Bacteria-enzyme combination method for fermentation of Chinese wolfberry fruits and application

Abstract

The invention relates to the field of extraction of effective components of natural plants, in particular to a bacterium-enzyme combination method for fermentation of Chinese wolfberry fruits and an application of the bacterium-enzyme combination method. The lycium barbarum polysaccharide is obtained by mixing lycium barbarum and water and then sequentially performing homogenization, enzymolysis, enzyme deactivation, fermentation, filtration and purification. Cellulase, maltase and amylase are adopted as a compound enzyme preparation, the cellulase firstly destroys cell walls to enable other components (polysaccharide, starch and protein) to be dissolved out, the amylase catalyzes the starch to be decomposed into the maltase, the maltase further catalyzes the maltose to be decomposed into glucose, and due to the addition of the amylase, the content of the maltose in the starch is reduced. The fermentation efficiency can also be improved. The streptococcus amylases, the streptococcus thermophilus and the bifidobacterium longum are used as the complex microbial inoculants, the streptococcus amylases can effectively decompose starch and non-starch polysaccharides, the streptococcus thermophilus and the bifidobacterium longum can further decompose other substances, the Chinese wolfberry fruits are fully utilized, and finally the purpose of improving the extraction rate and the purity of the Chinese wolfberry fruit polysaccharides is achieved.

Description

Technical Field

[0001] The invention relates to the field of extraction of effective components of natural plants, and in particular to a bacteria-enzyme combination method for wolfberry fermentation and its application. Background Art

[0002] Lycium barbarum is a plant of the genus Lycium of the Solanaceae family. Lycium barbarum is a general term for species of the genus Lycium, including commercial Lycium barbarum, plant Ningxia Lycium barbarum, and Chinese Lycium barbarum. Lycium barbarum has many functions, including immunomodulation, anti-oxidation, anti-aging, anti-fatigue, anti-tumor, anti-radiation damage, lowering blood lipids, lowering blood sugar, lowering blood pressure, protecting the liver, strengthening the brain, nourishing the kidneys, detoxifying, nourishing the skin, and protecting the reproductive system. Modern Chinese medicine research shows that the main active ingredients of Lycium barbarum are polysaccharides, pigments, flavonoids, proteins, betaine, amino acids, and carotenoids. Among them, Lycium barbarum polysaccharide is one of the main active ingredients of Lycium barbarum, which has multiple pharmacological effects and biological activities.

[0003] Lycium barbarum polysaccharide is a water-soluble polysaccharide extracted from wolfberry. Conventional extraction methods include water extraction and alcohol precipitation, microwave extraction, acid-base decomposition extraction, etc., but these methods often have problems such as complex steps, high energy consumption, low efficiency, and consumption of a large amount of resources, which limits the utilization of Lycium barbarum polysaccharide; for example, the water extraction and alcohol precipitation method for extracting Lycium barbarum polysaccharide has problems such as long extraction time and high temperature, which has an adverse effect on the biological activity of the polysaccharide. Although the prior art has optimized the extraction method of Lycium barbarum polysaccharide, there is still a low extraction rate. For example, according to the extraction method provided by Chinese patent CN115611993A, less than 4g of Lycium barbarum polysaccharide can be extracted from 100g of wolfberry. It can be seen that at this stage, it is necessary to find a method that can improve the extraction rate of Lycium barbarum polysaccharide. Summary of the invention

[0004] The purpose of the present invention is to provide a method and application of combining bacteria and enzymes for wolfberry fermentation, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a method for extracting wolfberry polysaccharides, which can significantly improve the extraction rate of wolfberry polysaccharides.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for preparing Lycium barbarum polysaccharides based on the combination of bacteria and enzymes, comprising the following steps:

[0007] After mixing wolfberry with water, homogenization, enzymolysis, enzyme inactivation, fermentation, filtration and purification are sequentially performed to obtain wolfberry polysaccharide;

[0008] The complex enzyme preparation used in the enzymolysis is cellulase, maltase and amylase;

[0009] The composite bacterial agent used in the fermentation is streptococcus amyloliquefaciens, streptococcus thermophilus and bifidobacterium longum.

[0010] Preferably, the mass ratio of the wolfberry to the water is 1:15-20.

[0011] Preferably, the dosage of the complex enzyme preparation is 0.6% of the mass of wolfberry;

[0012] The enzymatic activity of the cellulase in the composite enzyme preparation is 8000 U / mL, the enzymatic activity of the maltase is 6000 U / mL, and the enzymatic activity of the amylase is 8000 U / mL.

[0013] Preferably, the dosage of the composite bacterial agent is 4% of the mass of wolfberry;

[0014] The effective viable bacteria count of the composite bacterial agent is 1×10 11 CFU / mL.

[0015] Preferably, the ratio of the effective live bacteria counts of Streptococcus amyloliquefaciens, the thermophilic Streptococcus and Bifidobacterium longum in the composite bacterial agent is 1:1:1.

[0016] Preferably, the enzymatic hydrolysis temperature is 45°C and the time is 1-2h.

[0017] Preferably, the fermentation temperature is 60°C and the fermentation time is 2-3 hours.

[0018] Preferably, the purification comprises the steps of re-dissolving the crude extract obtained by filtration and then purifying it through a Sephadex G-100 chromatography column.

[0019] The present invention provides a Lycium barbarum polysaccharide prepared by the above method.

[0020] The present invention provides the use of the above-mentioned wolfberry polysaccharide in the preparation of antioxidant products.

[0021] The present invention discloses the following technical effects:

[0022] The present invention provides a method for preparing Lycium barbarum polysaccharide based on bacterial enzyme combination, comprising the following steps: after mixing Lycium barbarum with water, homogenizing, enzymolysis, enzyme inactivation, fermentation, filtration and purification are sequentially performed to obtain Lycium barbarum polysaccharide; the composite enzyme preparation used in the enzymolysis is cellulase, maltase and amylase; the composite bacterial agent used in the fermentation is Streptococcus amylolyticus, Streptococcus thermophilus and Bifidobacterium longum. The present invention adopts cellulase, maltase and amylase as composite enzyme preparation, wherein cellulase first destroys the cell wall, so that other components (polysaccharide, starch and protein) are dissolved, and amylase catalyzes starch decomposition into maltase, and maltase further catalyzes maltose decomposition into glucose, so that the extraction rate and purity of Lycium barbarum polysaccharide are increased, and due to the addition of amylase, the efficiency of fermentation can also be improved. The present invention uses streptococcus amyloliquefaciens, streptococcus thermophilus and bifidobacterium longum as composite bacterial agents, wherein the streptococcus amyloliquefaciens can effectively decompose starch and non-starch polysaccharides, and the streptococcus thermophilus and bifidobacterium can further decompose other substances, so as to make full use of wolfberry and finally achieve the purpose of improving the extraction rate and purity of wolfberry polysaccharides.

[0023] Furthermore, the method provided by the invention is simple and can be used for industrial production. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1

[0030] A method for preparing Lycium barbarum polysaccharides based on bacterial and enzyme combination, the steps are as follows:

[0031] 1. Homogenization: Mix wolfberry (1 kg) with water in a mass ratio of 1:15, and then homogenize to obtain a wolfberry homogenate;

[0032] 2. Enzymolysis: Mix the wolfberry homogenate with the composite enzyme preparation, perform enzymolysis at 45°C for 1 hour, and obtain an enzymolyzate; wherein the amount of the composite enzyme preparation is 0.6% of the mass of the wolfberry, the composite enzyme preparation is composed of cellulase, maltase and amylase, the enzymatic activity of the cellulase in the composite enzyme preparation is 8000U / mL, the enzymatic activity of the maltase is 6000U / mL, and the enzymatic activity of the amylase is 8000U / mL;

[0033] 3. Inactivation of enzyme and fermentation: After the enzymolysate is inactivated, a composite bacterial agent is added for fermentation to obtain a fermentation complex; wherein the inactivation time is 5 min and the temperature is 100° C.; the fermentation temperature is 60° C. and the time is 2 h; the amount of the composite bacterial agent added is 4% of the mass of wolfberry, and the composite bacterial agent is composed of amylolytic Streptococcus (strain number: CICC 24766, Latin name: Streptomyces diastaticus, purchased from: China Industrial Microbiological Culture Collection Administration Center), thermophilic Streptococcus (strain number: CGMCC 1.8748, Latin name: Streptococcus thermophilus, purchased from: China General Microbiological Culture Collection Center) and long Bifidobacterium (strain number: CGMCC 1.5082, Latin name: Bifidobacterium longum subsp.longum, purchased from China General Microbiological Culture Collection Center); the ratio of the effective viable counts of Streptococcus amylopectin, Streptococcus thermophilus and Bifidobacterium longum in the composite bacterial agent is 1:1:1, and the total viable count is 1×10 11 CFU / mL;

[0034] 4. Filtration: filtering the fermentation complex, precipitating and freeze-drying the filtrate obtained by filtration to obtain a crude extract;

[0035] 5. Purification: The crude extract was redissolved, purified by Sephadex G-100 chromatography column, recrystallized by ethanol, and then freeze-dried to obtain Lycium barbarum polysaccharide.

[0036] Example 2

[0037] The steps are the same as those in Example 1, except that the mass ratio of wolfberry to water is 1:18, the enzymatic hydrolysis time is 1.5 h, and the fermentation time is 2.5 h.

[0038] Example 3

[0039] The steps are the same as those in Example 1, except that the mass ratio of wolfberry to water is 1:20, the enzymatic hydrolysis time is 2 h, and the fermentation time is 3 h.

[0040] Comparative Example 1

[0041] The steps are the same as those in Example 1, except that the fermentation step is omitted.

[0042] Comparative Example 2

[0043] The steps are the same as those in Example 1, except that the steps of enzymolysis and enzyme inactivation are omitted.

[0044] Comparative Example 3

[0045] The steps are the same as those in Example 1, except that the composite enzyme preparation consists of cellulase, pectinase and amylase, the enzymatic activity of the cellulase in the composite enzyme preparation is 8000 U / mL, the enzymatic activity of the pectinase is 6000 U / mL, and the enzymatic activity of the amylase is 8000 U / mL.

[0046] Comparative Example 4

[0047] The steps are the same as those in Example 1, except that the composite enzyme preparation consists of cellulase, pectinase, protease and amylase, the enzymatic activity of the cellulase in the composite enzyme preparation is 8000U / mL, the enzymatic activity of the pectinase is 6000U / mL, the enzymatic activity of the protease is 8000U / mL, and the enzymatic activity of the amylase is 8000U / mL.

[0048] Comparative Example 5

[0049] The steps are the same as those in Example 1, except that the composite bacterial agent is composed of plant lactobacillus, thermophilic streptococcus (strain number: CGMCC 1.8748, Latin name: Streptococcus thermophilus, purchased from: China General Microbiological Culture Collection Center) and long bifidobacterium (strain number: CGMCC 1.5082, Latin name: Bifidobacterium longum subsp. longum, purchased from: China General Microbiological Culture Collection Center); the ratio of the effective viable counts of amylolytic streptococcus, the thermophilic streptococcus and the long bifidobacterium in the composite bacterial agent is 1:1:1, and the total viable count is 1×10 11 CFU / mL.

[0050] Comparative Example 6

[0051] The steps are the same as those in Example 1, except that the composite bacterial agent is composed of Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus (strain number: CGMCC 1.8748, Latin name: Streptococcus thermophilus, purchased from: China General Microbiological Culture Collection Center) and Bifidobacterium longum (strain number: CGMCC 1.5082, Latin name: Bifidobacterium longum subsp.longum, purchased from: China General Microbiological Culture Collection Center); the ratio of the effective viable counts of Streptococcus amylolyticus, the thermophilic Streptococcus and Bifidobacterium longum in the composite bacterial agent is 1:1:1, and the total viable count is 1×10 11 CFU / mL.

[0052] Experimental Example 1

[0053] The extraction rate and purity of the Lycium barbarum polysaccharide of Examples 1 to 3 and Comparative Examples 1 to 6 were investigated. Dextran was used as a standard and the phenol-sulfuric acid method was used to determine the purity of the Lycium barbarum polysaccharide obtained in each Example and Comparative Example. The extraction rate of Lycium barbarum polysaccharide was calculated using the formula: Lycium barbarum polysaccharide extraction rate (%) = mass of Lycium barbarum polysaccharide extracted / mass of Lycium barbarum × 100. The results are shown in Table 1.

[0054] Table 1 Investigation results of extraction rate and purity of Lycium barbarum polysaccharides under different treatments

[0055]

[0056]

[0057] As shown in Table 1, the extraction rate of Lycium barbarum polysaccharides in Examples 1 to 3 is as high as 26.5%, and the purity of Lycium barbarum polysaccharides is also above 89%. In contrast, the extraction rate of Lycium barbarum polysaccharides in Comparative Examples 1 to 6 is between 12.5% ​​and 21.8%, and the purity of Lycium barbarum polysaccharides is between 71.0% and 81.5%. Analyzing the reasons, comparative examples 1 and 2 omitted the steps of enzymolysis or fermentation, resulting in a decrease in the dissolution rate of Lycium barbarum polysaccharides in wolfberry, and ultimately resulting in a decrease in the extraction rate of Lycium barbarum polysaccharides; comparative examples 3 and 4 replaced the types of enzymes, and found that after replacing maltase with pectinase and protease, the extraction rate and purity of Lycium barbarum polysaccharides decreased, probably because cellulase first destroyed the cell wall, causing other components (polysaccharides, starch and protein) to dissolve, while amylase catalyzed the decomposition of starch into maltase, and maltase further catalyzed the decomposition of maltose into glucose, ultimately increasing the extraction rate and purity of Lycium barbarum polysaccharides, and due to the addition of amylase, the efficiency of fermentation can also be improved; comparative examples 3 and 4 omitted maltase, thus resulting in a decrease in the extraction rate and purity of Lycium barbarum polysaccharides. Comparative examples 5 and 6 changed the strain types of the fermentation process, but ultimately resulted in a decrease in the extraction rate and purity of Lycium barbarum polysaccharides, which shows that the selection of strains during the fermentation process has a great influence on the extraction rate and purity of Lycium barbarum polysaccharides.

[0058] Experimental Example 2 Antioxidant Experiment

[0059] The antioxidant activity of the Lycium barbarum polysaccharides prepared in Examples 1 to 3 and Comparative Examples 1 to 6 was measured by the DPPH method, with vitamin C used as a positive control. The clearance rate was calculated using the following formula. The results are shown in Table 2.

[0060] Table 2 Clearance rate of Lycium barbarum polysaccharides obtained by different treatments

[0061] deal with Clearance Example 1 88.6％ Example 2 90.3％ Example 3 87.5％ Comparative Example 1 70.4％ Comparative Example 2 71.6％ Comparative Example 3 73.6％ Comparative Example 4 75.2％ Comparative Example 5 73.9％ Comparative Example 6 75.9％ Positive Control 85.7％

[0062] As shown in Table 2, the clearance rates of Lycium barbarum polysaccharides prepared in Examples 1 to 3 were between 87.5% and 90.3%, which were significantly improved compared to Comparative Examples 1 to 6, and were comparable to or even higher than the positive control.

[0063] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing Lycium barbarum polysaccharides based on bacterial and enzyme combination, characterized in that: The following steps are involved: After mixing wolfberry with water, homogenization, enzymolysis, enzyme inactivation, fermentation, filtration and purification are sequentially performed to obtain wolfberry polysaccharide; The complex enzyme preparation used in the enzymolysis is cellulase, maltase and amylase; The composite bacterial agent used in the fermentation is streptococcus amyloliquefaciens, streptococcus thermophilus and bifidobacterium longum.

2. The method according to claim 1, characterized in that The mass ratio of the wolfberry to the water is 1:15-20.

3. The method according to claim 1, characterized in that The dosage of the complex enzyme preparation is 0.6% of the mass of wolfberry; The enzymatic activity of the cellulase in the composite enzyme preparation is 8000 U / mL, the enzymatic activity of the maltase is 6000 U / mL, and the enzymatic activity of the amylase is 8000 U / mL.

4. The method according to claim 1, characterized in that: The dosage of the composite bacterial agent is 4% of the mass of wolfberry; The effective viable bacteria count of the composite bacterial agent is 1×10 11 CFU / mL.

5. The method according to claim 1 or 4, characterized in that: The ratio of the effective live bacteria counts of Streptococcus amylopectin, Streptococcus thermophilus and Bifidobacterium longum in the composite bacterial agent is 1:1:

1.

6. The method according to claim 1, characterized in that The temperature of the enzymatic hydrolysis is 45° C. and the time is 1-2 h.

7. The method according to claim 1, characterized in that The fermentation temperature is 60°C and the fermentation time is 2-3 hours.

8. The method according to claim 1, characterized in that The purification comprises the steps of re-dissolving the crude extract obtained by filtration and then purifying it through a Sephadex G-100 chromatography column.

9. Lycium barbarum polysaccharide prepared by the method according to any one of claims 1 to 8.

10. Use of the Lycium barbarum polysaccharide according to claim 9 in the preparation of antioxidant products.

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