Pueraria ferment and preparation method and application thereof

Abstract

The application provides a pueraria ferment as well as a preparation method and application thereof. The preparation method of the plant ferment comprises the following steps: step 1, crushing dry pueraria root powder, mixing with water to obtain a pueraria root powder mixture; step 2, mixing the pueraria root powder mixture with a compound enzyme preparation to perform enzymolysis, to obtain an enzymolysis liquid; step 3, adding a carbon source into the enzymolysis liquid, mixing, and then performing a two-stage fermentation process; the first-stage fermentation is aerobic fermentation by using a bacterial agent containing yeast and acetic acid bacteria, and the second-stage fermentation is anaerobic fermentation by using a bacterial agent containing lactobacillus; and step 4, filtering the fermented mixture to obtain a clear solution, and the pueraria ferment is obtained. The obtained pueraria ferment has high contents of active ingredients such as puerarin and glucuronic acid, has good antioxidant capacity, has a health care effect, and has a liver protection effect.

Description

TECHNICAL FIELD

[0001] The present application relates to a kudzu root enzyme, a preparation method thereof and an application thereof. BACKGROUND

[0002] The main effective component in kudzu root is kudzu root isoflavones, mainly including puerarin, genistein, 3-hydroxy puerarin, daidzein, etc., which has the effects of anti-tumor, heat-relieving, intelligence-improving, and alcohol-resolving. The ancient book "Pharmacology" once recorded that kudzu root "stimulates appetite, and resolves alcohol toxicity". Puerarin, which accounts for the largest proportion in kudzu root isoflavones, is the only quantitative index component of kudzu root, and has the function of alcohol-resolving and liver-protecting. According to modern scientific research, puerarin can improve the activity of ethanol dehydrogenase and acetaldehyde dehydrogenase in the liver, strengthen the metabolic clearance capacity of the liver to alcohol, and enhance the activity of catalase, superoxide dismutase, and glutathione transferase to improve the free radical scavenging capacity, thereby having a certain relieving effect on inflammation caused by alcohol, reducing the activity of glutathione transaminase and glutamic transaminase, relieving the occurrence of alcoholic liver disease, and effectively reducing the expression of TGF-β to reduce liver damage caused by alcohol.

[0003] Plant enzyme refers to a liquid containing bioactive substances obtained by deep fermentation of plants. In theory, kudzu root enzyme can improve antioxidant activity and strengthen the alcohol-resolving and liver-protecting effects of kudzu root. In addition, kudzu root fermented by microorganisms can produce more phenolic substances, enzymes, organic acids, and other bioactive substances, further improve the free radical scavenging capacity of kudzu root, assist in relieving the occurrence of alcoholic liver disease, and improve the liver-protecting effect of kudzu root.

[0004] In the prior art, CN113826886A discloses a kudzu root enzyme obtained by using kudzu root as raw material, enzymolysis by amylase and glucoamylase, and then inoculating lactobacillus plantarum, and then fermenting at 30-40℃ for 24-48h. After fermentation, tagatose is added to adjust the sugar acid ratio, and then high-pressure sterilization is performed to obtain kudzu root enzyme. The process has the advantages of simplicity and short cycle, but the raw material selected is kudzu root, the content of puerarin in the final product is low, the microorganism inoculated is single, and the fermentation time is short, so that the content of lactic acid is low, the content of phenolic substances is not high, and the product has weak functionality.

[0005] CN112655950A discloses a comprehensive enzyme with radix puerariae, dried tangerine or orange peel and fruits and vegetables as raw materials. The invention mixes and crushes radix puerariae and dried tangerine or orange peel, heats and boils, and then mixes with fruit and vegetable to prepare a mixed solution. Garlic is distilled to prepare garlic hydrol and garlic essential oil. The mixed solution is inoculated with a strain and fermented for 6 months, filtered, added with garlic hydrol, and subjected to secondary fermentation for 2 months. After fermentation, garlic essential oil is added to prepare an emulsion, and subjected to third fermentation for 3 months. The fermented liquid is filtered, and the radix puerariae comprehensive enzyme is obtained by high-temperature sterilization and packaging. The method has many raw materials, and the radix puerariae is subjected to heating and boiling treatment, which causes loss of puerarin due to its thermal instability. The method has multiple fermentation stages, a long cycle, and a complex process, which is not conducive to industrial production. SUMMARY

[0006] The present application aims to provide a radix puerariae enzyme and a preparation method and application thereof, so as to solve the problems of the prior art, such as multiple raw materials, complex process, long cycle, poor process stability, uncontrollable reproducibility, and inability to shorten the cycle and simplify the process while ensuring the efficacy.

[0007] To solve the above-mentioned problems of the prior art, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect, the present application provides a radix puerariae enzyme, which is obtained by aerobic fermentation of an enzyme hydrolysate of radix puerariae using a mixed bacterial agent of Saccharomyces and Acetobacter, and then subjected to secondary anaerobic fermentation using a bacterial agent containing Lactobacillus.

[0009] Preferably, the pH of the radix puerariae enzyme is 3.7-4.0, the total acid content calculated as lactic acid is 1.6-2.1 wt%, and the puerarin content is 2.8-3.5 mg / g.

[0010] Preferably, in the radix puerariae enzyme, the glucuronide content is 0.15-0.2 mg / g, and / or the hydroxyl radical clearance rate is 80-88%, and / or the DPPH clearance rate is 84-93%, and / or the ABTS clearance rate is 64%-78%.

[0011] In a second aspect, the present application provides a preparation method of a radix puerariae enzyme, comprising the following steps:

[0012] Step 1: mixing dry radix puerariae powder with water to obtain a radix puerariae powder mixture;

[0013] Step 2: mixing the radix puerariae powder mixture with a complex enzyme preparation to carry out enzymolysis, to obtain an enzyme hydrolysate, wherein the complex enzyme preparation is a mixture of cellulase and amylase;

[0014] Step 3, the enzyme solution is mixed with a carbon source, and the mixture is subjected to a two-stage fermentation process. In the first stage, aerobic fermentation is performed using a bacterial agent containing yeast and acetic acid bacteria, and the inoculation amount of the bacterial agent is 1-2% of the mass of the mixture. In the second stage, anaerobic fermentation is performed using a bacterial agent containing lactobacillus, and the inoculation amount of the bacterial agent is 3-5% of the mass of the mixture.

[0015] Step 4, the fermented mixture is separated to obtain a clear solution, which is the gualou enzyme.

[0016] Preferably, in step 1, the dried gualou root powder is crushed into fine powder with a mesh size of 80-100, and the mass ratio of gualou root fine powder to water is 1:6-10, preferably 1:8-10.

[0017] Preferably, in step 2, the cellulase and amylase are mixed in a mass ratio of 1-5:1, preferably 1-2:1, and further preferably 2:1.

[0018] Alternatively, the amount of the complex enzyme preparation added is 1-2% of the mass of the gualou root powder mixture, preferably 1-1.5%, and further preferably 1.5%.

[0019] Preferably, in step 2, the temperature of the enzymatic hydrolysis is 50-60°C, preferably 50-55°C.

[0020] And / or, the pH of the enzymatic hydrolysis is 5.5-6.5, preferably 6.0-6.5.

[0021] And / or, the time of the enzymatic hydrolysis is 3-6h, preferably 5-6h.

[0022] Preferably, in step 3, the carbon source is sucrose, and the amount added is 8-10% of the mass of the enzyme solution.

[0023] Preferably, in step 3, the mass ratio of yeast and acetic acid bacteria in the first stage of fermentation is 1:1-2, preferably 1:1.5-1.6, the inoculation amount of the bacterial agent is 1.0-1.6% of the mass of the mixture, preferably 1.0-1.2%, the fermentation temperature is 28-30°C, preferably 30°C, and the fermentation time is 7-9d, preferably 7d.

[0024] And / or, in the second stage of fermentation, the inoculation amount of the bacterial agent is 3% of the mass of the mixture, the fermentation temperature is 35-37°C, preferably 37°C, and the fermentation time is 8-11d, preferably 11d.

[0025] Preferably, the yeast is Boidin's combined yeast, and the acetic acid bacteria is Gluconacetobacter.

[0026] and / or, the bacterial agent is Lactobacillus; preferably, the Lactobacillus is one or more of Lactobacillus plantarum, Lactobacillus acidophilus; further preferably, the Lactobacillus is a mixed bacterial agent of Lactobacillus plantarum and Lactobacillus acidophilus; more preferably, the Lactobacillus is a mixed bacterial agent of Lactobacillus plantarum and Lactobacillus acidophilus in a mass ratio of 1:1.

[0027] Preferably, in step 4, the mixed solution after fermentation is subjected to 200-300 mesh circulating filtration, preferably 200 mesh circulating filtration, to obtain a clear solution.

[0028] In a third aspect, the present application provides use of the above-mentioned Pueraria ferment in preparation of a liver-protecting product.

[0029] In a fourth aspect, the present application provides use of the above-mentioned Pueraria ferment in preparation of an antioxidant product.

[0030] The present application has the following beneficial effects:

[0031] In the present application, cellulase and amylase are used to enzymatically hydrolyze the Pueraria lobata to release as many active ingredients as possible, and produce some new small-molecule active substances and soluble fiber and other nutrients that are helpful for subsequent fermentation. Subsequent two-stage fermentation process is used to further promote the release and generation of active ingredients in Pueraria lobata, which helps to improve the multiple functions of the plant ferment, such as free radical scavenging, liver protection, and digestion promotion. DETAILED DESCRIPTION

[0032] In the specific embodiments of the present application, the source information of the instruments and reagents used is shown in Table 1.

[0033] Table 1

[0034]

[0035]

[0036] It should be further noted that, in the examples, the specific conditions not specified are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be purchased on the market.

[0037] The present application will be further described in detail below in conjunction with specific examples, which are an explanation of the present application rather than a limitation.

[0038] Example 1

[0039] The present example provides a preparation method of Pueraria ferment, comprising the following steps:

[0040] (1) Raw material screening and treatment: screening of dry root of kudzu vine without insect damage, mildew, tuber swelling, and less powder, washing, drying, cutting into small pieces, crushing, and passing through a 80-mesh screen to obtain kudzu vine root powder.

[0041] (2) Enzymolysis: 1 kg of kudzu vine root powder was taken, 6 times of water was added, and mixed well to obtain kudzu vine root residue suspension, and 1% of complex enzyme preparation (cellulase: amylase = 1:1) was added, the enzyme hydrolysis temperature was 50℃, the enzyme hydrolysis Ph was 5.5, and the enzyme hydrolysis time was 3h to obtain kudzu vine root enzyme hydrolysate.

[0042] (3) Inoculation: 8% of sucrose was added to the enzyme hydrolysate to obtain a mixed solution, which was sterilized and cooled to 28℃, and then 1:2 (mass ratio) of complex microbial agent (Bayer combined yeast: gluconic acid vinegar bacillus) was added for the first fermentation, the inoculation amount was 2% of the mixed solution, and aerobic fermentation was carried out for 9d; then the temperature was adjusted to 37℃ for the second fermentation, the inoculation amount of plant lactobacillus was 3% of the mixed solution, and anaerobic fermentation was carried out for 8d.

[0043] (4) Filtration and packaging: the solution obtained after fermentation was filtered through a 200-mesh circulating filter until the solution was clear, and then sterilized and packaged to obtain kudzu vine root enzyme.

[0044] Example 2

[0045] The embodiment provides a preparation method of kudzu vine root enzyme, which comprises the following steps:

[0046] (1) Raw material screening and treatment: screening of dry root of kudzu vine without insect damage, mildew, tuber swelling, and less powder, washing, drying, cutting into small pieces, crushing, and passing through a 80-mesh screen to obtain kudzu vine root powder.

[0047] (2) Enzymolysis: 1 kg of kudzu vine root powder was taken, 6 times of water was added, and mixed well to obtain kudzu vine root residue suspension, and 1% of complex enzyme preparation (cellulase: amylase = 1:1) was added, the enzyme hydrolysis temperature was 50℃, the enzyme hydrolysis Ph was 5.5, and the enzyme hydrolysis time was 3h to obtain kudzu vine root enzyme hydrolysate.

[0048] (3) Inoculation: 8% of sucrose was added to the enzyme hydrolysate to obtain a mixed solution, which was sterilized and cooled to 28℃, and then 1:2 (mass ratio) of complex microbial agent (Bayer combined yeast: gluconic acid vinegar bacillus) was added for the first fermentation, the inoculation amount was 2% of the mixed solution, and aerobic fermentation was carried out for 9d; then the temperature was adjusted to 37℃ for the second fermentation, the inoculation amount of plant lactobacillus was 3% of the mixed solution, and anaerobic fermentation was carried out for 8d.

[0049] (4) Filtration and packaging: the solution obtained after fermentation was filtered through a 200-mesh circulating filter until the solution was clear, and then sterilized and packaged to obtain kudzu vine root enzyme.

[0050] Example 3

[0051] The embodiment provides a preparation method of a kudzu vine enzyme, which comprises the following steps:

[0052] (1) raw material screening and treatment: screening of dry kudzu vine roots without insect damage, mildew, tuber swelling and less powder, washing, drying, cutting into small pieces, crushing, and passing through a 100-mesh screen to obtain kudzu vine root powder.

[0053] (2) enzyme hydrolysis: 1 kg of the kudzu vine root powder is taken, 10 times of water is added, and the mixture is uniformly mixed to obtain a kudzu vine root residue suspension, 1.5% of a composite enzyme preparation (cellulase: amylase = 2:1) is added, the enzyme hydrolysis temperature is 55°C, the enzyme hydrolysis Ph is 6.5, the enzyme hydrolysis is performed for 5 hours, and kudzu vine enzyme hydrolysis liquid is obtained.

[0054] (3) inoculation: 10% of sucrose of the enzyme hydrolysis liquid is added, and the mixture is uniformly mixed to obtain a mixed solution, the mixed solution is sterilized and cooled to 30°C, a composite inoculum (Bayer combined yeast: gluconic acid vinegar bacillus = 1:1.5, mass ratio) is added for first fermentation, the inoculation amount is 1% of the mixed solution, aerobic fermentation is performed for 7 days, then the temperature is adjusted to 37°C, and second fermentation is performed, the inoculum (plant lactobacillus: acidophilic lactobacillus = 1:1, mass ratio) is inoculated in an amount of 3% of the mixed solution, and anaerobic fermentation is performed for 11 days.

[0055] (4) filtration and packaging: the solution obtained after fermentation is circularly filtered through a 200-mesh screen until the solution is clear, then the solution is sterilized and packaged, and the kudzu vine enzyme is obtained.

[0056] Comparative Example 1

[0057] The comparative example provides a preparation method of a kudzu vine enzyme, which comprises the following steps:

[0058] (1) raw material screening and treatment: screening of dry kudzu vine roots without insect damage, mildew, tuber swelling and less powder, washing, drying, cutting into small pieces, crushing, and passing through a 100-mesh screen to obtain kudzu vine root powder.

[0059] (2) enzyme hydrolysis: 1 kg of the kudzu vine root powder is taken, 10 times of water is added, and the mixture is uniformly mixed to obtain a kudzu vine root residue suspension, 1.5% of a composite enzyme preparation (cellulase: amylase = 2:1) is added, the enzyme hydrolysis temperature is 55°C, the enzyme hydrolysis Ph is 6.5, the enzyme hydrolysis is performed for 5 hours, and kudzu vine enzyme hydrolysis liquid is obtained.

[0060] (3) fermentation: 8% of sucrose of the enzyme hydrolysis liquid is added, and the mixture is uniformly mixed to obtain a mixed solution, the mixed solution is sterilized and cooled to 30°C, a composite inoculum (Bayer combined yeast: gluconic acid vinegar bacillus = 1:1.5, mass ratio) is added for first fermentation, the inoculation amount is 1% of the mixed solution, aerobic fermentation is performed for 7 days, then the temperature is adjusted to 37°C, and second fermentation is performed, the inoculum (plant lactobacillus: acidophilic lactobacillus = 1:1, mass ratio) is inoculated in an amount of 3% of the mixed solution, and anaerobic fermentation is performed for 11 days.

[0061] (4) filtration and packaging: the solution obtained after fermentation is circularly filtered through a 200-mesh screen until the solution is clear, then the solution is sterilized and packaged, and the kudzu vine enzyme is obtained.

[0062] Comparative Example 2

[0063] The comparative example provides a preparation method of a kudzu vine enzyme, which comprises the following steps:

[0064] (1) Raw material screening and treatment: screening of dry root of Pueraria lobata Ohwi without insect damage, mildew, tuber swelling and less powder, washing, drying, cutting into small pieces, crushing, and passing through a 80 mesh screen to obtain Pueraria lobata Ohwi powder.

[0065] (2) Enzymolysis: taking 1 kg of Pueraria lobata Ohwi homogenate, adding 6 times of water to mix well to obtain Pueraria lobata Ohwi residue suspension, adding 1% of complex enzyme preparation (cellulase: amylase = 1:1) by mass of the suspension, and carrying out enzymolysis at 65℃ and Ph 4.5 for 6 hours to obtain Pueraria lobata Ohwi enzymolysis solution.

[0066] (3) Inoculation: adding 8% of sucrose by mass of the enzymolysis solution to mix well to obtain a mixed solution, sterilizing and cooling to 28℃, inoculating with complex microbial agent (Bayer combined yeast: Gluconacetobacter = 1:2) for the first fermentation, the inoculation amount being 2% of the mixed solution by mass, and carrying out aerobic fermentation for 9 days; adjusting the temperature to 37℃, and carrying out the second fermentation, the inoculation amount of Lactobacillus plantarum being 3% of the mixed solution by mass, and carrying out anaerobic fermentation for 8 days.

[0067] (4) Filtration and packaging: filtering the solution obtained after fermentation through a 200 mesh screen to make the solution clear, sterilizing, and packaging to obtain Pueraria lobata Ohwi ferment.

[0068] Comparative Example 3

[0069] The present comparative example provides a preparation method of Pueraria lobata Ohwi ferment, comprising the following steps:

[0070] (1) Raw material screening and treatment: screening of dry root of Pueraria lobata Ohwi without insect damage, mildew, tuber swelling and less powder, washing, drying, cutting into small pieces, crushing, and passing through a 100 mesh screen to obtain Pueraria lobata Ohwi powder.

[0071] (2) Enzymolysis: taking 1 kg of Pueraria lobata Ohwi powder, adding 10 times of water to mix well to obtain Pueraria lobata Ohwi residue suspension, adding 1% of complex enzyme preparation (cellulase: amylase = 2:1) by mass of the suspension, and carrying out enzymolysis at 55℃ and Ph 6.5 for 5 hours to obtain Pueraria lobata Ohwi enzymolysis solution.

[0072] (3) Inoculation: adding 10% of sucrose by mass of the enzymolysis solution to mix well to obtain a mixed solution, sterilizing and cooling to 30℃, inoculating with 1% of complex microbial agent (yellow wine yeast: Gluconacetobacter = 1:1.5) by mass of the mixed solution for the first fermentation, carrying out aerobic fermentation for 7 days, then adjusting the room temperature to 37℃, inoculating with 3% of microbial agent (Lactobacillus plantarum: Lactobacillus acidophilus = 1:1 by mass ratio) by mass of the mixed solution for the second fermentation, and carrying out anaerobic fermentation for 11 days to obtain Pueraria lobata Ohwi fermentation solution.

[0073] (4) Filtration and packaging: filtering the solution obtained after fermentation through a 200 mesh screen to make the solution clear, sterilizing, and packaging to obtain Pueraria lobata Ohwi ferment.

[0074] Comparative Example 4

[0075] The present comparative example provides a preparation method of a kudzu root enzyme, comprising the following steps:

[0076] (1) Raw material selection and treatment: select dry kudzu root without insect damage, mildew, tuber swelling, and less powder, wash, dry, cut into small pieces, crush, and pass through a 100-mesh sieve to obtain kudzu root powder.

[0077] (2) Enzymolysis: take 1 kg of kudzu root powder, add 10 times water to mix well to obtain kudzu root residue suspension, add 1.5% of complex enzyme preparation (cellulase: amylase = 2:1) by mass of the suspension, the enzyme hydrolysis temperature is 55°C, the enzyme hydrolysis Ph is 6.5, and the enzyme hydrolysis time is 5 h to obtain kudzu root enzyme hydrolysate.

[0078] (3) Inoculation: add 10% of sucrose by mass of the enzyme hydrolysate to mix well to obtain a mixed solution, sterilize, cool to room temperature (25°C), inoculate 4% of complex microbial agent (Bayer combined yeast: Gluconacetobacter: Lactobacillus plantarum = 1:1.5:7.5) by mass of the mixed solution, adjust the room temperature to 37°C, and anaerobic fermentation for 18 d to obtain kudzu root fermentation liquor.

[0079] (4) Filtration and packaging: the obtained solution after fermentation is filtered through a 200-mesh circulating filter until the solution is clear, then sterilized and packaged to obtain kudzu root enzyme.

[0080] Comparative Example 5

[0081] The present comparative example provides a preparation method of a kudzu root enzyme, comprising the following steps:

[0082] (1) Raw material selection and treatment: select dry kudzu root without insect damage, mildew, tuber swelling, and less powder, wash, dry, cut into small pieces, crush, and pass through a 100-mesh sieve to obtain kudzu root powder.

[0083] (2) Enzymolysis: take 1 kg of kudzu root powder, add 10 times water to mix well to obtain kudzu root residue suspension, add 1.5% of complex enzyme preparation (cellulase: amylase = 2:1) by mass of the suspension, the enzyme hydrolysis temperature is 55°C, the enzyme hydrolysis Ph is 6.5, and the enzyme hydrolysis time is 5 h to obtain kudzu root enzyme hydrolysate.

[0084] (3) Inoculation: add 10% of sucrose by mass of the enzyme hydrolysate to mix well to obtain a mixed solution, sterilize, cool to room temperature (25°C), inoculate 4% of complex microbial agent (Bayer combined yeast: Gluconacetobacter: Lactobacillus plantarum = 1:1.5:7.5) by mass of the mixed solution, adjust the room temperature to 37°C, and anaerobic fermentation for 18 d to obtain kudzu root fermentation liquor.

[0085] (4) Filtration and canning: the solution obtained after fermentation was filtered to be clear with 200 mesh, and then sterilized and canned to obtain the Pueraria lobata root enzyme.

[0086] Comparative Example 6

[0087] The present comparative example provides a preparation method of Pueraria lobata root enzyme, comprising the following steps:

[0088] (1) Raw material selection and treatment: Pueraria lobata root dry with no insect damage, mildew, long and slender root, and strong fibrous was selected, washed, dried, cut into small pieces, crushed, and passed through a 100 mesh screen to obtain Pueraria lobata root powder.

[0089] (2) Enzymolysis: 1 kg of Pueraria lobata root powder was taken, 10 times of water was added to mix well to obtain Pueraria lobata root residue suspension, and 1.5% of composite enzyme preparation (cellulase: amylase = 2:1) was added to the suspension, the enzymolysis temperature was 55°C, the enzymolysis Ph was 6.5, and the enzymolysis time was 5 h to obtain Pueraria lobata root enzymolysis solution.

[0090] (3) Inoculation: 10% of sucrose was added to the enzymolysis solution to mix well to obtain a mixed solution, sterilized and cooled to 30°C, and then composite inoculant (Bayer combined yeast: gluconic acid vinegar bacillus = 1:1.5, mass ratio) was added for the first fermentation, the inoculation amount was 1% of the mixed solution, and aerobic fermentation was performed for 7 d; then the temperature was adjusted to 37°C for the second fermentation, the inoculant (plant lactobacillus: acidophilic lactobacillus = 1:1, mass ratio) was inoculated in an amount of 3% of the mixed solution, and anaerobic fermentation was performed for 11 d.

[0091] (4) Filtration and canning: the solution obtained after fermentation was filtered to be clear with 200 mesh, and then sterilized and canned to obtain the Pueraria lobata root enzyme.

[0092] Comparative Example 7

[0093] The present comparative example provides a preparation method of Pueraria lobata root enzyme, comprising the following steps:

[0094] (1) Raw material selection and treatment: Pueraria lobata root dry with no insect damage, mildew, long and slender root, and strong fibrous was selected, washed, dried, cut into small pieces, crushed, and passed through a 100 mesh screen to obtain Pueraria lobata root powder.

[0095] (2) Enzymolysis: 1 kg of Pueraria lobata root powder was taken, 10 times of water was added to mix well to obtain Pueraria lobata root residue suspension, and 1.5% of composite enzyme preparation (cellulase: amylase = 2:1) was added to the suspension, the enzymolysis temperature was 55°C, the enzymolysis Ph was 6.5, and the enzymolysis time was 5 h to obtain Pueraria lobata root enzymolysis solution.

[0096] (3) Inoculation: the enzyme solution was sterilized and cooled to 30°C, and a compound microbial inoculum (Bayer combined yeast: Gluconacetobacter = 1:1.5, mass ratio) was added for the first fermentation, with an inoculation amount of 1% of the mass of the enzyme solution, aerobic fermentation for 7 days; then the temperature was adjusted to 37°C for the second fermentation, and the microbial inoculum (Lactobacillus plantarum: Lactobacillus acidophilus = 1:1, mass ratio) was inoculated at an amount of 3% of the mass of the enzyme solution, anaerobic fermentation for 11 days.

[0097] (4) Filtration and canning: the solution obtained after fermentation was filtered to clarify the solution, and then sterilized and canned to obtain the radix puerariae ferment.

[0098] Comparative Example 8

[0099] The present comparative example provides a preparation method of radix puerariae ferment, comprising the following steps:

[0100] (1) Raw material selection and treatment: dry radix puerariae roots without insect damage, mildew, tuber swelling, and little powder were selected, washed, dried, cut into small pieces, crushed, and passed through a 100-mesh sieve to obtain radix puerariae powder.

[0101] (2) Enzymolysis: 1 kg of the radix puerariae powder was taken, 10 times the amount of water was added, and mixed well to obtain a radix puerariae residue suspension, and a compound enzyme preparation (cellulase: amylase = 2:1) was added at an amount of 1.5% of the mass of the suspension, the enzyme hydrolysis temperature was 55°C, the enzyme hydrolysis Ph was 6.5, and the enzyme hydrolysis time was 5 h, to obtain a radix puerariae enzyme hydrolysate.

[0102] (3) Inoculation: 10% of the mass of the enzyme hydrolysate was added to the enzyme hydrolysate to obtain a mixed solution, which was sterilized and cooled to 30°C, and then a Bayer combined yeast was added for the first fermentation, with an inoculation amount of 1% of the mass of the mixed solution, aerobic fermentation for 7 days; then the temperature was adjusted to 37°C for the second fermentation, and the microbial inoculum (Lactobacillus plantarum: Lactobacillus acidophilus = 1:1, mass ratio) was inoculated at an amount of 3% of the mass of the mixed solution, anaerobic fermentation for 11 days.

[0103] (4) Filtration and canning: the solution obtained after fermentation was filtered to clarify the solution, and then sterilized and canned to obtain the radix puerariae ferment.

[0104] Comparative Example 9

[0105] The present comparative example provides a preparation method of radix puerariae ferment, comprising the following steps:

[0106] (1) Raw material selection and treatment: dry radix puerariae roots without insect damage, mildew, tuber swelling, and little powder were selected, washed, dried, cut into small pieces, crushed, and passed through a 100-mesh sieve to obtain radix puerariae powder.

[0107] (2) Enzymolysis: 1 kg of the Puerariae radix powder was mixed with 10 times of water to obtain a Puerariae radix residue suspension, and 1.5% of a compound enzyme preparation (cellulase: amylase = 2: 1) was added. The enzymeolysis temperature was 55°C, the enzymeolysis Ph was 6.5, and the enzymeolysis time was 5 h, thereby obtaining a Puerariae radix enzymeolysis solution.

[0108] (3) Inoculation: 10% of sucrose was added to the enzymeolysis solution to obtain a mixed solution, which was sterilized and cooled to 30°C. Then, a compound microbial agent (Bayer combined yeast: Gluconacetobacter = 1: 1.5, mass ratio) was added for the first fermentation, and the inoculation amount was 1% of the mixed solution. Aerobic fermentation was performed for 7 d. Then, the temperature was adjusted to 37°C for the second fermentation, and the microbial agent (Lactobacillus bulgaricus: Lactobacillus acidophilus = 1: 1, mass ratio) was inoculated at an amount of 3% of the mixed solution. Anaerobic fermentation was performed for 11 d.

[0109] (4) Filtration and packaging: the solution obtained after fermentation was filtered with a 200-mesh screen to clarify the solution, which was then sterilized and packaged, thereby obtaining the Puerariae radix enzyme.

[0110] Comparative Example 10

[0111] The present comparative example provides a preparation method of a Puerariae radix enzyme, which comprises the following steps:

[0112] (1) Raw material selection and treatment: Puerariae radix dry roots without insect damage, mildew, tuber swelling, and less powder were selected, washed, dried, cut into small pieces, and crushed to obtain Puerariae radix powder.

[0113] (2) Enzymolysis: 1 kg of the Puerariae radix powder was mixed with 10 times of water to obtain a Puerariae radix residue suspension, and 1.5% of a compound enzyme preparation (cellulase: amylase = 2: 1) was added. The enzymeolysis temperature was 55°C, the enzymeolysis Ph was 6.5, and the enzymeolysis time was 5 h, thereby obtaining a Puerariae radix enzymeolysis solution.

[0114] (3) Inoculation: 10% of sucrose was added to the enzymeolysis solution to obtain a mixed solution, which was sterilized and cooled to 25°C. Then, a compound microbial agent (Bayer combined yeast: Gluconacetobacter = 1: 1.5, mass ratio) was added for the first fermentation, and the inoculation amount was 1% of the mixed solution. Aerobic fermentation was performed for 7 d. Then, the temperature was adjusted to 37°C for the second fermentation, and the microbial agent (Lactobacillus bulgaricus: Lactobacillus acidophilus = 1: 1, mass ratio) was inoculated at an amount of 3% of the mixed solution. Anaerobic fermentation was performed for 11 d.

[0115] (4) Filtration and packaging: the solution obtained after fermentation was filtered with a 200-mesh screen to clarify the solution, which was then sterilized and packaged, thereby obtaining the Puerariae radix enzyme.

[0116] Comparative Example 11

[0117] The present comparative example provides a preparation method of a Puerariae radix enzyme, which comprises the following steps:

[0118] (1) Raw material screening and processing: Selecting dry root of kudzu vine without insect damage, tuber swelling, and little powder, washing, drying, cutting into small pieces, powdering, and passing through a 100-mesh sieve to obtain kudzu vine root powder.

[0119] (2) Enzymatic hydrolysis: Take 1 kg of kudzu vine root powder, add 10 times the amount of water to mix well to obtain a kudzu vine root residue suspension, add 1.5% of a composite enzyme preparation (cellulase: amylase = 2:1) by mass of the suspension, and enzymatically hydrolyze at 55°C and pH 6.5 for 5 hours to obtain kudzu vine root enzymatic hydrolysate.

[0120] (3) Inoculation: Add 10% sucrose by mass of the enzymatic hydrolysate to mix well to obtain a mixed solution, sterilize and cool to 30°C, inoculate with a composite inoculant (Bayer combined yeast: Gluconacetobacter = 1:1.5, mass ratio) for the first fermentation, the inoculation amount is 1% of the mixed solution, aerobic fermentation for 7 days; then adjust the temperature to 40°C for the second fermentation, inoculate with an inoculant (Lactobacillus plantarum: Lactobacillus acidophilus = 1:1, mass ratio) at an inoculation amount of 3% of the mixed solution, anaerobic fermentation for 11 days.

[0121] (4) Filtration and packaging: The solution obtained after fermentation is filtered with a 200-mesh screen to clarify the solution, then sterilized and packaged to obtain kudzu vine root ferment.

[0122] In addition, the present application also investigated the effect of three-stage fermentation process on kudzu vine root ferment at the initial stage of process exploration, but the three-stage process is too complex and does not meet the requirements of industrial application, and the fermentation result reduces the lactic acid content, which cannot meet the national relevant standards. After comprehensive consideration, the three-stage and above fermentation processes are completely not considered.

[0123] Related detection

[0124] The total acid, pH value, puerarin content, glucuronidic acid content, DPPH free radical scavenging capacity, hydroxyl radical scavenging capacity, ABTS free radical scavenging capacity, and sensory evaluation of kudzu vine root ferment obtained from kudzu vine root before fermentation and examples 1-3, comparative examples 1-11 were detected.

[0125] 1) The determination method of puerarin content is according to the RP-HPLC method in Chinese Pharmacopoeia, and the determination method of total acid content is according to the first method in national standard GB 12456-2021. The detection results are shown in Table 3.

[0126] 2) The determination method of glucuronidic acid is as follows:

[0127] 1. Preparation of standard curve of glucuronidic acid

[0128] A standard solution of glucuronic acid was prepared at a concentration of 2.00 mg / mL. 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mL of the standard solution was taken and placed in a 10 mL test tube with a stopper. 5 mL of sodium tetraborate / sulfuric acid solution was added to the test tube in an ice water bath, and the mixture was shaken well. After heating in the water bath for 5 min, the test tube was cooled to room temperature, and 100 μL of 1.5 mg / mL m-hydroxybenzene was added to the test tube and shaken well. A blank was prepared using 1 mL of distilled water. The absorbance was measured at 520 nm. The standard curve was prepared by plotting the mass concentration c (mg / mL) of the standard glucuronic acid solution on the horizontal axis and the absorbance on the vertical axis.

[0129] 2. Determination of the content of glucuronic acid in the sample

[0130] 0.1 mL of the fermentation broth of the Pueraria lobata roots of Examples 1-3 and Comparative Examples 1-11 was diluted 100-fold, and 1 mL of the diluted sample was taken and placed in a 10 mL test tube with a stopper. The test tube was treated according to the standard curve preparation method, and the absorbance of the Pueraria lobata root fermentation broth was measured at 520 nm. The content of glucuronic acid in the Pueraria lobata root fermentation broth was determined according to the standard curve of glucuronic acid.

[0131] 3) Sensory evaluation: 30 people were randomly selected, and the Pueraria lobata root enzyme obtained in Examples 1-3 and Comparative Examples 1-11 was evaluated according to the sensory evaluation standard shown in Table 2.

[0132] Table 2

[0133]

[0134]

[0135] The total acid content, pH value, puerarin content, glucuronic acid content, and sensory evaluation score of the Pueraria lobata root enzyme obtained in Examples 1-3 and Comparative Examples 1-11 are shown in Table 3.

[0136] Table 3

[0137]

[0138] The data in Table 3 show that the pH and total acid content of the Chai Gegen enzyme products prepared in Examples 1-3 of the present application are within the range of the industry standard (QB / T 5323-2018), and the content of puerarin and glucuronic acid in the Chai Gegen enzyme products prepared in Examples 1-3 of the present application is higher than that in the comparative examples, with the highest being 3.4 mg / g and 0.19 mg / g, respectively. Among them, Comparative Example 1 does not use natural fermentation of the inoculant, Comparative Examples 5 and 6 use Puerariae radix and Gemaom as raw materials, Comparative Example 7 does not add carbon source, Comparative Example 8 does not add Gluconacetobacter during aerobic fermentation, Comparative Example 9 replaces Lactobacillus plantarum with Lactobacillus bulgaricus, Comparative Example 10 changes the aerobic fermentation temperature, and Comparative Example 11 changes the anaerobic fermentation temperature. In combination with the above factors and the data in Table 3, it can be seen that the total acid content of Comparative Examples 1, 5-9 and 11 cannot meet the industry standard due to problems such as poor selection of raw materials, poor fermentation strains and conditions, and no addition of inoculants; the puerarin content in Comparative Examples 1, 2, 5 and 6 is low or zero due to problems such as no addition of inoculants, poor enzyme hydrolysis conditions, and poor selection of raw materials; and problems such as poor selection of raw materials, poor fermentation strains and conditions can lead to low pH value, low lactic acid content, and generation of irritating substances such as alcohol, resulting in low sensory evaluation scores of Comparative Examples 2-4 and 5-8.

[0139] 4) Application evaluation:

[0140] 1) DPPH free radical scavenging capacity determination

[0141] The Chai Gegen enzyme samples prepared in Examples 1-3 and Comparative Examples 1-4 were diluted with deionized water to 1 mg / mL to obtain a diluent, 1 mL of the diluent was taken, 1 mL of 0.1 mmol / L DPPH-anhydrous ethanol solution was added to each, mixed well, and reacted at room temperature under dark conditions for 30 min to obtain a sample solution. The absorbance value at a wavelength of 517 nm (denoted as A1) was measured, the absorbance value of the blank group was determined by using anhydrous ethanol instead of the sample solution (denoted as A0), and the absorbance value of the control group was determined by using anhydrous ethanol instead of the DPPH-anhydrous ethanol solution (denoted as A2). The DPPH free radical scavenging rate was calculated according to the following formula, and the detection results are shown in Table 4.

[0142] DPPH free radical scavenging rate (%) = [1-(A1-A2) / A0] x 100.

[0143] 2) Hydroxyl radical scavenging capacity determination

[0144] The samples of the Shai Gegen ferment prepared in Examples 1-3 and Comparative Examples 1-4 were diluted with deionized water to 1 mg / mL to obtain a diluted solution, 1 mL of the diluted solution was taken, 1 mL of a 6 mmol / L FeSO4 solution and 1 mL of a 2.4 mmol / L H2O2 solution were added thereto respectively, and mixed, after 10 min of reaction, 1 mL of a 6 mmol / L salicylic acid solution was added, and the mixture was reacted at 37°C for 30 min to obtain a sample solution, and the absorbance (A1) of the sample solution was measured at a wavelength of 510 nm. The absorbance (A0) of a blank group was measured by using distilled water instead of the sample solution, and the absorbance (A2) of a control group was measured by using distilled water instead of the H2O2 solution after reaction. The hydroxyl radical scavenging rate was calculated according to the following formula, and the test results are shown in Table 4.

[0145] The hydroxyl radical scavenging rate (%) = [1 - (A1 - A2) / A0] x 100.

[0146] 3. ABTS radical scavenging capacity determination

[0147] The samples of the Shai Gegen ferment prepared in Examples 1-3 and Comparative Examples 1-11 were diluted with deionized water to 1 mg / mL to obtain a diluted solution, 1 mL of a 7.4 mmol / L ABTS solution and 1 mL of a 2.6 mmol / L K2S2O4 solution were mixed, and the mixture was placed in the dark for 16 h to obtain an ABTS+ stock solution; before use, the ABTS+ stock solution was appropriately diluted with anhydrous ethanol solution, so that the absorbance of the mixture at 734 nm was in the range of 0.7±0.02, to obtain an ABTS+ working solution. 1.980 mL of the ABTS+ working solution was mixed with 20 μL of the sample diluted solution, and the mixture was reacted at 30°C for 10 min, and the absorbance A1 was measured at 734 nm; the absorbance A2 of a control group was measured by mixing distilled water and ethanol; the absorbance A0 of a blank group was measured by using distilled water instead of the sample diluted solution.

[0148] The ABTS radical scavenging rate (%) = [1 - (A1 - A2) / A0] x 100.

[0149] Table 4

[0150]

[0151] The data in Table 4 prove that the three antioxidant capacity indicators of the examples of the present application are all better than those of the comparative examples, and the antioxidant capacity of Example 3 is the strongest, which confirms that the Shai Gegen ferment prepared in the present application has stronger antioxidant capacity.

[0152] In addition, CN10591717A also discloses a comprehensive enzyme of kudzu root with more than 50 kinds of fruits and vegetables as raw materials. The invention method adopts a filling method of one layer of fresh kudzu root homogenate and one layer of brown sugar, and carries out natural fermentation. The fermentation time is 120-160 days. Meanwhile, one layer of fruits and vegetables and one layer of brown sugar are filled, and mixed bacteria are introduced for one-time fermentation. The fermentation time is 120-160 days. After fermentation, coarse filtration is carried out, and kudzu root residues are extracted by ethanol reflux to obtain kudzu root fermentation extract. The kudzu root fermentation extract is mixed with the fermentation liquor for two-time fermentation, and the fermentation time is 60-90 days. The kudzu root fruit and vegetable fermentation stock solution is obtained, and then concentrated and packaged to obtain kudzu root enzyme product. The invention method partially adopts natural fermentation, the process cycle is long, and it is easy to cause contamination of miscellaneous bacteria. In addition, the raw material types are many, organic reagents are used for reflux extraction for food production, the process is not green and environmentally friendly, and it is not conducive to industrialized production.

[0153] The cellulase and amylase complex enzyme preparation is used for enzymolysis of the kudzu root, so as to release the active ingredients in the kudzu root as much as possible and produce some new active substances and soluble fiber and other nutrients which are helpful to subsequent fermentation. Then, two-stage fermentation process is used to further promote the release of active ingredients and the generation of new active ingredients in the kudzu root. The gluconic acid vinegar bacillus can produce glucuronic acid, which can be combined with the carboxyl, amino and hydroxyl groups of toxic substances or other active substances in the liver, so as to reduce the toxicity and make it easier to be discharged from the body. In the process of mixed culture of the gluconic acid vinegar bacillus and the yeast, the yield of glucuronic acid can be further increased, and the liver protection effect of the kudzu root enzyme can be further improved. In addition, a variety of organic acids can be produced to improve the taste and promote the digestion and absorption of the human body. In the kudzu root enzyme, the content of puerarin is 2.8-3.5 mg / g, the content of glucuronic acid is 0.15-0.2 mg / g, the hydroxyl radical clearance rate is 80-88%, the DPPH clearance rate is 84-93%, and the ABTS clearance rate is 64%-78%. In the whole process, no organic reagent is used, the raw materials are simple and easy to obtain, the process is controllable, stable, reproducible, and can fully play the effect of the kudzu root enzyme on the basis of shortening the cycle as much as possible.

[0154] The above embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which belong to the protection scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. A Kudzu enzyme, characterized by, It is obtained by using the mixed bacterial agent of yeast and acetic acid bacteria to carry out aerobic fermentation on the enzyme solution of kudzu root, and then using the bacterial agent containing lactobacillus to carry out secondary anaerobic fermentation, and is obtained by the method comprising the following steps: Step 1, mix the dry powder of kudzu root with water to obtain a kudzu root powder mixture; Step 2, mix the kudzu root powder mixture obtained in step 1 with a complex enzyme preparation to carry out enzymolysis, to obtain an enzyme solution, wherein the complex enzyme preparation is a mixture of cellulase and amylase, the temperature of the enzyme solution is 50-55℃, the pH of the enzyme solution is 6.0-6.5, and the time of the enzyme solution is 5-6h, wherein the enzyme activity of the cellulase is 10000U / mL, and the enzyme activity of the amylase is 40000U / mL; Step 3, add a carbon source to the enzyme solution, and then carry out a two-stage fermentation process, the first stage is aerobic fermentation using a bacterial agent containing yeast and acetic acid bacteria, the inoculation amount of the bacterial agent in the first stage is 1-2% of the mass of the mixture, the second stage is anaerobic fermentation using a bacterial agent containing lactobacillus, the inoculation amount of the bacterial agent in the second stage is 3-5% of the mass of the mixture, wherein the fermentation temperature in the first stage is 28-30℃, and the fermentation time in the first stage is 7-8d, wherein the fermentation temperature in the second stage is 35-37℃, and the fermentation time in the second stage is 9-11d; Wherein, the yeast is boidinia, the acetic acid bacteria is gluconacetobacter, and the bacterial agent containing lactobacillus is a combination of lactobacillus plantarum and lactobacillus acidophilus, Step 4, separate the fermented mixture to obtain a clear solution, i.e. kudzu root enzyme, Wherein, the kudzu root enzyme contains lactic acid, puerarin and glucuronic acid; the total acid content calculated based on lactic acid is 1.6-2.1wt%, the puerarin content is 2.8-3.5mg / g, and the glucuronic acid content is 0.15-0.2mg / g, Wherein, in the kudzu root enzyme, the hydroxyl radical scavenging rate is 80-88%, and / or the DPPH scavenging rate is 84-93%, and / or the ABTS scavenging rate is 64-78%.

2. The Fenugreek enzyme as claimed in claim 1, wherein, The pH of the kudzu root enzyme is 3.7-4.

0.

3. The method of preparing the fenugreek ferment according to claim 1 or 2, characterized by, It comprises the following steps: Step 1, mix the dry powder of kudzu root with water to obtain a kudzu root powder mixture; Step 2, mix the kudzu root powder mixture obtained in step 1 with a complex enzyme preparation to carry out enzymolysis, to obtain an enzyme solution, wherein the complex enzyme preparation is a mixture of cellulase and amylase, the temperature of the enzyme solution is 50-55℃, the pH of the enzyme solution is 6.0-6.5, and the time of the enzyme solution is 5-6h, wherein the enzyme activity of the cellulase is 10000U / mL, and the enzyme activity of the amylase is 40000U / mL; Step 3, the enzyme solution is mixed with a carbon source, and the mixed solution is subjected to a two-stage fermentation process, the first stage of fermentation is aerobic fermentation using a bacterial agent containing yeast and acetic acid bacteria, the inoculation amount of the bacterial agent in the first stage is 1-2% of the mass of the mixed solution; the second stage of fermentation is anaerobic fermentation using a bacterial agent containing lactobacillus, the inoculation amount of the bacterial agent in the second stage is 3-5% of the mass of the mixed solution, wherein, in the first stage of fermentation, the fermentation temperature is 28-30℃, and the fermentation time is 7-8d; wherein, in the second stage of fermentation, the fermentation temperature is 35-37℃, and the fermentation time is 9-11d; The yeast is a bayer combined yeast, the acetic acid bacteria is gluconic acid acetobacter, and the bacterial agent containing lactobacillus is a combination of lactobacillus plantarum and lactobacillus acidophilus. Step 4, the fermented mixed solution is separated to obtain a clear solution, and the pueraria ferment is obtained. The pueraria ferment contains lactic acid, puerarin and glucuronic acid; the total acid content calculated based on lactic acid is 1.6-2.1wt%, the puerarin content is 2.8-3.5mg / g, and the glucuronic acid content is 0.15-0.2mg / g. The hydroxyl radical scavenging rate of the pueraria ferment is 80-88%, and / or the DPPH scavenging rate is 84-93%, and / or the ABTS scavenging rate is 64-78%.

4. The production method according to claim 3, characterized by, In step 1, the dried and powdered pueraria lobata is finely ground into a fine powder with a mesh size of 80-100, and the mass ratio of the fine pueraria lobata powder to water is 1:6-10.

5. The preparation method according to claim 4, characterized in that, The mass ratio of the fine pueraria lobata powder to water is 1:8-10.

6. The preparation method according to claim 3, characterized in that, In step 2, the cellulase and amylase are mixed in a mass ratio of 1-5:1, and the amount of the complex enzyme preparation added is 1-2% of the mass of the pueraria lobata powder mixture.

7. The production method according to claim 6, characterized by, In step 2, the cellulase and amylase are mixed in a mass ratio of 1-2:

1.

8. The production method according to claim 7, characterized by, In step 2, the cellulase and amylase are mixed in a mass ratio of 2:

1.

9. The preparation method according to claim 6, characterized in that, In step 2, the amount of the complex enzyme preparation added is 1-1.5% of the mass of the pueraria lobata powder mixture.

10. The method of claim 9, wherein, In step 2, the amount of the complex enzyme preparation added is 1.5% of the mass of the pueraria lobata powder mixture.

11. The preparation method according to claim 3, characterized in that, In step 3, the carbon source is sucrose, and the amount added is 8-10% of the mass of the enzyme solution.

12. The method of claim 3, wherein, In step 3, the mass ratio of yeast to acetic acid bacteria in the first stage of fermentation is 1:1-2, the inoculation amount of the bacterial agent is 1.0-1.6% of the mass of the mixed solution, and the inoculation amount of the bacterial agent in the second stage of fermentation is 3% of the mass of the mixed solution.

13. The method of claim 12, wherein, In step 3, the mass ratio of yeast to acetic acid bacteria in the first stage of fermentation is 1:1.5-1.

6.

14. The preparation method of claim 12, wherein the inoculation amount of the bacterial agent in the first stage of fermentation is 1.0-1.2% of the mass of the mixed solution.

15. The preparation method of claim 12, wherein the fermentation temperature in the first stage of fermentation is 30℃.

16. The preparation method of claim 12, wherein the fermentation time in the first stage of fermentation is 7d.

17. The method of claim 12, wherein, In step 3, the fermentation temperature in the second stage of fermentation is 37℃.

18. The method of claim 12, wherein, In step 3, the fermentation time in the second stage of fermentation is 11d.

19. The method of claim 3, wherein, The mass ratio of lactobacillus plantarum to lactobacillus acidophilus is 1:

1.

20. The method of claim 3, wherein, In step 4, the fermented mixed solution is subjected to circular filtration through a 200-300 mesh filter.

21. The method of claim 20, wherein, Circulation filtered through 200 mesh until a clear solution was obtained.

22. Use of the fenugreek enzyme according to claim 1 or 2 for the preparation of a liver-protecting product.

23. Use of the fenugreek enzyme according to claim 1 or 2 for the preparation of an antioxidant product.

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