CN114366787B - Chinese herbal medicine microbial fermentation preparation and application methods thereof - Google Patents
Chinese herbal medicine microbial fermentation preparation and application methods thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
- A61K36/815—Lycium (desert-thorn)
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- A—HUMAN NECESSITIES
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
- A61K36/258—Panax (ginseng)
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A61K36/185—Magnoliopsida (dicotyledons)
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- A61K36/185—Magnoliopsida (dicotyledons)
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Abstract
The invention provides a Chinese herbal medicine microbial fermentation preparation and a preparation method thereof, which belong to the technical field of Chinese herbal medicine fermentation and comprise the following steps: crushing and mixing the Chinese herbal medicine composition, adding water to adjust the humidity, and inoculating a first composite fermentation bacterium for fermentation culture to obtain a solid fermentation product; preparing a traditional Chinese medicine extracting solution by using the solid fermentation product as a raw material through a solvent extraction method; and (3) taking the traditional Chinese medicine extract as a fermentation substrate, and inoculating a second composite fermentation bacterium for fermentation culture to prepare the Chinese herbal medicine microbial fermentation preparation. The invention combines solid fermentation and liquid fermentation through secondary fermentation, so that effective substances are more soluble, the growth inhibition of the extracting solution to the later fermentation is reduced, and the extracting solution is used as a secondary fermentation substrate, so that the effective components and the active substances of the traditional Chinese medicines are extracted to the maximum extent and are efficiently absorbed and utilized by human bodies.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine fermentation, in particular to a Chinese herbal medicine microbial fermentation preparation and application methods thereof.
Background
The traditional Chinese medicine fermentation technology, also called traditional Chinese medicine bioconversion technology, is based on the traditional Chinese medicine fermentation processing method, combines modern microecology, bioengineering and fermentation engineering technology to biologically convert the extracted effective components of the traditional Chinese medicine, converts the macromolecular substances of the traditional Chinese medicine into small molecular components which can be directly absorbed by human intestinal tracts through microorganisms, and accelerates the absorption of the traditional Chinese medicine and quantitatively treats diseases. The modern traditional Chinese medicine fermentation process starts in the 80 s of the last century, is mostly single traditional Chinese medicine fermentation at the earliest, and mainly focuses on fungus self fermentation, such as ganoderma lucidum and cordyceps sinensis mycelium fermentation. At present, research on fermentation of traditional Chinese medicines has been developed from single medicines to compound medicines, and fermentation strains are expanded to beneficial bacteria such as photosynthetic bacteria, lactic acid bacteria, bacillus, yeast and the like besides fungi; the modern traditional Chinese medicine fermentation process is to add one or more of the preferred beneficial bacteria groups into the Chinese herbal medicine to prepare the Chinese herbal medicine microbial fermentation preparation containing the traditional Chinese medicine active ingredients, thalli and metabolites thereof.
The microorganism can produce various bioactive substances in the growth process, some microorganisms can secrete dozens of extracellular enzymes into a culture medium in the growth process, and intracellular enzymes produced by the biological activities of the microorganism are hundreds or thousands of, so that the abundant and powerful enzyme systems are substance bases for chemical reactions of traditional Chinese medicines, and can decompose and convert components of the medicines into new effective components, and the new components become substance bases for screening new active medicines or decompose toxic components to reduce toxic and side effects of the medicines; on the other hand, since microorganisms also form abundant and diverse secondary metabolites, some of them are drugs with good efficacy; or taking the active ingredients in the traditional Chinese medicine as precursors, forming a new compound through the metabolism of microorganisms, or reacting the secondary metabolites of the microorganisms with the ingredients in the traditional Chinese medicine to form a new compound, wherein the secondary metabolites of the microorganisms can also have compound synergistic effect with the active ingredients of the traditional Chinese medicine.
The traditional Chinese herbal medicine microbial fermentation preparation is prepared by adding a traditional Chinese medicine extract as a matrix into a strain for culture and fermentation, on one hand, liquid fermentation can shorten the fermentation time and improve the production efficiency, but the extraction efficiency is lower, the availability of the traditional Chinese medicine is low, on the other hand, the traditional Chinese medicine extract contains a large amount of active ingredients, most of the active ingredients have good antibacterial performance, the growth inhibition of zymophyte is easy to cause, and living bacteria in the preparation are easy to be inactivated due to the influence of external environment conditions, so that the activity is difficult to effectively maintain for a long time.
Disclosure of Invention
Aiming at least one of the problems, the invention provides a Chinese herbal medicine microbial fermentation preparation and application methods thereof.
The aim of the invention is realized by adopting the following technical scheme:
a preparation method of a Chinese herbal medicine microbial fermentation preparation comprises the following steps:
(1) Crushing and mixing the Chinese herbal medicine composition, adding water to adjust the humidity, and inoculating a first composite fermentation bacterium for fermentation culture to obtain a solid fermentation product;
(2) Preparing a traditional Chinese medicine extracting solution by using the solid fermentation product as a raw material through a solvent extraction method;
(3) And (3) taking the traditional Chinese medicine extract as a fermentation substrate, and inoculating a second composite fermentation bacterium for fermentation culture to prepare the Chinese herbal medicine microbial fermentation preparation.
Preferably, the first composite fermentation bacteria comprise photosynthetic bacteria, azotobacter, bacillus and lactobacillus, and the preparation method of the first composite fermentation bacteria comprises the following steps: respectively activating photosynthetic bacteria, azotobacter, bacillus and lactobacillus by using culture matrixes, performing expansion culture for 10-16 hours, and then performing expansion culture according to the number ratio of the viable bacteria of 5: (3-6): (8-12): (5-10) mixing.
Preferably, the second composite zymophyte comprises actinomycetes, lactobacillus, saccharomycetes and rhodotorula, and the preparation method of the second composite zymophyte comprises the following steps: activating actinomycetes, lactobacillus, saccharomycetes and rhodotorula respectively by using a culture medium, performing expansion culture for 10-16 hours respectively, and then performing expansion culture according to the number ratio of the viable bacteria of 5: (5-7): (2-4): (1-2) and mixing.
Preferably, the Chinese herbal medicine composition comprises wolfberry fruit, astragalus root, phellodendron bark, notoginseng, licorice, malt and tuckahoe.
Preferably, the second composite zymophyte further comprises a carrier.
Preferably, the carrier is a modified montmorillonite.
Preferably, the preparation method of the modified montmorillonite comprises the following steps:
(a) Stripping treatment
Weighing natural montmorillonite powder, dispersing in deionized water, adding 1wt.% polyethylene glycol, stirring at normal temperature for reaction for 6-10d, centrifuging at 6000-8000rpm for 10min, centrifuging for 2-3 times, and removing precipitate to obtain uniformly dispersed montmorillonite nanosheet suspension;
wherein, the dispersion ratio of the natural montmorillonite powder in deionized water is 1-2g/100mL;
(b) Surface modification
Heating the montmorillonite nanosheet suspension to 40-50 ℃, adding 30wt.% of hydrogen peroxide solution and tromethamine, fully stirring and mixing, continuing to perform heat preservation and stirring reaction for 1-20min, adding dopamine hydrochloride after the reaction is completed, dropwise adding initiator solution under stirring, continuing to perform stirring reaction for 1-10min after the dropwise adding is completed, sealing a reaction system, transferring into a constant-temperature oven at 160-180 ℃ for performing heat preservation reaction for 10-14h, cooling after the reaction is completed, separating out precipitate, washing with absolute ethyl alcohol and deionized water in sequence, and drying to obtain the surface modified montmorillonite nanosheets;
wherein, the mass ratio of montmorillonite nano-sheets in the montmorillonite nano-sheet suspension to the hydrogen peroxide solution, the tromethamine, the dopamine hydrochloride and the initiator is 1: (18-24): (5-6): (0.75-1): (0.2-0.4);
(c) Multi-hole assembly
Weighing polyvinyl alcohol, dispersing and dissolving the polyvinyl alcohol in dimethyl sulfoxide solution, heating and stirring the mixture until the polyvinyl alcohol is completely dissolved, continuously stirring and heating the mixture to 80-90 ℃, sequentially adding formaldehyde-based tetrastyrene and p-toluenesulfonic acid, fully stirring and mixing the mixture to obtain yellow solution, continuously keeping the temperature and stirring the mixture for 1-6 hours until the yellow color of the solution is removed, adding excessive acetone solvent until no white flocculent precipitate is separated out, separating the white flocculent precipitate, washing the white flocculent precipitate with acetone, and evaporating the acetone to obtain a tetraphenylethylene carboxylic acid derivative; weighing a nonionic surfactant and dissolving the nonionic surfactant in deionized water to obtain a solution A, stirring and heating to 80-90 ℃, adding the tetraphenylethylene carboxylic acid derivative, fully stirring and mixing, adding the surface-modified montmorillonite nano-sheets, dispersing, stirring and reacting overnight to obtain a suspension A, and freeze-drying the suspension A to obtain the pore-formed assembled montmorillonite nano-sheets;
wherein the dispersion ratio of the polyvinyl alcohol in the dimethyl sulfoxide solution is 1-10g/100mL, and the polyvinyl alcohol is mixed with
The mass ratio of the formaldehyde-based tetrastyrene to the p-toluenesulfonic acid is 1: (8-12): (0.1-0.15); the concentration of the solution A is 0.1-0.2g/100mL, and the mass ratio of the solution A to the tetraphenylethylene carboxylic acid derivative to the surface modified montmorillonite nano sheet is 100: (0.5-2): (3-5);
(d) Immobilization
Stirring and dispersing the porous assembled montmorillonite nano-sheets in tetrahydrofuran, adding an alkali solution of polydimethylsiloxane, fully mixing, steaming to remove the tetrahydrofuran, stirring and heating to 80-90 ℃, adding a curing agent for a small amount of times while stirring, continuing to perform heat preservation and stirring reaction for 12-16 hours after the addition, filtering out a precipitate after the reaction is finished, washing the precipitate with an alkaline solution and deionized water in sequence, and drying to obtain the modified montmorillonite;
wherein, the mass ratio of the pore-assembled montmorillonite nano-sheet to the polydimethylsiloxane is (2-3): 1.
the beneficial effects of the invention are as follows:
(1) The traditional Chinese medicine fermentation process is judged and controlled by subjective experience, has great subjectivity and randomness, and the stability of the quality is difficult to ensure, and the availability is not high; according to the invention, through secondary fermentation, solid fermentation and liquid fermentation are combined, fermentation treatment is carried out on Chinese herbal medicine cells by using first composite fermentation bacteria preferentially, and microorganisms break walls of the Chinese herbal medicine cells during fermentation, so that effective substances are more soluble, the concentration of active ingredients is further improved, meanwhile, macromolecular effective active substances which are difficult to be directly absorbed and utilized by a human body are primarily degraded, and the growth inhibition of extract on later fermentation is reduced; the Chinese herbal medicine is extracted after being fermented by the first composite fermentation bacteria, and the extracting solution is used as a secondary fermentation substrate, and as a plurality of intracellular and extracellular enzymes such as cellulase, lignin enzyme, amylase, protease, lipase and the like are generated in the growth metabolism process of the second composite fermentation bacteria, the Chinese herbal medicine has very strong capability of decomposing and converting substances, can generate abundant secondary metabolites or react with the components in the Chinese herbal medicine to form new compounds, can generate compound synergistic effect with the active components of the Chinese herbal medicine, and can furthest extract the active components and the active substances of the Chinese herbal medicine and be efficiently absorbed and utilized by human bodies.
(2) Antibacterial components contained in the traditional Chinese medicine extract are easy to cause growth inhibition of zymophyte, and living bacteria in the preparation are easily inactivated due to the influence of external environment conditions, so that the probiotic health care activity of the traditional Chinese medicine extract is difficult to be effectively maintained for a long time; according to the invention, a bacterial carrier is added into a second composite fermentation strain to provide protection for bacterial growth and colonization, the growth inhibition of antibacterial components on fermentation bacteria is reduced, furthermore, the modified carrier is prepared on the basis of natural montmorillonite, the loading and protection performance of the modified carrier on the fermentation bacteria are further improved, specifically, the natural montmorillonite is firstly subjected to delamination treatment to prepare a dispersible tablet layer, then a polydopamine modified layer grows on the surface by an initiating polymerization method, the rapid colonization of fermentation bacteria on silicate minerals can be promoted based on the good biological binding degree of polydopamine, and further, the micelle particles in a surfactant solution are used as a template agent, so that the modified nano-tablet is subjected to stacking construction of a pore structure on the surface of the micelle particles, and then the modified montmorillonite carrier is prepared by fixing polydimethylsiloxane, thereby reducing the activity influence of external environment on probiotics and improving the preparation stability.
Detailed Description
The invention will be further described with reference to the following examples.
Example 1
The embodiment relates to a preparation method of a Chinese herbal medicine microbial fermentation preparation, which comprises the following steps:
(1) The Chinese herbal medicine composition is prepared according to the following weight portion ratio: 8-10 parts of medlar, 10-12 parts of astragalus, 4-6 parts of phellodendron, 4-6 parts of pseudo-ginseng, 12-15 parts of liquorice, 15-16 parts of malt and 6-8 parts of poria cocos;
(2) Respectively activating photosynthetic bacteria (rhodopseudomonas palustris), azotobacter (azotobacter chroococcus), bacillus licheniformis or bacillus subtilis and lactobacillus rhamnosus by using culture matrixes, performing expansion culture for 12 hours, and then performing expansion culture according to the number ratio of the viable bacteria of 5:4:10:6, mixing to obtain the first composite zymophyte;
respectively activating actinomycetes (streptomyces), lactobacillus acidophilus or lactobacillus plantarum, saccharomycetes and rhodotorula through culture matrixes, performing expansion culture, respectively performing expansion culture for 12 hours, and then performing expansion culture according to the number ratio of the active bacteria of 5:6:3:1, mixing to obtain the second composite zymocyte;
the culture medium comprises, by weight, 4-6 parts of a carbon source, 9-12 parts of a nitrogen source and 0.3-0.4 part of inorganic salt;
(3) Crushing the Chinese herbal medicine composition, mixing, adding water to adjust the humidity to 20%, and inoculating a first composite fermentation bacterium for fermentation culture to obtain a solid fermentation product;
(4) With the fixingThe fermentation product is taken as a raw material, and water is added for extraction to prepare a traditional Chinese medicine extracting solution; adding 50 kg of Chinese medicinal extract and 30 kg of brown granulated sugar into 1t of purified water, mixing, sterilizing by ultraviolet rays, and performing aerobic fermentation in a sterile factory building at 28-30deg.C for 7d, wherein stirring is performed every 3 h; after the completion, inoculating a second composite fermentation bacterium to perform fermentation culture, and fermenting in a sealed anaerobic tank at 28-30 ℃ until the number of detected viable bacteria is more than 10 6 Fermenting for 15 days to obtain the Chinese herbal medicine microbial fermentation preparation.
Example 2
This example relates to a method for preparing a microbial fermentation preparation of chinese herbal medicine, which is different from example 1 in that: the second composite zymocyte also comprises a modified montmorillonite carrier, and specifically comprises the following steps:
(1) The Chinese herbal medicine composition is prepared according to the following weight portion ratio: 8-10 parts of medlar, 10-12 parts of astragalus, 4-6 parts of phellodendron, 4-6 parts of pseudo-ginseng, 12-15 parts of liquorice, 15-16 parts of malt and 6-8 parts of poria cocos;
(2) Respectively activating photosynthetic bacteria (rhodopseudomonas palustris), azotobacter (azotobacter chroococcus), bacillus licheniformis or bacillus subtilis and lactobacillus rhamnosus by using culture matrixes, performing expansion culture for 12 hours, and then performing expansion culture according to the number ratio of the viable bacteria of 5:4:10:6, mixing to obtain the first composite zymophyte;
respectively activating actinomycetes (streptomyces), lactobacillus acidophilus or lactobacillus plantarum, saccharomycetes and rhodotorula through culture matrixes, performing expansion culture, respectively performing expansion culture for 12 hours, and then performing expansion culture according to the number ratio of the active bacteria of 5:6:3:1, mixing to obtain the second composite zymocyte;
the culture medium comprises, by weight, 4-6 parts of a carbon source, 9-12 parts of a nitrogen source and 0.3-0.4 part of inorganic salt;
(3) Crushing the Chinese herbal medicine composition, mixing, adding water to adjust the humidity to 20%, and inoculating a first composite fermentation bacterium for fermentation culture to obtain a solid fermentation product;
(4) Taking the solid fermentation product as a raw material, and adding water for extraction to obtain a traditional Chinese medicine extracting solution; 50 kg of Chinese medicine extract and 30 kg of brown granulated sugar are added into 1t of purified water to be mixed,after ultraviolet sterilization, carrying out aerobic fermentation for 7d in a sterile factory building at 28-30 ℃ and stirring every 3 h; after the completion, inoculating a second composite fermentation bacterium to perform fermentation culture, and fermenting in a sealed anaerobic tank at 28-30 ℃ until the number of detected viable bacteria is more than 10 6 Fermenting for 10 days to obtain the Chinese herbal medicine microbial fermentation preparation;
the preparation method of the modified montmorillonite comprises the following steps:
(a) Stripping treatment
Weighing 3g of natural montmorillonite powder, dispersing the natural montmorillonite powder in 500mL of deionized water, adding polyethylene glycol with the final concentration of 1wt.% into the deionized water, stirring the mixture at normal temperature for reaction for 7d, centrifuging the mixture at 6000-8000rpm for 10min, repeating the centrifuging process for 3 times, and removing the precipitate to obtain uniformly dispersed montmorillonite nanosheet suspension;
(b) Surface modification
Heating the montmorillonite nanosheet suspension to 40-50 ℃, adding 10mL 30wt.% hydrogen peroxide solution and 2.5g tromethamine, fully stirring and mixing, continuing to perform heat preservation and stirring reaction for 1-20min, adding 0.4g dopamine hydrochloride after the reaction is completed, dropwise adding 0.1mol/L ammonium persulfate solution under stirring, continuing to perform stirring reaction for 10min after the dropwise adding is completed, sealing a reaction system, transferring the reaction system into a constant-temperature oven at 160-180 ℃ for heat preservation reaction for 10-14h, cooling after the reaction is completed, separating out precipitate, washing with absolute ethyl alcohol and deionized water sequentially, and drying to obtain the surface modified montmorillonite nanosheets;
(c) Multi-hole assembly
Weighing 6g of polyvinyl alcohol, dispersing and dissolving in 100mL of dimethyl sulfoxide solution, heating and stirring to dissolve completely, continuing stirring and heating to 80-90 ℃, then sequentially adding 100g of formaldehyde-based tetraphenyl ethylene and 1.3g of p-toluenesulfonic acid, fully stirring and mixing to obtain a yellow solution, continuing heat preservation and stirring for 4 hours until the yellow color of the solution is removed, adding excessive acetone solvent until no white flocculent precipitate is separated out, separating the white flocculent precipitate, washing with acetone, and evaporating acetone to obtain a tetraphenyl ethylene carboxylic acid derivative; weighing 0.01g of PEO-PPO-PEO triblock copolymer, dissolving in 100mL of deionized water to prepare solution A, stirring and heating to 80-90 ℃, adding the tetraphenylethylene carboxylic acid derivative, fully stirring and mixing, then adding the surface-modified montmorillonite nano-sheets, dispersing, stirring and reacting overnight to prepare suspension A, and freeze-drying the suspension A to prepare the pore-formed assembled montmorillonite nano-sheets;
(d) Immobilization
Stirring and dispersing the porous assembled montmorillonite nano-sheets in 10mL of tetrahydrofuran, adding an alkali solution of amino-terminated polydimethylsiloxane, fully mixing, steaming to remove the tetrahydrofuran, heating to 80-90 ℃ while stirring, adding a curing agent a small amount of times, continuing to perform heat preservation and stirring reaction for 12 hours after the addition, filtering out a precipitate after the reaction is finished, washing the precipitate with an alkaline solution and deionized water in sequence, and drying to obtain the modified montmorillonite;
wherein, the mass ratio of the pore-assembled montmorillonite nano-sheet to the polydimethylsiloxane is 2:1.
comparative example
The difference from example 1 is that: the second composite zymocyte also comprises a natural montmorillonite powder carrier.
Experimental example
1. The residual viable count (lg value) of the Chinese herbal medicine microbial fermentation preparations described in example 1, example 2 and comparative example under the same storage conditions was measured:
| preservation time | 0d | 30d | 60d | 90d |
| Example 1 | 7 | 6 | 5 | 3 |
| Example 2 | 7 | 7 | 7 | 6 |
| Comparative example | 7 | 7 | 6 | 5 |
2. Selecting animal model of hyperlipidemia rat, verifying, selecting 50 male rats with age of 8 weeks and weight of 200-225g, adaptively feeding for 3d, and randomly dividing into 2
The group (normal group: 10, high fat model group: 40) was used to construct a rat animal model for hyperlipidemia by using a high fat feed feeding method, the normal group fed a normal basal feed, the high fat model group fed a high fat feed, and fed tap water sterilized by ultraviolet rays for 30d, and after 30d, fasted for 12 hours, the orbital bleeding was performed to measure the total cholesterol, triglyceride and low density lipoprotein cholesterol content in the serum of the rat.
The high-fat model group was randomly divided into 4 groups, which were respectively a model group, a first treatment group, a second treatment group and a third treatment group, 10 animals were fed to the first treatment group and the second treatment group each 1 time per day, the fermented preparation described in example 1 and example 2 was fed to the third treatment group each 1 time per day, the normal group and the model group were fed with 1 time per day of an equivalent amount of physiological saline, and after 60d of feeding time, the feeding was fasted for 12 hours, and the orbital bleeding was performed to measure the total cholesterol, triglyceride and low-density lipoprotein cholesterol content in the serum of the rat.
The test results are shown in the following table:
finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (2)
1. The preparation method of the traditional Chinese medicine microbial fermentation preparation for treating hyperlipidemia is characterized by comprising the following steps:
(1) Crushing and mixing the traditional Chinese medicine composition, adding water to adjust the humidity, and inoculating a first composite fermentation bacterium for fermentation culture to obtain a solid fermentation product;
the traditional Chinese medicine composition is prepared from medlar, astragalus, phellodendron, pseudo-ginseng, liquorice, malt and poria cocos; the Chinese medicinal composition is prepared from (by weight parts) Lycii fructus 8-10, astragali radix 10-12, phellodendri cortex 4-6, notoginseng radix 4-6, glycyrrhrizae radix 12-15, malt 15-16 and Poria 6-8;
the first composite fermentation bacteria are prepared from photosynthetic bacteria, azotobacter, bacillus and lactobacillus, and the preparation method of the first composite fermentation bacteria comprises the following steps: respectively activating photosynthetic bacteria, azotobacter, bacillus and lactobacillus by using culture matrixes, performing expansion culture for 10-16 hours, and then performing expansion culture according to the number ratio of the viable bacteria of 5: (3-6): (8-12): (5-10) mixing;
(2) Preparing a traditional Chinese medicine extracting solution by using the solid fermentation product as a raw material through a solvent extraction method;
(3) Taking the traditional Chinese medicine extract as a fermentation substrate, and inoculating a second composite fermentation bacterium for fermentation culture to prepare the traditional Chinese medicine microbial fermentation preparation;
the second composite zymophyte is prepared from actinomycetes, lactobacillus, saccharomycetes and rhodotorula, and the preparation method of the second composite zymophyte comprises the following steps: activating actinomycetes, lactobacillus, saccharomycetes and rhodotorula respectively by using a culture medium, performing expansion culture for 10-16 hours respectively, and then performing expansion culture according to the number ratio of the viable bacteria of 5: (5-7): (2-4): (1-2) mixing;
the second composite zymocyte also comprises a carrier;
the carrier is modified montmorillonite;
the preparation method of the modified montmorillonite comprises the following steps:
(a) Stripping treatment
Weighing natural montmorillonite powder, dispersing in deionized water, adding 1wt.% polyethylene glycol, stirring at normal temperature for reaction for 6-10d, centrifuging at 6000-8000rpm for 10min, centrifuging for 2-3 times, and removing precipitate to obtain uniformly dispersed montmorillonite nanosheet suspension;
wherein, the dispersion ratio of the natural montmorillonite powder in deionized water is 1-2g/100mL;
(b) Surface modification
Heating the montmorillonite nanosheet suspension to 40-50 ℃, adding 30wt.% of hydrogen peroxide solution and tromethamine, fully stirring and mixing, continuing to perform heat preservation and stirring reaction for 1-20min, adding dopamine hydrochloride after the reaction is completed, dropwise adding initiator solution under stirring, continuing to perform stirring reaction for 1-10min after the dropwise adding is completed, sealing a reaction system, transferring into a constant-temperature oven at 160-180 ℃ for performing heat preservation reaction for 10-14h, cooling after the reaction is completed, separating out precipitate, washing with absolute ethyl alcohol and deionized water in sequence, and drying to obtain the surface modified montmorillonite nanosheets;
wherein, the mass ratio of montmorillonite nano-sheets in the montmorillonite nano-sheet suspension to the hydrogen peroxide solution, the tromethamine, the dopamine hydrochloride and the initiator is 1: (18-24): (5-6): (0.75-1): (0.2-0.4);
(c) Multi-hole assembly
Weighing polyvinyl alcohol, dispersing and dissolving the polyvinyl alcohol in dimethyl sulfoxide solution, heating and stirring the mixture until the polyvinyl alcohol is completely dissolved, continuously stirring and heating the mixture to 80-90 ℃, sequentially adding formaldehyde-based tetrastyrene and p-toluenesulfonic acid, fully stirring and mixing the mixture to obtain yellow solution, continuously keeping the temperature and stirring the mixture for 1-6 hours until the yellow color of the solution is removed, adding excessive acetone solvent until no white flocculent precipitate is separated out, separating the white flocculent precipitate, washing the white flocculent precipitate with acetone, and evaporating the acetone to obtain a tetraphenylethylene carboxylic acid derivative; weighing a nonionic surfactant and dissolving the nonionic surfactant in deionized water to obtain a solution A, stirring and heating to 80-90 ℃, adding the tetraphenylethylene carboxylic acid derivative, fully stirring and mixing, adding the surface-modified montmorillonite nano-sheets, dispersing, stirring and reacting overnight to obtain a suspension A, and freeze-drying the suspension A to obtain the pore-formed assembled montmorillonite nano-sheets;
wherein the dispersion ratio of the polyvinyl alcohol in the dimethyl sulfoxide solution is 1-10g/100mL, and the mass ratio of the polyvinyl alcohol to the formaldehyde-based tetrastyrene to the p-toluenesulfonic acid is 1: (8-12): (0.1-0.15); the concentration of the solution A is 0.1-0.2g/100mL, and the mass ratio of the solution A to the tetraphenylethylene carboxylic acid derivative to the surface modified montmorillonite nano sheet is 100: (0.5-2): (3-5);
(d) Immobilization
Stirring and dispersing the porous assembled montmorillonite nano-sheets in tetrahydrofuran, adding an alkali solution of polydimethylsiloxane, fully mixing, steaming to remove the tetrahydrofuran, stirring and heating to 80-90 ℃, adding a curing agent for a small amount of times while stirring, continuing to perform heat preservation and stirring reaction for 12-16 hours after the addition, filtering out a precipitate after the reaction is finished, washing the precipitate with an alkaline solution and deionized water in sequence, and drying to obtain the modified montmorillonite;
wherein, the mass ratio of the pore-assembled montmorillonite nano-sheet to the polydimethylsiloxane is (2-3): 1.
2. the fermented preparation of Chinese medicinal microorganism for treating hyperlipidemia according to claim 1.
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