阅读说明：本技术 一种从鲜蒜中提取的含硫物质和大蒜素及其提取方法 (Sulfur-containing substance and garlicin extracted from fresh garlic and extraction method thereof ) 是由 杨正庭 王鹏 钟斌 朱颖秋 王庚申 于 2021-06-23 设计创作，主要内容包括：本发明提出一种从鲜蒜中提取的含硫物质和大蒜素及其提取方法,属于天然物质提取、分离和纯化技术领域,能够解决现有的提取工艺提取成分单一,无法实现多种物质联合提取的技术问题。该从鲜蒜中提取含硫物质和大蒜素的方法,包括：(1)大蒜素提取液制备；(2)内源酶转化；(3)含硫物质和大蒜素的制备；其中,含硫物质包括S-烯丙基-L-半胱氨酸和S-烯丙基-巯基-L-半胱氨酸。本发明提出的从鲜蒜中提取含硫物质和大蒜素的方法具有操作简单、产品纯度高且能够实现多种物质联合提取等特点。本发明能够应用于大蒜提取物联合制备方面。(The invention provides a sulfur-containing substance and allicin extracted from fresh garlic and an extraction method thereof, belongs to the technical field of natural substance extraction, separation and purification, and can solve the technical problems that the existing extraction process has single extraction component and can not realize combined extraction of various substances. The method for extracting sulfur-containing substances and allicin from fresh garlic comprises the following steps: (1) preparing an allicin extracting solution; (2) endogenous enzyme transformation; (3) preparing sulfur-containing substances and allicin; wherein the sulfur-containing substance comprises S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine. The method for extracting the sulfur-containing substances and the allicin from the fresh garlic has the characteristics of simple operation, high product purity, capability of realizing combined extraction of various substances and the like. The invention can be applied to the combined preparation of garlic extracts.)

1. A method for extracting sulfur-containing substances and allicin from fresh garlic is characterized by comprising the following steps:

preparing an allicin extracting solution: peeling and cleaning fresh garlic, crushing into mashed garlic, extracting by adopting an extraction solvent, and filtering to obtain an allicin extracting solution;

endogenous enzyme transformation: converting the allicin extracting solution under different temperature and pH conditions to obtain a reaction solution containing sulfur-containing substances and allicin;

preparation of sulfur-containing substances and allicin: after passing through macroporous resin, the reaction solution containing the sulfur-containing substances and the allicin is sequentially eluted by using different eluents, and macroporous resin eluents are respectively collected and concentrated and dried to prepare the sulfur-containing substances and the allicin;

wherein the sulfur-containing substance comprises S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine.

2. The method of claim 1, wherein the step of converting the endogenous enzyme comprises maintaining the S-allyl-L-cysteine at 25-35 deg.C and pH 4-5 for 2-6h and S-allyl-mercapto-L-cysteine at 55-65 deg.C and pH 5.5-7.5 for 1-3 h.

3. The method for extracting sulfur-containing substances and allicin from fresh garlic as claimed in claim 1, wherein in the step of preparing the allicin extract, the extraction solvent is purified water with pH of 6.0-7.0, and the weight ratio of the extraction solvent to the fresh garlic is (5-10): 1.

4. The method as claimed in claim 1, wherein the macroporous resin is a high specific surface area resin with a specific surface area of 1000-1200m²/g。

5. The method for extracting sulfur-containing substances and allicin from fresh garlic as claimed in claim 1, wherein in the step of preparing the sulfur-containing substances and allicin, the flow rate of the reaction solution containing the sulfur-containing substances and allicin through the macroporous resin is 2-6 column volumes/hour.

6. The method for extracting sulfur-containing substances and allicin from fresh garlic as claimed in claim 1, wherein in the step of preparing the sulfur-containing substances and allicin, when the eluted product is S-allyl-L-cysteine, the eluent is 5-50% ethanol aqueous solution, the elution volume is 2-6 column volumes, and the elution speed is 1-3 column volumes/hour.

7. The method according to claim 1, wherein in the step of preparing the sulfur-containing materials and allicin, when the eluted product is S-allyl-mercapto-L-cysteine, the eluent is 5-50% ethanol aqueous solution, the elution volume is 2-6 column volumes, and the elution rate is 1-3 column volumes/hour.

8. The method according to claim 1, wherein the step of preparing the sulfur-containing materials and allicin comprises eluting with 70-95% ethanol aqueous solution at 2-6 column volumes and 1-3 column volumes/hr when the product is allicin.

9. A sulfur-containing material obtained by the method for extracting sulfur-containing material and allicin from fresh garlic according to any one of claims 1 to 8, wherein the sulfur-containing material comprises S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine;

wherein the product purity of the S-allyl-L-cysteine is 42.88-44.14%, and the product purity of the S-allyl-mercapto-L-cysteine is 62.23-63.42%.

10. Allicin prepared by the method for extracting sulfur-containing substances and allicin from fresh garlic according to any one of claims 1-8, wherein the product purity of the allicin is 62.2-68.4%.

Technical Field

The invention belongs to the technical field of natural substance extraction, separation and purification, and particularly relates to a sulfur-containing substance and allicin extracted from fresh garlic and an extraction method thereof.

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.

S-allyl-L-cysteine (SAC) is a water-soluble non-protein amino acid in garlic, and has effects of resisting oxidation, preventing senile dementia, resisting bacteria, diminishing inflammation, reducing cholesterol, preventing cancer, resisting tumor, lowering blood pressure, resisting diabetes, etc.; allicin (Allicin) is a sulfur-containing compound generated by alliin under the catalysis of alliinase, can block the formation of nitrosamine, has the effects of resisting cancer and reducing blood fat, and has obvious inhibiting effect on various viruses, bacteria, fungi and helicobacter pylori; S-allyl-mercapto-L-cysteine (SAMC) is a water-soluble allicin derivative obtained by natural biotransformation of allicin, and has a low content in raw garlic.

At present, patent applications (US2005260250A, US2007275876a11) disclose that SAMC is prepared by adopting a reaction of thiosulfinate and cystine, and particularly, SAMC is prepared by a long-time aging or cystine adding reaction mode, but the preparation processes only simply extract one or two components in garlic, so that the combined preparation of the three substances is difficult to realize, and the defects of complicated preparation steps, long time consumption and the like exist. Therefore, how to develop a preparation method which has simple process and can realize the combined extraction of a plurality of substances is an important way for solving the problems.

Disclosure of Invention

The invention provides a preparation process which is simple to operate, high in product purity and capable of realizing combined extraction of multiple substances, aiming at the technical problems that the existing extraction process is single in extraction component and cannot realize combined extraction of multiple substances.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for extracting sulfur-containing substances and allicin from fresh garlic comprises the following steps:

preparing an allicin extracting solution: peeling and cleaning fresh garlic, crushing into mashed garlic, extracting by adopting an extraction solvent, and filtering to obtain an allicin extracting solution;

endogenous enzyme transformation: converting the allicin extracting solution under different temperature and pH conditions to obtain a reaction solution containing sulfur-containing substances and allicin;

preparation of sulfur-containing substances and allicin: after passing through macroporous resin, the reaction solution containing the sulfur-containing substances and the allicin is sequentially eluted by using different eluents, and macroporous resin eluents are respectively collected and concentrated and dried to prepare the sulfur-containing substances and the allicin;

wherein the sulfur-containing substance comprises S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine.

Preferably, in the step of converting the endogenous enzyme, the conversion conditions for S-allyl-L-cysteine are 25 to 35 ℃ and pH 4 to 5, and the incubation is carried out for 2 to 6 hours, and the conversion conditions for S-allyl-mercapto-L-cysteine are 55 to 65 ℃ and pH 5.5 to 7.5, and the incubation is carried out for 1 to 3 hours.

Preferably, in the step of preparing the allicin extract, the extraction solvent is purified water with the pH value of 6.0-7.0, and the weight ratio of the extraction solvent to the fresh garlic is (5-10): 1.

Preferably, in the step of preparing the sulfur-containing material and the allicin, the macroporous resin is a high specific surface area resin with a specific surface area of 1000-1200m²/g。

Preferably, in the step of preparing the sulfur-containing substance and allicin, the flow rate of the reaction liquid containing the sulfur-containing substance and allicin through the macroporous resin is 2 to 6 column volumes/hour.

Preferably, in the step of preparing the sulfur-containing substance and allicin, when the eluted product is S-allyl-L-cysteine, the eluent is 5-50% ethanol aqueous solution, the elution volume is 2-6 column volumes, and the elution speed is 1-3 column volumes/hour.

Preferably, in the step of preparing the sulfur-containing substance and allicin, when the eluted product is S-allyl-mercapto-L-cysteine, the eluent is 5-50% ethanol aqueous solution, the elution volume is 2-6 column volumes, and the elution rate is 1-3 column volumes/hour.

Preferably, in the step of preparing the sulfur-containing substance and allicin, when the eluted product is allicin, the eluent is 70-95% ethanol aqueous solution, the elution volume is 2-6 column volumes, and the elution speed is 1-3 column volumes/hour.

The invention also provides a sulfur-containing substance prepared by the method for extracting the sulfur-containing substance and the allicin from the fresh garlic according to any one of the preferable technical schemes, wherein the sulfur-containing substance comprises S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine;

wherein the product purity of the S-allyl-L-cysteine is 42.88-44.14%, and the product purity of the S-allyl-mercapto-L-cysteine is 62.23-63.42%.

The invention also provides the allicin prepared by the method for extracting the sulfur-containing substances and the allicin from the fresh garlic according to any one of the preferable technical schemes, wherein the purity of the allicin product is 62.2-68.4%.

Compared with the prior art, the invention has the advantages and positive effects that:

1. the invention provides a method for extracting sulfur-containing substances and allicin from fresh garlic, which utilizes the principle that the fresh garlic contains different GGT invertases (namely glutamyl transpeptidase), has different activities under different temperature and pH conditions and different conversion precursors, and respectively converts GSAC, GS1PC and GSAMC in the garlic by adjusting the conditions of temperature, pH and the like so as to obtain reaction liquid containing S-allyl-L-cysteine, S-allyl-mercapto-L-cysteine and allicin, and finally prepares three garlic extract products with high added values after the treatment of resin adsorption, elution, drying and the like;

2. the method for extracting the sulfur-containing substances and the allicin from the fresh garlic solves the technical problems that the existing extraction method has complicated operation steps, consumes longer time and can not realize the combined preparation of various substances;

3. the invention provides a method for extracting sulfur-containing substances and allicin from fresh garlic, which comprises the steps of carrying out a large number of screening tests on the principle that different GGT invertases (namely glutamyltranspeptidase) are contained in the fresh garlic, and finally optimizing to obtain a whole set of conversion system suitable for S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine under the conditions of different activities and different conversion precursors under different temperature and pH conditions;

4. the method for extracting sulfur-containing substances and allicin from fresh garlic utilizes the principles that the fresh garlic contains different GGT invertases (namely glutamyl transpeptidase), has different activities under different temperature and pH conditions and different transformation precursors, realizes the preparation of various extracts, does not use organic solvents and other auxiliary preparations in the whole process, and has natural substance components, no organic solvent residue and high safety;

5. the purity of S-allyl-L-cysteine prepared by the method for extracting sulfur-containing substances and allicin from fresh garlic reaches 42.88-44.14 percent, the purity of S-allyl-mercapto-L-cysteine reaches 62.23-63.42 percent, and the purity of allicin reaches 62.2-68.4 percent.

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 method for extracting sulfur-containing substances and allicin from fresh garlic, which comprises the following steps:

s1, preparing an allicin extracting solution: peeling and cleaning fresh garlic, crushing into mashed garlic, extracting by adopting an extraction solvent, and filtering to obtain an allicin extracting solution;

s2, endogenous enzyme transformation: converting the allicin extracting solution under different temperature and pH conditions to obtain a reaction solution containing sulfur-containing substances and allicin;

s3, preparation of sulfur-containing substances and allicin: after passing through macroporous resin, the reaction solution containing the sulfur-containing substances and the allicin is sequentially eluted by using different eluents, and macroporous resin eluents are respectively collected and concentrated and dried to prepare the sulfur-containing substances and the allicin;

wherein the sulfur-containing substance comprises S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine.

In the technical scheme, the invention provides a method for extracting sulfur-containing substances and allicin from fresh garlic, which utilizes the principle that the fresh garlic contains different GGT invertases (namely glutamyl transpeptidase), has different activities under different temperature and pH conditions and different conversion precursors, and converts GSAC, GS1PC and GSAMC in the garlic respectively by adjusting the conditions of temperature, pH and the like to obtain reaction liquid containing S-allyl-L-cysteine, S-allyl-mercapto-L-cysteine and allicin, and finally prepares three garlic extract products with high added values after resin adsorption, elution, drying and the like.

In a preferred embodiment, in the step of converting the endogenous enzyme, the conversion conditions for S-allyl-L-cysteine are 25-35 ℃ at pH 4-5 and incubation for 2-6h, and the conversion conditions for S-allyl-mercapto-L-cysteine are 55-65 ℃ at pH 5.5-7.5 and incubation for 1-3 h.

In the preferred embodiment, the invention finally optimizes and obtains a whole set of conversion system suitable for S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine by a large number of screening tests under the principles that fresh garlic contains different GGT convertases (namely glutamyl transpeptidase) and has different activities and conversion precursors under different temperature and pH conditions, and substances obtained by conversion by using the conversion system have the characteristics of good quality, high purity and the like.

In a preferred embodiment, in the step of preparing the allicin extractive solution, the extraction solvent is purified water with pH of 6.0-7.0, and the weight ratio of the extraction solvent to the fresh garlic is (5-10): 1.

In a preferred embodiment, in the step of preparing the sulfur-containing material and the allicin, the macroporous resin is a high specific surface area resin with a specific surface area of 1000-1200m²/g。

In the above preferred embodiment, the reason why the resin with a high specific surface area is selected in the present invention in carrying out the production of the sulfur-containing substance and allicin is that: the traditional amino acid separation mainly adopts anion and cation exchange resin, but the anion and cation exchange resin can not adsorb the allicin sulphosulfinate; the specific surface area of the common macroporous adsorption resin is insufficient, so that the adsorption of sulfur-containing substances and allicin is insufficient. The active resin with high specific surface area is used for more sufficient adsorption, so that the sulfur-containing substances and the allicin can be adsorbed simultaneously, the resin dosage can be effectively reduced, and the subsequent separation can be realized only by alcohol solutions with different concentrations. Therefore, compared with the traditional anion-cation exchange resin, the resin with high specific surface area selected in the embodiment of the invention can effectively reduce the consumption of acid and alkali in the regeneration process, simultaneously save the regeneration time and improve the efficiency.

In a preferred embodiment, in the step of preparing the sulfur-containing substance and allicin, the flow rate of the reaction liquid containing the sulfur-containing substance and allicin through the macroporous resin is 2 to 6 column volumes/hour.

In a preferred embodiment, in the step of preparing the sulfur-containing substance and allicin, when the eluted product is S-allyl-L-cysteine, the eluent is 5-50% ethanol aqueous solution, the elution volume is 2-6 column volumes, and the elution speed is 1-3 column volumes/hour.

In the above preferred embodiment, the reason why 5-50% aqueous ethanol is used as the eluent when eluting S-allyl-L-cysteine is that: compared with methanol and acetone, the ethanol has relatively low smell and toxicity, relatively low polarity and relatively high elution strength, and can reduce the consumption of organic solvent. S-allyl-L-cysteine is absorbed in resin, and the S-allyl-L-cysteine can be sufficiently resolved by 5-50% ethanol aqueous solution according to polar similarity and compatibility.

In a preferred embodiment, in the step of preparing the sulfur-containing substance and allicin, when the eluted product is S-allyl-mercapto-L-cysteine, the eluent is 5-50% aqueous ethanol, the elution volume is 2-6 column volumes, and the elution rate is 1-3 column volumes/hour.

In the preferred embodiment described above, the reason for using 5-50% aqueous ethanol as the eluent in eluting S-allyl-mercapto-L-cysteine is that: compared with methanol and acetone, the ethanol has smaller smell and toxicity, smaller polarity than methanol and acetone, higher elution strength and can effectively reduce the dosage of ethanol organic solvent, S-allyl-mercapto-L-cysteine is absorbed in resin, and S-allyl-mercapto-L-cysteine is fully resolved by 5-50% ethanol aqueous solution according to similar and compatible polarity.

In a preferred embodiment, in the step of preparing the sulfur-containing substance and allicin, when the eluted product is allicin, the eluent is 70-95% ethanol aqueous solution, the elution volume is 2-6 column volumes, and the elution speed is 1-3 column volumes/hour.

In the above preferred embodiment, the reason why 70-95% ethanol aqueous solution is used as the eluent when eluting allicin is that: compared with methanol and acetone, the ethanol has relatively small smell and toxicity, relatively small polarity and relatively high elution strength, and can reduce the consumption of ethanol as organic solvent. 70-95% ethanol water solution can fully resolve allicin, and simultaneously realize regeneration of macroporous resin. Meanwhile, the concentration of the ethanol water solution is also of great importance, the consumption of organic solvents which can not be used for resolving the allicin or are used is obviously increased due to the low concentration of the ethanol water solution, and the difficulty in realizing the subsequent production amplification is increased due to the high concentration of the ethanol water solution.

The invention also provides a sulfur-containing substance prepared by the method for extracting the sulfur-containing substance and the allicin from the fresh garlic, which is described in any one of the preferred embodiments, wherein the sulfur-containing substance comprises S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine;

wherein the product purity of the S-allyl-L-cysteine is 42.88-44.14%, and the product purity of the S-allyl-mercapto-L-cysteine is 62.23-63.42%.

The invention also provides the allicin prepared by the method for extracting the sulfur-containing substances and the allicin from the fresh garlic, which is disclosed by any one of the preferred embodiments, wherein the product purity of the allicin is 62.2-68.4%.

In order to more clearly and specifically describe the sulfur-containing substances and allicin extracted from fresh garlic and the method for extracting the same provided in the embodiments of the present invention, the following description will be given with reference to specific embodiments.

Example 1

The embodiment provides a method for extracting sulfur-containing substances and allicin from fresh garlic, which comprises the following steps:

(1) preparing an allicin extracting solution: peeling fresh garlic, cleaning, crushing into mashed garlic, adding 5 times of purified water with pH of 6.0-7.0 by volume into the mashed garlic, extracting to obtain an extracting solution, and filtering the obtained extracting solution by a plate frame to obtain an allicin extracting solution;

(2) endogenous enzyme transformation: performing heat preservation for 2h at 25 ℃ and pH 4 for the allicin extract to convert natural GSAC in the garlic into S-allyl-L-cysteine (SAC), adjusting the conversion conditions, performing heat preservation for 2h at 55 ℃ and pH 6 for the allicin extract to convert GSAMC in the garlic into S-allyl-mercapto-L-cysteine (SAMC), and obtaining a reaction solution containing sulfur-containing substances and allicin;

(3) after the reaction liquid containing the sulfur-containing substances and the allicin passes through the LX-68 high specific surface area resin, controlling the flow rate of the reaction liquid containing the sulfur-containing substances and the allicin to be 2 column volumes/hour, adsorbing SAC, SAMC and the allicin on the high specific surface area resin, eluting the LX-68 high specific surface area resin by using 10% ethanol water solution, eluting 5 column volumes at the elution speed of 2 column volumes/hour, collecting SAMC-rich eluent, and concentrating and drying to obtain a high-content SAMC product;

(4) eluting the LX-68 resin with high specific surface area by using 30 percent ethanol water solution, eluting at the speed of 2 column volumes/hour and 4 column volumes, collecting the SAC-rich eluent, and concentrating and drying the SAC-rich eluent to obtain a high-content SAC product;

(5) eluting the LX-68 resin with high specific surface area by using 70 percent ethanol water solution, eluting at the speed of 2 column volumes/hour and 4 column volumes, collecting the eluate rich in the allicin, and concentrating and drying to obtain the product with high allicin content.

Example 2

The embodiment provides a method for extracting sulfur-containing substances and allicin from fresh garlic, which comprises the following steps:

(1) preparing an allicin extracting solution: peeling fresh garlic, cleaning, crushing into mashed garlic, adding 10 times of purified water with pH of 6.0-7.0 by volume into the mashed garlic, extracting to obtain an extracting solution, and filtering the obtained extracting solution by a plate frame to obtain an allicin extracting solution;

(2) endogenous enzyme transformation: maintaining the temperature of the allicin extract at 30 ℃ and pH5 for 4h to convert natural GSAC in the garlic into SAC, adjusting the conversion conditions, maintaining the temperature of the allicin extract at 55 ℃ and pH 6.5 for 3h to convert GSAMC in the garlic into SAMC, and obtaining a reaction solution containing sulfur-containing substances and allicin;

(3) after the reaction liquid containing the sulfur-containing substances and the allicin passes through the LSA-10 high specific surface area resin, controlling the flow rate of the reaction liquid containing the sulfur-containing substances and the allicin to be 4 column volumes/hour, adsorbing SAC, SAMC and the allicin on the LSA-10 high specific surface area resin, eluting the LSA-10 high specific surface area resin by using 20% ethanol aqueous solution, eluting 4 column volumes at the elution speed of 3 column volumes/hour, collecting SAMC-rich eluent, and concentrating and drying to obtain a high-content SAMC product;

(4) eluting the LSA-10 resin with high specific surface area by using 40% ethanol water solution, eluting at the speed of 3 column volumes/hour, eluting 4 column volumes, collecting the SAC-rich eluent, and concentrating and drying to obtain a high-content SAC product;

(5) and (3) eluting the LSA-10 high specific surface area resin by using 95% ethanol water solution, wherein the elution speed is 1 column volume/hour, eluting 2 column volumes, collecting the eluate rich in the allicin, and concentrating and drying to obtain the high-content allicin product.

Example 3

The embodiment provides a method for extracting sulfur-containing substances and allicin from fresh garlic, which comprises the following steps:

(1) preparing an allicin extracting solution: peeling fresh garlic, cleaning, crushing into mashed garlic, adding 8 times of purified water with the pH value of 6.0-7.0 into the mashed garlic for extraction to obtain an extracting solution, and filtering the obtained extracting solution by using a plate frame to obtain an allicin extracting solution;

(2) endogenous enzyme transformation: maintaining the temperature of the allicin extract at 35 deg.C and pH 4 for 6h to convert natural GSAC in Bulbus Allii into SAC, adjusting conversion conditions, maintaining the temperature of the allicin extract at 65 deg.C and pH 6.5 for 2.5h to convert GSAMC in Bulbus Allii into SAMC, and obtaining reaction solution containing sulfur-containing substances and allicin;

(3) after the reaction liquid containing the sulfur-containing substances and the allicin passes through XDA-200B high specific surface area resin, controlling the flow rate of the reaction liquid containing the sulfur-containing substances and the allicin to be 4 column volumes/hour, adsorbing SAC, SAMC and the allicin on the XDA-200B high specific surface area resin, eluting the XDA-200B high specific surface area resin by using 20% ethanol aqueous solution, eluting at the speed of 3 column volumes/hour, eluting 4 column volumes, collecting SAMC-rich eluent, and concentrating and drying the eluent to obtain a high-content SAMC product;

(4) eluting XDA-200B high specific surface area resin by using 40% ethanol water solution, wherein the elution speed is 3 column volumes/hour, eluting 4 column volumes, collecting SAC-rich eluent, and concentrating and drying the eluent to obtain a high-content SAC product;

(5) eluting XDA-200B resin with high specific surface area with 95% ethanol water solution at the speed of 1 column volume/hr for 2 column volumes, collecting eluate rich in allicin, concentrating, and drying to obtain high-content allicin product.

Comparative example 1

The comparative example provides an extraction method of S-allyl-L-cysteine, which specifically comprises the following steps:

(1) soaking cleaned Bulbus Allii at 5-50 deg.C and pH of 5-10 for 1-3d0-50mmol/L CaCl₂The amount of the soaking solution is based on submerging the garlic bulbs;

(2) grinding soaked Bulbus Allii, and mixing with CaCl₂The solution forms a homogeneous reaction system;

(3) performing enzyme reaction at 15-50 deg.C and pH of 5-10 at 20-500rpm for 2-12 hr to obtain garlic product rich in S-allyl-L-cysteine.

Comparative example 2

The comparative example provides a method for extracting sulfur-containing substances with strong taste from garlic, which comprises the following steps:

(1) weighing intact garlic bulbs, and adding distilled water according to the weight-volume ratio of 1: 2-10;

(2) heating at 80-100 deg.C for 10-60 min, and mashing with tissue mashing machine;

(3) heating at 80-100 deg.c for 10-60 min, and centrifuging at 5000r/min for 30 min;

(4) taking the supernatant, and concentrating to 1/4-1/7 times of the original volume by rotary evaporation;

(5) concentrating, and separating with strong acid cation exchange resin;

(6) concentrating the separated liquid to 1/4-1/7 times of the original volume by using a rotary evaporator;

(7) freeze drying the supernatant to obtain off-white powder, namely the sulfur-containing substances with thick taste in the garlic; the sulfur-containing substances with thick taste in Bulbus Allii include S-allyl-L-cysteine sulfoxide, S-allyl-L-cysteine, S-methyl-L-cysteine, S-propenyl-L-cysteine, and gamma-L-glutamyl-S-allyl-L-cysteine.

SAC, SAMC and allicin yield determination

The present invention has carried out the determination of the yield of SAC, SAMC and allicin prepared in the above examples and comparative examples, and the specific test methods and results are as follows:

the test method comprises the following steps:

(1) the method for detecting the yield of the allicin product comprises the following steps: the determination of allicin in the garlic NY/T2643-2014 and the products is as follows:

a chromatographic column: c18, 250mm by 4.6mm, 5 μm, or equivalent size chromatography column;

mobile phase: acetonitrile and 1% formic acid solution, gradient elution procedure is shown in table 1;

flow rate: 1.0 ml/min;

column temperature: 40 ℃;

detection wavelength: 245 nm.

TABLE 1 gradient elution procedure for allicin yield determination experiments

(2) The method for detecting the yield of SAC and SAMC products comprises the following steps:

a chromatographic column: c18, 250mm by 4.6mm, 5 μm, or equivalent size chromatography column;

mobile phase: acetonitrile and aqueous solution, gradient elution procedure is shown in table 2;

flow rate: 1.0 ml/min;

column temperature: 40 ℃;

detection wavelength: 214nm

Sample introduction amount: 20 μ L.

TABLE 2 gradient elution procedure for SAC, SAMC yield determination experiments

Time, min	1% formic acid solution,%	Acetonitrile, is%
			0-5	90	10
5-17	90-70	10-30
			17-20	70	30
20-30	70-50	30-50
			30-35	50	50
35-40	50-90	50-10
			50	90	10

The test results, as shown in tables 3-5:

TABLE 3 SAC yield measurement results obtained in examples

TABLE 4 results of SAMC yield measurement obtained in examples

TABLE 5 results of determination of allicin yield in each example

As can be seen from the data shown in tables 3-5, the content of SAC, SAMC and allicin before loading the column on fresh garlic is 20ug/g fresh garlic, 2ug/g fresh garlic and 24mg/g fresh garlic, and the column loading yield, the product content and the drying yield of the SAC product prepared by the extraction method provided in the embodiments 1-3 of the present invention are about 81%, the product content is 42.88% -44.14% and the drying yield is about 0.461% -0.504%; the column-loading yield of the SAMC product reaches about 82 percent, the product content is 62.23 to 63.42 percent, and the drying yield is about 0.164 to 0.195 percent; the column-loading yield of the allicin product is as high as about 56%, the product content is 62.2% -68.4%, and the drying yield is 1.996% -2.197%, so that the extraction method provided by the embodiment of the invention can realize the combined preparation of SAC, SAMC and allicin, and the obtained product has high yield and good quality.

Endogenous enzyme transformation conditions screening assays

In the embodiment of the invention, in order to optimize a whole set of transformation systems suitable for SAC and SAMC in fresh garlic, screening tests are carried out on transformation conditions, and specific test methods and test results are as follows:

(1) SAC transformation conditions screening assay:

the experimental conditions are as follows: the temperature is 25 ℃, 40 ℃, 60 ℃, 80 ℃, the pH is 2, 4, 6 and 8, and the conversion time is 2h, 3h, 4h, 5h and 6 h;

the experimental method specifically comprises the following steps:

a1, peeling fresh garlic, cleaning, crushing into mashed garlic, adding 10 times of purified water with pH of 6.0-7.0, extracting to obtain extractive solution, and filtering with plate-and-frame filter to obtain garlicin extractive solution;

a2, performing a multi-factor experiment of endogenous enzyme conversion by referring to each parameter set by the experimental conditions, and respectively obtaining reaction solutions;

and A3, after the reaction solution passes through macroporous resin, eluting by using an eluant, collecting macroporous resin eluent, concentrating and drying to obtain an extract product, and testing the content of the extract product, wherein the specific test result is shown in the following table.

TABLE 6 SAC transformation conditions screening test results statistics

(2) SAMC transformation conditions screening test:

the experimental conditions are as follows: the temperature is 25 ℃, 40 ℃, 60 ℃, 80 ℃, the pH is 2, 4, 6 and 8, and the conversion time is 1h, 2h and 3 h;

the experimental method specifically comprises the following steps:

a1, peeling fresh garlic, cleaning, crushing into mashed garlic, adding 10 times of purified water with pH of 6.0-7.0, extracting to obtain extractive solution, and filtering with plate-and-frame filter to obtain garlicin extractive solution;

a2, performing a multi-factor experiment of endogenous enzyme conversion by referring to each parameter set by the experimental conditions, and respectively obtaining reaction solutions;

and A3, after the reaction solution passes through macroporous resin, eluting by using an eluant, collecting macroporous resin eluent, concentrating and drying to obtain an extract product, and testing the content of the extract product, wherein the specific test result is shown in the following table.

TABLE 7 SAMC transformation conditions screening test results statistics

As can be seen from the above screening test and the test data shown in tables 6-7, the conversion efficiency is optimal when the conversion temperature is 25-35 deg.C, pH is 4-5, and the temperature is maintained for 2-6h, for example, when the temperature is 25 deg.C, pH is 4, and the temperature is 6h, the conversion efficiency is as high as 2360ug/g fresh garlic; when the conversion temperature is 55-65 ℃, the pH value is 5.5-7.5, and the heat preservation is carried out for 1-3h, the effect of converting the natural GSAMC into SAMC is ideal, for example, when the temperature is 60 ℃, the pH value is 6, and the heat preservation time is 3h, the conversion efficiency is optimal, and the content of SAMC can reach 1450ug/g fresh garlic.

Therefore, SAC, SAMC and allicin products prepared by the method for extracting sulfur-containing substances and allicin from fresh garlic provided by the embodiment of the invention have good quality and high yield, and can solve the technical problems of complicated operation steps, long time consumption and incapability of realizing combined preparation of a plurality of substances in the existing extraction method; the embodiment of the invention also optimizes and obtains a whole set of conversion system suitable for S-allyl-L-cysteine and S-allyl-mercapto-L-cysteine by a large number of screening tests under the principles that the fresh garlic contains different GGT convertases (namely glutamyl transpeptidase) and has different activities and conversion precursors under different temperature and pH conditions.