A Chinese herbal medicine composition for promoting the growth of probiotics and its application

Abstract

The invention discloses a Chinese herbal medicine composition for promoting the growth of probiotics and an application thereof; the Chinese herbal medicine composition comprises shiitake mushrooms, hericium erinaceus, enoki mushrooms and bamboo fungus; the invention also conducts in vitro experiments based on the Chinese herbal medicine composition in a "multi-bacteria, multi-prebiotics and multi-drug" mode to study the interaction between different components and explore their synergistic effects on solid beverages; finally, a product formula consisting of shiitake mushrooms, hericium erinaceus, enoki mushrooms, bamboo fungus, Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, inulin, oligofructose and oligoxylose is obtained; Chinese herbal medicine is used as raw material, and the combined effect of probiotics and prebiotics is utilized; the synergistic effect of the three is more significant, and the combined fermentation technology improves its activity value, providing a new concrete scheme for the concept of synbiotic preparations; a new functional food that meets the dietary needs of modern people is obtained.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a Chinese herbal medicine composition for promoting the growth of probiotics and an application thereof. Background Art

[0002] Probiotics alter the efficacy of traditional Chinese medicines or enhance synergistic effects by influencing the metabolism, conversion, and absorption of their active ingredients. After microbial fermentation, the active macromolecules in these herbs are converted into small molecules that can be directly absorbed by the animal's intestines. These molecules are then fully metabolized within the animal body, preventing drug residues. Prebiotics produced by these fermented herbs promote microbial growth, which in turn promotes the absorption and utilization of the herbs. Furthermore, prebiotics can promote the growth and abundance of beneficial bacteria in the intestine, thereby maintaining intestinal health. These three factors complement and synergize to better exert their immune-modulating effects. The combination of probiotics, prebiotics, and traditional Chinese medicines is an innovative approach to the development of functional foods and traditional Chinese medicines, and a new approach to the modernization of traditional Chinese medicine research.

[0003] Currently, there are many probiotic and prebiotic beverages and probiotic fermented Chinese medicine products on the market. For example, the patent by Gu Jinsong et al. [1] describes a probiotic composition and its application, a probiotic product and its preparation method, which realizes the combined effect of probiotics and polysaccharides. However, there are relatively few products that use probiotic fermented Chinese medicine and prebiotics to make solid beverages.

[0004] Most existing probiotic products are single probiotics with additives or sweeteners added to improve the taste. For example, Tang Zhenxing et al. [2] invented a product that uses sodium alginate to encapsulate probiotics. The disadvantages of this product are that there are still many unknown factors in the long-term impact of additives on health; secondly, the vitality of probiotics cannot be guaranteed, making it difficult to fully exert their activity.

[0005] [1] Gu Jinsong, Qi Cuihua, Zhang Yu, et al. Compositions prepared from probiotic fermented Chinese medicine preparations and their preparation methods and applications: CN201210527023.9[P].CN102934809A.

[0006] [2] Tang Zhenxing, Xu Zheng, Liu Ying, et al. A probiotic product and its preparation method: CN202210204681.8[P]. 2022. Summary of the Invention

[0007] The purpose of the present invention is to provide a Chinese herbal medicine complex that can promote the growth of probiotics, and to prepare a "multi-bacteria, multi-prebiotic, and multi-drug" probiotic product based on the Chinese herbal medicine complex by compounding probiotics and prebiotics. The product improves the utilization rate of Chinese herbal medicines, and the prebiotics can also maintain the vitality of probiotics at the same time. The three components complement each other, give full play to their functional activities, and improve the body's immunity; at the same time, the three ingredients in the formula are all natural products and will not impose a large burden on the human body.

[0008] The technical solution adopted by the present invention is:

[0009] A first aspect of the present invention provides a Chinese herbal medicine composition, comprising an aqueous extract of Lentinus edodes, an aqueous extract of Hericium erinaceus, an aqueous extract of Flammulina velutipes, and an aqueous extract of Dictyophora dasyphylla.

[0010] Preferably, the mass ratio of the shiitake mushroom water extract, the hericium erinaceus water extract, the enoki mushroom water extract, and the bamboo fungus water extract is (1-3): (0.5-1): (0.5-1): (1-3).

[0011] Preferably, the preparation method of the water extract comprises: adding water to the Chinese herbal medicines Lentinus edodes, Hericium erinaceus, Flammulina velutipes and / or Dictyophora japonica for extraction, filtering and centrifuging, and collecting the supernatant.

[0012] Preferably, the material-liquid ratio of the Chinese herbal medicines shiitake mushroom, hericium erinaceus, enoki mushroom and / or bamboo fungus to water is 1:25-40.

[0013] Preferably, the extraction conditions are: extraction temperature of 70 to 90° C.; and / or extraction time of 1 to 4 hours.

[0014] Preferably, the filtering condition is to use a filter cloth with a pore size of 150 to 250 meshes for filtering.

[0015] Preferably, the filtering is performed 1 to 3 times.

[0016] Preferably, the centrifugal conditions are: a centrifugal speed of 4000 to 6000 rpm; and / or a centrifugal time of 8 to 12 min.

[0017] Preferably, the concentrations of the shiitake mushroom water extract, the hericium erinaceus water extract, the enoki mushroom water extract, and the bamboo fungus water extract are all 150-250 mg / mL.

[0018] Preferably, the aqueous extract may be further sterilized after preparation, and the sterilization conditions include but are not limited to high temperature and high pressure sterilization.

[0019] The second aspect of the present invention provides use of the composition according to the first aspect of the present invention in promoting the growth of probiotics and / or preparing a product that promotes the growth of probiotics.

[0020] Preferably, the probiotics include at least one of Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus rhamnosus.

[0021] Preferably, the viable count of Lactobacillus acidophilus is 1×10 10 ~1×10 12 CFU / g.

[0022] Preferably, the Bifidobacterium lactis is 1×10 10 ~1×10 12 CFU / g.

[0023] Preferably, the Lactobacillus rhamnosus is 1×10 10 ~1×10 12 CFU / g.

[0024] Preferably, the mass ratio of Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus rhamnosus is (10-20):(5-15):1.

[0025] Preferably, the Lactobacillus acidophilus includes but is not limited to Lactobacillus acidophilus NCFM.

[0026] Preferably, the Bifidobacterium lactis includes but is not limited to Bifidobacterium lactis Bi-07.

[0027] Preferably, the Lactobacillus rhamnosus includes but is not limited to Lactobacillus rhamnosus HN001.

[0028] The third aspect of the present invention provides a composite composition, wherein the raw material components of the composite composition include the Chinese herbal medicine composition described in the first aspect of the present invention and probiotics.

[0029] Preferably, the complex composition further comprises prebiotics.

[0030] Preferably, the mass ratio of the Chinese herbal medicine composition, probiotics, and prebiotics is 600-1000:0.5-1.5:100-250;

[0031] Preferably, the mass ratio of the Chinese herbal medicine composition, probiotics, and prebiotics is 700-800:0.9-1.1:140-200;

[0032] Preferably, the mass ratio of the Chinese herbal composition, probiotics and prebiotics is 350:0.97:70.

[0033] Preferably, the prebiotics include at least two of inulin, fructooligosaccharides and xylo-oligosaccharides.

[0034] Preferably, the prebiotics include inulin and oligofructose.

[0035] Preferably, the mass ratio of inulin, fructooligosaccharide and xylo-oligosaccharide is (2-4): (0.5-1.5):1.

[0036] Preferably, the mass ratio of inulin to oligofructose is (2-4):1.

[0037] Preferably, the probiotics include at least one of Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus rhamnosus.

[0038] Preferably, the mass ratio of Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus rhamnosus is (10-20):(5-15):1.

[0039] Preferably, the viable count of Lactobacillus acidophilus is 1×10 10 ~1×10 12 CFU / g.

[0040] Preferably, the viable count of the Bifidobacterium lactis is 1×10 10 ~1×10 12 CFU / g.

[0041] Preferably, the viable count of the Lactobacillus rhamnosus is 1×10 10 ~1×10 12 CFU / g.

[0042] A fourth aspect of the present invention provides a method for promoting the growth of probiotics, comprising: adding the Chinese herbal medicine composition according to the first aspect of the present invention to a probiotic culture medium.

[0043] Preferably, the culture medium further comprises prebiotics.

[0044] Preferably, the mass ratio of the shiitake mushroom water extract, the hericium erinaceus water extract, the enoki mushroom water extract, and the bamboo fungus water extract is (1-3): (0.5-1): (0.5-1): (1-3).

[0045] Preferably, the concentrations of the shiitake mushroom water extract, the hericium erinaceus water extract, the enoki mushroom water extract, and the bamboo fungus water extract are all 150-250 mg / mL.

[0046] Preferably, the probiotics include at least one of Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus rhamnosus.

[0047] Preferably, the mass ratio of Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus rhamnosus is (10-20):(5-15):1.

[0048] Preferably, the viable count of Lactobacillus acidophilus is 1×10 10 ~1×10 12 CFU / g.

[0049] Preferably, the viable count of the Bifidobacterium lactis is 1×10 10 ~1×10 12 CFU / g.

[0050] Preferably, the viable count of the Lactobacillus rhamnosus is 1×10 10 ~1×10 12 CFU / g.

[0051] Preferably, the prebiotics include at least two of inulin, fructooligosaccharides and xylo-oligosaccharides.

[0052] Preferably, the prebiotics include inulin and oligofructose.

[0053] Preferably, the mass ratio of inulin, fructooligosaccharide and xylo-oligosaccharide is (2-4): (0.5-1.5):1.

[0054] Preferably, the mass ratio of inulin to oligofructose is (2-4):1.

[0055] Preferably, the culture medium includes but is not limited to MRS medium, LB medium, M17 medium and the like.

[0056] The fifth aspect of the present invention provides a method for preparing the composite composition according to the third aspect of the present invention, comprising: adding raw material components of the composite composition to a basal culture medium for fermentation.

[0057] Preferably, the volume ratio of the Chinese herbal medicine to the basal culture medium in the raw material components of the composite composition is 1:(0.5-2).

[0058] Preferably, the inoculation amount of the probiotics in the raw material components of the composite composition is 1-4%.

[0059] Preferably, the final concentration of the prebiotics in the raw material components of the complex composition is 5 to 15 mg / mL.

[0060] Preferably, the fermentation time is 8 to 32 hours.

[0061] Preferably, the fermentation time is 12 to 24 hours.

[0062] Preferably, the fermentation condition is anaerobic fermentation.

[0063] The sixth aspect of the present invention provides use of the Chinese herbal medicine composition described in the first aspect of the present invention or the composite composition described in the third aspect of the present invention in preparing a product.

[0064] Preferably, the function of the product includes enhancing immunity.

[0065] Preferably, the product further comprises at least one of a pharmaceutically or food-acceptable carrier, excipient, and diluent.

[0066] Preferably, the dosage form of the product is at least one selected from the group consisting of solution, suspension, granule, emulsion, powder, lozenge, pill, buccal lozenge, tablet, chewing gum, syrup and capsule.

[0067] Preferably, the product comprises at least one of food, medicine, and feed additive.

[0068] Preferably, the food includes at least one of beverages, biscuits, candies, cakes, and dairy products.

[0069] The seventh aspect of the present invention provides a product, which includes the Chinese herbal medicine composition described in the first aspect of the present invention or the composite composition described in the third aspect of the present invention.

[0070] Preferably, the function of the product includes enhancing immunity.

[0071] Preferably, the product further comprises at least one of a pharmaceutically or food-acceptable carrier, excipient, and diluent.

[0072] Preferably, the dosage form of the product is at least one selected from the group consisting of solution, suspension, granule, emulsion, powder, lozenge, pill, buccal lozenge, tablet, chewing gum, syrup and capsule.

[0073] Preferably, the product comprises at least one of food, medicine, and feed additive.

[0074] Preferably, the food includes at least one of beverages, biscuits, candies, cakes, and dairy products.

[0075] The beneficial effects of the present invention are:

[0076] In order to fully explore the medicinal value of Chinese herbal medicines and improve their bioavailability while retaining the active ingredients of the raw materials, the present invention selects a variety of Chinese herbal medicines to obtain their water extracts. This extraction method can efficiently extract the active ingredients and nutrients of the herbs, fully retain the biological activity and medicinal value of the Chinese herbal medicines, and promote the growth of probiotics; the present invention also adopts the "multi-bacteria, multi-prebiotics and multi-drugs" model to conduct in vitro experiments to study the interaction between different components and explore their synergistic effects on solid beverages; finally, a product formula consisting of shiitake mushrooms, hericium erinaceus, enoki mushrooms, bamboo fungus, Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus rhamnosus, inulin, oligofructose and oligoxylose is obtained; Chinese herbal medicines are used as raw materials and the combined effects of probiotics and prebiotics are utilized. The synergistic effect of the Chinese herbal medicine combination, probiotics and prebiotics is more significant, and their immune regulatory function is better exerted; and the combined fermentation technology improves their active value, providing a new concrete solution for the concept of synergistic preparations; a new functional food that meets the dietary needs of modern people is obtained; not only the active value and utilization rate of Chinese herbal raw materials are improved, but also new ideas are provided for the reuse of Chinese medicinal resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is the technical roadmap of the present invention.

[0078] Figure 2 This is the growth curve of Bi-07 in different Chinese herbal medicine water extracts.

[0079] Figure 3 This is the growth curve of HN001 in different Chinese herbal medicine water extracts.

[0080] Figure 4 This is the growth curve of NCFM in different Chinese herbal medicine water extracts.

[0081] Figure 5 The reduction of polysaccharides before and after fermentation of Bi-07 and Chinese herbal medicine combination.

[0082] Figure 6 The reduction of polysaccharides before and after fermentation of HN001 and Chinese herbal medicine combination.

[0083] Figure 7 The reduction of polysaccharides before and after fermentation of NCFM and Chinese herbal medicine combination.

[0084] Figure 8 This is the growth curve of the water extract of Chinese herbal medicine formula fermented by mixed bacteria.

[0085] Figure 9 The pH value of the water extract of the mixed fermentation Chinese herbal medicine formula.

[0086] Figure 10 It is the reduction of polysaccharide before and after mixed fermentation.

[0087] Figure 11 It is the production of short-chain fatty acids before and after mixed fermentation.

[0088] Figure 12 This is the growth curve of the mixed bacterial solution in the prebiotic group with different proportions.

[0089] Figure 13 The pH value of the fermentation broth produced by mixed bacteria and prebiotic groups in different proportions.

[0090] Figure 14 The short-chain fatty acids produced by mixed fermentation of prebiotic groups with different proportions.

[0091] Figure 15 Thymus index.

[0092] Figure 16 CON A is the stimulation index of spleen lymphocyte transformation induced by CON A. DETAILED DESCRIPTION

[0093] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0094] The present invention adopts the experimental form of "multi-bacteria, multi-prebiotics and multi-drugs", which greatly improves the utilization rate of Chinese herbal medicine. The prebiotics in the formula can also maintain the vitality of probiotics at the same time. The three components complement each other and give full play to their functional activities. At the same time, the three ingredients in the formula are all natural products and will not cause a large burden on the human body. Figure 1 The technical route is used to screen the best Chinese herbal medicines, probiotics and prebiotics, and the synergistic benefits between the ingredients can enhance the body's immunity.

[0095] Probiotics Bi-07:HN001:NCFM were purchased from Danisco (China) Co., Ltd. and stored at 4°C.

[0096] MRS medium formula: peptone 10.0 g / L, beef powder 8.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L.

[0097] Example 1 Screening of the best Chinese herbal medicine

[0098] In order to screen the best Chinese herbal medicines, this example used three probiotics to ferment a variety of Chinese herbal medicines, and tested the three probiotics (NCFM viable count of 2×10 11 CFU / g, the number of viable bacteria of Bi-07 is 3×10 11 CFU / g, the number of viable bacteria of HN001 is 4.5×10 11 The optimal Chinese herbal medicine selection process was determined by measuring the growth of 1000 CFU / g and the polysaccharide content before and after fermentation. The Chinese herbal medicines included: Ganoderma lucidum, bamboo fungus, Ganoderma lucidum spore powder + Hericium erinaceus + Flammulina velutipes (1:1:1), Astragalus membranaceus + Morinda officinalis (1:1), Tremella fuciformis, Poria cocos, and Lentinus edodes. The ratios of the Chinese herbal medicines are by mass, the same below. The specific steps are as follows:

[0099] 1. Preparation of Chinese herbal medicine aqueous extract

[0100] The crushed dried Chinese herbal medicine was mixed with water at a material-liquid ratio of 1:30, extracted at 80°C for 2 hours, filtered through a 200-mesh nylon filter cloth, and the filtrate was collected. The process was repeated twice, the filtrates were combined, and the mixture was centrifuged at 5000 rpm for 10 minutes. The supernatant was collected and concentrated to a constant volume to obtain a 200 mg / mL aqueous extract. The extract was sterilized at 121°C and high pressure in an autoclave for 15 minutes to obtain aqueous extracts of Ganoderma lucidum, Dictyophora japonica, Ganoderma lucidum spore powder + Hericium erinaceus + Flammulina velutipes (1:1:1), Astragalus membranaceus + Morinda officinalis (1:1), Tremella fuciformis, Poria cocos, and Lentinus edodes.

[0101] 2. Fermentation of Chinese herbal medicine water extract

[0102] The prepared aqueous extracts of Ganoderma lucidum, Dictyophora dasyphylla, Ganoderma lucidum spore powder + Hericium erinaceus + Flammulina velutipes (1:1:1), Astragalus membranaceus + Morinda officinalis (1:1), Tremella fuciformis, Poria cocos, and Lentinus edodes were diluted to the concentration of the Chinese herbal medicine aqueous extract determined in step 1. In the Chinese herbal medicine addition group, 7 mL of the corresponding concentration of Chinese herbal medicine aqueous extract and 2% inoculum were added to 7 mL of MRS medium. A control group was set up, and the Chinese herbal medicine aqueous extract was replaced with distilled water. Three parallel tubes were used in each group, for a total of 32 groups. This process was carried out in an anaerobic environment at 37°C and 5% CO2, and the groups were grouped as shown in the following table;

[0103] Table 1

[0104] Grouping Fermentation system Chinese medicine supplement group 7mL culture medium + 7mL Chinese medicine extract + 2% bacterial solution Growth control group 7mL culture medium + 7mL distilled water + 2% bacterial solution Chinese medicine control group 7mL culture medium + 7mL Chinese medicine extract + 2% distilled water Blank control group 7mL culture medium + 7mL distilled water + 2% distilled water

[0105] 3. Growth curve determination

[0106] During the fermentation process, the OD600 value of each group was measured at 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, and 32 h. The measurement was stopped if the plateau phase was reached. The growth curves of Bi-07, HN001, and NCFM in different groups of herbal water extracts were plotted with the culture time as the horizontal axis and the OD600 value as the vertical axis.

[0107] Wherein: OD600 = OD600 value of the Chinese medicine added group - background OD600 value of the Chinese medicine water extract.

[0108] 4. Determination of polysaccharide content before and after fermentation

[0109] The polysaccharide content was determined by the phenol-sulfuric acid method combined with the DNS method.

[0110] (1) Preparation of phenol solution

[0111] Melt the phenol in a 50°C water bath, aspirate with a pipette, weigh 6 g into a light-proof beaker, dissolve in 194 mL of warm water, and store at 4°C in the dark.

[0112] (2) Preparation of standard curve for phenol-sulfuric acid method

[0113] Accurately weigh 5 mg of anhydrous glucose standard (Glu), dilute to a 50 mL volumetric flask with distilled water, prepare a 0.1 mg / mL glucose standard solution, pipette 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose standard solution into test tubes, add distilled water to each tube to make the total liquid volume 1 mL, add 1.0 mL of 3% phenol and 4.0 mL of concentrated sulfuric acid (add slowly), shake well, cool, and let it stand at room temperature for 30 min. Then measure the absorbance at a wavelength of 490 nm. Plot the absorbance value as the ordinate and the standard concentration (mg / mL) as the abscissa to obtain the phenol-sulfuric acid method standard curve.

[0114] (3) Preparation of DNS standard curve

[0115] Accurately weigh 50 mg of anhydrous glucose standard (Glu) and dilute to a 50 mL volumetric flask with distilled water to prepare a 1 mg / mL glucose standard solution. Pipette 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the glucose standard solution into test tubes, respectively, and add distilled water to make the total liquid volume reach 1 mL. Add 2 mL of DNS reagent, boil in a boiling water bath for 5 min, and terminate the reaction in an ice bath. Detect the absorbance at 540 nm and plot the DNS standard curve with the absorbance value as the ordinate and the standard concentration (mg / mL) as the abscissa.

[0116] (4) Preparation and determination of test solution

[0117] The mixed culture broth before fermentation and the mixed culture broth after fermentation were centrifuged at 8000 rpm / min for 10 min, the supernatant was taken, ethanol was added to a final concentration of 80%, and the mixture was precipitated overnight at 5000 rpm / min. The supernatant was discarded, and the ethanol was evaporated by vacuum concentration. The ethanol was redissolved and diluted to an appropriate concentration with distilled water. The total sugar content in the sample solution was determined by the phenol-sulfuric acid method. 1 mL of the sample solution was placed in a test tube, 1.0 mL of 3% phenol and 4.0 mL of concentrated sulfuric acid (added slowly) were added, the solution was shaken and cooled, and the solution was placed at room temperature for 30 min. The absorbance was then measured at a wavelength of 490 nm, and the total sugar content was calculated by substituting the absorbance into the standard curve equation of the phenol-sulfuric acid method.

[0118] The reducing sugar content in the sample solution was detected by the DNS method. 1 mL of the diluted sample solution was taken into a test tube, and 2 mL of DNS solution was added. The reaction was stopped by boiling in water for 5 min and then by ice bath. The absorbance at 540 nm was detected and substituted into the DNS standard curve to calculate the reducing sugar content.

[0119] Polysaccharide content (mg / mL) = total sugar content - reducing sugar content.

[0120] See the results Figures 2 to 7 .

[0121] According to Figures 2 to 4 From the growth curves of the three probiotics, it can be seen that the Chinese herbal medicines with better promoting effects on NCFM bacteria are Ganoderma lucidum spore powder + Hericium erinaceus + Flammulina velutipes (1:1:1) > Lentinus edodes > Dictyophora japonica > Astragalus membranaceus + Morinda officinalis (1:1); the Chinese herbal medicines with better promoting effects on Bi-07 bacteria are Ganoderma lucidum spore powder + Hericium erinaceus + Flammulina velutipes (1:1:1) > Lentinus edodes > Dictyophora japonica > Tremella fuciformis; the Chinese herbal medicines with better promoting effects on HN001 bacteria are: Astragalus membranaceus + Morinda officinalis (1:1) > Lentinus edodes > Dictyophora japonica > Tremella fuciformis. Among the three strains, HN001 had the best growth. Among the seven Chinese herbal medicines or combinations, Ganoderma lucidum spore powder + Hericium erinaceus + Flammulina velutipes (1:1:1) was the Chinese herbal medicine group with the best promoting effect on NCFM and Bi-07 bacteria; the best promoting effect on HN001 bacteria was Astragalus membranaceus + Morinda officinalis (1:1); Lentinus edodes and Dictyophora japonica ranked second and third in promoting effect on the three strains.

[0122] according to Figures 5 to 7 From the polysaccharide content before and after fermentation, it can be seen that the reduction of polysaccharide content in the fermentation broth of Bi-07 bacteria is in the order of shiitake mushroom > bamboo fungus > Ganoderma lucidum spore powder + Hericium erinaceus + Flammulina velutipes (1:1:1) > Tremella fuciformis; the reduction of polysaccharide content in the fermentation broth of HN001 bacteria is in the order of Tremella fuciformis > shiitake mushroom > Astragalus + Morinda officinalis (1:1) > Ganoderma lucidum spore powder + Hericium erinaceus + Flammulina velutipes (1:1:1) > Poria cocos > bamboo fungus > Ganoderma lucidum; the reduction of polysaccharide content in the fermentation broth of NCFM bacteria is in the order of Tremella fuciformis > bamboo fungus > Ganoderma lucidum + Hericium erinaceus + Flammulina velutipes (1:1:1) > shiitake mushroom > Astragalus + Morinda officinalis (1:1) > Ganoderma lucidum > Poria cocos.

[0123] The growth curves illustrate the growth of different bacteria in different herbal extracts. The reduction in polysaccharide content indicates the extent to which different bacteria utilize the extracts. Based on the growth curves of the three bacteria, it can be seen that the top four herbal extracts promoting the growth of the three bacteria are all from Lentinus edodes and Dictyophora indicum. Ganoderma lucidum spore powder + Hericium erinaceus + Flammulina velutipes (1:1:1), Astragalus membranaceus + Morinda officinalis (1:1), and Tremella fuciformis rank in the top four for promoting the growth of the two bacteria. Considering that Ganoderma lucidum spore powder, Astragalus membranaceus, and Morinda officinalis are not medicinal or edible Chinese herbs, the Hericium erinaceus-Enoki mushroom combination (1:1) and Tremella fuciformis were selected. In summary, Lentinus edodes, Hericium erinaceus:Enoki mushroom (1:1), Dictyophora indicum, and Tremella fuciformis were selected for subsequent experiments.

[0124] Example 2 Screening of the best Chinese herbal medicine combination

[0125] The freeze-dried bacterial powders of Bi-07, HN001, and NCFM were weighed and dissolved in PBS according to the ratio (NCFM:Bi-07:HN001=150:100:11.1) to obtain a mixed bacterial solution. The viable bacterial count of NCFM was 2×10 11 CFU / g, the number of viable bacteria of Bi-07 is 3×10 11 CFU / g, the viable bacterial count of HN001 was 4.5×10 11 CFU / g, add 7mL of water extract formula of different Chinese herbal medicine combinations and 7mL MRS liquid culture medium at a 2% (v / v) inoculation volume; set up a control group, replace the Chinese herbal medicine water extract with distilled water, and use three parallel tubes in each group to measure the absorbance value; take the same system into a Hungate anaerobic tube for measuring gas production.

[0126] The groups are as follows:

[0127] Table 2

[0128]

[0129] 1. Growth curve determination

[0130] As in Example 1, the OD600 values ​​of each group were measured at 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, and 32 h, and the growth curves of the mixed bacterial solution in different groups of Chinese herbal medicine water extracts were plotted.

[0131] 2. Determination of polysaccharide content before and after fermentation

[0132] As in Example 1, the phenol-sulfuric acid method combined with the DNS method was used to draw a standard curve and determine the polysaccharide content.

[0133] The mixed culture broth before fermentation and the mixed culture broth after fermentation were centrifuged at 8000 rpm / min for 10 min, the supernatant was taken, ethanol was added to a final concentration of 80%, the mixture was precipitated overnight, and the mixture was centrifuged at 5000 rpm / min for 10 min. The supernatant was discarded, and the ethanol was evaporated by vacuum concentration. The ethanol was redissolved and diluted to an appropriate concentration with distilled water. The total sugar content in the sample solution was determined by the phenol-sulfuric acid method. 1 mL of the sample solution was placed in a test tube, 1.0 mL of 3% phenol and 4.0 mL of concentrated sulfuric acid (added slowly) were added, the solution was shaken and cooled, and the solution was placed at room temperature for 30 min. The absorbance was then measured at a wavelength of 490 nm, and the total sugar content was calculated by substituting the absorbance into the standard curve equation of the phenol-sulfuric acid method.

[0134] The reducing sugar content in the sample solution was detected by the DNS method. 1 mL of the diluted sample solution was taken into a test tube, and 2 mL of DNS solution was added. The reaction was stopped by boiling in water for 5 min and then by ice bath. The absorbance at 540 nm was detected and substituted into the DNS standard curve to calculate the reducing sugar content.

[0135] Polysaccharide content (mg / mL) = total sugar content - reducing sugar content.

[0136] 3. Determination of acid and gas production

[0137] Take samples every 4 hours and use a pH measuring pen to measure the pH value and analyze the acid production.

[0138] The gas production was measured every 4 h using the syringe calibration method: a disposable syringe was used to measure the amount of gas during the fermentation process. The anaerobic tube was removed at the corresponding time point, and the syringe needle tip was inserted into the tube through the double-ended rubber stopper of the anaerobic tube. Air was pumped to the bottom and waited for rebound. The final height to which the piston rose was the gas production (mL).

[0139] 4. Determination of short-chain fatty acids

[0140] Samples were taken before and after fermentation and stored at -20°C to determine the content of short-chain fatty acids.

[0141] Take 1 mL of fermentation sample and centrifuge at 13000 rpm / min for 5 minutes. Pass the supernatant through a 0.22 μm aqueous filter. Pipette 0.5 mL of the filtrate, add 0.2 mL of 10% (v / v) sulfuric acid, and vortex for 1 minute to mix. Add 0.4 mL of anhydrous ether, vortex and mix, let stand for 2 minutes, and centrifuge at 13000 rpm / min for 2 minutes. Pass the supernatant through a 0.22 μm organic filter membrane into a sample injection bottle for determination by Agilent 7890B gas chromatograph.

[0142] The external standard method was used to prepare the standard curve. First, each short-chain fatty acid was accurately weighed in the same centrifuge tube, and the weight of each short-chain fatty acid was recorded. It was dissolved in ether solvent to prepare a mother liquor, and then gradient diluted to 2, 4, 6, 8, and 10 times to prepare a mixed standard solution. Three parallel solutions were set for each gradient, and the samples were passed through a 0.22 μm organic filter membrane in an injection bottle for determination by an Agilent 7890B gas chromatograph.

[0143] See the results Figures 8 to 11 .

[0144] Depend on Figure 8 The mixed bacteria growth curve showed that the growth of mixed bacteria in the Chinese herbal formula groups of Lentinus edodes + Hericium erinaceus: Flammulina velutipes + Tremella fuciformis and Hericium erinaceus: Flammulina velutipes + Dictyophora fuciformis were significantly higher than that in the Lentinus edodes + Hericium erinaceus: Flammulina velutipes + Dictyophora fuciformis group. However, compared with the control group, the four Chinese herbal formula combinations all promoted the growth of mixed bacteria. Figure 9 As can be seen from the pH values, the pH values ​​of all Chinese medicine combinations gradually decreased with the extension of fermentation time, but there was no significance; no obvious gas production was found in all groups. Figure 11 The results of the determination of short-chain fatty acids only detected acetic acid, and the amount of acetic acid produced and Figure 10 The differences in polysaccharides were the highest in shiitake mushrooms + hericium erinaceus: enoki mushrooms + bamboo fungus. Based on the above four indicators, a Chinese herbal medicine water extract group with a mass ratio of 1:0.5:0.5:1 of shiitake mushroom water extract, hericium erinaceus water extract, enoki mushroom water extract, and bamboo fungus water extract was selected for prebiotic screening experiments.

[0145] Example 3 Screening for the best prebiotic combination

[0146] Weigh proportionally lyophilized bacterial powders of Bi-07, HN001, and NCFM and dissolve them in PBS to create a mixed bacterial solution. Add MRS sugar-free liquid medium, an optimally formulated Chinese herbal medicine extract, and prebiotics. The mass ratio of Chinese herbal medicine:probiotics:prebiotics was 350:0.97:70, with a final prebiotic concentration of 10 mg / mL. A control group without prebiotics was established, with three replicates per group. The experimental groups are shown in the table below.

[0147] Table 3

[0148]

[0149] 1. Growth curve determination

[0150] As in Example 1, the growth curves of the mixed bacterial solution in different proportions of prebiotic groups were plotted.

[0151] 2. Acid production determination

[0152] Similar to Example 2, the acid production of mixed bacteria in prebiotic groups with different ratios was analyzed.

[0153] 3. Determination of short-chain fatty acids

[0154] Similar to Example 2, the short-chain fatty acid content produced by mixed fermentation of prebiotic groups with different ratios was analyzed.

[0155] from Figure 12 From the perspective of mixed bacteria growth, the growth of groups A and C was better, both significantly higher than that of group B, indicating that inulin + oligofructose and inulin + oligofructose + oligoxylose had a better effect on promoting the growth of probiotics. Figure 13 From the perspective of the pH value of the fermentation liquid, the pH of each group was significantly lower than that of the control group, but there was no difference between the groups. Figure 14 In terms of the production of short-chain fatty acids, group A produced more acetic acid than groups B and C, but less than the blank control group.

[0156] Based on the screening of the three components of Chinese herbal medicine, probiotics and prebiotics, the product formula was finally selected with a mass ratio of shiitake mushroom, hericium erinaceus, enoki mushroom and bamboo fungus of 1:0.5:0.5:1, a mass ratio of probiotics of NCFM:Bi-07:HN001=150:100:11.1, and a mass ratio of prebiotics of inulin: oligofructose=3:1. The formula mass ratio was Chinese medicine: probiotics: prebiotics=350:0.97:70, and this ratio was used in subsequent immune experiments.

[0157] Example 4

[0158] Except for the normal group, all groups were treated with cyclophosphamide to create an immunodeficiency model. The probiotic group was gavaged with compound probiotics (Lactobacillus acidophilus, Bifidobacterium lactis, and Lactobacillus rhamnosus), the prebiotic group was gavaged with compound prebiotics (inulin and oligofructose), the Chinese medicine liquid group was gavaged with a water extract of a combination of Chinese herbal medicines (Shiitake mushrooms + Hericium erinaceus: Flammulina velutipes + Dictyophora flavescentis), and the compound product group was gavaged with a water extract of Chinese medicines, probiotics, and prebiotics.

[0159] Healthy five-week-old BALB / c female mice weighing 16-19g were randomly divided into a normal group, a model group, a positive control group, a probiotic group, a Chinese medicine group, a prebiotic group, and a combination formula group, with 12 animals per group. Except for the normal group, mice in all other groups received intraperitoneal injections of cyclophosphamide at a dose of 100 mg / kg for three consecutive days to establish an immunocompromised model. The normal group received intraperitoneal injections of normal saline. The positive control group received oral administration of levamisole hydrochloride.

[0160] The positive control group received 40 mg / kg of levamisole hydrochloride by gavage for 7 consecutive days; the probiotic group received 39.6 mg / kg of probiotics daily by gavage. The traditional Chinese medicine group received 118.8 mg / kg (based on the Chinese herbal medicine) of the traditional Chinese medicine extract daily by gavage for 7 consecutive days; the prebiotic group received 224.13 mg / kg of the prebiotic daily by gavage for 7 consecutive days; and the combined formula group received 39.6 mg / kg of probiotics, 118.8 mg / kg (based on the Chinese herbal medicine) of the traditional Chinese medicine extract, and 224.13 mg / kg of the prebiotic daily by gavage for 7 consecutive days.

[0161] The immune system is an essential defense mechanism for living things, consisting of immune organs, immune cells, and immune molecules. The thymus is the main immune organ in animals, and the thymus index can, to a certain extent, reflect the body's nonspecific immune capacity. T lymphocytes are extremely important immune cells in the body and participate in cellular immunity. The spleen, as an organ where T lymphocytes gather and participate in immune responses, can exert immunoregulatory effects through T cell-mediated cellular immunity. Measuring the in vitro proliferation response of lymphocytes is one of the important indicators for detecting lymphocyte function. CON A-induced spleen lymphocyte proliferation can be used to detect the proliferation level of T lymphocytes. Therefore, the thymus index and CON A-induced spleen lymphocyte transformation stimulation index were used to detect the effects of each group.

[0162] Thymus index statistics are shown in Figure 15 The results showed that compared with the normal group, the thymus index of the model group was significantly reduced (p<0.0001), indicating that the model was successful and the immune function of the mice was significantly suppressed. The thymus index of the positive control group and the compound product group was significantly increased (p<0.05), and no obvious effect was observed in other components. This shows that the compound product group has a synergistic effect compared with other single components, and has a certain effect on improving the immune dysfunction caused by CTX.

[0163] The statistical results of CON A-induced spleen lymphocyte transformation stimulation index are shown in Figure 16 The results showed that CON A-induced splenic lymphocyte proliferation in the model group was significantly inhibited compared with the normal group (p<0.05), indicating that CTX suppressed the specific immune function of the mice and successfully established the model. Compared with the model group, the probiotic group showed a trend of increasing CON A-induced splenic lymphocyte proliferation, but the difference was not significant. The combined product group significantly increased CON A-induced splenic lymphocyte proliferation (p<0.01), indicating that the combined product had a synergistic effect with CON A compared with the other single components, promoting splenic lymphocyte proliferation in immunocompromised mice and enhancing the body's immune function.

[0164] The above specific embodiments provide a detailed description of the present invention. However, the present invention is not limited to the above embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with each other unless there is a conflict.

Claims

1. A Chinese herbal medicine composition for promoting the growth of probiotics, the Chinese herbal medicine composition is composed of an aqueous extract of shiitake mushrooms, an aqueous extract of hericium erinaceus, an aqueous extract of Flammulina velutipes and an aqueous extract of bamboo fungus, wherein the mass ratio of the aqueous extract of shiitake mushrooms, the aqueous extract of hericium erinaceus, the aqueous extract of Flammulina velutipes and the aqueous extract of bamboo fungus is 1:0.5:0.5:1; The preparation method of the shiitake mushroom water extract comprises: adding water to the Chinese herbal medicine shiitake mushroom for extraction, filtering and centrifuging, and collecting the supernatant, wherein the material-liquid ratio of the Chinese herbal medicine shiitake mushroom to water is 1:30; The preparation method of the water extract of Hericium erinaceus comprises: adding water to the Chinese hericium erinaceus for extraction, filtering and centrifuging, and collecting the supernatant, wherein the solid-liquid ratio of the Chinese hericium erinaceus to water is 1:30; The method for preparing the Flammulina velutipes water extract comprises: adding water to the Chinese herbal medicine Flammulina velutipes for extraction, filtering and centrifuging, and collecting the supernatant, wherein the solid-liquid ratio of the Chinese herbal medicine Flammulina velutipes to water is 1:30; The preparation method of the bamboo fungus water extract comprises: adding water to the Chinese herbal medicine bamboo fungus for extraction, filtering and centrifuging, and collecting the supernatant, wherein the solid-liquid ratio of the Chinese herbal medicine bamboo fungus to water is 1:30; The extraction conditions are: extraction temperature is 80°C; and, extraction time is 2h; The probiotics are a mixture of Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus rhamnosus.

2. Use of the Chinese herbal medicine composition according to claim 1 in promoting the growth of probiotics and / or preparing a product that promotes the growth of probiotics; the probiotics are a mixture of Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus rhamnosus.

3. A composite composition, wherein the raw material components of the composite composition include the Chinese herbal medicine composition according to claim 1 and probiotics; the probiotics are a mixed bacteria of Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus rhamnosus; and the preparation method of the composite composition comprises: The raw material components of the complex composition are added into the basic culture medium for fermentation.

4. The composite composition according to claim 3, characterized in that The raw material components of the composite composition also include prebiotics, which include at least two of inulin, oligofructose and oligoxylose; the mass ratio of the Chinese herbal composition, probiotics and prebiotics is 600-1000:0.5-1.5:100-250.

5. A method for preparing the composite composition according to any one of claims 3 to 4, comprising: The raw material components of the complex composition are added into the basic culture medium for fermentation.

6. Use of the Chinese herbal medicine composition according to claim 1 or the composite composition according to any one of claims 3 to 4 in the preparation of products.

7. A product comprising the Chinese herbal composition according to claim 1 or the composite composition according to any one of claims 3 to 4.

8. A method for promoting the growth of probiotics, comprising: Adding the Chinese herbal medicine composition according to claim 1 to the culture medium of probiotics; The probiotics are a mixture of Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus rhamnosus.

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