包括：本发明提出一种大蒜提取物的联合制备方法,属于天然物质提取、分离和纯化领域,能够解决现有技术存在提取工艺制得的成分单一,未能真正实现大蒜原料充分利用的问题。该制备方法包括：1)大蒜提取液制备；2)蒜氨酸酶制备；3)大蒜素制备；4)大蒜多糖和大蒜氨基酸制备。本发明能够应用于大蒜提取物的联合制备方面。(The invention provides a combined preparation method of a garlic extract, belongs to the field of natural substance extraction, separation and purification, and can solve the problems that the components prepared by an extraction process are single and the full utilization of garlic raw materials cannot be really realized in the prior art. The preparation method comprises the following steps: 1) preparing garlic extract; 2) preparing alliinase; 3) preparing allicin; 4) garlic polysaccharide and garlic amino acid. The invention can be applied to the combined preparation of garlic extract.)
1. A combined preparation method of garlic extract is characterized by comprising the following steps:
preparing garlic extract: peeling and cleaning fresh garlic, pulping in a solvent, and filtering to obtain garlic extract;
preparing alliinase: separating the garlic extract by adopting ultrafiltration membrane equipment to obtain trapped fluid and dialysate, and carrying out salting-out precipitation, dissolution, desalination and freeze drying treatment on the trapped fluid to obtain alliinase;
preparing allicin: after passing the dialysate through macroporous resin, collecting macroporous resin effluent, eluting the macroporous resin by using an eluent, collecting macroporous resin eluent, and concentrating and drying to obtain allicin;
preparing garlic polysaccharide and garlic amino acid: passing the macroporous resin effluent through cation exchange resin, eluting with purified water for 1-3 column volumes, collecting cation exchange resin effluent and eluate, adjusting pH to neutral, concentrating, and drying to obtain Bulbus Allii polysaccharide;
eluting the cation exchange resin by using an eluent, collecting the eluent of the cation exchange resin, and concentrating and drying to obtain the garlic amino acid.
2. The joint preparation method of garlic extract as claimed in claim 1, wherein in the garlic extract preparation step, the fresh garlic is pulped in a solvent to 60-200 mesh at a weight ratio of solvent to fresh garlic of 1-5: 1.
3. The combined preparation method of garlic extract as claimed in claim 2, wherein the solvent is water or phosphate buffer solution with pH of 6.0-7.5.
4. The integrated preparation method of garlic extract according to claim 1, wherein in the alliinase preparation step, the ultrafiltration membrane apparatus is an ultrafiltration membrane apparatus having a molecular weight cut-off of 2 to 8 ten thousand daltons.
5. The combined preparation method of garlic extract as claimed in claim 1, wherein in the step of preparing alliinase, the agent for salting-out precipitation is ammonium sulfate in an amount of 20-60% by mass of the retentate, and the precipitate obtained by salting-out precipitation is crude alliinase;
the dissolving reagent is phosphate buffer solution with pH of 6.0-7.5, and desalting is carried out by dialyzing with polyurethane bag in flowing ice water tank at 0-5 deg.C for 30-60 h.
6. The integrated preparation method of garlic extract according to claim 1, wherein in the garlicin preparation step, the macroporous resin used is medium-polarity or weak-polarity macroporous resin, and the flow rate of the dialysate passing through the macroporous resin is 2-10 column volumes/h;
in the preparation steps of the garlic polysaccharide and the garlic amino acid, the cation exchange resin is strong polarity or weak polarity cation exchange resin, and the flow rate of the effluent of the macroporous resin passing through the cation exchange resin is 2-10 column volumes/h.
7. The combined preparation method of garlic extract as claimed in claim 1, wherein in the allicin preparation step, the eluent is 20-95% methanol or ethanol aqueous solution by mass, the elution volume is 3-10 column volumes, and the elution speed is 1-5 column volumes/h;
in the preparation steps of the garlic polysaccharide and the garlic amino acid, an eluent is an ammonia water solution with the mass concentration of 0.1-1%, the elution volume is 3-20 column volumes, and the elution speed is 1-5 column volumes/h.
8. The combined preparation method of garlic extract as claimed in claim 1, wherein in the step of preparing garlic polysaccharide and garlic amino acid, before the concentration and drying treatment of the cation exchange resin eluent, the method further comprises detecting the garlic amino acid content by paper chromatography.
9. The combined preparation method of garlic extract as claimed in claim 8, wherein the paper chromatography is specifically:
developing agent: n-butanol: formic acid: water 15:3:2 (v/v);
color developing agent: 0.4% ninhydrin ethanol solution;
color development and inspection: heating at 50-80 deg.C until the spots are clear, and inspecting with visible light.
10. The combined preparation method of garlic extract as claimed in claim 1, further comprising, after the preparing step of allicin, before the preparing steps of garlic polysaccharide and garlic amino acid: detecting the allicin content by an HPLC method, wherein the chromatographic conditions are as follows:
a chromatographic column: c18 column, 4.6 m.times.150 mm, 5 μm;
detection wavelength: 245-260 nm;
mobile phase: the mobile phase is a mixed solution of methanol and 1% formic acid water with the volume ratio of 60: 40;
flow rate: 0.5-0.8 mL/min;
column temperature: 25-35 ℃;
sample introduction amount: 20 μ L.
Technical Field
The invention belongs to the technical field of natural substance extraction, separation and purification, and particularly relates to a combined preparation method of a garlic extract.
Background
Garlic (Allium Sativum L.) has a long history as a plant used as both medicine and food, and a large number of researches show that the garlic has various effects of resisting bacteria, diminishing inflammation, resisting thrombus, reducing blood fat, resisting tumors, enhancing immunity and the like, is rich in various components such as saccharides, proteins, sulfur-containing organic compounds, amino acids and the like, and is also a material basis for the garlic to play various biological effects.
Alliinase is also called C-S lyase, can efficiently cleave C-S bonds, can catalyze various allyl cysteine sulfoxides including alliin into allyl disulfide sulfoxides, is mostly applied to the aspects of tumor treatment and the like at present, and has wide application prospect in the biological medicine industry; allicin is a sulfur-containing compound generated by alliin under the catalytic action of alliinase, can be further converted into sulfur-containing compounds such as ajoene, diallyl disulfide and diallyl trisulfide under proper conditions, can block the formation of nitrosamine, has the effects of resisting cancer and reducing blood fat, and also has obvious inhibiting effect on various viruses, bacteria, fungi and helicobacter pylori; the garlic polysaccharide is mainly fructan heteropolysaccharide, belongs to inulin polysaccharide, accounts for about 40% of dry matter content, has monosaccharide composition mainly comprising fructose and glucose, has multiple effects, and has effects of regulating human immunity, controlling blood lipid, reducing blood sugar, preventing and treating constipation, resisting oxidation, protecting liver injury, etc.; the garlic amino acid is unique non-protein sulfur-containing amino acid in garlic, accounts for about 2-15% of the dry matter weight, mainly comprises arginine, alliin, lysine, serine and other sulfur-containing amino acids, and has various pharmacological effects of resisting tumor, synergistically lowering blood pressure, resisting bacteria and killing viruses, removing free radicals, protecting liver, resisting diabetes and the like.
At present, patent application (CN1556102A) proposes a method for jointly preparing alliin and garlic polysaccharide, but the preparation process only simply extracts one or two components in garlic, and does not realize full utilization of garlic raw material components, so how to develop a joint preparation method capable of extracting multiple garlic extracts is an important solution for realizing full utilization of garlic raw materials.
Disclosure of Invention
The invention provides a combined preparation method of a garlic extract, which is simple and convenient to operate, low in cost, high in raw material utilization rate and high in extract yield, aiming at the technical problems that the existing extraction process is single in extraction component and cannot really realize full utilization of garlic raw materials.
In order to achieve the purpose, the invention adopts the technical scheme that:
a combined preparation method of garlic extract comprises the following steps:
preparing garlic extract: peeling and cleaning fresh garlic, pulping in a solvent, and filtering to obtain garlic extract;
preparing alliinase: separating the garlic extract by adopting ultrafiltration membrane equipment to obtain trapped fluid and dialysate, and carrying out salting-out precipitation, dissolution, desalination and freeze drying treatment on the trapped fluid to obtain alliinase;
preparing allicin: after passing the dialysate through macroporous resin, collecting macroporous resin effluent, eluting the macroporous resin by using an eluent, collecting macroporous resin eluent, and concentrating and drying to obtain allicin;
preparing garlic polysaccharide and garlic amino acid: passing the macroporous resin effluent through cation exchange resin, eluting with purified water for 1-3 column volumes, collecting cation exchange resin effluent and eluate, adjusting pH to neutral, concentrating, and drying to obtain Bulbus Allii polysaccharide;
eluting the cation exchange resin by using an eluent, collecting the eluent of the cation exchange resin, and concentrating and drying to obtain the garlic amino acid.
Preferably, in the preparation step of the garlic extract, the fresh garlic is pulped in a solvent to 60-200 meshes, and the weight ratio of the solvent to the fresh garlic is 1-5: 1.
Preferably, the solvent is water or a phosphate buffer solution having a pH of 6.0 to 7.5.
Preferably, in the step of preparing the alliinase, the ultrafiltration membrane equipment is ultrafiltration membrane equipment with the molecular weight cutoff of 2-8 ten thousand daltons.
Preferably, in the preparation step of the alliinase, the reagent used for salting-out precipitation is ammonium sulfate, the addition amount of the ammonium sulfate is 20-60% of the mass of the trapped fluid, and the precipitate obtained by salting-out precipitation is a crude alliinase;
the dissolving reagent is phosphate buffer solution with pH of 6.0-7.5, and desalting is carried out by dialyzing with polyurethane bag in flowing ice water tank at 0-5 deg.C for 30-60 h.
Preferably, in the step of preparing the allicin, the macroporous resin is medium-polarity or weak-polarity macroporous resin, and the flow rate of the dialysate passing through the macroporous resin is 2-10 column volumes/h;
in the preparation steps of the garlic polysaccharide and the garlic amino acid, the cation exchange resin is strong polarity or weak polarity cation exchange resin, and the flow rate of the effluent of the macroporous resin passing through the cation exchange resin is 2-10 column volumes/h.
Preferably, in the preparation step of the allicin, the eluent is methanol or ethanol water solution with the mass concentration of 20-95%, the elution volume is 3-10 column volumes, and the elution speed is 1-5 column volumes/h;
in the preparation steps of the garlic polysaccharide and the garlic amino acid, an eluent is an ammonia water solution with the mass concentration of 0.1-1%, the elution volume is 3-20 column volumes, and the elution speed is 1-5 column volumes/h.
Preferably, in the step of preparing the garlic polysaccharide and the garlic amino acid, before the concentration and drying treatment of the cation exchange resin eluent, the method further comprises the step of detecting the content of the garlic amino acid by a paper chromatography.
Preferably, the paper chromatography is specifically:
developing agent: n-butanol: formic acid: water 15:3:2 (v/v);
color developing agent: 0.4% ninhydrin ethanol solution;
color development and inspection: heating at 50-80 deg.C until the spots are clear, and inspecting with visible light.
Preferably, after the step of preparing the allicin, the step of preparing the garlic polysaccharide and the garlic amino acid further comprises the following steps: detecting the allicin content by an HPLC method, wherein the chromatographic conditions are as follows:
a chromatographic column: c18 column, 4.6 m.times.150 mm, 5 μm;
detection wavelength: 245-260 nm;
mobile phase: the mobile phase is a mixed solution of methanol and 1% formic acid water with the volume ratio of 60: 40;
flow rate: 0.5-0.8 mL/min;
column temperature: 25-35 ℃;
sample introduction amount: 20 μ L.
Compared with the prior art, the invention has the advantages and positive effects that:
1. the invention provides a combined preparation method of garlic extracts, which organically combines an ultrafiltration method, different types of adsorption column adsorption methods and the like, not only prepares four garlic extracts comprising alliinase, garlicin, garlic amino acid, garlic polysaccharide and the like, but also ensures the purity and quality of the substances to the maximum extent, really realizes the full utilization of garlic raw materials and obtains various products with high added values;
2. the invention provides a combined preparation method of garlic extract, the macroporous adsorption resin selected by the method has lower cost and simple operation, and is more suitable for industrial production;
3. the invention provides a combined preparation method of garlic extract, and the dialysate flowing out through macroporous resin no longer has unpleasant garlic special smell, and simultaneously retains active substances such as amino acid, polysaccharide and the like, so that the product acceptance can be improved.
Drawings
Fig. 1 is a process flow diagram of a combined preparation method of garlic extract according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention provides a combined preparation method of garlic extract, which comprises the following steps:
s1, preparing garlic extract: peeling and cleaning fresh garlic, pulping in a solvent, and filtering to obtain garlic extract;
s2, preparing alliinase: separating the garlic extract by adopting ultrafiltration membrane equipment to obtain trapped fluid and dialysate, and carrying out salting-out precipitation, dissolution, desalination and freeze drying treatment on the trapped fluid to obtain alliinase;
s3, preparing allicin: after passing the dialysate through macroporous resin, collecting macroporous resin effluent, eluting the macroporous resin by using an eluent, collecting macroporous resin eluent, and concentrating and drying to obtain allicin;
s4, preparing garlic polysaccharide and garlic amino acid: passing the macroporous resin effluent through cation exchange resin, eluting with purified water for 1-3 column volumes, collecting cation exchange resin effluent and eluate, adjusting pH to neutral, concentrating, and drying to obtain Bulbus Allii polysaccharide;
eluting the cation exchange resin by using an eluent, collecting the eluent of the cation exchange resin, and concentrating and drying to obtain the garlic amino acid.
In the preparation process, the extraction and purification of the alliinase are mainly realized by two steps of ultrafiltration and precipitation, specifically, the separation of the alliinase and polysaccharide is realized by using a molecular sieve, the enzyme activity can be improved by two times preliminarily, the enzyme activity is further improved by using the precipitation, and finally the enzyme activity is improved by about 10 times.
In a preferred embodiment, in the preparation step of the garlic extract, the fresh garlic is pulped in a solvent to 60-200 meshes, and the weight ratio of the solvent to the fresh garlic is 1-5: 1.
In a preferred embodiment, the solvent is water or a phosphate buffered solution at pH 6.0-7.5.
In a preferred embodiment, in the preparation step of alliinase, the ultrafiltration membrane equipment is ultrafiltration membrane equipment with a molecular weight cut-off of 2-8 ten thousand daltons, wherein the molecular weight cut-off of the ultrafiltration membrane equipment can be specifically selected from 2, 3, 4, 5, 6, 7 or 8 ten thousand daltons, and any value within the above range can be selected according to actual needs and falls within the protection scope of the present invention.
In a preferred embodiment, in the preparation step of the alliinase, the reagent for salting out and precipitating is ammonium sulfate, the addition amount of the ammonium sulfate is 20-60% of the mass of the trapped fluid, and the precipitate obtained by salting out and precipitating is a crude alliinase;
the dissolving reagent is phosphate buffer solution with pH of 6.0-7.5, and desalting is carried out by dialyzing with polyurethane bag in flowing ice water tank at 0-5 deg.C for 30-60 h.
In a preferred embodiment, in the step of preparing the allicin, the macroporous resin used is medium-polarity or weak-polarity macroporous resin, and the flow rate of the dialysate passing through the macroporous resin is 2-10 column volumes/h;
in the preparation steps of the garlic polysaccharide and the garlic amino acid, the cation exchange resin is strong polarity or weak polarity cation exchange resin, and the flow rate of the effluent of the macroporous resin passing through the cation exchange resin is 2-10 column volumes/h.
In the above preferred embodiment, the macroporous resin is medium-polarity or weak-polarity macroporous resin, such as AB-8, D101, HPD-100, etc., and the cation exchange resin is strong-polarity or weak-polarity cation exchange resin, such as D201, D301, 732, etc.
In a preferred embodiment, in the step of preparing the allicin, the eluent is a methanol or ethanol aqueous solution with the mass concentration of 20-95%, the elution volume is 3-10 column volumes, and the elution speed is 1-5 column volumes/h;
in the preparation steps of the garlic polysaccharide and the garlic amino acid, an eluent is an ammonia water solution with the mass concentration of 0.1-1%, the elution volume is 3-20 column volumes, and the elution speed is 1-5 column volumes/h.
In a preferred embodiment, in the step of preparing garlic polysaccharide and garlic amino acid, before the concentration and drying treatment of the cation exchange resin eluent, the method further comprises the step of detecting the content of garlic amino acid by using a paper chromatography.
In a preferred embodiment, the paper chromatography is specifically:
developing agent: n-butanol: formic acid: water 15:3:2 (v/v);
color developing agent: 0.4% ninhydrin ethanol solution;
color development and inspection: heating at 50-80 deg.C until the spots are clear, and inspecting with visible light.
In a preferred embodiment, after the step of preparing the allicin, the step of preparing the garlic polysaccharide and the garlic amino acid further comprises the following steps: detecting the allicin content by an HPLC method, wherein the chromatographic conditions are as follows:
a chromatographic column: c18 column, 4.6 m.times.150 mm, 5 μm;
detection wavelength: 245-260 nm;
mobile phase: the mobile phase is a mixed solution of methanol and 1% formic acid water with the volume ratio of 60: 40;
flow rate: 0.5-0.8 mL/min;
column temperature: 25-35 ℃;
sample introduction amount: 20 μ L.
In order to more clearly and specifically describe the combined preparation method of garlic extract provided in the embodiments of the present invention, the following description will be given with reference to specific embodiments.
Comparative example 1
The comparative example provides a joint preparation method of garlic extract, which comprises the following specific steps:
(1) preparing garlic extract: soaking fresh garlic in 3 times of drinking water for 1 hr, peeling with running water, rinsing, adding 5 times of water, pulping in a pulping machine to obtain 200 mesh garlic pulp, and filtering with vacuum filtration equipment to obtain clear garlic extract;
(2) preparing alliinase: separating the garlic extract by using ultrafiltration membrane equipment of 10 ten thousand daltons to obtain trapped fluid and dialysate, wherein alliinase is in the trapped fluid, stirring and adding 30% ammonium sulfate into the trapped fluid, standing to separate out precipitate, centrifuging by using a centrifugal machine to obtain alliinase precipitate, dissolving the precipitate by using phosphate buffer solution with pH of 5.0, putting the alliinase solution into a polyurethane bag, dialyzing in a flowing ice water tank at 0-5 ℃ for 48 hours to remove salt, and freeze-drying for 20-40 hours to obtain the alliinase;
(3) preparing allicin: enabling the dialysate prepared in the step (2) to pass through AB-8 type macroporous adsorption resin, controlling the flow rate to be 15 column volumes/h, enabling allicin to be adsorbed on the resin, eluting the macroporous resin with 95% ethanol solution, controlling the flow rate to be 8 column volumes/h, collecting macroporous resin eluent, detecting and collecting the content of the allicin in the macroporous resin eluent by adopting an HPLC method, and concentrating and drying by using a rotary evaporator to prepare the allicin;
(4) preparing garlic polysaccharide and garlic amino acid: passing the effluent of macroporous resin through 732 type cation exchange resin, controlling flow rate at 15 column volume/h to adsorb garlic amino acid on the resin, washing the resin with 3 column volumes of purified water, collecting the effluent and eluate of cation exchange resin, and adding appropriate amount of Na2CO3Adjusting pH of the polysaccharide solution to neutral, concentrating, and drying to obtain Bulbus Allii polysaccharide;
eluting the cation exchange resin with 2% ammonia water solution, controlling the flow rate at 8 column volumes/h, collecting the cation exchange resin eluate, detecting the amino acid content of garlic by using a paper chromatography, and concentrating and drying to obtain the garlic amino acid.
Comparative example 2
The comparative example provides a joint preparation method of garlic extract, which comprises the following specific steps:
(1) preparing garlic extract: soaking fresh garlic in 3 times of drinking water for 1 hr, peeling with running water, rinsing, adding 5 times of water, pulping in a pulping machine to obtain 200 mesh garlic pulp, and filtering with vacuum filtration equipment to obtain clear garlic extract;
(2) preparing alliinase: separating the garlic extract by using ultrafiltration membrane equipment with the temperature of 5000 daltons to obtain trapped fluid and dialysate, wherein alliinase is in the trapped fluid, stirring and adding 30% ammonium sulfate into the trapped fluid, standing to separate out precipitate, centrifuging by using a centrifugal machine to obtain alliinase precipitate, dissolving the precipitate by using phosphate buffer with the pH value of 8.0, putting the alliinase solution into a polyurethane bag, dialyzing in a flowing ice water tank with the temperature of 0-5 ℃ for 48 hours to remove salt, and freeze-drying for 20-40 hours to obtain the alliinase;
(3) preparing allicin: enabling the dialysate prepared in the step (2) to pass through AB-8 type macroporous adsorption resin, controlling the flow rate to be 1 column volume/h, enabling allicin to be adsorbed on the resin, eluting the macroporous resin with 95% ethanol solution, controlling the flow rate to be 0.5 column volume/h, collecting macroporous resin eluent, detecting and collecting the content of allicin in the macroporous resin eluent by adopting an HPLC method, and concentrating and drying by using a rotary evaporator to prepare the allicin;
(4) preparing garlic polysaccharide and garlic amino acid: passing the effluent of macroporous resin through 732 type cation exchange resin, controlling flow rate at 1 column volume/h to adsorb garlic amino acid on the resin, washing the resin with 3 column volumes of purified water, collecting the effluent and eluate of cation exchange resin, and adding appropriate amount of Na2CO3Adjusting pH of the polysaccharide solution to neutral, concentrating, and drying to obtain Bulbus Allii polysaccharide;
eluting the cation exchange resin with 0.05% ammonia water solution, controlling the flow rate at 0.5 column volume/h, collecting the cation exchange resin eluate, detecting the amino acid content of garlic by using a paper chromatography, and concentrating and drying to obtain the garlic amino acid.
Comparative example 3
The comparative example provides a joint preparation method of garlic extract, which comprises the following specific steps:
(1) and (3) processing dehydrated garlic slices: soaking fresh dehydrated garlic slices in 8 times of drinking water for 1-2h, and boiling for 10 min. Steaming to soften and inactivate alliinase;
(2) preparing a transparent garlic extracting solution: making the steamed garlic slices into 100-150-mesh garlic pulp by using a beater, and filtering by using 250-mesh filter cloth to obtain the garlic crude extract. Centrifuging to remove suspended impurities, and filtering the obtained clear liquid with organic membrane with retention force of 1 ten thousand to obtain transparent extractive solution of Bulbus Allii;
(3) adsorbing alliin in the concentrated extract by cation exchange resin: passing the transparent extractive solution of Bulbus Allii through cation exchange resin adsorption column. After adsorption saturation, eluting with deionized water until no sugar and protein are produced. Collecting the filtrate and deionized eluent to obtain garlic polysaccharide solution;
(4) refining, concentrating and drying the garlic polysaccharide liquid: adding 0.3% bentonite, gelatin and silica sol into Bulbus Allii polysaccharide solution, standing for half an hour, filtering with plate and frame filter to obtain refined Bulbus Allii polysaccharide solution, vacuum concentrating at below 60 deg.C until soluble solid content is 50%, drying in oven at 60 deg.C, and pulverizing to obtain Bulbus Allii polysaccharide;
(5) preparing alliin: eluting the cation exchange column with 0.2-0.7 mol ammonia water solution, and collecting eluate with pH of 3.8-8.5 to obtain alliin solution. Concentrating and drying to obtain alliin powder.
Example 1
The embodiment provides a combined preparation method of garlic extract, which comprises the following specific steps:
(1) preparing garlic extract: soaking fresh garlic in 3 times of drinking water for 1 hr, peeling with running water, rinsing, adding 5 times of water, pulping in a pulping machine to obtain 200 mesh garlic pulp, and filtering with vacuum filtration equipment to obtain clear garlic extract;
(2) preparing alliinase: separating the garlic extract by using an ultrafiltration membrane device with 8 ten thousand daltons to obtain trapped fluid and dialysate, wherein alliinase is in the trapped fluid, stirring and adding 30% ammonium sulfate into the trapped fluid, standing to separate out precipitate, centrifuging by using a centrifugal machine to obtain alliinase precipitate, dissolving the precipitate by using a phosphate buffer solution with the pH value of 6.5, placing the alliinase solution in a polyurethane bag, dialyzing in a flowing ice water tank at the temperature of 0-5 ℃ for 48 hours to remove salt, and freeze-drying for 20-40 hours to obtain the alliinase;
(3) preparing allicin: enabling the dialysate prepared in the step (2) to pass through AB-8 type macroporous adsorption resin, controlling the flow rate to be 10 column volumes/h, enabling allicin to be adsorbed on the resin, eluting the macroporous resin with 95% ethanol solution, controlling the flow rate to be 5 column volumes/h, collecting macroporous resin eluent, detecting and collecting the content of the allicin in the macroporous resin eluent by adopting an HPLC method, and concentrating and drying by using a rotary evaporator to prepare the allicin;
(4) preparing garlic polysaccharide and garlic amino acid: passing the macroporous resin effluent through 732 type cation exchange resin, and controlling the flow rate to 10 columnsVolume/h, adsorbing garlic amino acid on resin, washing the resin with 3 column volumes of purified water, collecting cation exchange resin effluent and eluate, and adding appropriate amount of Na2CO3Adjusting pH of the polysaccharide solution to neutral, concentrating, and drying to obtain Bulbus Allii polysaccharide;
eluting the cation exchange resin with 1% ammonia water solution, controlling the flow rate at 5 column volume/h, collecting the cation exchange resin eluate, detecting the amino acid content of garlic by using a paper chromatography, and concentrating and drying to obtain the garlic amino acid.
Example 2
The embodiment provides a combined preparation method of garlic extract, which comprises the following specific steps:
(1) preparing garlic extract: soaking fresh garlic in 3 times of drinking water for 1 hr, peeling with running water, rinsing, adding 4 times of water, pulping in a pulping machine to obtain 200 mesh garlic pulp, and filtering with vacuum filtration equipment to obtain clear garlic extract;
(2) preparing alliinase: separating the garlic extract by using 5 ten thousand dalton ultrafiltration membrane equipment to obtain trapped fluid and dialysate, wherein alliinase is in the trapped fluid, stirring and adding 40% ammonium sulfate into the trapped fluid, standing to separate out precipitate, centrifuging by using a centrifugal machine to obtain alliinase precipitate, dissolving the precipitate by using a phosphate buffer solution with the pH value of 6.0, placing the alliinase solution in a polyurethane bag, dialyzing in a flowing ice water tank at the temperature of 0-5 ℃ for 48h to remove salt, and freeze-drying for 20-40h to obtain the alliinase;
(3) preparing allicin: enabling the dialysate prepared in the step (2) to pass through AB-8 type macroporous adsorption resin, controlling the flow rate to be 5 column volumes/h, enabling allicin to be adsorbed on the resin, eluting the macroporous resin with 80% ethanol solution, controlling the flow rate to be 3 column volumes/h, collecting macroporous resin eluent, detecting and collecting the content of the allicin in the macroporous resin eluent by adopting an HPLC method, and concentrating and drying by using a rotary evaporator to prepare the allicin;
(4) preparing garlic polysaccharide and garlic amino acid: after the effluent of the macroporous resin passes through 732 type cation exchange resin, the flow rate is controlled to be 8 column volumes/h and largeAdsorbing alliin on resin, washing with 2 column volumes of purified water, collecting cation exchange resin effluent and eluate, and adding appropriate amount of Na2CO3Adjusting pH of the polysaccharide solution to neutral, concentrating, and drying to obtain Bulbus Allii polysaccharide;
eluting the cation exchange resin with 0.5% ammonia water solution, controlling the flow rate at 3 column volumes/h, collecting the cation exchange resin eluate, detecting the amino acid content of garlic by using a paper chromatography, and concentrating and drying to obtain the garlic amino acid.
Example 3
The embodiment provides a combined preparation method of garlic extract, which comprises the following specific steps:
(1) preparing garlic extract: adding fresh garlic into drinking water with the mass of 3 times of that of the fresh garlic, soaking for 1 hour, peeling and rinsing with running water, adding water with the mass of 3 times of that of garlic cloves, crushing the garlic into garlic pulp with the size of 100 meshes in a pulping machine, and filtering the garlic pulp by using reduced pressure suction filtration equipment to obtain clear garlic extract for later use;
(2) preparing alliinase: separating the garlic extract by using ultrafiltration membrane equipment of 3 ten thousand daltons to obtain trapped fluid and dialysate, wherein alliinase is in the trapped fluid, stirring and adding 50% ammonium sulfate into the trapped fluid, standing to separate out precipitate, centrifuging by using a centrifugal machine to obtain alliinase precipitate, dissolving the precipitate by using phosphate buffer solution with the pH value of 7.0, putting the alliinase solution into a polyurethane bag, dialyzing in a flowing ice water tank at the temperature of 0-5 ℃ for 48h to remove salt, and freeze-drying for 20-40h to obtain the alliinase;
(3) preparing allicin: enabling the dialysate prepared in the step (2) to pass through AB-8 type macroporous adsorption resin, controlling the flow rate to be 3 column volumes/h, enabling allicin to be adsorbed on the resin, eluting the macroporous resin with 50% ethanol solution, controlling the flow rate to be 2 column volumes/h, collecting macroporous resin eluent, detecting and collecting the content of the allicin in the macroporous resin eluent by adopting an HPLC method, and concentrating and drying by using a rotary evaporator to prepare the allicin;
(4) preparing garlic polysaccharide and garlic amino acid: passing the effluent of macroporous resin through 732 type cation exchange resin, and controlling the flow rate at 3 column volumes/h to make the garlic amino acidAdsorbing on resin, washing with 2 column volumes of purified water, collecting cation exchange resin effluent and eluate, and adding appropriate amount of Na2CO3Adjusting pH of the polysaccharide solution to neutral, concentrating, and drying to obtain Bulbus Allii polysaccharide;
eluting the cation exchange resin with 0.1% ammonia water solution, controlling the flow rate at 5 column volume/h, collecting the cation exchange resin eluate, detecting the amino acid content of garlic by using a paper chromatography, and concentrating and drying to obtain the garlic amino acid.
And (3) performance testing:
the invention also detects the enzyme activity, purity, content and the like of various extracts prepared by the preparation methods of the embodiment and the comparative example, and the specific detection results are as follows:
TABLE 1 alliinase assay results of examples and comparative examples
Alliinase specific activity (U/g)
Alliinase recovery/%)
Example 1
1065
81.32％
Example 2
1741
78.39％
Example 3
1653
80.11％
Comparative example 1
105
8.56％
Comparative example 2
234
10.13％
Comparative example 3
/
/
As can be seen from the results shown in Table 1, the specific activity of the alliinase prepared by the preparation method of the invention in the examples 1-3 is up to 1741U/g, and the recovery rate is up to 81.32%, while the specific activity of the alliinase prepared by the comparative examples 1-2 is only about 100-200U/g, and the recovery rate is only 8% -10%.
TABLE 2 allicin assay results of examples and comparative examples
Allicin purity/%)
Allicin recovery/%)
Example 1
83.53％
72.84％
Example 2
86.89％
75.55％
Example 3
84.06％
70.35％
Comparative example 1
56.48％
35.22％
Comparative example 2
15.91％
23.46％
Comparative example 3
/
/
As can be seen from the results shown in Table 2, the purity of allicin obtained by the preparation method of examples 1-3 of the present invention was 86.89% at the highest, and the recovery rate was 75.55% at the highest, whereas the allicin obtained by the preparation method of comparative examples 1-2 was only about 15-50% at the recovery rate of 20-30%, and thus it can be seen that the purity and the recovery rate of allicin obtained by the preparation method of the present invention were significantly higher than those of the comparative examples.
TABLE 3 determination results of garlic polysaccharide in each example and comparative example
Content of garlic polysaccharide/%)
Garlic polysaccharide recovery/%)
Example 1
50.51％
85.69％
Example 2
53.64％
88.37％
Example 3
55.19％
89.91％
Comparative example 1
45.30％
72.58％
Comparative example 2
42.19％
75.37％
Comparative example 3
95％
50-70％
As can be seen from the results shown in Table 3, the content of the garlic polysaccharide obtained by the preparation method of examples 1-3 of the present invention was as high as 55.19%, and the recovery rate was 89.91%, whereas the purity of the allicin obtained by comparative examples 1-2 was only about 40%, and the recovery rate was about 70%, and thus it was found that the content and the recovery rate of the garlic polysaccharide obtained by the preparation method of the present invention were higher.
TABLE 4 amino acid determination results of garlic in each example and comparative example
Garlic amino acid content/%)
Garlic amino acid recovery/%)
Example 1
87.59％
62.36％
Example 2
89.13％
62.59％
Example 3
91.65％
63.85％
Comparative example 1
71.52％
49.36％
Comparative example 2
68.84％
45.62％
Comparative example 3
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/
As can be seen from the results shown in table 4, the content of the amino acid in the garlic obtained by the preparation method of examples 1 to 3 of the present invention was as high as 91.65%, and the recovery rate was as high as 63%, while the content of the amino acid in the garlic obtained by the comparative examples 1 to 2 was only about 60 to 70%, and the recovery rate was about 40%.
According to the content, the preparation method provided by the invention organically combines an ultrafiltration method, adsorption column methods of different types and the like, so that the four garlic extracts including alliinase, allicin, garlic amino acid, garlic polysaccharide and the like are prepared, the purity and the quality of the substances are ensured to the maximum extent, and the full utilization of the garlic raw materials is really realized.