A method for preparing a garlic active ingredient-enriched substance by utilizing a deep eutectic solvent coupled fermentation

Abstract

This invention discloses a method for preparing garlic active ingredient concentrates using deep eutectic solvent coupled fermentation, comprising the following steps: (1) taking garlic, pretreating it, and preparing garlic slurry; (2) selectively subjecting the garlic slurry to enzyme activity regulation and / or edible microbial fermentation according to the target components to obtain garlic treated slurry; (3) preparing binary, ternary, or quaternary deep eutectic solvents using hydrogen bond acceptors and hydrogen bond donors as the main raw materials; (4) mixing the garlic slurry or garlic treated slurry with the deep eutectic solvent for extraction to obtain an extraction mixture; (5) separating and purifying the extraction mixture to obtain the garlic active ingredient concentrates. This method uses garlic as raw material and, through selective enzyme activity regulation and edible microbial fermentation, deep eutectic solvent directional extraction, separation, and purification, enables active ingredients with different polarities, molecular weights, and stability to be released, transformed, enriched, and recovered within the same overall process framework.

Description

Technical Field

[0001] This invention belongs to the field of natural product deep processing technology, and in particular relates to a method for preparing garlic active ingredient enrichment by deep eutectic solvent coupled fermentation. Background Technology

[0002] Fresh garlic has a high water content and strong enzyme activity. Reactions involving alliinase, peroxidase, and polyphenol oxidation affect the conversion and stability of sulfur-containing compounds, polyphenols, and flavonoids. Its active components exhibit a wide range of polarities: alliin, S-allyl cysteine, amino acids, nucleosides, organic acids, polysaccharides, and fructans are predominantly hydrophilic; allicin, ajoene, diallyl disulfide, diallyl trisulfide, and some volatile sulfides are predominantly low to medium polar; polyphenols, flavonoids, and saponins are moderately polar and affected by pH, hydrogen bonding, and solvent viscosity. Therefore, single water extraction, ethanol extraction, or conventional organic solvent extraction is insufficient to extract all these components simultaneously.

[0003] Deep eutectic solvents (DES), as a type of designable solvent, have been used for the extraction of various plant active ingredients; edible microbial fermentation has also been used for the processing of plant raw materials. However, existing technologies typically use DES extraction and fermentation conversion separately, or only design processes for a single component, lacking synergistic control schemes for multiple components in fresh garlic.

[0004] Therefore, existing methods such as water extraction, alcohol extraction, conventional organic solvent extraction, single DES extraction, or fermentation still suffer from problems such as narrow coverage of target components, easy degradation of sulfur-containing components, difficulty in simultaneous release of components with different polarities, and insufficient DES background interference and subsequent fractionation recovery. There is a need to establish a preparation method suitable for fresh garlic that combines enzyme activity regulation, fermentation transformation, selective DES extraction, and fractionation enrichment. Summary of the Invention

[0005] Objective of the Invention: To address the problems existing in the prior art, this invention provides a method for preparing garlic active ingredient concentrates using deep eutectic solvent coupled fermentation. This method uses garlic as raw material and, through low-temperature raw material treatment, enzyme activity regulation, edible microbial fermentation, directional extraction with binary / ternary / quaternary deep eutectic solvents, solid-liquid separation, resin adsorption, membrane separation, liquid-liquid partitioning, alcohol precipitation, ion exchange, HILIC separation, and detection and evaluation, enables the release, transformation, enrichment, and recovery of active ingredients with different polarities, molecular weights, and stabilityes within the same overall process framework.

[0006] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing garlic active ingredient concentrates using deep eutectic solvent coupled fermentation, comprising the following steps:

[0008] (1) Take garlic, pre-treat it, and prepare garlic slurry;

[0009] (2) Based on the target components, the garlic slurry is optionally subjected to enzyme activity regulation and / or edible microbial fermentation treatment to obtain garlic-treated slurry;

[0010] (3) Using hydrogen bond acceptors and hydrogen bond donors as the main raw materials, prepare binary, ternary or quaternary deep eutectic solvents;

[0011] (4) The garlic slurry obtained in step (1) or the garlic treatment slurry obtained in step (2) is mixed with the deep eutectic solvent obtained in step (3) for extraction to obtain an extraction mixture;

[0012] (5) The extracted mixture is separated and purified to obtain the garlic active ingredient enrichment.

[0013] As a specific implementation plan, in step (1), the garlic is white-skinned garlic, purple-skinned garlic or single-clove garlic; the garlic is fresh garlic that has not been dried, fermented with black garlic or cooked at high temperature within 0-30 days after harvest, and the fresh garlic is stored at 4-15℃ for a short period of 0-30 days and warmed to 10-25℃ before use;

[0014] The pretreatment includes removing mud, root plates and inedible impurities, peeling, washing and draining;

[0015] The method for preparing garlic paste includes slicing, crushing, homogenizing, colloid milling or low-temperature pulverization to obtain garlic paste.

[0016] As a specific implementation plan, in step (2), the method of enzyme activity regulation includes: standing at 20-40℃ for 5-90 min to activate alliinase, or short-term treatment at 60-95℃ for 0.5-10 min to inactivate alliinase and polyphenol oxidation-related enzymes.

[0017] For example, when the target is an active sulfide, the garlic slurry is left to stand at 20-40℃ for 5-90 min; when the target is allicin, S-allyl cysteine ​​or γ-glutamyl cysteine ​​derivatives, a short-time heat treatment at 60-95℃ for 0.5-10 min, ice bath pulverization, nitrogen protection or ascorbic acid protection are used.

[0018] The method for fermenting edible microorganisms includes: inoculating with one or more of lactic acid bacteria, yeast, or Bacillus, and fermenting for 6-72 h at 20-37℃ and pH 3.0-6.5.

[0019] Preferably, fermentation is carried out at 28-37℃ and pH 3.0-6.5 for 18-36 h.

[0020] As a further embodiment, the lactic acid bacteria are selected from one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus acidophilus; the yeast is selected from Saccharomyces cerevisiae, Saccharomyces cerevisiae, or a combination thereof; the Bacillus is selected from Bacillus subtilis, Bacillus coagulans, or a combination thereof; and the inoculation amount is 10^5-10^8 CFU / g garlic slurry.

[0021] As a specific implementation scheme, in step (3), the hydrogen bond acceptor is selected from one or more of the following: choline chloride, betaine, betaine hydrochloride, L-proline, L-carnitine, acetylcholine chloride, glycine betaine, choline bicarbonate, tetraalkylammonium salt, imidazoline salt, amino acid salt, and organic acid salt.

[0022] The hydrogen bond donor is selected from one or more of the following: lactic acid, malic acid, citric acid, tartaric acid, acetic acid, levulinic acid, glycerol, propylene glycol, 1,3-propanediol, ethylene glycol, polyethylene glycol, urea, acetamide, nicotinamide, glucose, fructose, sucrose, trehalose, xylitol, sorbitol, mannitol, erythritol, ascorbic acid, caffeic acid, ferulic acid, gallic acid, chlorogenic acid, and amino acids.

[0023] The raw materials of the deep eutectic solvent may also optionally include viscosity-adjusting components, antioxidant components, pH-adjusting components and / or stabilizing components;

[0024] The eutectic solvent is a clear, homogeneous liquid with a viscosity of 10-3000 mPa·s at 25°C and a pH of 2.0-8.0. After separation, the eutectic solvent can be recovered through water dilution, resin adsorption, antisolvent precipitation, membrane concentration, or vacuum concentration, and reused in the next batch of extraction. After 1-5 cycles, the yield of the target component remains at no less than 80%.

[0025] As a specific implementation plan, in step (3), the preparation method of the deep eutectic solvent includes: mixing the raw materials, stirring at 40-100℃ for 0.5-6 h, and adjusting the water mass fraction to 0-70% to obtain the solvent;

[0026] As a specific implementation plan, in step (4), the extraction method includes: adding a deep eutectic solvent at a liquid-solid ratio of 5-100 mL / g, extracting at 10-90℃, low-temperature stirring, ultrasonic assistance, microwave assistance, enzyme assistance or a combination thereof for 5-360 min;

[0027] As a specific implementation plan, in step (5), the separation and purification methods include one or a combination of centrifugation, filtration, membrane separation, liquid-liquid partitioning, macroporous resin adsorption, polyamide resin adsorption, ion exchange, HILIC column separation, ethanol precipitation, dialysis, antisolvent precipitation, vacuum concentration or freeze drying.

[0028] As one implementation scheme, in steps (2)-(5), when the target is polyphenol-flavonoid enrichment, the deep eutectic solvent is selected from organic acid-choline type, betaine-organic acid type, trihydric acid type or antioxidant quaternary DES, with a water content of 20-45%; the extraction method includes: liquid-solid ratio of 10-40 mL / g, temperature of 35-55℃, ultrasonic power of 200-600 W, extraction for 20-70 min; the separation and purification method includes: elution with D101, AB-8 or polyamide resin with 30-70% ethanol;

[0029] Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2), or a quaternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2):(0.05-0.2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, L-proline, L-carnitine and acetylcholine chloride, and the hydrogen bond donor is selected from one of lactic acid, malic acid, propylene glycol, glycerol, citric acid and ascorbic acid;

[0030] More preferably, the deep eutectic solvent is selected from choline chloride-lactic acid-glycerol, choline chloride-malic acid-propylene glycol, betaine-lactic acid-glycerol, betaine-citric acid-glycerol, L-proline-lactic acid-glycerol, L-carnitine-malic acid-propylene glycol, acetylcholine chloride-lactic acid-glycerol, choline chloride-lactic acid-glycerol-ascorbic acid, or betaine-malic acid-glycerol-ascorbic acid.

[0031] As one implementation scheme, in steps (2)-(5), when the target is an active sulfide enrichment, the garlic slurry is first allowed to stand at 20-40℃ for 5-90 min, and then extracted at 15-45℃ under light-proof, low-oxygen or inert gas protection conditions using a weakly acidic or acidic deep eutectic solvent at a liquid-to-solid ratio of 10-40 mL / g. The slurry is then enriched by partitioning with ethyl acetate, n-hexane, MCT oil or vegetable oil phase. The active sulfide includes one or more of allicin, ajoene, diallyl disulfide, diallyl trisulfide or vinyl dithiocyclohexene.

[0032] Preferably, the deep eutectic solvent is selected from a binary system of hydrogen bond acceptor-hydrogen bond donor with a mass ratio of 1:(1-2), or a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2), or a quaternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2):(0.05-0.2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, and L-carnitine, and the hydrogen bond donor is selected from one of lactic acid, propylene glycol, glycerol, and ascorbic acid;

[0033] More preferably, the deep eutectic solvent is selected from choline chloride-lactic acid, betaine-lactic acid-propylene glycol, choline chloride-lactic acid-glycerol-ascorbic acid, betaine-lactic acid-glycerol-ascorbic acid, or L-carnitine-lactic acid-propylene glycol.

[0034] As one implementation scheme, in steps (2)-(5), when the target is a sulfur-containing precursor enrichment, the garlic slurry is first subjected to short-term heat treatment at 60-95℃ for 0.5-10 min, low-temperature pulverization or antioxidant protection, and then extracted at 10-40℃ for 20-120 min using an amino acid-organic acid type, polyol type or low acidity quaternary protective deep eutectic solvent at a liquid-to-solid ratio of 10-40 mL / g, and enriched by microporous membrane filtration, cation exchange or HILIC column; the sulfur-containing precursor includes one or more of allicin, S-allyl cysteine, S-allyl mercaptocysteine ​​or γ-glutamyl cysteine ​​derivatives;

[0035] Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2), or a quaternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2):(0.05-0.2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, and L-proline, and the hydrogen bond donor is selected from one of malic acid, citric acid, propylene glycol, glycerol, and ascorbic acid;

[0036] More preferably, the eutectic solvent is selected from L-proline-malic acid-glycerol-ascorbic acid, betaine-malic acid-glycerol, or choline chloride-citric acid-propylene glycol.

[0037] As one implementation scheme, in steps (2)-(5), when the target is a polysaccharide / fructan enrichment, a sugar alcohol type, a polyol type, or a neutral weak acid deep eutectic solvent is used, with a water content of 40-70%, a liquid-to-solid ratio of 10-60 mL / g, a temperature of 45-85℃, and an extraction time of 60-240 min. The extract is then precipitated with 3-4 times the volume of ethanol, deproteinized with Sevag, dialyzed, and recovered by ultrafiltration fractionation at 1 kDa, 3 kDa, or 10 kDa. The polysaccharide / fructan enrichment includes one or more of polysaccharides, fructans, or oligofructoses.

[0038] Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, L-proline and glycine betaine, and the hydrogen bond donor is selected from one of sorbitol, glucose, fructose, glycerol and trehalose;

[0039] More preferably, the eutectic solvent is selected from betaine-glycerol-sorbitol, choline chloride-glycerol-glucose, L-proline-glycerol-fructose, or glycine-betaine-glycerol-trehalose.

[0040] As one implementation scheme, in steps (2)-(5), when the target is a steroidal saponin / sterol / triterpenoid enrichment, a low-moisture polyol-organic acid type, choline-alcohol type or quaternary weak hydrophobic deep eutectic solvent with a water content of 0-35% and a liquid-to-solid ratio of 10-50 mL / g is used for extraction at 35-70℃ for 60-180 min. The enriched product is then partitioned with n-butanol, ethyl acetate or a low-polarity solvent and enriched by macroporous resin, ODS or membrane separation. The enriched product includes one or more of steroidal saponins, sterols, triterpenes or low-polarity volatile sulfides.

[0041] Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine and L-carnitine, and the hydrogen bond donor is selected from one of levulinic acid, propylene glycol, lactic acid and glycerol;

[0042] More preferably, the deep eutectic solvent is selected from betaine-levulinic acid-propylene glycol, choline chloride-lactic acid-glycerol, L-carnitine-levulinic acid-propylene glycol, or choline chloride-propylene glycol-levulinic acid.

[0043] As one implementation scheme, when the target is to enrich small molecule amino acids / nucleosides / organic acids, a high-water-content organic acid-polyol type or amino acid type antioxidant deep eutectic solvent with a water content of 40-60% and a liquid-to-solid ratio of 10-40 mL / g is used for extraction at 10-45℃ for 30-120 min. The resulting extract is then ultrafiltered at 3 kDa and separated using a strong cation exchange, strong anion exchange, or HILIC column. The HILIC column is an amide type, zwitterionic type, or silica gel type hydrophilic interaction chromatographic column, and the mobile phase is acetonitrile-aqueous phase containing 5-20 mmol / L ammonium formate or ammonium acetate, with the acetonitrile volume fraction gradually decreasing from 80-90% to 50-65%, used to enrich one or more of free amino acids, γ-glutamyl peptides, nucleosides, nucleotides, or organic acids.

[0044] Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(0.2-2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, L-proline and glycine betaine, and the hydrogen bond donor is selected from one of malic acid, propylene glycol, ascorbic acid, lactic acid and glycerol;

[0045] More preferably, the deep eutectic solvent is selected from choline chloride-lactic acid-glycerol, betaine-malic acid-glycerol, L-proline-glycerol-ascorbic acid, choline chloride-malic acid-propylene glycol, glycine betaine-lactic acid-glycerol, or L-proline-malic acid-glycerol.

[0046] Secondly, the present invention provides a garlic active ingredient enrichment prepared by the above method.

[0047] As a specific implementation scheme, the garlic active ingredient enrichment is one or more of the following: polyphenol-flavonoid enrichment, active sulfide enrichment, sulfur-containing precursor enrichment, steroidal saponin / sterol / triterpenoid enrichment, polysaccharide / fructan enrichment, small molecule amino acid / nucleoside / organic acid enrichment, or selenium-containing organic matter enrichment.

[0048] As a further embodiment, the polyphenol-flavonoid concentrate contains 30.0-43.5 mg GAE / g fresh garlic and 3.9-5.85 mg RE / g fresh garlic; the active sulfide concentrate contains 1.5-2.4 mg / g fresh garlic; the sulfur-containing precursor concentrate contains 6.0-7.95 mg / g fresh garlic and 1.05-1.65 mg / g fresh garlic and S-allyl cysteine ​​and fructan and 67.5-84 mg / g fresh garlic and 43.5-57 mg / g fresh garlic and fructan ...

[0049] The enriched material may be a liquid concentrate, a resin-eluting concentrate, a freeze-dried powder, a spray-dried powder, a microcapsule powder, an edible film additive, a cosmetic raw material liquid, or a feed additive raw material.

[0050] Thirdly, the present invention provides the application of the garlic active ingredient concentrate in the preparation of antioxidant food raw materials, food preservation coatings, plant-derived antibacterial agents, functional food dietary fiber raw materials, cosmetic or daily chemical antioxidant raw materials.

[0051] As a specific implementation scheme, based on a total mass of 100 parts for the corresponding products, the antioxidant food raw material contains 0.05-0.30 parts of polyphenol-flavonoid enrichment, with the remainder being a food matrix; the food preservation coating contains 0.02-0.20 parts of active sulfide enrichment and 1.0-2.0 parts of chitosan, with the remainder being a film-forming matrix; the functional food dietary fiber raw material contains 0.10-1.00 parts of polysaccharide / fructan enrichment, with the remainder being a food or dietary fiber matrix.

[0052] The cosmetic or daily chemical antioxidant raw material contains 0.02-0.20 parts of polyphenol-flavonoid concentrate, with the remainder being a cosmetic or daily chemical matrix including glycerin, butylene glycol, sodium hyaluronate, carbomer or combinations thereof, and the resulting system has a pH of 5.0-6.5.

[0053] The method described in this invention can perform targeted control based on the differences in various functional components in fresh garlic: for active sulfides such as allicin, ajoene, diallyl disulfide, diallyl trisulfide, and vinyl dithiocyclohexene, the production rate is increased and degradation is reduced by moderate enzyme activation after crushing, low temperature and light protection, and low oxygen extraction; for alliin, S-allyl cysteine, S-allyl mercaptocysteine, and γ-glutamyl cysteine ​​derivatives, the retention rate or conversion rate is improved by short-term enzyme inactivation, antioxidant protection, or fermentation conversion; for polyphenols, flavonoids, saponins, polysaccharides, fructans, oligofructose, free amino acids, nucleosides, organic acids, sterols, triterpenes, and selenium-containing organic compounds, fractional enrichment is achieved by regulating the composition (mass ratio) of DES, water content, pH, viscosity, temperature, and separation path.

[0054] The present invention has the following innovations: (1) It establishes a continuous process of "enzyme activity regulation-fermentation conversion-DES targeted extraction-gradual enrichment-detection confirmation" for fresh garlic, taking into account the characteristics of garlic's heat sensitivity, enzymatic conversion and coexistence of multipolar components; (2) It constructs binary, ternary and quaternary deep eutectic solvent systems, and achieves selective release of multiple target components through hydrogen bond acceptors, hydrogen bond donors, water content, pH and viscosity adjustment; (3) It incorporates polyphenols, flavonoids, saponins, polysaccharides, sulfur-containing compounds, sulfur-containing precursors, small molecule amino acids, nucleosides, organic acids, sterols, triterpenes and selenium-containing organics into the same overall technical system; (4) It improves the purity of target enrichment and reduces DES background interference through resin, membrane, liquid-liquid partition, alcohol precipitation, ion exchange and HILIC separation; (5) It forms multiple enrichment product forms that can be used in the fields of food, daily chemical, cosmetic, preservation and feed.

[0055] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0056] (1) The target components are widely covered, and one or more garlic active ingredient enrichments can be obtained based on the same raw materials;

[0057] (2) The extraction conditions are mild, which can reduce the loss of easily degradable components such as allicin, S-allyl cysteine, allicin, polyphenols and flavonoids;

[0058] (3) The solvent system is highly designable, and the HBA / HBD combination, water content and extraction window can be adjusted according to the polarity, molecular weight and stability of the active ingredients;

[0059] (4) The separation route is clear, and the purity and detection accuracy can be improved through multi-stage enrichment;

[0060] (5) DES can be recycled through water dilution, resin adsorption, antisolvent precipitation, membrane concentration or vacuum concentration, reducing the amount of traditional organic solvents used.

[0061] (6) The enriched product can be prepared into food, cosmetics, daily chemical products, fresh-keeping products, plant-derived antibacterial products and feed additives according to its composition and safety level. Detailed Implementation

[0062] The technical solution of the present invention is illustrated below with detailed examples. However, it is worth noting that the scope of protection of the present invention is not limited to the specific embodiments described.

[0063] If specific techniques or conditions are not explicitly described in the examples, operations can be performed according to the techniques or conditions described in relevant literature in the field, or by following the relevant product instructions. Reagents or instruments, unless otherwise specified by manufacturer, are conventional products that can be purchased through legal channels.

[0064] Unless otherwise specified, all contents in the embodiments of this invention are based on the fresh weight of fresh garlic; the mass ratio is the mass ratio of the amounts of each eutectic solvent raw material added; the water content is the mass fraction of water in the eutectic solvent system; and the liquid-solid ratio is the ratio of the volume of the eutectic solvent or extraction solvent to the fresh weight of garlic. In the embodiments, "mg GAE / g fresh garlic" represents the content per gram of fresh garlic in gallic acid equivalents, "mg RE / g fresh garlic" represents the content per gram of fresh garlic in rutin equivalents, and "μmol TE / g enrichment" represents the antioxidant capacity per gram of enrichment in Trolox equivalents.

[0065] I. Preparation of Deep Eutectic Solvent

[0066] The hydrogen bond acceptor and hydrogen bond donor were mixed at a predetermined mass ratio and stirred at 40-100°C for 0.5-6 h until a clear, homogeneous liquid was formed. The water content was then adjusted to 0-70% according to the target composition. The DES / NADES system used in the examples consisted of the hydrogen bond acceptor, hydrogen bond donor, water, and antioxidant components explicitly listed in the table. Unless otherwise specified in the table, no additional solubilizing, pH adjusting, viscosity adjusting, or stabilizing components were added; the optional components mentioned above were only used to adjust the viscosity, pH, or stability of the system and were not essential for achieving the extraction and enrichment of the target composition. After preparation, the pH and viscosity of each system were measured at 25℃. The specific results were within the following ranges: polyphenol-flavonoid system: pH 2.5-4.5, viscosity 50-1500 mPa·s; active sulfide system: pH 2.0-4.5, viscosity 80-1800 mPa·s; sulfur-containing precursor system: pH 3.0-5.5, viscosity 50-1500 mPa·s; polysaccharide / fructan system: pH 4.0-7.0, viscosity 10-500 mPa·s; steroidal saponin / sterol / triterpenoid system: pH 2.5-5.5, viscosity 100-2500 mPa·s; small molecule amino acid / nucleoside / organic acid system: pH 3.0-6.5, viscosity 10-600 mPa·s. In this invention, the construction principle of the deep eutectic solvent is as follows: the basic ionic or zwitterionic environment is determined through hydrogen bond acceptors; acidity, polarity, viscosity, and solubility are adjusted through hydrogen bond donors; mass transfer efficiency and enzyme activity stability are adjusted through water content; and appropriate separation pathways are selected according to the target components. Binary DES is used to establish basic solubility; ternary DES is used to balance polarity, acidity, and viscosity; and quaternary DES is used to simultaneously consider solubility, stability, mass transfer, and selective enrichment. Unless otherwise specified, all extraction rates and contents are based on the fresh weight of fresh garlic.

[0067] For applications in food, health food, cosmetics, or daily chemical products, NADES composed of food-grade, pharmaceutical-grade, or cosmetically permitted raw materials such as choline chloride, betaine, L-proline, L-carnitine, glycerol, lactic acid, malic acid, citric acid, glucose, fructose, sorbitol, and ascorbic acid is preferred. Systems such as tetraalkylammonium salts or imidazoline salts can be used as process screening or industrial intermediate extraction systems; the final product should undergo thorough separation and safety confirmation before being used in its intended application.

[0068] II. Classification of active ingredients in fresh garlic and corresponding processing windows are shown in Table 1:

[0069] Table 1. Classification of active ingredients in fresh garlic and corresponding processing windows

[0070]

[0071] III. The methods for detecting active ingredients, blank subtraction, and quality control are as follows:

[0072] The DES system itself may affect spectrophotometry and the chromatographic baseline; therefore, DES blanks, reagent blanks, sample blanks, and spiked recoveries are set up for each batch of tests. Spectrophotometric samples need to be diluted 10-50 times according to their color and absorbance range, and the DES blank of the same dilution factor should be subtracted. Chromatographic methods require evaluation of standard curve linearity, limit of detection, limit of quantitation, precision, repeatability, and sample stability.

[0073] Table 2. Testing Methods and Quality Control

[0074]

[0075] IV. Preparation of Garlic Paste:

[0076] Take fresh white garlic, remove moldy, severely sprouted or mechanically damaged cloves, remove mud, root plate, outer skin and inedible impurities, wash and drain, then slice, crush, homogenize and pulverize at low temperature to obtain fresh garlic paste, and conduct the following experiments.

[0077] Example 1: Preparation, separation and detection of polyphenol-flavonoid concentrates

[0078] Take 100 g of fresh garlic slurry and extract it according to the DES system and process conditions in Table 3. After extraction, centrifuge at 10,000 rpm for 10 min, take the supernatant, dilute it with water 5-10 times, and pass it sequentially through a D101, AB-8, or polyamide resin column. Load the sample at a flow rate of 1-2 BV / h, wash with water for 2 BV to remove sugars, organic acids, and DES background, then elute with 30-70% ethanol for 2-4 BV. Concentrate the eluent under reduced pressure at 35-45℃ and freeze-dry to obtain the polyphenol-flavonoid concentrate.

[0079] Total polyphenols were detected using the Folin-Ciocalteu method, with gallic acid as the standard, at 760 nm. Total flavonoids were detected using the aluminum salt colorimetric method, with rutin as the standard, at 510 nm. Phenolic acids and flavonoid glycosides were detected using HPLC-DAD or UPLC-DAD with a C18 column, a mobile phase of 0.1% formic acid and acetonitrile, gradient elution, and detection wavelengths of 280, 320, and 360 nm. Antioxidant capacity was verified using DPPH, ABTS, and FRAP.

[0080] Table 3 Specific Examples of Polyphenol-Flavone Accumulators

[0081]

[0082]

[0083] Example 2: Preparation and detection of active sulfide concentrates and sulfur-containing precursor concentrates

[0084] For active sulfide concentrates, fresh garlic slurry is first allowed to stand at 20-40℃ for 5-90 min to allow alliinase to fully act and generate allicin and other sulfides. Then, weakly acidic DES is added at a liquid-to-solid ratio of 10-40 mL / g, and extraction is carried out at 15-45℃ under light-proof, low-oxygen, or nitrogen-protected conditions for 20-90 min. For sulfur-containing precursor concentrates, excessive enzymatic conversion is first inhibited by short-term heat treatment at 60-95℃ for 0.5-10 min or low-temperature pulverization. Then, low-acidity or antioxidant DES is added at a liquid-to-solid ratio of 10-40 mL / g, and extraction is carried out at 10-40℃ for 20-120 min.

[0085] Allicin was detected by HPLC-DAD at a wavelength of 240 nm; volatile or semi-volatile sulfides such as DADS, DATS, and ajoene were detected by GC-MS. Alliin, SAC, SAMC, and γ-glutamyl peptide were detected by HPLC, OPA / FMOC-derived HPLC, or HILIC-LC-MS. Samples should be kept at low temperature and protected from light from preparation to injection; detection of active sulfide samples is preferably completed within 2 hours.

[0086] Table 4 Specific Examples of Sulfur-Containing Compounds and Sulfur-Containing Precursors

[0087]

[0088]

[0089] Example 3 Preparation of polysaccharide / fructan enrichment and steroidal saponin / sterol / triterpenoid enrichment

[0090] For polysaccharide / fructan enrichment, high-water-content sugar alcohol or polyol DES is preferred, with a liquid-to-solid ratio of 10-60 mL / g, and extraction at 45-85℃ for 60-240 min. The extract is diluted with water and then precipitated with 3-4 times its volume of ethanol. The precipitate is then deproteinized using Sevag, dialyzed, and ultrafiltered to obtain the polysaccharide / fructan enrichment. For steroidal saponins / sterols / triterpenes enrichment, low-water-content polyol-organic acid or weakly hydrophobic quaternary DES is preferred, with a liquid-to-solid ratio of 10-50 mL / g, and extraction at 35-70℃ for 60-180 min. After extraction, enrichment is performed using n-butanol, ethyl acetate, macroporous resin, or ODS.

[0091] Polysaccharides were determined using the phenol-sulfuric acid method, and oligosaccharide distribution was determined by HPLC-RI or HPAEC-PAD; saponins were detected using the vanillin-perchloric acid colorimetric method, HPLC-ELSD, or LC-MS; sterols and triterpenes were detected using HPLC-ELSD, GC-MS, or LC-MS.

[0092] Table 5 Specific Examples of Polysaccharides / Fructans and Saponins / Sterols

[0093]

[0094] Example 4: Preparation, separation, and detection of small molecule amino acid / nucleoside / organic acid enrichments

[0095] Take 100 g of fresh garlic slurry and extract with high-water-content organic acid-polyol or amino acid-based antioxidant DES as listed in Table 6, at a liquid-to-solid ratio of 10-40 mL / g, stirring at 10-45℃ for 30-120 min. After extraction, centrifuge at 10000 rpm for 10 min to obtain a clear extract. Dilute the clear extract with water until the DES mass fraction is below 10%, and remove macromolecular polysaccharides and proteins by ultrafiltration at 3 kDa. Select a strong cation exchange column (SCX) to enrich amino acids and γ-glutamyl peptides, or a strong anion exchange column (SAX) to enrich organic acids and some nucleotides, depending on the target components. Wash the SCX column with water for 2-3 BV, then elute with a gradient of 0.05-0.50 mol / L ammonium acetate or ammonium formate; wash the SAX column with water for 2-3 BV, then elute with a gradient of 0.10-0.80 mol / L ammonium formate.

[0096] The HILIC column can be an amide, zwitterionic, or silica-based hydrophilic interaction column. Mobile phase A is an aqueous phase containing 5-20 mmol / L ammonium formate or ammonium acetate, and mobile phase B is acetonitrile. The gradient program is 0-3 min 85-90% B, 3-20 min decreasing to 55-65% B, 20-25 min holding, and 25-30 min returning to the initial ratio. Detection can be performed using DAD, ELSD, or LC-MS; amino acids can be detected after OPA derivatization or directly using HILIC-LC-MS.

[0097] Table 6 Specific Examples of Small Molecule Amino Acid / Nucleoside / Organic Acid Enrichments

[0098]

[0099] Example 5: Specific Implementation of Pre-fermentation Treatment Coupled with DES Extraction

[0100] In this embodiment, fermentation of edible microorganisms was carried out before DES extraction, and the fermentation temperature, initial pH, and fermentation time specified in Table 7 were used as fermentation control conditions. The fermentation time was taken as the fermentation endpoint. After fermentation, the fermented slurry was directly used for DES extraction. F1-F6 were respectively used as non-fermentation controls P1, P3, G3, G5, P8, and G4. Except for the addition of a pre-extraction fermentation step, the DES composition (mass ratio), liquid-solid ratio, extraction temperature, extraction time, and subsequent separation and detection conditions were the same as the corresponding controls to evaluate the effect of fermentation treatment on the release, transformation, or stability of the target components.

[0101] Table 7. Examples of DES extraction coupled with pre-fermentation treatment and control cases.

[0102]

[0103] Example 6: Integrated process of one-pot extraction and multi-stage separation

[0104] Take 1000 g of fresh garlic slurry, first let it stand at 30℃ for 20 min to activate alliinase, then add choline chloride-lactic acid-glycerol-ascorbic acid quaternary DES (mass ratio 1:2:1:0.1, water content 30%), mix at a liquid-solid ratio of 20 mL / g, and sonicate at 35℃ and 400 W for 30 min. After centrifugation, obtain the extract and residue.

[0105] The extract was processed in three streams: the first stream was fractionated twice with ethyl acetate at a 1:1 volume ratio, the organic phases were combined, and concentrated under low temperature and reduced pressure to obtain an active sulfide concentrate; the second stream involved diluting the aqueous phase and loading it onto D101 macroporous resin, washing with water for 2 BV, and then eluting with 50% ethanol for 3 BV to obtain a polyphenol-flavonoid-saponin concentrate; the third stream involved precipitating the resin flow-through with 4 times the volume of ethanol, and the precipitate was dialyzed and ultrafiltered to obtain a polysaccharide / fructan concentrate. The supernatant was ultrafiltered at 3 kDa and then enriched with SCX / HILIC to obtain small molecule amino acids / nucleosides / organic acids.

[0106] This one-pot extraction route can form more than four types of concentrates after one extraction step, making it suitable for industrial scale-up. Detection results showed that the polyphenol-flavonoid concentrate had a TPC of 38.22 mg GAE / g and a TFC of 4.74 mg RE / g; the active sulfide concentrate had allicin of 1.97 mg / g and DADS of 0.56 mg / g; the polysaccharide / fructan concentrate had a polysaccharide of 72.9 mg / g and a fructan of 48.6 mg / g; and the small molecule amino acid / nucleoside / organic acid concentrate had free amino acids of 25.2 mg / g and nucleosides of 1.83 mg / g.

[0107] Example 7: Application of the extract

[0108] This embodiment provides feasible application formulations and evaluation indicators. All formulations below are based on a total product mass of 100 parts. Components not listed are made up to 100 parts using a matrix, carrier, or water permitted for the corresponding application. The application examples below are only used to illustrate the possible applications of the enriched material of this invention and do not limit the specific dosage form, excipients, or usage ratio of the enriched material.

[0109] Table 8 Examples of Extract Applications

[0110]

[0111]

[0112] Example 8: DES Recycling Cycle and Scale-up Verification

[0113] The resin flow-through and washing liquid from the P1 system extraction were combined and concentrated under reduced pressure to remove some water. DES was then recovered via activated carbon decolorization or membrane concentration. Lactic acid, glycerol, and water were added to the recovered DES to restore the original mass ratio and water content, and the recovered DES was reused for the next batch of fresh garlic extraction. After five consecutive cycles, the TPC retention rates for the 1st, 3rd, and 5th cycles were 100%, 91.8%, and 84.6%, respectively, while the TFC retention rates were 100%, 90.9%, and 83.7%, respectively.

[0114] After extraction in the S3 system, the aqueous phase was partitioned with ethyl acetate to remove sulfides, and the residual DES was recovered by membrane concentration. After three cycles, the allicin extraction rate was 88.5%, and the DADS retention rate was 86.2%. Therefore, DES can be recovered and recycled through water dilution, resin adsorption, antisolvent precipitation, membrane concentration, or vacuum concentration to reduce solvent consumption.

[0115] For industrial scale-up, jacketed stirred tanks, low-temperature colloid mills, continuous centrifuges, ceramic or organic membrane systems, macroporous resin column arrays, and low-temperature vacuum concentration equipment can be used. For food or cosmetic applications, residual DES, ethanol, ethyl acetate, heavy metals, microbial limits, and allergen risks must be controlled according to target regulations.

[0116] Example 9: Grouping and Summarizing of Main Hydrogen Bond Acceptors

[0117] To illustrate the substitutability and scalability of the deep eutectic solvent system of this invention, the main hydrogen bond acceptors are grouped by category. Choline, betaine, amino acid, and quaternary ammonium salts can all form feasible systems with organic acids, polyols, sugar alcohols, antioxidant components, etc.; among them, choline, betaine, and amino acid salts are preferred for use in food, functional food, cosmetics, and daily chemical raw materials, while tetraalkylammonium salts and imidazodium salts are preferred as industrial intermediates or screening systems.

[0118] Table 9. Grouping of main hydrogen bond acceptors and their applicable directions

[0119]

[0120] Example 10: Representative Preferred Combination

[0121] Table 10 Representative Preferred Combinations

[0122]

[0123] Example 11 Comparative Example

[0124] To verify the necessity of deep eutectic solvent-coupled fermentation, water extraction, ethanol extraction, simple fermentation, deep eutectic solvent extraction without fermentation, and high-temperature strong acid treatment were set as comparative examples. The results showed that the coupling process of the present invention was superior to the corresponding controls in terms of the retention rate of polyphenols, flavonoids, sulfur-containing compounds, and small molecule conversion.

[0125] Table 11 Comparison of scale and technical effects

[0126]

[0127] As shown in the comparative examples, the water extraction system is mild but releases insufficient polyphenols and flavonoids. The ethanol system has a certain extraction capacity for moderately polar components but is not conducive to the simultaneous extraction of polysaccharides and small-molecule aqueous components. Although simple fermentation can promote the release of SAC or small molecules, it is difficult to fully enrich polyphenols and flavonoids. High-temperature and strong acid treatment can increase the release of some sugars but significantly reduces the retention rate of active sulfides. This invention, through the combination of fermentation, enzyme activity regulation, deep eutectic solvent, and fractionation, can achieve a more balanced technical effect across different target directions.

[0128] Example 13 Safety, Residue Control and Product Form

[0129] For food and health food ingredients, edible or existing natural deep eutectic solvent components are preferred, such as choline chloride, betaine, L-proline, glycerol, lactic acid, malic acid, citric acid, glucose, fructose, sorbitol, and ascorbic acid. DES residues in the product can be controlled through processes such as washing, resin adsorption, membrane separation, dialysis, ultrafiltration, vacuum concentration, or spray drying.

[0130] For cosmetic or daily chemical raw materials, in addition to controlling microbial limits, heavy metals, and solvent residues, pH, color difference, odor, centrifugal stability, thermal stability, and low-temperature stability should also be examined. Polyphenol-flavonoid concentrates exhibit good stability in pH 5.0-6.5 systems and are suitable for formulation with glycerol, butylene glycol, sodium hyaluronate, carbomer, xanthan gum, or other commonly used matrices.

[0131] For food preservation or plant-derived antibacterial applications, active sulfide concentrates can be formulated with chitosan, sodium alginate, gelatin, glycerin, or vegetable oils to form spray solutions, impregnation solutions, edible films, or microcapsule powders. For use as feed additives, low-temperature spray drying, vacuum drying, or freeze-drying are recommended to minimize the loss of allicin and volatile sulfur compounds.

[0132] Example 14: Product Form and Stability Study

[0133] The enriched products obtained in this invention can be prepared as liquid concentrates, resin-eluted concentrates, lyophilized powders, spray-dried powders, microcapsule powders, edible film additives, or cosmetic raw material solutions, depending on the application. In this embodiment, polyphenol-flavonoid enriched products, active sulfide enriched products, and polysaccharide / fructan enriched products were selected for stability studies.

[0134] Table 12 Product Form and Stability Assessment

[0135]

[0136] Stability results show that polyphenol-flavonoid concentrates are preferably in lyophilized powder or light-protected concentrated form, active sulfides are preferably enriched in the oil phase or stored at low temperature and protected from light, and polysaccharide / fructan concentrates are preferably in lyophilized powder or spray-dried powder form. The product form can be selected according to the downstream application scenario.

[0137] Example 15: Scale-up Verification

[0138] To verify the scale-up applicability of this invention, the polyphenol-flavonoid and polysaccharide / fructan directions were scaled up from 100 g of garlic slurry to 5 kg of garlic slurry. During the scale-up process, the composition of the eutectic solvent, liquid-solid ratio, water content, and separation path were kept constant, and the stirring rate or ultrasonic input method was adjusted according to the equipment load. The results showed that the target content remained above 90% of the results of the small-scale test after scale-up, indicating that this invention has a good foundation for process scale-up.

[0139] Table 13 Scale-up Verification Examples

[0140]

[0141] During scale-up validation, key control points should be placed on temperature uniformity, slurry dispersibility, extract viscosity, and solid-liquid separation efficiency. For high-viscosity systems, staged water addition, circulating stirring, jacketed temperature control, or online filtration can be used to avoid localized overheating and insufficient mass transfer.

[0142] Example 16: Boundary of Process Parameter Combinations

[0143] To further illustrate the process boundaries of this invention, this embodiment sets low-temperature, room-temperature, medium-temperature, and high-water-content conditions within the scope defined by the claims. For different target directions, the entire system is not fixed by a single liquid-solid ratio, a single temperature, or a single water content; instead, appropriate combinations are selected based on the polarity and stability of the target components. For the polyphenol-flavonoid direction, moderate water content and moderate sonication are emphasized; for the reactive sulfide direction, low-temperature light protection and low-oxygen protection are emphasized; for the sulfur-containing precursor direction, short-term enzyme inactivation and antioxidant protection are emphasized; and for the polysaccharide / fructan direction, high water content and longer mass transfer time are emphasized.

[0144] Table 14 Examples of Process Parameter Combinations

[0145]

[0146] As shown in Table 14, the target enrichment can be obtained within a relatively wide parameter range, but different optimal windows exist for different target directions. This setting provides flexibility for actual production, avoiding a decrease in the yield or insufficient stability of a certain type of component due to a fixed single parameter.

[0147] In summary, this invention establishes a deep eutectic solvent-coupled fermentation preparation system using fresh garlic as raw material. Through enzyme activity regulation, edible microbial fermentation, DES-directed extraction, multi-stage separation and enrichment, and detection and evaluation, it can obtain enriched compounds of polyphenols-flavonoids, active sulfides, sulfur-containing precursors, polysaccharides / fructans, steroidal saponins / sterols / triterpenes, small molecule amino acids / nucleosides / organic acids, and other active garlic components. This method features mild conditions, adjustable components, and a clearly defined separation pathway, enabling the high-value utilization of various active components in garlic.

[0148] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for preparing garlic active ingredient concentrates using deep eutectic solvent coupled fermentation, characterized in that, Includes the following steps: (1) Take garlic, pre-treat it, and prepare garlic slurry; (2) Based on the target components, the garlic slurry is optionally subjected to enzyme activity regulation and / or edible microbial fermentation treatment to obtain garlic-treated slurry; (3) Using hydrogen bond acceptors and hydrogen bond donors as the main raw materials, prepare binary, ternary or quaternary deep eutectic solvents; (4) Mix the garlic slurry obtained in step (1) or the garlic treatment slurry obtained in step (2) with the deep eutectic solvent obtained in step (3) to extract the mixture. (5) The extracted mixture is separated and purified to obtain the garlic active ingredient enrichment.

2. The method for preparing garlic active ingredient enrichment using deep eutectic solvent coupled fermentation according to claim 1, characterized in that, In step (1), the garlic is white-skinned garlic, purple-skinned garlic or single-clove garlic; the garlic is fresh garlic that has not been dried, fermented with black garlic or cooked at high temperature after harvesting, and the fresh garlic is stored at 4-15℃ for a short period of 0-30 days and warmed up to 10-25℃ before use; The pretreatment includes removing mud, root plates and inedible impurities, peeling, washing and draining; The method for preparing garlic paste includes slicing, crushing, homogenizing, colloid milling or low-temperature pulverization to obtain garlic paste. In step (2), the method for regulating enzyme activity includes: standing at 20-40℃ for 5-90 min to activate alliinase, or short-term treatment at 60-95℃ for 0.5-10 min to inactivate alliinase and polyphenol oxidation-related enzymes; The method for fermenting edible microorganisms includes: inoculating with one or more of lactic acid bacteria, yeast, or Bacillus, and fermenting at 20-37℃ and pH 3.0-6.5 for 6-72 h; Preferably, fermentation is carried out at 28-37℃ and pH 3.0-6.5 for 18-36 h; Preferably, the lactic acid bacteria are selected from one or more of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus acidophilus; the yeast is selected from Saccharomyces cerevisiae, Saccharomyces cerevisiae, or a combination thereof; the Bacillus is selected from Bacillus subtilis, Bacillus coagulans, or a combination thereof; the inoculation amount is 10^5-10^8 CFU / g garlic slurry; In step (3), the hydrogen bond acceptor is selected from one or a combination of several of the following: choline chloride, betaine, betaine hydrochloride, L-proline, L-carnitine, acetylcholine chloride, glycine betaine, choline bicarbonate, tetraalkylammonium salt, imidazolyl salt, amino acid salt, and organic acid salt. The hydrogen bond donor is selected from one or more of the following: lactic acid, malic acid, citric acid, tartaric acid, acetic acid, levulinic acid, glycerol, propylene glycol, 1,3-propanediol, ethylene glycol, polyethylene glycol, urea, acetamide, nicotinamide, glucose, fructose, sucrose, trehalose, xylitol, sorbitol, mannitol, erythritol, ascorbic acid, caffeic acid, ferulic acid, gallic acid, chlorogenic acid, and amino acids. The raw materials of the deep eutectic solvent may also optionally include viscosity-adjusting components, antioxidant components, pH-adjusting components and / or stabilizing components; The deep eutectic solvent is a clear and homogeneous liquid with a viscosity of 10-3000 mPa·s at 25°C and a pH of 2.0-8.

0. The method for preparing the deep eutectic solvent includes: mixing the raw materials, stirring at 40-100℃ for 0.5-6 h, and adjusting the water content to 0-70% by mass to obtain the solvent. In step (4), the extraction method includes: adding a deep eutectic solvent at a liquid-solid ratio of 5-100 mL / g, and performing extraction at 10-90℃, low-temperature stirring, ultrasonic assistance, microwave assistance, enzyme assistance or a combination thereof for 5-360 min; In step (5), the separation and purification methods include one or a combination of centrifugation, filtration, membrane separation, liquid-liquid partitioning, macroporous resin adsorption, polyamide resin adsorption, ion exchange, HILIC column separation, ethanol precipitation, dialysis, antisolvent precipitation, vacuum concentration or freeze drying.

3. The method for preparing garlic active ingredient concentrates using deep eutectic solvent coupled fermentation according to claim 1, characterized in that, In steps (2)-(5), when the target is polyphenol-flavonoid enrichment, the deep eutectic solvent is selected from organic acid-choline type, betaine-organic acid type, trihydric acid type or antioxidant quaternary DES, with a water content of 20-45%; the extraction method includes: liquid-solid ratio of 10-40 mL / g, temperature of 35-55℃, ultrasonic power of 200-600 W, extraction for 20-70 min; the separation and purification method includes: elution with D101, AB-8 or polyamide resin with 30-70% ethanol; Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2), or a quaternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2):(0.05-0.2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, L-proline, L-carnitine and acetylcholine chloride, and the hydrogen bond donor is selected from one of lactic acid, malic acid, propylene glycol, glycerol, citric acid and ascorbic acid; More preferably, the deep eutectic solvent is selected from choline chloride-lactic acid-glycerol, choline chloride-malic acid-propylene glycol, betaine-lactic acid-glycerol, betaine-citric acid-glycerol, L-proline-lactic acid-glycerol, L-carnitine-malic acid-propylene glycol, acetylcholine chloride-lactic acid-glycerol, choline chloride-lactic acid-glycerol-ascorbic acid, or betaine-malic acid-glycerol-ascorbic acid.

4. The method for preparing garlic active ingredient enrichment using deep eutectic solvent coupled fermentation according to claim 1, characterized in that, In steps (2)-(5), when the target is an active sulfide enrichment, the garlic slurry is first allowed to stand at 20-40℃ for 5-90 min, and then extracted at 15-45℃ under light-proof, low-oxygen or inert gas protection conditions using a weakly acidic or acidic deep eutectic solvent at a liquid-to-solid ratio of 10-40 mL / g. The slurry is then enriched by partitioning with ethyl acetate, n-hexane, MCT oil or vegetable oil phase. The active sulfide includes one or more of allicin, ajoene, diallyl disulfide, diallyl trisulfide or vinyl dithiocyclohexene. Preferably, the deep eutectic solvent is selected from a binary system of hydrogen bond acceptor-hydrogen bond donor with a mass ratio of 1:(1-2), or a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2), or a quaternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2):(0.05-0.2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, and L-carnitine, and the hydrogen bond donor is selected from one of lactic acid, propylene glycol, glycerol, and ascorbic acid; More preferably, the deep eutectic solvent is selected from choline chloride-lactic acid, betaine-lactic acid-propylene glycol, choline chloride-lactic acid-glycerol-ascorbic acid, betaine-lactic acid-glycerol-ascorbic acid, or L-carnitine-lactic acid-propylene glycol.

5. The method for preparing garlic active ingredient concentrates using deep eutectic solvent coupled fermentation according to claim 1, characterized in that, In steps (2)-(5), when the target is a sulfur-containing precursor enrichment, the garlic slurry is first subjected to short-term heat treatment at 60-95℃ for 0.5-10 min, low-temperature pulverization or antioxidant protection, and then extracted at 10-40℃ for 20-120 min using an amino acid-organic acid type, polyol type or low acidity quaternary protective deep eutectic solvent at a liquid-to-solid ratio of 10-40 mL / g, and enriched by microporous membrane filtration, cation exchange or HILIC column; the sulfur-containing precursor includes one or more of allicin, S-allyl cysteine, S-allyl mercaptocysteine ​​or γ-glutamyl cysteine ​​derivatives; Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2), or a quaternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2):(0.05-0.2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, and L-proline, and the hydrogen bond donor is selected from one of malic acid, citric acid, propylene glycol, glycerol, and ascorbic acid; More preferably, the eutectic solvent is selected from L-proline-malic acid-glycerol-ascorbic acid, betaine-malic acid-glycerol, or choline chloride-citric acid-propylene glycol.

6. The method for preparing garlic active ingredient concentrates using deep eutectic solvent coupled fermentation according to claim 1, characterized in that, In steps (2)-(5), when the target is a polysaccharide / fructan enrichment, a sugar alcohol, polyol, or neutral weak acid eutectic solvent is used, with a water content of 40-70%, a liquid-to-solid ratio of 10-60 mL / g, a temperature of 45-85℃, and an extraction time of 60-240 min. The extract is then precipitated with 3-4 times the volume of ethanol, deproteinized with Sevag, dialyzed, and recovered by ultrafiltration at 1 kDa, 3 kDa, or 10 kDa. The polysaccharide / fructan enrichment includes one or more of polysaccharides, fructans, or oligofructoses. Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, L-proline and glycine betaine, and the hydrogen bond donor is selected from one of sorbitol, glucose, fructose, glycerol and trehalose; More preferably, the eutectic solvent is selected from betaine-glycerol-sorbitol, choline chloride-glycerol-glucose, L-proline-glycerol-fructose, or glycine-betaine-glycerol-trehalose.

7. The method for preparing garlic active ingredient concentrates using deep eutectic solvent coupled fermentation according to claim 1, characterized in that, In steps (2)-(5), when the target is steroidal saponins / sterols / triterpenes, a low-moisture polyol-organic acid type, choline-alcohol type, or quaternary weakly hydrophobic deep eutectic solvent with a water content of 0-35% and a liquid-to-solid ratio of 10-50 mL / g is used. Extraction is carried out at 35-70℃ for 60-180 min, followed by partitioning with n-butanol, ethyl acetate, or a low-polarity solvent, and enrichment is achieved by macroporous resin, ODS, or membrane separation. The enriched substances include one or more of steroidal saponins, sterols, triterpenes, or low-polarity volatile sulfides. Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(1-2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine and L-carnitine, and the hydrogen bond donor is selected from one of levulinic acid, propylene glycol, lactic acid and glycerol; More preferably, the deep eutectic solvent is selected from betaine-levulinic acid-propylene glycol, choline chloride-lactic acid-glycerol, L-carnitine-levulinic acid-propylene glycol, or choline chloride-propylene glycol-levulinic acid.

8. The method for preparing garlic active ingredient enrichment using deep eutectic solvent coupled fermentation according to claim 1, characterized in that, In steps (2)-(5), when the target is a small molecule amino acid / nucleoside / organic acid enrichment, a high-water-content organic acid-polyol type or amino acid type antioxidant deep eutectic solvent with a water content of 40-60% and a liquid-to-solid ratio of 10-40 mL / g is used for extraction at 10-45℃ for 30-120 min. After ultrafiltration at 3 kDa, the resulting extract is separated by strong cation exchange, strong anion exchange or HILIC column. The HILIC column is an amide type, zwitterionic type or silica type hydrophilic interaction chromatographic column, and the mobile phase is acetonitrile-aqueous phase containing 5-20 mmol / L ammonium formate or ammonium acetate. The volume fraction of acetonitrile is gradiented from 80-90% to 50-65% to enrich one or more of free amino acids, γ-glutamyl peptides, nucleosides, nucleotides or organic acids. Preferably, the deep eutectic solvent is selected from a ternary system of hydrogen bond acceptor-hydrogen bond donor-hydrogen bond donor with a mass ratio of 1:(1-2):(0.2-2); wherein the hydrogen bond acceptor is selected from one of choline chloride, betaine, L-proline and glycine betaine, and the hydrogen bond donor is selected from one of malic acid, propylene glycol, ascorbic acid, lactic acid and glycerol; More preferably, the deep eutectic solvent is selected from choline chloride-lactic acid-glycerol, betaine-malic acid-glycerol, L-proline-glycerol-ascorbic acid, choline chloride-malic acid-propylene glycol, glycine betaine-lactic acid-glycerol, or L-proline-malic acid-glycerol.

9. A garlic active ingredient concentrate prepared by the method according to any one of claims 1-8.

10. The application of the garlic active ingredient concentrate according to claim 9 in the preparation of antioxidant food raw materials, food preservation coatings, plant-derived antibacterial agents, functional food dietary fiber raw materials, cosmetic or daily chemical antioxidant raw materials.

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