CN108185140A - Pueraria lobata composite fermentation object and its preparation method and application - Google Patents

Abstract

The invention discloses a kudzu root compound fermented product, a preparation method and application thereof. The culture medium containing the kudzu root compound enzymatic hydrolyzate is used for three fermentations under different conditions, and then the fermented product is subjected to solid-liquid separation to obtain a fermentation stock solution, which is the kudzu root compound fermented product. The preparation method of the invention has a short fermentation time, thereby being more favorable for industrialized production. In addition, the fermentation method of the present invention can also improve the active ingredients in the obtained fermented product, and significantly improve the drug efficacy, especially the antidiarrheal effect.

Description

Compound fermented product of kudzu root and its preparation method and application

technical field

The invention relates to a kudzu root compound fermented product and its preparation method and application, in particular to a kudzu root compound fermented product with anti-diarrhea effect, its preparation method and application.

Background technique

Puerarin contains isoflavone components puerarin, puerarin xyloside, daidzein, daidzein, β-sitosterol, arachidic acid, and a large amount of starch. Radix Puerariae is used to treat superficial syndromes of fever, strong pain in the nape and back, opaque measles, fever and thirst, yin deficiency and thirst, heat diarrhea and dysentery, and diarrhea due to spleen deficiency.

In order to further develop and utilize traditional Chinese medicine resources, research on traditional Chinese medicine fermentation technology has been carried out in recent years. For example, CN104606487A discloses a fermented Chinese medicine preparation, wherein the raw materials of the Chinese medicine are Alisma, Trichosanthes, Coptidis, Codonopsis, Pueraria, Rhizoma Polygonatum, Astragalus, Cortex Phellodendron, Bitter Gourd, Lycium barbarum, Schisandra, Ganoderma lucidum, Poria, Earthworm, Chinese yam, Atractylodes atractylodes, Silkworm, Polyporus, Atractylodes Rhizoma, Anemarrhena. The fermented Chinese medicine preparation provided in this document has good effects in treating and curing diabetes.

For another example, CN106306512 A discloses a fermented Chinese herbal medicine feed additive for the treatment of swine diarrhea. Prepare Polygonum multiflorum, Shengdi, Ten Great Merits, Viola, Agrimony, Cuscuta, Scrophulariae, Xu Changqing, Poria, Bupleurum, Clove and Alisma. The additive has strong animal disease resistance, promotes animal growth, improves survival rate and reduces diarrhea rate.

Although the above-mentioned literatures have all carried out fermentation research on compound recipes containing Pueraria lobata, these technologies either have complex raw material components or insufficient antidiarrheal efficacy.

Therefore, how to further develop and utilize Puerariae resources and improve the utilization rate of Puerariae is a subject worth studying.

Contents of the invention

The object of the present invention is to provide a preparation method of kudzu root compound fermented product or kudzu root compound fermented dry powder, which can use only three kinds of traditional Chinese medicine raw materials for fermentation, the fermentation time is short, and the medicinal effect of the obtained fermented product is greatly improved. Another object of the present invention is to provide a compound fermented product of kudzu root, which has improved antidiarrheal effect. Another object of the present invention is the use of the above-mentioned kudzu root compound fermentation product. The purpose of the present invention is achieved through the following technical solutions.

The first aspect of the present invention provides a preparation method of kudzu root compound fermented product, which comprises the following steps:

The first fermentation step: using yeast to carry out anaerobic fermentation in the medium at 26-32°C for 10-20 hours to obtain the first fermented product, wherein the medium contains kudzu root compound enzymatic hydrolyzate;

The second fermentation step: raising the temperature of the first fermented product to 33-40°C, and using lactic acid bacteria to carry out static fermentation at this temperature for 30-40 hours to obtain the second fermented product;

The third fermentation step: raising the temperature of the second fermented product to 50-70° C., and standing and fermenting at this temperature for 25-35 hours to obtain the third fermented product;

Solid-liquid separation step: the obtained third fermented product is subjected to solid-liquid separation to obtain a fermentation stock solution, which is a kudzu root compound fermented product.

According to the preparation method of the present invention, preferably, the kudzu root compound enzymatic hydrolyzate is obtained by using pectinase, cellulase, xylanase and trypsin at a pH value of 6.0-7.2 and a temperature of 45-60°C. It is obtained by enzymatically hydrolyzing the aqueous suspension containing kudzu root, astragalus and poria cocos for 2 to 6 hours.

According to the preparation method of the present invention, preferably, the dosage of the pectinase is 0.05-0.3wt% of the total weight of the aqueous suspension, and the dosage of the cellulase is 0.05-0.3wt% of the total weight of the aqueous suspension %, the dosage of xylanase is 0.05-0.3wt% of the total weight of the aqueous suspension, and the dosage of the trypsin is 0.05-0.15wt% of the total weight of the aqueous suspension.

According to the preparation method of the present invention, preferably, the weight ratio of pectinase, cellulase, xylanase and trypsin is 1:1:1:1.

According to the preparation method of the present invention, preferably, in the aqueous suspension containing Pueraria Radix, Radix Astragali and Poria Cocos, 30-55 parts by weight of Pueraria Radix, 30-40 parts by weight of Radix Astragali, and 10-20 parts by weight of Poria Cocos , and the content of kudzu root is greater than that of astragalus.

According to the preparation method of the present invention, preferably, the medium further comprises 0.1-0.5wt% yeast extract, 1-5wt% ammonium salt, 0.01-0.2wt% magnesium salt, 0.3-0.8wt% phosphate and 0.01 -0.2 wt% zinc salt; the above weight percentages are based on the total weight of the culture medium.

According to the preparation method of the present invention, preferably, the inoculum amount of the yeast in the first fermentation step is 1×10 ⁷ cfu to 9×10 ⁷ cfu per ml of the medium, and the yeast in the second fermentation step The inoculum amount of lactic acid bacteria is 1×10 ⁶ cfu to 9×10 ⁶ cfu per ml of the culture medium.

The second aspect of the present invention provides a method for preparing kudzu root compound fermented dry powder, which comprises adopting the preparation method described in the first aspect of the present invention to obtain a kudzu root compound fermented product, and then concentrating and drying the kudzu root compound fermented product to obtain Fermented dry powder.

The third aspect of the present invention provides a kudzu root compound fermented product, which is the kudzu root compound fermented product obtained according to the preparation method described in the first aspect of the present invention or the kudzu root compound fermented dry powder obtained by the preparation method described in the second aspect of the present invention.

The fourth aspect of the present invention provides the use of the kudzu root compound fermentation product of the present invention in the preparation of medicine, food or feed with antidiarrheal effect.

The invention reduces the fermentation time by adopting the method of enzymatic hydrolysis combined with three times of intensified fermentation, thereby being more conducive to industrial production. In addition, the enhanced fermentation of the present invention can also improve the active ingredients in the obtained fermented product, and significantly improve the drug efficacy, especially the antidiarrheal effect.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto.

The "pueraria compound fermented product" described in the present invention refers to a fermented product prepared by biological fermentation using a compound Chinese medicine containing pueraria root, and is sometimes referred to herein as "pueraria root compound enzyme", or simply referred to as "composite fermented product" or "composite fermented product". Enzyme".

Aiming at the problem of low drug efficacy when using Pueraria root to treat diarrhea, the present invention proposes a plan based on enzymatic hydrolysis of Pueraria root compound traditional Chinese medicine components, combined with three times of fermentation for intensive treatment, and completes the present invention based on this plan. Specifically, the present invention includes the following contents.

The first aspect of the present invention provides a method for preparing a kudzu root compound fermentation product, which includes three fermentation steps and a solid-liquid separation step. Each step is described in detail below.

<First fermentation step>

The first fermentation step is the anaerobic fermentation step of yeast, including using yeast to carry out anaerobic fermentation in the medium at 26-32°C for 10-20 hours to obtain the first fermented product, wherein the medium contains kudzu root compound enzymatic hydrolysis things.

Using yeast to ferment kudzu root compound enzymatic hydrolyzate under anaerobic conditions can produce effective metabolites that can promote the proliferation of beneficial intestinal bacteria. The inoculation amount of yeast in the first fermentation is 1×10 ⁷ cfu～9×10 ⁷ cfu per ml of the culture medium, preferably 1.5×10 ⁷ cfu～7×10 ⁷ cfu, more preferably 3×10 ⁷ cfu～6 ×10 ⁷ cfu. As the yeast, any strain known in the art can be used, and it is not particularly specified. Highly active dry yeast produced by Angel Yeast Co., Ltd. is preferred, and this strain is particularly beneficial for the biological transformation of the active ingredients of kudzu root.

The fermentation temperature of the first fermentation is 26-32°C, preferably 27-31°C, more preferably 28°C. This temperature range is beneficial to the fermentation of yeast and the biological transformation of the active ingredients of kudzu root. If the first fermentation temperature is too high, it will affect the activity of yeast.

The fermentation time of the first fermentation is 10-20 h, preferably 11-18 h, more preferably 16 h. This fermentation time can make the effect of the yeast fully exerted, and the content of high active ingredients in the obtained fermented product is high.

The culture medium in the first fermentation step needs to contain the complex hydrolyzate of kudzu root. The kudzu root compound enzymatic hydrolyzate is an enzyme-treated product after enzyme treatment releases components that are beneficial to fermentation or decomposes components that are not conducive to fermentation. Preferably, the kudzu root compound enzymatic hydrolyzate is a mixture obtained by treating the kudzu root compound medicinal material with a combination of pectinase, cellulase, xylanase and trypsin. The four enzymes can act simultaneously and efficiently under the conditions described in this application to promote enzymolysis, and there is no adverse effect among the enzyme activities.

The kudzu root compound medicinal material includes kudzu root and other traditional Chinese medicines, and the other traditional Chinese medicines are preferably astragalus and/or poria cocos. Pueraria root compound medicinal material is the combination of Pueraria root, Radix Astragali and Poria cocos. According to one embodiment of the present invention, the Pueraria root compound medicinal material only consists of Pueraria root, Radix Astragali and Poria cocos.

In the present invention, Pueraria lobata (Willd.) ohwi contains isoflavone components puerarin, puerarin xyloside, daidzein, daidzein, β-sitosterol, arachidic acid, and a large amount of starch. In traditional Chinese medicine, it is used for superficial fever, stiff neck and back pain, opaque measles, fever and thirst, yin deficiency and thirst, heat diarrhea and dysentery, and diarrhea due to spleen deficiency. The present invention strengthens spleen-deficiency diarrhea by using kudzu root as the monarch drug and rational compatibility.

In the present invention, Astragalus membranaceus (Fisch.) Bunge.) contains saponins, sucrose, polysaccharides, various amino acids, folic acid, selenium, zinc, copper and other trace elements. Traditional Chinese medicine believes that the effects of Astragalus are beneficial to Qi consolidation, diuresis, detumescence, detoxification, and muscle growth. Astragalus in the present invention can be selected arbitrarily and is not particularly limited. Honey-roasted Radix Astragalus is preferred, because it can complement Pueraria radix more effectively and improve the medicinal effect.

In the present invention, Poria cocos (Poria cocos (Schw.) Wolf) contains pachyan, B-pachyan, pachynic acid, lecithin, sterol, and the like. Traditional Chinese medicine believes that it has the effects of detoxifying and anti-cancer, diuresis and drinking, invigorating the spleen and reducing phlegm, calming the mind and calming the nerves.

Among the three kinds of medicinal materials, Astragalus is used as the minister, and Puerariae root is used to assist the monarch, and Poria cocos is used as the minister. The monarch, minister and assistant envoy are reasonably compatible. Preferably, Pueraria root is 30-55 parts by weight, preferably 35-50 parts by weight; Astragalus is 30-40 parts by weight, preferably 32-48 parts by weight; Poria cocos is 10-20 parts by weight, preferably 12-18 parts by weight. In the present invention, the content of kudzu root is preferably greater than that of astragalus. The combination of the three medicinal materials can act synergistically during fermentation, and the antidiarrheal effect is better.

The conditions of the enzyme treatment (sometimes referred to herein as "enzyme hydrolysis") of the present invention are not particularly limited, for example, the enzyme containing the compound drug can be enzymatically hydrolyzed at a pH value of 6.0 to 7.2 and a temperature of 45 to 60°C. Water suspension 2 ~ 6h. Preferably, the pH value of the enzyme treatment is 6.5-6.9, more preferably 6.8, and this pH value range can ensure that various enzymes of the present invention maintain high activity at the same time. Preferably, the enzyme treatment temperature is 50-60°C, more preferably 50°C. The enzyme in the present invention needs to be resistant to higher temperature, because too low enzymatic hydrolysis temperature is not conducive to the release of antidiarrheal components. The enzyme treatment time is preferably 3 to 5 hours, for example, 4.5 hours.

The amounts of pectinase, cellulase, xylanase and trypsin used in the enzyme treatment may be 0.05-0.3 wt%, based on the total weight of the suspension. Preferably respectively 0.06～0.2wt%, more preferably respectively 0.08～0.15wt%, also preferably respectively respectively 0.1～0.12wt%, the consumption of trypsin can be 0.03～0.2wt%, preferably 0.05～0.15wt%, more Preferably it is 0.1 to 0.15 wt%. The combination of four enzymes provides raw materials such as amino acids, monosaccharides, oligosaccharides and other beneficial ingredients for fermentation culture. The weight ratio of enzymes is not particularly specific, for example, the weight ratio of pectinase, cellulase, xylanase and trypsin may be 1:1:1:1.

Each medicinal material in the kudzu root compound medicinal material used for the enzyme treatment may be the whole plant or the root, which is not particularly limited. Preferably, the kudzu root, astragalus root, and poria cocos used for the enzyme treatment are all powders, and the fineness of the powders is not particularly limited, and finer powders are preferred, so as to facilitate the enzymatic hydrolysis. Preferably, the above three powders are mixed with water to prepare an aqueous suspension, which is used for enzyme treatment. The weight ratio of the total weight of the three powders to water is not particularly limited, preferably 1:5-15, for example, 1:10. As water, pure water, deionized water and distilled water can be used. The pH of the water is preferably 6.0-6.5, and water within this pH range is more conducive to enzymatic hydrolysis.

The medium in the first fermentation step may also contain nutrients. Preferably, the nutrient may be selected from at least one component selected from yeast extract, ammonium salt, zinc salt, magnesium salt and phosphate. Based on the total weight of the medium, the medium may contain 0.1-0.5wt% yeast extract, 1-5wt% ammonium salt, 0.01-0.2wt% magnesium salt, 0.3-0.8wt% phosphate and 0.01-0.2wt% zinc salt . Commercially available yeast paste can be used. The dosage of yeast extract is preferably 0.2-0.3 wt%. The ammonium salt can be selected from at least one of ammonium sulfate, ammonium nitrate or ammonium carbonate, preferably ammonium sulfate. The amount of ammonium salt used is preferably 1.5-3.5 wt%, more preferably 1.5-2.5 wt%. The magnesium salt may be selected from at least one of magnesium sulfate, magnesium chloride, magnesium nitrate, and magnesium carbonate, preferably magnesium sulfate. The dosage of the magnesium salt is preferably 0.03-0.15 wt%, more preferably 0.05-0.1 wt%. Phosphate may be selected from at least one of sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium phosphate, and magnesium phosphate, preferably potassium dihydrogen phosphate. The amount of phosphate used is preferably 0.35-0.65 wt%, more preferably 0.35-0.5 wt%. The zinc salt can be selected from at least one of zinc sulfate, zinc chloride, and zinc nitrate, and is preferably zinc chloride. The nutrient may include 0.03-0.2wt%, preferably 0.05-0.15wt%, more preferably 0.08-0.1wt% of zinc salt. Nutrients and ratios within the above range are more conducive to meeting the nutrient element requirements for the fermentation and cultivation of yeast and lactic acid bacteria, and can reduce the fermentation time.

The nutritional substances of the present invention may also contain potassium salts. The potassium salt and the phosphate are preferably the same substance, such as potassium phosphate, dipotassium hydrogenphosphate or potassium dihydrogenphosphate, preferably potassium dihydrogenphosphate.

<Second Fermentation Step>

The second fermentation step is a step of adding lactic acid bacteria for intensified fermentation, including raising the temperature of the first fermented product to 33-40° C., and using lactic acid bacteria for static fermentation at this temperature for 30-40 hours to obtain the second fermented product. The second fermented product is an enhanced fermented product or an enhanced enzyme. It not only has the general function of ordinary enzymes, but also has stronger potency especially against diarrhea.

The lactic acid bacteria of the present invention can be selected from one or more of Lactobacillus bulgaricus and Streptococcus thermophilus, preferably a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus. The weight ratio of Lactobacillus bulgaricus and Streptococcus thermophilus can be 1:10-10:1, preferably 1:5-5:1, more preferably 1:1. The inoculation amount of lactic acid bacteria is 1×10 ⁶ cfu to 9×10 ⁶ cfu per ml of the culture medium, preferably 2×10 ⁶ cfu to 8×10 ⁶ cfu, more preferably 5×10 ⁶ cfu to 8×10 ⁶ cfu. The inoculum of lactic acid bacteria needs to be greater than the inoculum of saccharomyces during the first fermentation. If the inoculum amount of lactic acid bacteria is too low, the amount of yeast in the first fermentation will be relatively too much, which will affect the proliferation of lactic acid bacteria and further affect the strengthening effect of the fermented product.

The temperature of the second fermentation step needs to be higher than the temperature of the first fermentation step, preferably in the range of 33-40°C, more preferably 35-39°C, even more preferably 37°C. Such a temperature is close to the ambient temperature of the human body, simulating the action temperature of lactic acid bacteria in the human body, so that the active ingredients transformed or released from traditional Chinese medicine are more conducive to the absorption of the human body.

The time for the second fermentation is not particularly limited, and is generally 30-40 hours, preferably 35-39 hours, more preferably 32 hours. If the fermentation time is too long, the rate of biotransformation will decrease, and other by-products can be generated at the same time, which are not conducive to the efficacy of the drug. On the other hand, if the fermentation time is too short, the reaction will not be sufficient, and some active ingredients of the medicine will not be produced, so that the medicine effect of the obtained fermented product will be reduced, and resources will be wasted.

The second fermentation needs static fermentation. Static fermentation is more similar to the environment of the probiotic flora in the body, so it is preferred.

<The third fermentation step>

The third fermentation step is a high-temperature fermentation step of yeast and lactic acid bacteria, which includes raising the temperature of the second fermented product to 50-70° C., and standing and fermenting at this temperature for 25-35 hours to obtain the third fermented product.

By making yeast and lactic acid bacteria continue to ferment simultaneously at 50-70° C., the content of active ingredients can be more effectively promoted to be rapidly produced in a short time. The range of the second fermentation temperature is preferably 55-65°C, more preferably 65°C. At this temperature, the third fermented product with desired properties can be obtained only by static fermentation for 20-35 hours, preferably 21-32 hours, more preferably 26-30 hours, which greatly shortens the fermentation time in the traditional method.

<Solid-liquid separation step>

Solid-liquid separation is used to remove the solid residue of the product produced during fermentation and recover the original fermentation liquid. Any known method can be used for solid-liquid separation, including but not limited to centrifugation, filtration, precipitation and the like. Preferably, the solid-liquid separation can be performed by centrifugation, for example, by using a centrifuge. The specific conditions of the centrifugation are not particularly limited, and can be appropriately adjusted according to needs. The fermentation stock solution obtained by separation is the kudzu root compound fermentation product.

The second aspect of the present invention provides a method for preparing a kudzu root compound fermented dry powder. The above preparation method is used to obtain a kudzu root compound fermented product, and then the kudzu root compound fermented product is concentrated and dried to obtain a fermented dry powder. Drying and/or concentration steps can be used to remove the effects of small molecular components in the medium on drug efficacy. Preferably, a concentration step is included before the drying step. Baking powder is obtained by concentration and drying. Concentration and drying can be carried out by reducing pressure, which will not be repeated here.

The third aspect of the present invention provides a kudzu root compound fermentation product, which is the kudzu root compound fermentation product or kudzu root compound fermentation dry powder prepared by the preparation method described in the present invention. According to one embodiment of the present invention, the water content of the fermented dry powder is lower than 5wt%, preferably lower than 4.5wt%, the activity of superoxide dismutase SOD is greater than or equal to 420±5U/g, and the number of viable lactic acid bacteria is greater than or equal to 3.3 ±0.5×10 ⁸ CFU/g, and the puerarin content is greater than 5wt%.

The fourth aspect of the present invention provides the use of kudzu root compound fermentation products in the preparation of medicines, food or feed additives with antidiarrheal effect. Wherein, the addition amount of the kudzu root compound fermented product in medicine, food or feed additive is not particularly limited.

Examples of drugs include, but are not limited to, solid dosage forms such as powders and pills, and liquid dosage forms such as suspensions, mixtures, and emulsifiers. Examples of food include but are not limited to candy, beverages (such as various non-alcoholic beverages), bread, biscuits, wine, and various health products. Examples of feed include, but are not limited to, feed additives. Feed additives refer to small or trace substances added during feed production, processing and use, which are used in small amounts but have significant effects in feed.

The preparation method of embodiment 1-pueraria root compound fermentation product

1) Pulverization: Pueraria Radix, Radix Astragali and Poria Cocos are pulverized respectively No. 2 sieve of Pharmacopoeia, raw materials are mixed by the ratio of 50 parts by weight of Radix Puerariae, 35 parts by weight of Radix Astragali and 15 parts by weight of Poria Cocos, then add 10 times the pure water of raw material mass ratio, A suspension is prepared.

2) Preparation of enzymatic solution: based on the total weight of the suspension, add 0.1wt% pectinase (activity unit 500,000 U/g), 0.1wt% cellulase (activity unit 500,000 U/g), 0.1 wt% xylanase (activity unit 500,000 U/g) and trypsin were used in an amount of 0.1 wt% (activity unit 500,000 U/g) for enzymolysis. The pH is 6.8, the temperature is 52°C, and the enzymatic hydrolysis time is 4.5h.

3) Appropriate supplementation of nutrients: 0.3wt% of yeast extract, 1.5wt% of ammonium sulfate, 0.05wt% of zinc chloride, 0.05wt% of magnesium sulfate, 0.4wt% of potassium dihydrogen phosphate, and sterilization of the fermentation substrate.

4) Inoculation and fermentation: inoculate yeast 3×10 ⁷ cfu/mL, anaerobically ferment at 28°C for 16 hours, inoculate lactic acid bacteria starter at 5×10 ⁶ cfu/mL, and then ferment at 37°C for 32 hours , and finally maintain the temperature at 65° C., let stand for 30 hours, and carry out fermentation.

5) Centrifugation: After the fermentation is finished, centrifugation is carried out to separate the fermented product stock solution and solid residue. The stock solution of the fermented product is the kudzu root compound fermented product.

Embodiment 2- kudzu root compound fermentation powder

1) Pulverization: Pueraria Radix, Radix Astragali and Poria Cocos are pulverized respectively No. 2 sieve of Pharmacopoeia, raw materials are mixed by the ratio of 50 parts by weight of Radix Puerariae, 35 parts by weight of Radix Astragali and 15 parts by weight of Poria Cocos, then add 10 times the pure water of raw material mass ratio, A suspension is prepared.

2) Preparation of enzymatic solution: add 0.1wt% pectinase (activity unit 500,000 U/g), 0.1wt% cellulase (activity unit 500,000 U/g), 0.1wt% xylan Enzyme (activity unit 500,000 U/g) and 0.15 wt% trypsin (activity unit 500,000 U/g) were used for enzymolysis. The pH is 6.8, the temperature is 50°C, and the enzymatic hydrolysis time is 4.5h.

3) Appropriate supplementation of nutrients: 0.3wt% of yeast extract, 1.5wt% of ammonium sulfate, 0.05wt% of zinc chloride, 0.05wt% of magnesium sulfate, 0.4wt% of potassium dihydrogen phosphate, and sterilization of the fermentation substrate.

4) Inoculation and fermentation: Inoculate yeast 6×10 ⁷ cfu/mL, anaerobic fermentation at 28°C for 16 hours, inoculate lactic acid bacteria starter 8×10 ⁶ cfu/mL, and then stand for fermentation at 37°C for 32 hours , and finally maintain the temperature at 65° C., let stand for 30 hours, and carry out fermentation.

5) Centrifugation: After the fermentation is finished, centrifugation is carried out to separate and obtain the fermented product stock solution and solid residue;

6) Concentration: Concentrate the separated fermented material stock solution under reduced pressure, and dry under reduced pressure to obtain fermented powder, wherein the water content is 4 wt%.

Embodiment 3- Pueraria root compound fermented beverage

Mix the kudzu root compound fermented product prepared in Example 1 with water at a weight ratio of 1:6, add 15wt% sucrose, 0.5%wt citric acid, and perform homogenization-degassing-pasteurization-filling-cooling, etc. step, the finished beverage can be obtained.

Embodiment 4- Pueraria root composite fermented product wine

The kudzu root compound fermentation product that embodiment 1 makes is mixed with 45 degree high-quality liquor, drinking water with the weight ratio of 1:8:4, and carry out the steps such as homogeneous-degassing-pasteurization-filling-cooling, can The kudzu root compound fermented product blended wine was obtained.

The change of puerarin content before and after embodiment 5-fermentation

Preparation of reference substance solution: Accurately weigh an appropriate amount of puerarin reference substance, add 30% ethanol to make a solution containing 80 μg per 1 ml, and obtain it.

Preparation of the test solution:

Fermented product sample: take by weighing 0.35g of fermented powder of embodiment 2 (equivalent to 2.0g of kudzu root, 1.4g of astragalus and 0.6g of Poria cocos), accurately weighed, put in a stoppered Erlenmeyer flask, accurately add 50ml of 30% ethanol, weigh Determine the weight, heat to reflux for 30 minutes, let cool, then weigh again, make up the lost weight with 30% ethanol, shake well, filter, take 1mL of the filtrate in a 10mL volumetric flask, and dilute to the mark with 30% ethanol Line, that is.

Water extract sample: Take 2.0g of kudzu root, 1.4g of astragalus root and 0.6g of poria cocos, soak in water for 30min, decoct and extract twice, add 10 times the amount of water each time, extract for 2 hours each time, filter, combine the filtrates, and concentrate to Dry powder, put in a stoppered Erlenmeyer flask, accurately add 50ml of 30% ethanol, weigh, heat and reflux for 30 minutes, let cool, weigh again, make up the lost weight with 30% ethanol, shake well, filter, Take 1 mL of the continued filtrate in a 10 mL volumetric flask, and dilute to the mark with 30% ethanol to obtain the final product.

test methods:

Precisely draw 10 μl each of the reference substance solution and the test solution, inject it into the liquid chromatograph, measure it, and obtain it. Chromatographic conditions: the mobile phase is methanol and water (25:75), the detection wavelength is 250nm, and the reference substance puerarin is dissolved in 30% ethanol solution. Chromatographic column: Diamonsil C18 column (250mm×4.6mm, 5μm), flow rate 1.0mL/min, running time 30min, injection volume 20μL.

Table 1 Determination results of puerarin content in fermented product sample and water extract sample

sample Puerarin content in sample solution mg/mL Fermentation sample 0.0452 water extract sample 0.0286

Embodiment 6-anti-diarrheal effect drug efficacy experiment

Preparation of the drug:

Fermented powder: get 3.5g of fermented powder obtained in Example 2 (equivalent to the original drug of 20g of Pueraria Radix, 14g of Radix Astragali and 6g of Poria cocos), add distilled water and be settled to 120ml, to get final product (according to the volume of 20ml/ kg to calculate the volume).

Preparation of water extract: Take 20g of kudzu root, 14g of astragalus and 6g of poria cocos, soak in water for 30min, decoct and extract twice, add 10 times the amount of water each time, extract for 2 hours each time, filter, combine the filtrates, and concentrate by rotary evaporation. Dilute to 120ml with distilled water, that is, too.

Pueraria root extract: Take 40g of Pueraria root, soak in water for 30min, decoct and extract twice, add 10 times the amount of water each time, and extract for 2 hours each time, filter, combine the filtrates, concentrate by rotary evaporation, and dilute to 120ml with distilled water, Instantly.

Astragalus extract: take 40g of Astragalus, soak in water for 30min, decoct and extract twice, add 10 times the amount of water each time, extract for 2 hours each time, filter, combine the filtrates, concentrate by rotary evaporation, and dilute to 120ml with distilled water, Instantly.

Poria cocos extract: take 40g Poria cocos, soak in water for 30min, decoct and extract twice, add 10 times the amount of water each time, extract for 2 hours each time, filter, combine the filtrates, concentrate by rotary evaporation, and distill the volume to 120ml with distilled water, Instantly.

Senna leaf suspension: before use, dry leaves (vein removed), add boiling distilled water to grind evenly, and then add cold distilled water to prepare 8% senna leaf suspension.

experimental method:

Take 60 mice, half male and half female, body weight (20 ± 2.0) g, randomly divided into 6 groups, 10 mice in each group, respectively blank control group, fermentation powder, water extract group, kudzu root group, astragalus group and Poria cocos group, Continuous administration for 5 days, each group was given the corresponding medicinal solution by intragastric administration, and the blank control group was given the same volume of normal saline. ml/only, divided into cages, one cage for each mouse, and absorbent paper (filter paper) was laid under the cage, and the number of defecation of each animal was recorded, and the following indicators were observed and statistically analyzed.

Loose feces rate: the ratio of the number of loose feces discharged by each animal to the total number of feces, and each grain or pile of feces on the filter paper is regarded as one defecation.

Loose stool grade: graded by the area diameter (cm) of the filter paper contaminated by loose stool, divided into four grades: grade 1 (<10), grade 2 (1-1.9), grade 3 (2-3), grade 4 (>3 ), when counting, first count the series of each pile of loose stools one by one, then add all the series of loose stools of the mouse, and divide by the number of loose stools to get the average series of loose stools

Diarrhea index: the product of the loose stool rate and the average grade of loose stool.

Table 2. Results of anti-diarrhea experiment

group Number of animals (n) Dose (g/kg) Diarrhea index Blank control group 10 -- 1.00±0.17 ^△△ Baking powder group 10 6.7 0.44±0.15 ^** Water Extract Group 10 6.7 0.60±0.10 ^**△ Pueraria group 10 6.7 0.75±0.12 ^**△△ Astragalus group 10 6.7 0.98±0.15 ^△△ Poria cocos group 10 6.7 0.71±0.15 ^**△△

Note: Compared with blank group, *P<0.05**P<0.01; compared with baking powder group, △P<0.05, △△P<0.01.

According to the statistical results of the experimental data, compared with the blank group, the diarrhea index of the mice in the fermentation powder group, the water extract group, the Pueraria root group and the Poria cocos group decreased significantly (P<0.01). Compared with the fermentation powder, the spleen diarrhea index of the water extract group, Pueraria root group, Poria cocos group and Astragalus group significantly decreased (P<0.05). This shows that the fermented powder group is better than the water extract group, kudzu root group, poria cocos group and astragalus group in reducing the diarrhea index of mice.

The present invention is not limited to the above-mentioned embodiments. Without departing from the essence of the present invention, any deformation, improvement, and replacement conceivable by those skilled in the art fall within the scope of the present invention.

Claims (10)

1. a preparation method of kudzu root compound fermentation product, is characterized in that, comprises the following steps: The first fermentation step: using yeast to carry out anaerobic fermentation in the medium at 26-32°C for 10-20 hours to obtain the first fermented product, wherein the medium contains kudzu root compound enzymatic hydrolyzate; The second fermentation step: raising the temperature of the first fermented product to 33-40°C, and using lactic acid bacteria to carry out static fermentation at this temperature for 30-40 hours to obtain the second fermented product; The third fermentation step: raising the temperature of the second fermented product to 50-70° C., and standing for fermentation at this temperature for 25-35 hours to obtain the third fermented product; Solid-liquid separation step: the obtained third fermented product is subjected to solid-liquid separation to obtain a fermentation stock solution, which is a kudzu root compound fermented product. 2. The preparation method according to claim 1, characterized in that, the kudzu root compound enzymatic hydrolyzate is obtained by using pectinase, cellulase, xylanase and trypsin at a pH value of 6.0～7.2 and 45～ It is prepared by enzymatically hydrolyzing the water suspension containing kudzu root, astragalus and poria cocos at 60°C for 2-6 hours. 3. preparation method according to claim 2 is characterized in that, the consumption of described pectinase is 0.05～0.3wt% of the total weight of water suspension, and the consumption of described cellulase is 0.3% of the total weight of water suspension. 0.05-0.3 wt%, the dosage of xylanase is 0.05-0.3 wt% of the total weight of the water suspension and the dosage of the trypsin is 0.05-0.15 wt% of the total weight of the water suspension. 4. preparation method according to claim 3 is characterized in that, the weight ratio of pectinase, cellulase, xylanase and trypsin is 1:1:1:1. 5. The preparation method according to claim 1, characterized in that, in the aqueous suspension containing Pueraria Radix, Radix Astragali and Poria Cocos, Radix Puerariae is 30～55 parts by weight, Radix Astragali is 30～40 parts by weight, and Poria Cocos is 10 parts by weight. ~20 parts by weight, and the content of kudzu root is greater than that of astragalus. 6. The preparation method according to claim 1, wherein the culture medium further comprises 0.1-0.5wt% yeast extract, 1-5wt% ammonium salt, 0.01-0.2wt% magnesium salt, 0.3-0.8wt% Phosphate and 0.01-0.2 wt% zinc salt; the above weight percentages are all based on the total weight of the culture medium. 7. The preparation method according to claim 1, characterized in that the inoculation amount of the yeast in the first fermentation step is 1×10 ⁷ cfu to 9×10 ⁷ cfu per ml of the medium, and the second fermentation The inoculation amount of the lactic acid bacteria in the step is 1×10 ⁶ cfu to 9×10 ⁶ cfu per ml of the culture medium. 8. A method for preparing a kudzu root composite fermentation dry powder, comprising the steps of: adopting the preparation method according to any one of claims 1 to 7 to obtain a kudzu root composite fermentation product, and then subjecting the kudzu root composite fermentation product to Concentrated and dried to obtain fermented dry powder. 9. A kudzu root compound fermentation product, characterized in that it is the kudzu root compound fermentation product obtained according to the preparation method described in any one of claims 1 to 7 or the kudzu root compound fermentation product obtained according to the preparation method described in claim 8 dry powder. 10. the purposes of kudzu root composite fermentation product according to claim 9 in the preparation medicine, food or feed that have antidiarrheal effect.