CN117925444A - Chinese herbal medicine composition for promoting growth of probiotics and application thereof - Google Patents
Chinese herbal medicine composition for promoting growth of probiotics and application thereof Download PDFInfo
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- CN117925444A CN117925444A CN202311675802.8A CN202311675802A CN117925444A CN 117925444 A CN117925444 A CN 117925444A CN 202311675802 A CN202311675802 A CN 202311675802A CN 117925444 A CN117925444 A CN 117925444A
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- chinese herbal
- probiotics
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Classifications
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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Abstract
The invention discloses a Chinese herbal medicine composition for promoting growth of probiotics and application thereof; the Chinese herbal medicine composition comprises mushrooms, hericium erinaceus, flammulina velutipes and bamboo fungi; the invention also carries out in vitro experiments based on the Chinese herbal medicine composition by adopting a mode of 'multi-bacteria multi-prebiotics multi-medicine', researches the interaction relation among different components and explores the synergy of the components on the solid beverage; finally, a product formula consisting of lentinus edodes, hericium erinaceus, flammulina velutipes, bamboo fungus, lactobacillus acidophilus, bifidobacterium lactis, lactobacillus rhamnosus, inulin, fructo-oligosaccharide and xylo-oligosaccharide is obtained; the Chinese herbal medicines are used as raw materials, and the combined action of probiotics and prebiotics is utilized; the synergistic effect of the three components is more obvious, the active value of the three components is improved by combining the fermentation technology, and a new concrete scheme is provided for the concept of a synbiotic preparation; a novel functional food meeting the dietary requirements of modern people is obtained.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a Chinese herbal medicine composition for promoting growth of probiotics and application thereof.
Background
The probiotics change the efficacy of the Chinese herbal medicines or increase the synergistic effect by influencing the metabolism, transformation, absorption and other effects of the effective components of the Chinese herbal medicines. After microbial fermentation, active macromolecular substances in the Chinese herbal medicine are converted into small molecular substances which can be directly absorbed by animal intestinal tracts, and the small molecular substances are fully metabolized in animal bodies, so that medicine residues are avoided. The prebiotics produced by the Chinese herbal medicines after fermentation promote the propagation of microorganisms, and the microorganisms can promote the absorption and utilization of the Chinese herbal medicines. Meanwhile, the prebiotics can promote the growth of beneficial bacteria in the intestinal tract, increase the quantity of the beneficial bacteria and play a role in keeping the health of the intestinal tract. The three components complement each other and cooperate to better play the role of regulating immunity. The combination of probiotics, prebiotics and traditional Chinese medicines is an innovation point of functional food and traditional Chinese medicine research and development, and is also a new idea of traditional Chinese medicine modernization research.
There are many probiotics, prebiotic beverages and probiotic fermented traditional Chinese medicine products on the market at present, for example, gu Jinsong [1] et al patent describes a probiotic composition and application thereof, a probiotic product and a preparation method thereof, and the combined action of probiotics and polysaccharide is realized. However, there are relatively few products that utilize probiotics to ferment traditional Chinese medicine and prebiotics to make solid beverages.
Most of the existing probiotic products have improved taste by adding additives or sweeteners to the probiotic, for example Tang Zhenxing [2] et al describe a product in which the probiotic is embedded with sodium alginate, which has the disadvantage that there are many unknown factors for the long-term health effects of the additives; secondly, the activity of probiotics cannot be guaranteed, and the activity of probiotics is difficult to fully exert.
[1] Gu Jinsong, Qi Cuihua, zhangyu, etc. composition prepared by fermenting Chinese medicinal preparation with probiotics, and its preparation method and application are CN201210527023.9[ P ]. CN102934809A.
[2] Tang Zhenxing, xu Zheng, liu Ying, et cetera, a probiotic preparation and a method for preparing the same are disclosed in CN202210204681.8[ P ].2022.
Disclosure of Invention
The invention aims to provide a Chinese herbal medicine compound capable of promoting the growth of probiotics, and a multi-bacteria multi-prebiotic multi-medicine probiotic product is prepared based on the Chinese herbal medicine compound and the probiotics, the utilization rate of Chinese herbal medicine is improved, the probiotics can also keep the activity of the probiotics, and the three components supplement each other, so that the functional activity of the probiotic is fully exerted, and the immunity of an organism is improved; meanwhile, the three components in the formula are natural products, so that a large burden can not be generated on a human body.
The technical scheme adopted by the invention is as follows:
In a first aspect of the present invention, a herbal composition is provided, 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 indusiata.
Preferably, the mass ratio of the mushroom water extract, the hericium erinaceus water extract, the flammulina velutipes water extract and the bamboo fungus water extract is (1-3): (0.5-1): (0.5-1): (1-3).
Preferably, the preparation method of the water extract comprises the following steps: extracting Lentinus Edodes, hericium Erinaceus, needle Mushroom and/or Dictyophora Indusiata with water, filtering, centrifuging, and collecting supernatant.
Preferably, the feed liquid ratio of the Chinese herbal medicines of the mushrooms, the hericium erinaceus, the flammulina velutipes and/or the bamboo fungus to the water is 1:25-40.
Preferably, the conditions of the extraction are: the extraction temperature is 70-90 ℃; and/or the extraction time is 1-4 h.
Preferably, the filtering condition is that filtering is performed by using filter cloth with the pore diameter of 150-250 meshes.
Preferably, the number of times of filtration is 1 to 3.
Preferably, the centrifugation conditions are: the centrifugal rotation speed is 4000-6000 rpm; and/or the centrifugation time is 8-12 min.
Preferably, the concentration of the mushroom water extract, the hericium erinaceus water extract, the flammulina velutipes water extract and the bamboo fungus water extract is 150-250 mg/mL.
Preferably, the aqueous extract may be further sterilized after preparation, including but not limited to high temperature autoclaving.
In a second aspect of the invention there is provided the use of a composition according to the first aspect of the invention for promoting the growth of a probiotic and/or for the preparation of a product for promoting the growth of a probiotic.
Preferably, the probiotics comprise at least one of lactobacillus acidophilus, bifidobacterium lactis and lactobacillus rhamnosus.
Preferably, the viable count of the lactobacillus acidophilus is 1×10 10～1×1012 CFU/g.
Preferably, the bifidobacterium lactis is 1X 10 10～1×1012 CFU/g.
Preferably, the lactobacillus rhamnosus is 1×10 10～1×1012 CFU/g.
Preferably, the mass ratio of lactobacillus acidophilus, bifidobacterium lactis and lactobacillus rhamnosus is (10-20): (5-15): 1.
Preferably, the lactobacillus acidophilus includes, but is not limited to, lactobacillus acidophilus NCFM.
Preferably, the bifidobacterium lactis includes, but is not limited to, bifidobacterium lactis Bi-07.
Preferably, the lactobacillus rhamnosus includes, but is not limited to lactobacillus rhamnosus HN001.
In a third aspect of the present invention, there is provided a composite composition comprising the herbal composition of the first aspect of the present invention and a probiotic.
Preferably, the composite composition further comprises a prebiotic.
Preferably, the mass ratio of the Chinese herbal medicine composition to the probiotics to the prebiotics is 600-1000:0.5-1.5:100-250;
preferably, the mass ratio of the Chinese herbal medicine composition to the probiotics to the prebiotics is 700-800:0.9-1.1:140-200;
preferably, the mass ratio of the Chinese herbal medicine composition to the probiotics to the prebiotics is 350:0.97:70.
Preferably, the prebiotic comprises at least two of inulin, fructo-oligosaccharide, xylo-oligosaccharide.
Preferably, the prebiotic comprises inulin, fructooligosaccharides.
Preferably, the mass ratio of the inulin to the fructo-oligosaccharide to the xylo-oligosaccharide is (2-4): (0.5-1.5): 1.
Preferably, the mass ratio of the inulin to the fructo-oligosaccharide is (2-4): 1.
Preferably, the probiotics comprise at least one of lactobacillus acidophilus, bifidobacterium lactis and lactobacillus rhamnosus.
Preferably, the mass ratio of lactobacillus acidophilus, bifidobacterium lactis and lactobacillus rhamnosus is (10-20): (5-15): 1.
Preferably, the viable count of the lactobacillus acidophilus is 1×10 10～1×1012 CFU/g.
Preferably, the viable count of the bifidobacterium lactis is 1X 10 10～1×1012 CFU/g.
Preferably, the viable count of the lactobacillus rhamnosus is 1×10 10～1×1012 CFU/g.
In a fourth aspect of the invention, there is provided a method of promoting the growth of probiotics comprising: the Chinese herbal medicine composition according to the first aspect of the invention is added to the probiotic culture medium.
Preferably, the medium further comprises a prebiotic.
Preferably, the mass ratio of the mushroom water extract, the hericium erinaceus water extract, the flammulina velutipes water extract and the bamboo fungus water extract is (1-3): (0.5-1): (0.5-1): (1-3).
Preferably, the concentration of the mushroom water extract, the hericium erinaceus water extract, the flammulina velutipes water extract and the bamboo fungus water extract is 150-250 mg/mL.
Preferably, the probiotics comprise at least one of lactobacillus acidophilus, bifidobacterium lactis and lactobacillus rhamnosus.
Preferably, the mass ratio of lactobacillus acidophilus, bifidobacterium lactis and lactobacillus rhamnosus is (10-20): (5-15): 1.
Preferably, the viable count of the lactobacillus acidophilus is 1×10 10～1×1012 CFU/g.
Preferably, the viable count of the bifidobacterium lactis is 1X 10 10～1×1012 CFU/g.
Preferably, the viable count of the lactobacillus rhamnosus is 1×10 10～1×1012 CFU/g.
Preferably, the prebiotic comprises at least two of inulin, fructo-oligosaccharide, xylo-oligosaccharide.
Preferably, the prebiotic comprises inulin, fructooligosaccharides.
Preferably, the mass ratio of the inulin to the fructo-oligosaccharide to the xylo-oligosaccharide is (2-4): (0.5-1.5): 1.
Preferably, the mass ratio of the inulin to the fructo-oligosaccharide is (2-4): 1.
Preferably, the medium includes, but is not limited to, MRS medium, LB medium, M17 medium, and the like.
In a fifth aspect of the present invention, there is provided a method for preparing the composite composition according to the third aspect of the present invention, comprising: and adding the raw material components of the composite composition into a basic culture medium for fermentation.
Preferably, the volume ratio of the Chinese herbal medicine to the basic culture medium in the raw material components of the composite composition is 1 (0.5-2).
Preferably, the probiotic bacteria in the raw material components of the composite composition are inoculated in an amount of 1-4%.
Preferably, the final concentration of prebiotics in the feed components of the composite composition is 5-15 mg/mL.
Preferably, the fermentation time is 8-32 hours.
Preferably, the fermentation time is 12-24 hours.
Preferably, the conditions of the fermentation are anaerobic fermentation.
In a sixth aspect of the present invention there is provided the use of a herbal composition according to the first aspect of the present invention or a composite composition according to the third aspect of the present invention in the preparation of a product.
Preferably, the function of the product comprises enhancing immunity.
Preferably, the product further comprises at least one of a pharmaceutically or food acceptable carrier, excipient, diluent.
Preferably, the product is in a dosage form selected from at least one of solutions, suspensions, granules, emulsions, powders, lozenges, pills, troches, tablets, chewing gums, slurries and capsules.
Preferably, the product comprises at least one of a food, a pharmaceutical, a feed additive.
Preferably, the food product comprises at least one of a beverage, a biscuit, a candy, a pastry, a dairy product.
In a seventh aspect of the present invention, there is provided a product comprising the herbal composition of the first aspect of the present invention or the complex composition of the third aspect of the present invention.
Preferably, the function of the product comprises enhancing immunity.
Preferably, the product further comprises at least one of a pharmaceutically or food acceptable carrier, excipient, diluent.
Preferably, the product is in a dosage form selected from at least one of solutions, suspensions, granules, emulsions, powders, lozenges, pills, troches, tablets, chewing gums, slurries and capsules.
Preferably, the product comprises at least one of a food, a pharmaceutical, a feed additive.
Preferably, the food product comprises at least one of a beverage, a biscuit, a candy, a pastry, a dairy product.
The beneficial effects of the invention are as follows:
In order to fully discover the medicinal value of the Chinese herbal medicine and improve the bioavailability of the Chinese herbal medicine while keeping the active ingredients of the raw materials, the invention selects various Chinese herbal medicines to obtain the water extract of the Chinese herbal medicine, and the extraction mode can efficiently extract the active ingredients and the nutritional ingredients of the Chinese herbal medicine, fully keep the biological activity and the medicinal value of the Chinese herbal medicine and promote the growth of probiotics; in the invention, an in vitro experiment is carried out by adopting a mode of 'multi-bacteria multi-prebiotic multi-drug', the interaction relation among different components is researched, and the synergy of the components on the solid beverage is researched; finally, a product formula consisting of lentinus edodes, hericium erinaceus, flammulina velutipes, bamboo fungus, lactobacillus acidophilus, bifidobacterium lactis, lactobacillus rhamnosus, inulin, fructo-oligosaccharide and xylo-oligosaccharide is obtained; the Chinese herbal medicines are used as raw materials, and the combined action of probiotics and prebiotics is utilized. The synergy of the Chinese herbal medicine composition, the probiotics and the prebiotics is more remarkable, and the immunoregulation function of the Chinese herbal medicine composition is better exerted; the combined fermentation technology improves the activity value of the plant extract and provides a new concrete scheme for the concept of a synbiotic preparation; a novel functional food meeting the dietary requirements of modern people is obtained; not only improves the activity value and the utilization rate of the Chinese herbal medicine raw materials, but also provides a new idea for reutilization of Chinese herbal medicine resources.
Drawings
FIG. 1 is a technical roadmap of the invention.
FIG. 2 shows the growth curves of Bi-07 in aqueous extracts of different Chinese herbal medicines.
FIG. 3 shows the growth curves of HN001 in different aqueous extracts of Chinese herbal medicines.
FIG. 4 shows the growth curves of NCFM in various aqueous extracts of herbs.
FIG. 5 shows the polysaccharide reduction before and after fermentation of Bi-07 and Chinese herbal medicine combinations.
FIG. 6 shows the polysaccharide reduction before and after HN001 and Chinese herbal medicine combined fermentation.
FIG. 7 shows the polysaccharide reduction before and after fermentation of NCFM and Chinese herbal medicine combination.
FIG. 8 shows the growth curve of the water extract of the mixed fermentation Chinese herbal medicine formula.
FIG. 9 shows the pH of the aqueous extract of the mixed fermentation herbal formulation.
FIG. 10 shows the polysaccharide reduction before and after mixed fermentation.
FIG. 11 shows the short chain fatty acid production before and after mixed fermentation.
FIG. 12 shows growth curves of mixed bacteria solutions at different proportions of prebiotics.
FIG. 13 shows pH of fermentation broths fermented with mixed bacteria in different proportions of prebiotics.
Figure 14 shows the short chain fatty acid production of different proportions of prebiotic tuples by mixed fermentation.
Fig. 15 is a thymus index.
FIG. 16 is a graph showing the CON A-induced spleen lymphocyte transformation stimulation index.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
The invention adopts the experimental form of 'multi-bacteria multi-prebiotics multi-medicine', greatly improves the utilization rate of Chinese herbal medicines, and the prebiotics in the formula can also simultaneously maintain the activity of the probiotics, and the three components supplement each other to fully exert the functional activity of the probiotics; meanwhile, the three components in the formula are natural products, so that a large burden can not be generated on a human body. The best Chinese herbal medicine, probiotics and prebiotics are obtained through screening by the technical route of fig. 1, and the ingredients are synergistically enhanced, so that the immunity of the organism can be enhanced.
Probiotic Bi-07: HN001: NCFM was purchased from Denmark (China) Inc., and stored at 4 ℃.
MRS culture medium formula: 10.0g/L peptone, 8.0g/L beef powder, 4.0g/L yeast powder, 20.0g/L glucose, 2.0g/L dipotassium hydrogen phosphate, 2.0g/L diammonium hydrogen citrate, 5.0g/L sodium acetate, 0.2g/L magnesium sulfate, 0.04g/L manganese sulfate and 1.0g/L Tween 80.
EXAMPLE 1 screening of the best Chinese herbal medicine
In order to screen the optimal Chinese herbal medicine, the present example used three probiotics to ferment a plurality of Chinese herbal medicines, and examined the growth of three probiotics (NCFM viable count of 2×10 11 CFU/g, bi-07 viable count of 3×10 11 CFU/g, HN001 viable count of 4.5×10 11 CFU/g) and polysaccharide content before and after fermentation, thereby determining the screening of the optimal Chinese herbal medicine. Wherein the Chinese herbal medicine comprises: ganoderma lucidum, dictyophora Indusiata, ganoderma lucidum spore powder, hericium erinaceus, needle mushroom (1:1:1), radix astragali, radix Morindae officinalis (1:1), tremella, poria, and Lentinus Edodes, wherein the weight ratio of the Chinese herbal medicines is the same as below; the method comprises the following specific steps:
1. Preparation of Chinese herbal medicine water extract
Mixing the crushed dry Chinese herbal medicines with water according to a feed liquid ratio of 1:30, extracting for 2 hours at 80 ℃, passing through 200 meshes of Long Lvbu, collecting filtrate, repeating twice, combining the filtrate, centrifuging at 5000rpm for 10min, collecting supernatant, concentrating and fixing volume to obtain 200mg/mL water extract, sterilizing at 121 ℃ for 15min at high temperature and high pressure in a sterilizing pot to obtain ganoderma lucidum, bamboo fungus, ganoderma lucidum spore powder, hericium erinaceus, flammulina velutipes (1:1:1), astragalus membranaceus, morinda officinalis (1:1), tremella, poria cocos and lentinus edodes water extract.
2. Fermentation of water extract of Chinese herbal medicine
Diluting the prepared ganoderma lucidum, bamboo fungus, ganoderma lucidum spore powder, hericium erinaceus, flammulina velutipes (1:1), astragalus membranaceus, morinda officinalis (1:1), tremella, poria cocos and lentinus edodes water extract to the concentration of the traditional Chinese medicine water extract determined in the step 1, adding 7mL of the traditional Chinese medicine water extract with corresponding concentration and 2% inoculum size bacteria liquid into 7mL of MRS culture medium, setting a control group, replacing the traditional Chinese medicine water extract with distilled water, and carrying out the process in an anaerobic environment of 37 ℃ and 5% CO 2 in a total of 32 groups, wherein the steps are as follows;
TABLE 1
| Grouping | Fermentation system |
| Traditional Chinese medicine adding group | 7ML of culture medium, 7mL of traditional Chinese medicine water extract and 2% of bacterial liquid |
| Growth control group | 7ML culture medium, 7mL distilled water and 2% bacterial liquid |
| Traditional Chinese medicine control group | 7ML of culture medium, 7mL of traditional Chinese medicine extract and 2% distilled water |
| Blank control group | 7ML of culture medium, 7mL of distilled water and 2% distilled water |
3. Growth curve determination
In the fermentation process, OD600 values of each group are measured at 0h, 4h, 8h, 12h, 16h, 20h, 24h, 28h and 32h, if the platform period is reached, the measurement can be stopped, the culture time is taken as an abscissa, the OD600 value is taken as an ordinate, and growth curves of Bi-07, HN001 and NCFM in water extracts of different groups of Chinese herbal medicines are drawn.
Wherein: OD600 = chinese drug addition group OD600 value-chinese drug aqueous extract background OD600 value.
4. Polysaccharide content determination before and after fermentation
And determining the polysaccharide content by adopting a phenol-sulfuric acid method and a DNS method.
(1) Preparation of phenol solution
Heating in a water bath at 50 ℃ to melt phenol, sucking out by a suction pipe, weighing 6g in a light-resistant beaker, dissolving in 194mL warm water, and preserving at 4 ℃ in a light-resistant manner.
(2) Production of standard curve of phenol-sulfuric acid method
Accurately weighing 5mg of anhydrous glucose standard substance (Glu), metering distilled water to a 50mL volumetric flask, preparing 0.1mg/mL glucose standard solution, respectively sucking 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0mL glucose standard solution into test tubes, respectively supplementing distilled water to enable the total liquid amount to reach 1mL, adding 1.0mL of 3% phenol and 4.0mL concentrated sulfuric acid (slowly adding), shaking uniformly, cooling, standing at room temperature for 30min, measuring absorbance under the condition of 490nm wavelength, and plotting the absorbance value as ordinate and each standard concentration (mg/mL) as abscissa to obtain a phenol sulfuric acid method standard curve.
(3) Production of DNS standard curve
Accurately weighing 50mg of anhydrous glucose standard substance (Glu), metering distilled water to a 50mL volumetric flask, preparing 1mg/mL glucose standard solution, respectively sucking 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0mL of glucose standard solution into test tubes, respectively adding distilled water to enable the total liquid amount to reach 1mL, adding 2mL of DNS reagent, boiling water bath for 5min, stopping reaction in ice bath, detecting absorbance at 540nm, and plotting each standard concentration (mg/mL) as an abscissa to obtain a DNS standard curve.
(4) Preparation and determination of test solutions
Respectively taking mixed culture solution before fermentation and mixed culture solution after fermentation, centrifuging at 8000rpm/min for 10min, taking supernatant, adding ethanol to a final concentration of 80%, precipitating with ethanol overnight, centrifuging at 5000rpm/min for 10min, discarding supernatant, vacuum concentrating, volatilizing ethanol, adding distilled water, re-dissolving, diluting to a proper concentration, performing phenol sulfuric acid method to determine the total sugar content in the sample solution, taking 1mL of sample solution, adding 1.0mL of 3% phenol and 4.0mL of concentrated sulfuric acid (slowly adding), shaking, cooling, standing at room temperature for 30min, measuring absorbance at a wavelength of 490nm, and substituting into a standard curve equation of the phenol sulfuric acid method to calculate the total sugar content.
Detecting the content of reducing sugar in the sample solution by a DNS method, taking 1mL of diluted sample solution and a test tube, adding 2mL of DNS solution, stopping the reaction in boiling water bath for 5min in ice bath, detecting absorbance at 540nm, and substituting into a DNS standard curve to calculate the content of the reducing sugar.
Polysaccharide content (mg/mL) =total sugar content-reducing sugar content.
The results are shown in FIGS. 2 to 7.
As can be seen from the growth curves of the three probiotics shown in fig. 2-4, the traditional Chinese medicine with better NCFM (NCFM) bacteria promoting effect is ganoderma lucidum spore powder, hericium erinaceus, flammulina velutipes (1:1:1) > lentinus edodes > bamboo fungus > radix astragali and morinda officinalis (1:1); the traditional Chinese medicine with better promoting effect on Bi-07 bacteria is ganoderma lucidum spore powder, hericium erinaceus and flammulina velutipes (1:1:1) > lentinus edodes > bamboo fungus > tremella; the traditional Chinese medicine with better promoting effect on HN001 bacteria comprises the following components: radix astragali+radix Morindae officinalis (1:1) > Lentinus Edodes > Dictyophora Indusiata > Tremella. Among the three strains, HN001 has best growth condition, and among 7 Chinese herbal medicines or combinations, ganoderma lucidum spore powder, hericium erinaceus and flammulina velutipes (1:1:1) are the Chinese herbal medicine group with best promotion effect on NCFM bacteria and Bi-07 bacteria; the best promoting effect on HN001 bacteria is that astragalus and morinda root (1:1); the promotion effect of the mushrooms and the bamboo fungus on the three bacteria is all arranged in the second and the third.
According to the polysaccharide content before and after fermentation in the figures 5-7, the polysaccharide content in the Bi-07 strain fermentation liquid can be reduced by mushroom > Dictyophora Indusiata > ganoderma lucidum spore powder + hericium erinaceus + flammulina velutipes (1:1:1) > tremella; the reduction amount of polysaccharide content in HN001 bacterial fermentation broth is tremella > mushroom > radix astragali+morinda officinalis (1:1) > ganoderma lucidum spore powder+hericium erinaceus+flammulina velutipes (1:1:1) > poria cocos > bamboo fungus > ganoderma lucidum; the reduction amount of polysaccharide content in NCFM bacterial fermentation liquor is tremella, dictyophora Indusiata, ganoderma lucidum, hericium erinaceus, flammulina velutipes (1:1:1) > lentinus edodes, astragalus membranaceus, morinda officinalis (1:1) > ganoderma lucidum and poria cocos.
The growth curve shows the growth condition of different bacteria in different Chinese herbal medicine water extracts, the reduction of polysaccharide content shows the utilization degree of different bacteria to different Chinese herbal medicine water extracts, the growth condition can be seen according to the growth curve of three bacteria, two Chinese herbal medicines of mushrooms and bamboo fungus are all used for four of the promotion effect ranks of the three bacteria, and the ganoderma lucidum spore powder+hericium erinaceus+flammulina velutipes (1:1:1), the astragalus membranaceus+morinda officinalis (1:1) and the tremella are used for four of the promotion effect ranks of the two bacteria, so that the hericium erinaceus-flammulina velutipes combination (1:1) and tremella are selected in consideration of the fact that the ganoderma lucidum spore powder, the astragalus membranaceus and the morinda officinalis are not medicinal and edible homologous Chinese herbal medicines. In summary, mushrooms and hericium erinaceus are selected: needle mushroom=1:1, bamboo fungus, tremella and subsequent experiments were performed.
EXAMPLE 2 screening of optimal Chinese herbal medicine combinations
Weighing the freeze-dried bacterial powder of Bi-07, HN001 and NCFM according to a proportion (NCFM: bi-07:HN001=150:100:11.1), dissolving the freeze-dried bacterial powder in PBS, compounding to obtain mixed bacterial liquid, wherein the viable count of NCFM is 2X 10 11 CFU/g, the viable count of Bi-07 is 3X 10 11 CFU/g, the viable count of HN001 is 4.5X 10 11 CFU/g, and adding the mixed bacterial liquid into 7mL of water extract formula of different Chinese herbal medicine combinations and 7mL of MRS liquid culture medium according to an inoculum size of 2% (v/v); setting a control group, and replacing the water extract of the Chinese herbal medicine with distilled water, wherein each group is provided with three parallel tubes for measuring absorbance values; the same system was also taken into a Hungate anaerobic tube for measuring gas production.
Grouping the following table;
TABLE 2
1. Growth curve determination
OD600 values of each group are measured in the same way as in examples 1,0h, 4h, 8h, 12h, 16h, 20h, 24h, 28h and 32h, and growth curves of the mixed bacterial liquid in water extracts of different groups of Chinese herbal medicines are drawn.
2. Polysaccharide content determination before and after fermentation
A standard curve was drawn and the polysaccharide content was determined by the phenol-sulfuric acid method in combination with the DNS method as in example 1.
Centrifuging the mixed culture solution before fermentation and the mixed culture solution after fermentation at 8000rpm/min for 10min, taking supernatant, adding ethanol to a final concentration of 80%, precipitating with ethanol overnight, centrifuging at 5000rpm/min for 10min, discarding supernatant, concentrating in vacuum to volatilize ethanol, adding distilled water for re-dissolving and diluting to a proper concentration, measuring the total sugar content in the sample solution by a phenol sulfuric acid method, taking 1mL of the sample solution into a test tube, adding 1.0mL of 3% phenol and 4.0mL of concentrated sulfuric acid (slowly adding), shaking and cooling, standing at room temperature for 30min, measuring absorbance at a wavelength of 490nm, and substituting the absorbance into a standard curve equation of the phenol sulfuric acid method to calculate the total sugar content.
Detecting the content of reducing sugar in the sample solution by a DNS method, taking 1mL of diluted sample solution and a test tube, adding 2mL of DNS solution, stopping the reaction in boiling water bath for 5min in ice bath, detecting absorbance at 540nm, and substituting into a DNS standard curve to calculate the content of the reducing sugar.
Polysaccharide content (mg/mL) =total sugar content-reducing sugar content.
3. Determination of acid and gas production
The pH value is measured by a pH measuring pen every 4 hours for sampling, and the acid production condition is analyzed.
The gas production condition is measured by a syringe graduation method at intervals of 4 hours: and (3) measuring the gas quantity in the fermentation process by adopting a disposable injector, taking out the anaerobic pipe at a corresponding time point, enabling the needle point of the injector to enter the pipe through the rubber plug part at the two ends of the anaerobic pipe, exhausting air to the bottom to wait for rebound, and finally obtaining the rising height of the piston as gas yield (mL).
4. Determination of short-chain fatty acids
Sampling before and after fermentation, preserving at-20deg.C, and determining short chain fatty acid content.
Taking 1mL of fermentation sample, centrifuging at 13000rpm/min for 5min, filtering the supernatant with a 0.22 mu m water filter head, sucking 0.5mL of filtrate, adding 0.2mL of 10% (v/v) sulfuric acid, stirring for 1min, mixing uniformly, adding 0.4mL of anhydrous diethyl ether, stirring uniformly, standing for 2min, centrifuging at 13000rpm/min for 2min, filtering the supernatant with a 0.22 mu m organic filter membrane in a sample injection bottle, and measuring by an Agilent 7890B gas chromatograph.
The standard curve is prepared by adopting an external standard method, each short chain fatty acid is firstly accurately weighed in the same centrifuge tube, the weight of each short chain fatty acid is recorded, the short chain fatty acid is dissolved in an diethyl ether solvent to prepare mother liquor, the mother liquor is diluted to 2, 4, 6, 8 and 10 times in a gradient manner, a mixed standard solution is prepared, three parallel gradients are arranged in each gradient manner, an organic filter membrane with the thickness of 0.22 mu m is arranged in a sample injection bottle, and the concentration is measured by an Agilent7890B gas chromatograph.
The results are shown in FIGS. 8 to 11.
As can be seen from the mixed growth curve of fig. 8, the mushroom and the hericium erinaceus: needle mushroom + tremella and hericium erinaceus: the growth conditions of the needle mushroom, bamboo fungus and tremella traditional Chinese medicine formula mixed bacteria are obviously higher than those of mushroom, hericium erinaceus: the flammulina velutipes and bamboo fungus groups are compared with the control groups, and the combination of the four Chinese herbal medicine formulas promotes the growth of mixed bacteria; as can be seen from the pH conditions of fig. 9, the pH gradually decreased with the increase of fermentation time, but not significantly; no significant gas generation was observed for all groups. Only acetic acid was detected as a result of measurement of short chain fatty acid in FIG. 11, and both the acetic acid production amount and the polysaccharide difference in FIG. 10 were Lentinus edodes + Hericium erinaceus: the golden mushroom and the bamboo fungus are the highest, and the four indexes are combined, and a prebiotic screening experiment is performed by selecting a Chinese herbal medicine liquid extract group with the mass ratio of the mushroom liquid extract to the hericium erinaceus liquid extract to the golden mushroom liquid extract to the bamboo fungus liquid extract of 1:0.5:0.5:1.
Example 3 screening of optimal prebiotic combinations
And weighing the freeze-dried bacterial powder of Bi-07, HN001 and NCFM according to a proportion, dissolving the freeze-dried bacterial powder in PBS, and compounding to obtain the mixed bacterial liquid. Adding MRS sugar-free liquid culture medium, and optimal formula of Chinese herbal medicine water extract and prebiotics, wherein the Chinese herbal medicine: probiotics: the mass ratio of the prebiotics is 350:0.97:70, the final concentration of the prebiotics reaches 10mg/mL, a control group without adding the prebiotics is arranged, three tubes are arranged in parallel in each group, and the experiment groups are as follows;
TABLE 3 Table 3
1. Growth curve determination
The growth curves of the mixed bacterial solutions in the different proportions of the prebiotics were plotted as in example 1.
2. Acid production assay
The acid production of the mixed bacteria in different proportions of the prebiotics was analyzed as in example 2.
3. Short chain fatty acid assay
The short chain fatty acid content produced by the mixed fermentation of different proportions of the prebiotics was analyzed as in example 2.
From the aspect of mixed bacteria growth in FIG. 12, A, C groups are better in growth condition and are obviously higher than that of B groups, which indicates that inulin+fructo-oligosaccharide and inulin+fructo-oligosaccharide+xylo-oligosaccharide have better promotion effect on the growth of probiotics; from the fermentation broth pH of fig. 13, each group had significantly lower pH than the control group, but there was no difference between the groups; from the short chain fatty acid production profile of fig. 14, group a produced acetic acid higher than B, C but lower than the placebo.
And (3) combining screening conditions of three components of Chinese herbal medicines, probiotics and prebiotics, and finally selecting mushrooms: hericium erinaceus, flammulina velutipes and bamboo fungus have a mass ratio of 1:0.5:0.5:1, and probiotics have a mass ratio of NCFM: bi-07: HN001 = 150:100:11.1, prebiotic mass ratio of inulin: fructo-oligosaccharide=3:1 comprises the following components in percentage by mass: probiotics: prebiotics = 350:0.97:70, followed by immunization experiments with this ratio.
Example 4
The treatment of cyclophosphamide in all groups except the normal group causes a model with low immunity, the probiotics group is a stomach-filling compound probiotic (lactobacillus acidophilus, bifidobacterium lactis and lactobacillus rhamnosus), the probiotics group is a stomach-filling compound prebiotic (inulin and fructo-oligosaccharide), the traditional Chinese medicine liquid group is a water extract of a stomach-filling Chinese herbal medicine combination (lentinus edodes+hericium erinaceus: flammulina velutipes+bamboo fungus), and the composite product group is a stomach-filling traditional Chinese medicine water extract, probiotics and prebiotics.
Healthy BALB/c female mice with five weeks of age are taken, the weight is 16-19g, and after being adaptively fed for one week, the mice are randomly divided into a normal group, a model group, a positive control group, a probiotic group, a traditional Chinese medicine group, a probiotic group and a compound formula group, wherein each group comprises 12 animals. Except for the normal group, the mice in other groups are continuously injected with cyclophosphamide for 3 days in the abdominal cavity according to the dosage of 100mg/kg to construct an immunocompromised model, and the normal group is injected with physiological saline in the abdominal cavity. The positive control group was subjected to levamisole hydrochloride gastric lavage treatment.
The positive control group is continuously irrigated with levamisole hydrochloride for 7 days according to the ratio of 40 mg/kg; the probiotic group was perfused with 39.6mg/kg of gastric probiotics per day. The traditional Chinese medicine group is filled with the traditional Chinese medicine liquid extract according to 118.8mg/kg (calculated by the traditional Chinese medicine) for 7 days; the prebiotics are infused with the stomach prebiotics according to 224.13mg/kg each day for 7 days; the compound formula comprises 39.6mg/kg probiotics+118.8 mg/kg (calculated by Chinese medicinal materials) of Chinese medicinal liquid extract+ 224.13mg/kg prebiotics for gastric lavage every day, and the gastric lavage is carried out for 7 days.
The immune system is a necessary defense mechanism for organisms and consists of immune organs, immune cells and immune molecules. Thymus is the main immune organ in the animal body, and the height of thymus index can reflect the nonspecific immunity ability of the organism to a certain extent. T lymphocytes are extremely important immune cells of an organism, participate in cellular immunity, spleen serves as an organ for T lymphocyte aggregation and participate in immune response, can play a role in regulating immunity through T cell mediated cellular immunity, and measurement of lymphocyte in-vitro proliferation response is one of important indexes for detecting lymphocyte functions. Each group was thus tested for effect by thymus index and CON a-induced spleen lymphocyte transformation stimulation index.
The thymus index statistical result is shown in fig. 15, and the results show that compared with the normal group, the thymus index of the model group is obviously reduced (p < 0.0001), which indicates that the model is successful, the immune function of the mice is obviously inhibited, the thymus index (p < 0.05) is obviously improved by the positive control group and the composite product group, and other components have no obvious effect, so that the composite product group has a synergistic effect compared with other single components, and has a certain effect on improving the immune function hypofunction caused by CTX.
The statistical results of the index of the stimulation of the transformation of the spleen lymphocytes induced by CON A are shown in FIG. 16, and the results show that the proliferation of the spleen lymphocytes induced by CON A in the model group is significantly inhibited (p < 0.05) compared with the normal group, which indicates that CTX inhibits the specific immune function of mice and the modeling is successful. Compared with the model group, the probiotics group has the trend of improving the proliferation of the spleen lymphocytes induced by CON A, but has no significance; the compound product group obviously improves the proliferation of the spleen lymphocytes induced by the CON A (p < 0.01), and shows that compared with other single components, the compound product has a synergistic effect, can promote the proliferation of the spleen lymphocytes of the immunocompromised mice by the synergistic effect of the CON A, and enhances the immune function of the organism.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A Chinese herbal medicine composition comprises a mushroom water extract, a hericium erinaceus water extract, a flammulina velutipes water extract and a bamboo fungus water extract.
2. The Chinese herbal medicine composition according to claim 1, wherein the mass ratio of the mushroom aqueous extract, the hericium erinaceus aqueous extract, the flammulina velutipes aqueous extract and the bamboo fungus aqueous extract is (1-3): (0.5-1): (0.5-1): (1-3).
3. Use of a herbal composition according to claim 1 or 2 for promoting the growth of probiotics and/or for the preparation of a product promoting the growth of probiotics.
4. A composite composition comprising the herbal composition of claim 1 or 2 and a probiotic.
5. The composite composition of claim 4 wherein the raw material components of the composite composition further comprise a prebiotic.
6. The composite composition according to claim 5, wherein the mass ratio of the Chinese herbal medicine composition, the probiotics and the prebiotics is 600-1000:0.5-1.5:100-250; and/or the concentration of the mushroom water extract, the hericium erinaceus water extract, the flammulina velutipes water extract and the bamboo fungus water extract in the Chinese herbal medicine composition is 150-250 mg/mL.
7. A method of preparing the composite composition of any one of claims 4 to 6, comprising: and adding the raw material components of the composite composition into a basic culture medium for fermentation.
8. Use of the herbal composition of any one of claims 1 to 2 or the composite composition of any one of claims 4 to 6 in the preparation of a product.
9. A product comprising the herbal composition of any one of claims 1 to 2 or the composite composition of any one of claims 4 to 6.
10. A method of promoting growth of probiotics comprising: the herbal composition of claim 1 or 2 is added to a culture medium for probiotics.
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