Method for extracting leek flavor precursor substance s-hydrocarbyl cysteine sulfoxide

Abstract

The application belongs to the technical field of biochemistry and particularly relates to a leek flavor precursor S-hydrocarbyl cysteine sulfoxide extraction method, which comprises the following steps: S1, sampling: selecting fresh and complete leek leaves; S2, grinding and crushing, and adding pre-cooled 0.1 mol / L, pH 6.8 phosphate buffer solution, stirring and dissolving to obtain leek solution; S3, separation of S-hydrocarbyl cysteine sulfoxide and alliinase; S4, extraction: adding petroleum ether to the solution containing S-hydrocarbyl cysteine sulfoxide at a volume ratio of 1:1-3, fully stirring, standing and separating, and then taking the water phase; and S5, column chromatography: first separating by using a cation exchange column, and then separating by using a C18 preparation column to obtain methyl alliin and alliin. The application has the advantages that the extraction rate and purity of methyl alliin and alliin can be effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically relating to a method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor. Background Technology

[0002] S-alk(en)ylcysteine ​​sulfoxide (CSO), a flavor precursor in chives, is one of the most important sulfur-containing secondary metabolites involved in the formation of the characteristic aroma of Allium species. S-alk(en)ylcysteine ​​sulfoxide is decomposed by alliinase, generating various volatile organic sulfur compounds, enzymatic pyruvate, and other metabolites, ultimately releasing the unique pungent and savory flavor of chives. In chives, methyl alliin (S-methylcysteine ​​sulfoxide) is the most abundant, followed by alliin (S-allylcysteine ​​sulfoxide), with trace amounts of isoalliin (S-propenylcysteine ​​sulfoxide).

[0003] Methylalliin plays a role in supporting joint health and reducing inflammation and pain. After enzymatic hydrolysis, alliin is converted into active substances such as allicin, which possess antibacterial, cardiovascular protective, antioxidant, and anti-inflammatory properties. However, current technologies suffer from low extraction rates and low purity of both methylalliin and alliin. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, in order to improve the extraction rate and purity of methylalliin and allicin.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] A method for extracting S-alkylcysteine ​​sulfoxide, a precursor of leek flavor, includes the following steps:

[0007] S1. Sampling: Select fresh, intact chive leaves;

[0008] S2. Grinding and pulverizing: Place the leek leaves in a mortar, add liquid nitrogen to the mortar, and grind the leek leaves thoroughly to obtain leek powder; weigh the leek powder, put it into a container, and add pre-cooled 0.1mol / L, pH 6.8 phosphate buffer at a material-to-liquid ratio of 1:5 to 10, stir to dissolve, and obtain leek solution;

[0009] S3, separation of S-alkylcysteine ​​sulfoxide and alliinase: S-alkylcysteine ​​sulfoxide and alliinase in the leek solution obtained in step S2 were separated under low temperature conditions to obtain a solution containing S-alkylcysteine ​​sulfoxide.

[0010] S4. Extraction: Add petroleum ether to the solution containing S-hydroxycysteine ​​sulfoxide at a volume ratio of 1:1 to 3, stir thoroughly, allow to stand and separate into layers, and take the aqueous phase.

[0011] S5, Column Chromatography:

[0012] 1) Load the aqueous phase obtained from the extraction onto a cation exchange column and elute with water containing 0.1% formic acid for 2 column volumes to remove uncharged and negatively charged impurities and adsorb the target analyte onto the cation exchange column; then elute with 1 mol / L ammonia and collect 0.2 column volumes of the eluent obtained from the ammonia elution.

[0013] 2) Load the eluent collected in 1) into a C18 column;

[0014] 3) Elute with the first eluent for 1 column volume; then elute with the second eluent for 0.5 column volumes; then elute with the third eluent for 2 column volumes.

[0015] The first eluent is water and methanol containing 0.1% vol, with a volume ratio of 95:5; the second eluent is water and methanol containing 0.1% vol, with a volume ratio of 90:10; and the third eluent is methanol.

[0016] 4) Collect 0.3 column volumes of eluent from the first eluent (containing methylalliin) and 0.1 column volumes of eluent from the second eluent (containing alliin). The flavor precursor of chives, S-hydroxycysteine ​​sulfoxide, consists of methylalliin, alliin, and isoalliin. Since isoalliin is present in extremely small amounts, only trace amounts, this invention does not extract isoalliin.

[0017] In the extraction method of S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, provided by the present invention, preferably, the separation of S-hydroxycysteine ​​sulfoxide and alliinase in step S3 is carried out by filtering the leek solution with filter paper under low temperature conditions to obtain a first filtrate and a filter residue, and then subjecting the first filtrate to ultrafiltration to obtain a permeate; the permeate contains S-hydroxycysteine ​​sulfoxide and does not contain alliinase.

[0018] In the extraction method of S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, provided by the present invention, it is even more preferably that the molecular weight cutoff of the ultrafiltration membrane is 2 kDa.

[0019] In the extraction method of S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, provided by the present invention, more preferably, the separation of S-hydroxycysteine ​​sulfoxide and alliinase in step S3 is as follows: pre-cooled acetone is added to the leek solution while stirring, so that the acetone is fully dissolved in the leek solution, causing the alliinase to denature and precipitate. The solution is then filtered with filter paper or centrifuged to obtain a third filtrate or supernatant. The third filtrate or supernatant is then used to remove the acetone using a rotary evaporator to obtain a solution containing S-hydroxycysteine ​​sulfoxide.

[0020] In the extraction method of S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, provided by the present invention, it is further preferred that the amount of acetone added is 30-50% Vol of the leek solution.

[0021] In the extraction method of S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, provided by the present invention, it is further preferred that the centrifugation speed is 10000-20000 r / min.

[0022] In the extraction method of S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, provided by the present invention, it is further preferred that the temperature during rotary evaporation is 55°C and the vacuum degree is 50-100 mbar.

[0023] By employing the above technical solution, the extraction rate is effectively guaranteed by first separating S-alkylcysteine ​​sulfoxide and alliinase, preventing alliin from decomposing S-alkylcysteine ​​sulfoxide. Extraction removes oils and some substances soluble in petroleum ether, followed by column chromatography, effectively improving the purity of S-alkylcysteine ​​sulfoxide (methylalliin and alliin). Detailed Implementation

[0024] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Example 1

[0026] This embodiment provides a method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, comprising the following steps:

[0027] S1. Sampling: Select fresh, intact leaves of Pingjiu No. 4 chives;

[0028] S2. Grinding and pulverizing: Place the leek leaves in a pre-cooled mortar, add liquid nitrogen to the mortar, and grind the leek leaves thoroughly to obtain leek powder; weigh 20g of leek powder, put it into a container, add 100mL of pre-cooled 0.1mol / L, pH 6.8 phosphate buffer, stir to dissolve, and obtain leek solution;

[0029] Separation of S3, S-alkylcysteine ​​sulfoxide and alliinase: At 4℃, the leek solution obtained in step S2 was filtered with filter paper to obtain the first filtrate and the residue. The first filtrate was ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 2 kDa. The permeate was collected to obtain 79 mL of solution containing S-alkylcysteine ​​sulfoxide.

[0030] S4. Extraction: Add 90 mL of petroleum ether to the solution containing S-hydroxycysteine ​​sulfoxide, stir thoroughly, let stand for separation, and take the aqueous phase.

[0031] S5, Column Chromatography:

[0032] 1) The aqueous phase obtained from the extraction was loaded onto an SP cation exchange column with a column volume of 2 L. Elution was performed with water containing 0.1% formic acid for 2 column volumes to remove uncharged and negatively charged impurities and to adsorb the target analyte onto the cation exchange column. Elution was then performed with 1 mol / L ammonia solution, and the first 0.2 column volumes of eluent (400 mL) were collected. The collected eluent was concentrated using a reverse osmosis membrane to obtain 60 mL of concentrate.

[0033] 2) Load the concentrate from step 1) into a preparative C18 column with a column volume of 1L;

[0034] 3) Elute with the first eluent for 1 column volume; then elute with the second eluent for 0.5 column volumes; then elute with the third eluent for 2 column volumes.

[0035] The first eluent is water and methanol containing 0.1% vol, with a volume ratio of 95:5; the second eluent is water and methanol containing 0.1% vol, with a volume ratio of 90:10; the third eluent is methanol.

[0036] 4) Collect the first 0.3 column volumes of eluent from the first eluent, i.e., collect 300 mL of the eluent. Concentrate the eluent using a reverse osmosis membrane to obtain 53 mL of concentrate. Freeze-dry the concentrate to obtain 46 mg of solids, which is methyl allicin with an extraction rate of 0.23%.

[0037] Collect the first 0.1 column volume of eluent from the second eluent, i.e., collect 100 mL of eluent. Concentrate the eluent using a reverse osmosis membrane to obtain 25 mL of concentrate. Freeze-dry the concentrate to obtain 12 mg of solids, which is allicin, with an extraction rate of 0.06%.

[0038] Example 2

[0039] This embodiment provides a method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, comprising the following steps:

[0040] S1. Sampling: Select fresh, intact leaves of Pingjiu No. 4 chives;

[0041] S2. Grinding and pulverizing: Place the leek leaves in a pre-cooled mortar, add liquid nitrogen to the mortar, and grind the leek leaves thoroughly to obtain leek powder; weigh 30g of leek powder, put it into a container, add 210mL of pre-cooled 0.1mol / L, pH 6.8 phosphate buffer, stir to dissolve, and obtain leek solution;

[0042] Separation of S3, S-alkylcysteine ​​sulfoxide and alliinase: At 4℃, 100mL of acetone was added to the leek solution obtained in step S2 while stirring to ensure that the acetone was fully dissolved in the leek solution, causing the alliinase to denature and precipitate. The solution was filtered through filter paper to obtain a filtrate. The acetone was removed from the filtrate using a rotary evaporator at a temperature of 55℃ and a vacuum of 50-100mbar to obtain 185mL of a solution containing S-alkylcysteine ​​sulfoxide.

[0043] S4. Extraction: Add 370 mL of petroleum ether to the solution containing S-hydroxycysteine ​​sulfoxide, stir thoroughly, let stand for separation, and take the aqueous phase.

[0044] S5, Column Chromatography:

[0045] 1) The aqueous phase obtained from the extraction was loaded onto an SP cation exchange column with a column volume of 2 L. Eluting was performed with water containing 0.1% formic acid for 2 column volumes to remove uncharged and negatively charged impurities and to adsorb the target analyte onto the cation exchange column. Eluting was then performed with 1 mol / L ammonia solution, and the first 0.2 column volumes of eluent (400 mL) were collected. The collected eluent was concentrated using a reverse osmosis membrane to obtain 72 mL of concentrate.

[0046] 2) Load the concentrate from step 1) into a preparative C18 column with a column volume of 1L;

[0047] 3) Elute with the first eluent for 1 column volume; then elute with the second eluent for 0.5 column volumes; then elute with the third eluent for 2 column volumes.

[0048] The first eluent is water and methanol containing 0.1% vol, with a volume ratio of 95:5; the second eluent is water and methanol containing 0.1% vol, with a volume ratio of 90:10; the third eluent is methanol.

[0049] 4) Collect the first 0.3 column volumes of eluent from the first eluent, i.e., collect 300 mL of the eluent. Concentrate the eluent using a reverse osmosis membrane to obtain 50 mL of concentrate. Freeze-dry the concentrate to obtain 49 mg of solids, which is methyl allicin with an extraction rate of 0.163%.

[0050] The first 0.1 column volume of eluent eluted from the second eluent was collected, i.e., 100 mL of eluent was collected. The eluent was concentrated using a reverse osmosis membrane to obtain 32 mL of concentrate. The concentrate was freeze-dried to obtain 10 mg of solids, which was allicin, with an extraction rate of 0.033%.

[0051] Example 3

[0052] This embodiment provides a method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, comprising the following steps:

[0053] S1. Sampling: Select fresh, intact leaves of Pingjiu No. 4 chives;

[0054] S2. Grinding and pulverizing: Place the leek leaves in a pre-cooled mortar, add liquid nitrogen to the mortar, and grind the leek leaves thoroughly to obtain leek powder; weigh 40g of leek powder, put it into a container, add 400mL of pre-cooled 0.1mol / L, pH 6.8 phosphate buffer, stir to dissolve, and obtain leek solution;

[0055] Separation of S3, S-alkylcysteine ​​sulfoxide and alliinase: At 4℃, 200mL of acetone was added to the leek solution obtained in step S2 while stirring to ensure that the acetone was fully dissolved in the leek solution and that the alliinase was denatured and precipitated. The centrifuge temperature was set to 4℃ and the speed to 15000r / min, and the centrifugation was carried out for 20 minutes. The supernatant was collected, and the acetone was removed from the supernatant by rotary evaporation. The supernatant was then concentrated at a temperature of 55℃ and a vacuum of 50mbar to obtain 135mL of a solution containing S-alkylcysteine ​​sulfoxide.

[0056] S4. Extraction: Add 405 mL of petroleum ether to the solution containing S-hydroxycysteine ​​sulfoxide, stir thoroughly, let stand for separation, and take the aqueous phase.

[0057] S5, Column Chromatography:

[0058] 1) The aqueous phase obtained from the extraction was loaded onto an SP cation exchange column with a column volume of 2 L. Elution was performed with water containing 0.1% formic acid for 2 column volumes to remove uncharged and negatively charged impurities and to adsorb the target analyte onto the cation exchange column. Elution was then performed with 1 mol / L ammonia solution, and the first 0.2 column volumes of eluent (400 mL) were collected. The collected eluent was concentrated using a reverse osmosis membrane to obtain 75 mL of concentrate.

[0059] 2) Load the concentrate obtained in step 1) into a preparative C18 column with a column volume of 1L;

[0060] 3) Elute with the first eluent for 1 column volume; then elute with the second eluent for 0.5 column volumes; then elute with the third eluent for 2 column volumes.

[0061] The first eluent is water and methanol containing 0.1% vol, with a volume ratio of 95:5; the second eluent is water and methanol containing 0.1% vol, with a volume ratio of 90:10; the third eluent is methanol.

[0062] 4) Collect the first 0.3 column volumes of eluent from the first eluent, i.e., collect 300 mL of eluent. Concentrate the eluent using a reverse osmosis membrane to obtain 83 mL of concentrate. Freeze-dry the concentrate to obtain 72 mg of solids, which are methyl allicin with an extraction rate of 0.18%.

[0063] The first 0.1 column volume of eluent eluted from the second eluent was collected, i.e., 100 mL of eluent was collected. The eluent was concentrated using a reverse osmosis membrane to obtain 36 mL of concentrate. The concentrate was freeze-dried to obtain 23 mg of solids, which was allicin, with an extraction rate of 0.058%.

[0064] Example 4

[0065] This embodiment provides a method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, comprising the following steps:

[0066] S1. Sampling: Select fresh, intact leaves of Pingjiu No. 4 chives;

[0067] S2. Grinding and pulverizing: Place the leek leaves in a pre-cooled mortar, add liquid nitrogen to the mortar, and grind the leek leaves thoroughly to obtain leek powder; weigh 40g of leek powder, put it into a container, add 320mL of pre-cooled 0.1mol / L, pH 6.8 phosphate buffer, stir to dissolve, and obtain leek solution;

[0068] Separation of S3, S-alkylcysteine ​​sulfoxide and alliinase: At 4℃, 145 mL of acetone was added to the leek solution obtained in step S2 while stirring to ensure that the acetone was fully dissolved in the leek solution and that the alliinase was denatured and precipitated. The centrifuge was set at 4℃ and 18000 r / min for 20 minutes. The supernatant was collected and the acetone was removed by rotary evaporator. The supernatant was then concentrated at 55℃ and 100 mbar to obtain 110 mL of a solution containing S-alkylcysteine ​​sulfoxide.

[0069] S4. Extraction: Add 200 mL of petroleum ether to the solution containing S-hydroxycysteine ​​sulfoxide, stir thoroughly, let stand for separation, and take the aqueous phase.

[0070] S5, Column Chromatography:

[0071] 1) The aqueous phase obtained from the extraction was loaded onto an SP cation exchange column with a column volume of 2 L. Elution was performed with water containing 0.1% formic acid for 2 column volumes to remove uncharged and negatively charged impurities and to adsorb the target analyte onto the cation exchange column. Elution was then performed with 1 mol / L ammonia solution, and the first 0.2 column volumes of eluent (400 mL) were collected. The collected eluent was concentrated using a reverse osmosis membrane to obtain 82 mL of concentrate.

[0072] 2) Load the concentrate obtained in step 1) into a preparative C18 column with a column volume of 1L;

[0073] 3) Elute with the first eluent for 1 column volume; then elute with the second eluent for 0.5 column volumes; then elute with the third eluent for 2 column volumes.

[0074] The first eluent is water and methanol containing 0.1% vol, with a volume ratio of 95:5; the second eluent is water and methanol containing 0.1% vol, with a volume ratio of 90:10; the third eluent is methanol.

[0075] 4) Collect the first 0.3 column volumes of eluent from the first eluent, i.e., collect 300 mL of eluent. Concentrate the eluent using a reverse osmosis membrane to obtain 76 mL of concentrate. Freeze-dry the concentrate to obtain 70 mg of solids, which are methyl allicin with an extraction rate of 0.175%.

[0076] The first 0.1 column volume of eluent eluted from the second eluent was collected, i.e., 100 mL of eluent was collected. The eluent was concentrated using a reverse osmosis membrane to obtain 42 mL of concentrate. The concentrate was freeze-dried to obtain 19 mg of solids, which was allicin, with an extraction rate of 0.0475%.

[0077] Example 5

[0078] This embodiment provides a method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, comprising the following steps:

[0079] S1. Sampling: Select fresh, intact leaves of Pingjiu No. 4 chives;

[0080] S2. Grinding and pulverizing: Place the leek leaves in a pre-cooled mortar, add liquid nitrogen to the mortar, and grind the leek leaves thoroughly to obtain leek powder; weigh 40g of leek powder, put it into a container, add 320mL of pre-cooled 0.1mol / L, pH 6.8 phosphate buffer, stir to dissolve, and obtain leek solution;

[0081] Separation of S3, S-alkylcysteine ​​sulfoxide and alliinase: At 4℃, 96 mL of acetone was added to the leek solution obtained in step S2 while stirring to ensure that the acetone was fully dissolved in the leek solution, causing the alliinase to denature and precipitate. The centrifuge temperature was set to 4℃ and the speed to 20000 r / min, and the centrifugation was carried out for 20 minutes. The supernatant was collected, and the acetone was removed from the supernatant using a rotary evaporator. The supernatant was then concentrated at a temperature of 55℃ and a vacuum degree of 100 mbar to obtain 125 mL of a solution containing S-alkylcysteine ​​sulfoxide.

[0082] S4. Extraction: Add 125 mL of petroleum ether to the solution containing S-hydroxycysteine ​​sulfoxide, stir thoroughly, let stand for separation, and take the aqueous phase.

[0083] S5, Column Chromatography:

[0084] 1) The aqueous phase obtained from the extraction was loaded onto an SP cation exchange column with a column volume of 2 L. Elution was performed with water containing 0.1% formic acid for 2 column volumes to remove uncharged and negatively charged impurities and to adsorb the target analyte onto the cation exchange column. Elution was then performed with 1 mol / L ammonia solution, and the first 0.2 column volumes of eluent (400 mL) were collected. The collected eluent was concentrated using a reverse osmosis membrane to obtain 61 mL of concentrate.

[0085] 2) Load the concentrate obtained in step 1) into a preparative C18 column with a column volume of 1L;

[0086] 3) Elute with the first eluent for 1 column volume; then elute with the second eluent for 0.5 column volumes; then elute with the third eluent for 2 column volumes.

[0087] The first eluent is water and methanol containing 0.1% vol, with a volume ratio of 95:5; the second eluent is water and methanol containing 0.1% vol, with a volume ratio of 90:10; the third eluent is methanol.

[0088] 4) Collect the first 0.3 column volumes of eluent from the first eluent, i.e., collect 300 mL of eluent. Concentrate the eluent using a reverse osmosis membrane to obtain 58 mL of concentrate. Freeze-dry the concentrate to obtain 67 mg of solids, which is methyl allicin with an extraction rate of 0.168%.

[0089] Collect the first 0.1 column volume of eluent from the second eluent, i.e., collect 100 mL of eluent. Concentrate the eluent using a reverse osmosis membrane to obtain 40 mL of concentrate. Freeze-dry the concentrate to obtain 26 mg of solids, which is allicin, with an extraction rate of 0.065%.

[0090] Test case

[0091] 1. Weigh 10 mg of methylalliin obtained in Examples 1 to 5 respectively, dissolve and dilute to 1 mg / mL with 80% Vol acetonitrile aqueous solution. Purity is determined by high performance liquid chromatography (HPLC) under the following chromatographic conditions:

[0092] Chromatographic column: Waters XBridge BEH Amide (4.6×250mm, 5μm). Sample loading volume: 10μL; column temperature: 30℃; mobile phase: A) acetonitrile, B) water (containing 0.1% vol formic acid), with the following gradients: 0-12 min, A:B=85:15; 12-15 min, A:B=65:35; 15-25 min, A:B=60:40. Detection wavelength: 210nm. Three consecutive injections were performed, and purity was calculated using the area normalization method. The results are shown in Table 1.

[0093] Table 1. Purity of methylalliin obtained in each example

[0094]

[0095] As shown in Table 1, the purity of methylalliin obtained in each embodiment was lowest in Example 2 and highest in Example 4, at 98.13% and 98.87%, respectively. This demonstrates that the extraction method of the present invention effectively extracts methylalliin with high purity.

[0096] 2. Weigh 10 mg of alliin obtained in Examples 1 to 5 respectively, dissolve and dilute to 1 mg / mL with 80% Vol acetonitrile aqueous solution. Purity is determined by high performance liquid chromatography (HPLC) under the following chromatographic conditions:

[0097] Chromatographic column: Waters XBridge BEH Amide (4.6×250mm, 5μm). Sample loading volume: 10μL; column temperature: 30℃; mobile phase: A) acetonitrile, B) water (containing 0.1% vol formic acid), with the following gradients: 0-12 min, A:B=85:15; 12-15 min, A:B=65:35; 15-25 min, A:B=85:15. Detection wavelength: 210nm. Three consecutive injections were performed, and purity was calculated using the area normalization method. The results are shown in Table 2.

[0098] Table 2. Purity of alliin obtained in each example

[0099]

[0100] As shown in Table 2, the purity of alliin obtained in each embodiment was lowest in Example 4 and highest in Example 2, at 98.07% and 99.27%, respectively. This demonstrates that the method of the present invention can effectively extract alliin from leeks and obtain high-purity alliin.

[0101] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A method for extracting S-hydroxycysteine ​​sulfoxide, a flavor precursor of chives, characterized in that, Includes the following steps: S1. Sampling: Select fresh, intact chive leaves; S2. Grinding and pulverizing: Place the leek leaves in a mortar, add liquid nitrogen to the mortar, and grind the leek leaves thoroughly to obtain leek powder; weigh the leek powder, put it into a container, and add pre-cooled 0.1mol / L, pH 6.8 phosphate buffer at a material-to-liquid ratio of 1:5 to 10, stir to dissolve, and obtain leek solution; S3, separation of S-alkylcysteine ​​sulfoxide and alliinase: S-alkylcysteine ​​sulfoxide and alliinase in the leek solution obtained in step S2 were separated under low temperature conditions to obtain a solution containing S-alkylcysteine ​​sulfoxide. S4. Extraction: Add petroleum ether to the solution containing S-hydroxycysteine ​​sulfoxide at a volume ratio of 1:1 to 3, stir thoroughly, allow to stand and separate into layers, and take the aqueous phase. S5, Column Chromatography: 1) Load the aqueous phase obtained from the extraction onto a cation exchange column and elute with water containing 0.1% formic acid for 2 column volumes to remove uncharged and negatively charged impurities and adsorb the target analyte onto the cation exchange column; then elute with 1 mol / L ammonia and collect 0.2 column volumes of the eluent obtained from the ammonia elution. 2) Load the eluent collected in step 1) into a C18 column; 3) Elute with the first eluent for 1 column volume; then elute with the second eluent for 0.5 column volumes; then elute with the third eluent for 2 column volumes. The first eluent is water and methanol containing 0.1% vol, with a volume ratio of 95:5; the second eluent is water and methanol containing 0.1% vol, with a volume ratio of 90:10; and the third eluent is methanol. 4) Collect 0.3 column volumes of eluent from the first eluent and 0.1 column volumes of eluent from the second eluent.

2. The method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, according to claim 1, is characterized in that, The separation of S-alkylcysteine ​​sulfoxide and alliinase in step S3 is achieved by filtering the leek solution with filter paper under low temperature conditions to obtain a first filtrate and a filter residue. The first filtrate is then subjected to ultrafiltration to obtain a permeate. The permeate contains S-alkylcysteine ​​sulfoxide but does not contain alliinase.

3. The method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, according to claim 2, is characterized in that, The ultrafiltration membrane has a molecular weight cutoff of 2 kDa.

4. The method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, according to claim 1, is characterized in that, The separation of S-alkylcysteine ​​sulfoxide and alliinase in step S3 is as follows: pre-cooled acetone is added to the leek solution while stirring to fully dissolve the acetone in the leek solution, causing the alliinase to denature and precipitate. The solution is then filtered with filter paper or centrifuged to obtain a third filtrate or supernatant. The third filtrate or supernatant is then used to remove the acetone using a rotary evaporator to obtain a solution containing S-alkylcysteine ​​sulfoxide.

5. The method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, according to claim 4, is characterized in that, The amount of acetone added is 30-50% Vol of the leek solution.

6. The method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, according to claim 5, is characterized in that, The centrifuge speed is 10000-20000 r / min.

7. The method for extracting S-hydroxycysteine ​​sulfoxide, a precursor of leek flavor, according to claim 6, is characterized in that, The rotary evaporation process takes place at a temperature of 55°C and a vacuum level of 50–100 mbar.

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