Composition for reducing blood sugar and blood fat, and preparation method and application of composition
A ginsenoside, a rare technology, applied in the field of bioengineering, can solve the problems of polluting the environment, unable to directly eat, unable to meet new drugs and other problems
Inactive Publication Date: 2020-10-23
SHAANXI GIANT BIOTECHNOLOGY CO LTD
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AI-Extracted Technical Summary
Problems solved by technology
However, their hydrolysis conditions are very severe, and aglycones are prone to dehydration or cyclization, which changes the structure of aglycones and increases by-products, making the products difficult to purify and polluting the environment.
The method of preparing rare ginsenosides by microbial transformation method ha...
Method used
[0055] Wherein, probiotics are active microorganisms beneficial to the host by colonizing in the human body and changing the composition of the flora in a certain part of the host. By regulating the immune function of the host mucosa and system or by regulating the balance of intestinal flora, it promotes nutrient absorption and maintains intestinal health, thereby producing single microorganisms or mixed microo...
Abstract
The invention discloses a composition for reducing blood sugar and blood fat as well as a preparation method and application of the composition. The composition for reducing blood sugar and blood fatcomprises rare ginsenoside and probiotic powder, wherein the probiotic powder comprises eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus. The composition provided by the invention can give full play to the effects of reducing blood sugar and blood fat of rare ginsenoside and eurotium cristatum, and has the effects of mutual promotion and synergistic interaction compared with thepure eurotium cristatum or converted rare ginsenoside. Moreover, the composition can realize multiple treatment effects with one medicine, can reduce the medicine taking types of patients suffering from diabetes and hyperlipidemia at the same time, enhances the medical compliance of the patients, and is convenient for the patients to take medicines; and meanwhile, the multiple effects of regulating the proportion of probiotics in intestinal flora, supplementing probiotics, promoting absorption and the like can be achieved at the same time.
Application Domain
Metabolism disorderUnknown materials +1
Technology Topic
Medication takingGinsenoside +11
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Examples
- Experimental program(16)
Example Embodiment
[0054] The present invention provides a preparation method of rare ginsenosides, wherein the preparation method includes fermentation of active raw materials by probiotics.
[0055] Among them, probiotics are a kind of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of a certain part of the host's flora. By adjusting the host mucosa and the immune function of the system or by adjusting the balance of the intestinal flora, it promotes nutrient absorption to maintain intestinal health, thereby producing single microorganisms that are beneficial to health or mixed microorganisms with clear composition.
[0056] In a preferred specific embodiment of the present invention, the probiotics include Santocystis cristatum, Bacillus subtilis and Lactobacillus bulgaricus.
[0057] Said saccharomycetes cristata, commonly known as "Golden Flower", belongs to a fungus belonging to the genus saccharomycete of the saccharomycete family. It is composed of ascomyces and hyphae and has low nutritional requirements. It can be used on potato dextrose agar medium, Bengal red medium grows, has strong adaptability, and can utilize a variety of nitrogen and carbon sources. It is the dominant strain in the fermentation process of Fuzhuan tea. It obtains nutrients from the tea and produces various enzymes through its own metabolism to catalyze the tea. Various substances are transformed to form the unique color, aroma and taste quality of Fuzhuan tea, so it is an important index for evaluating the quality of Fuzhuan tea.
[0058] The said saccharomyces cristatus is selected from the mycelium, spores or a combination of the two obtained by large-scale fermentation and cultivation of saccharomyces cristatum. The mycelium and spores can be processed by physical, biological or chemical methods. The broken state in which cell wall morphology is broken to release intracellular contents may also be a non-broken state without treatment, preferably a broken state. The physical, biological, and chemical method of breaking the cell wall refers to the breaking method frequently used by those skilled in the art, such as: high-pressure homogenization, ultrasonic breaking, biological enzymatic breaking and acid-base breaking, etc.;
[0059] The bacterial powders of Bacillus subtilis and Lactobacillus bulgaricus are respectively obtained by fermentation and culture of Bacillus subtilis and Lactobacillus bulgaricus.
[0060] In a preferred embodiment of the present invention, wherein the active raw material is selected from one or more of ginseng, panax notoginseng, panax notoginseng flower, American ginseng, Gynostemma pentaphyllum or their extracts, preferably ginseng or its extracts Things.
[0061] In a more preferred specific embodiment of the present invention, wherein the preparation method includes:
[0062] Activate and expand the culture of Sansevisia cristatum, Bacillus subtilis and Lactobacillus bulgaricus to obtain the seed liquid;
[0063] Fermentation: Inoculate the seed liquor of Sansacella cristatum, Bacillus subtilis and Lactobacillus bulgaricus into the fermenter respectively, and add active raw materials to the fermenter to obtain the fermented liquid;
[0064] The fermentation broth is subjected to solid-liquid separation, and the supernatant is collected and concentrated to obtain rare ginsenosides.
[0065] In a more preferred embodiment of the present invention, in the fermentation step, the seed liquid of Pseudocystis cristatum is first inoculated into the culture medium in the fermentor; then the active raw materials are added for fermentation; and then Lactobacillus bulgaricus, The Bacillus subtilis seed liquid is respectively inoculated into the culture medium in the fermentor and further fermented to obtain the fermentation liquid, wherein the fermentation temperature is 28-30°C, the fermentation pH is 4-8, preferably 5-7, and in the fermentation step, The amount of dissolved oxygen is ≥20%, preferably 50-70%.
[0066] In a more preferred embodiment of the present invention, the steps of activation and expansion of the strain of Astrodon cristata are: inoculating the strain of Astrodon cristata on a PDA culture slant, and culture it at 28°C Activate, wait for the hyphae to grow over the slope, wash the hyphae with 5ml sterile saline, inoculate 0.2ml into a 500ml Erlenmeyer flask containing 200ml PDA liquid medium, and place it on a shaker at 28°C and 160rpm for 120h ; Then transfer the seeds from the secondary shaker flask at a 10% inoculum amount and place them on a shaker at 28°C and 160 rpm for 72 hours;
[0067] The steps for the activation and expansion of Lactobacillus bulgaricus strains are as follows: inoculate the Lactobacillus bulgaricus strain on the modified MRS agar medium plate, place it at 37°C for activation and culture for 48 hours, scrape 1 loop with an inoculation loop and inoculate it with 80mL MRS Place the liquid culture medium in a 250mL Erlenmeyer flask and place it on a shaker at 30°C and 160r/min for 48 hours.
[0068] The steps of activation and expansion of Bacillus subtilis strains are as follows: inoculate Bacillus subtilis strains on agar medium (the composition of the medium is: 1L distilled water+20g glucose+15g peptone+0.5g beef extract+20g agar) slant, Place it at 30°C for 48h activation and culture, use an inoculating loop to scrape 1 loop and inoculate it into a 250mL Erlenmeyer flask containing 80mL liquid medium (the composition of the medium: 1L distilled water + 20g glucose + 15g peptone + 0.5g beef extract). Place it on a shaker at 30°C and 160r/min for 36 hours.
[0069] In a preferred embodiment of the present invention, wherein, in the fermentation step, the inoculation amount of P. cristatus is 5-15%, preferably, the fermentation temperature is 28-30°C, and more preferably, The fermentation pH is 4-7, and more preferably, the fermentation time is 66-78h.
[0070] In a preferred embodiment of the present invention, wherein, in the fermentation step, the inoculation amount of Lactobacillus bulgaricus is 2-5%, preferably, the fermentation temperature is 28-30°C, and more preferably, the fermentation pH For 5-8.
[0071] In a preferred embodiment of the present invention, wherein, in the fermentation step, the inoculation amount of Bacillus subtilis is 2-5%, preferably, the fermentation temperature is 28-30°C, and more preferably, the fermentation pH For 5-8.
[0072] In a more preferred embodiment of the present invention, in the fermentation step, the fermentation medium (1% sugar and milk powder) is put into the fermenter for high-pressure steam sterilization at a volume of about 60%. After cooling the fermentation medium, insert 10% of the expanded saccharomycetes, and add active raw materials throughout the fermentation process, control DO not less than 20%, temperature 28-30℃, pH5-6, culture for 66-78h, and then The expanded Lactobacillus bulgaricus and Bacillus subtilis strains were connected at the inoculum of 3% respectively, and the temperature of the fermentation culture was controlled at 28-30℃, pH 6-7, rotation speed controlled at 140-160r/min, and controlled The dissolved oxygen is not less than 20%, and the culture is continued for 60-80 hours to obtain a composite fermentation broth.
[0073] Wherein, the DO is the amount of oxygen dissolved in water (unit: mg/L). The percentage of air saturation is often used in fermentation to indicate the dissolved oxygen concentration (the 100% air saturation concentration of oxygen in the fermentation broth at 28°C is only about 0.25 mmol/L).
[0074] In a preferred embodiment of the present invention, in the solid-liquid separation step, the supernatant contains rare ginsenosides. In the solid-liquid separation step, solid-liquid separation can be achieved by plate and frame, ultrafiltration, ceramic membrane and other equipment, and then the solids are collected to obtain probiotics, and the supernatant is concentrated, separated and purified to obtain rare saponin.
[0075] In a preferred embodiment of the present invention, the active raw materials are carried out in a flow-feeding manner, by mixing the active raw materials and ultrapure water into a suitable mother liquor, wherein the flow-through amount of the active raw materials is based on the solid active material. Judging by the mass and the volume after the addition, the final ratio of active raw materials is 5%-50% (w/v), preferably 10%-30%, more preferably 25%, added through the entire fermentation process Into the mixed culture medium.
[0076] The present invention provides a method for preparing probiotic powder, wherein the preparation method includes fermenting active raw materials with probiotic bacteria.
[0077] In a preferred specific embodiment of the present invention, the probiotics include Santocystis cristatum, Bacillus subtilis and Lactobacillus bulgaricus.
[0078] Said saccharomycetes cristata, commonly known as "Golden Flower", belongs to a fungus belonging to the genus saccharomycete of the saccharomycete family. It is composed of ascomyces and hyphae and has low nutritional requirements. It can be used on potato dextrose agar medium, Bengal red medium grows, has strong adaptability, and can utilize a variety of nitrogen and carbon sources. It is the dominant strain in the fermentation process of Fuzhuan tea. It obtains nutrients from the tea and produces various enzymes through its own metabolism to catalyze the tea. Various substances are transformed to form the unique color, aroma and taste quality of Fuzhuan tea, so it is an important index to evaluate the quality of Fuzhuan tea.
[0079] The said saccharomyces cristatus is selected from the mycelium, spores or a combination of the two obtained by large-scale fermentation and cultivation of saccharomyces cristatum. The mycelium and spores can be processed by physical, biological or chemical methods. The broken state in which cell wall morphology is broken to release intracellular contents may also be a non-broken state without treatment, preferably a broken state. The physical, biological, and chemical method of breaking the cell wall refers to the breaking method frequently used by those skilled in the art, such as: high-pressure homogenization, ultrasonic breaking, biological enzymatic breaking and acid-base breaking, etc.;
[0080] The bacterial powders of Bacillus subtilis and Lactobacillus bulgaricus are respectively obtained by fermentation and culture of Bacillus subtilis and Lactobacillus bulgaricus.
[0081] In a preferred embodiment of the present invention, wherein the active raw material is selected from one or more of ginseng, panax notoginseng, panax notoginseng flower, American ginseng, Gynostemma pentaphyllum or their extracts, preferably ginseng or its extracts Things.
[0082] In a preferred embodiment of the present invention, the preparation method includes:
[0083] Activate and expand the culture of Sansevisia cristatum, Bacillus subtilis and Lactobacillus bulgaricus to obtain the seed liquid;
[0084] Fermentation: Inoculate the seed liquor of Sansacella cristatum, Bacillus subtilis and Lactobacillus bulgaricus into the fermenter respectively, and add active raw materials to the fermenter to obtain the fermented liquid;
[0085] The fermentation broth is subjected to solid-liquid separation, and the solids are collected and lyophilized to obtain probiotic powder.
[0086] In a more preferred embodiment of the present invention, in the fermentation step, the seed liquid of Pseudocystis cristatum is first inoculated into the culture medium in the fermentor; then the active raw materials are added for fermentation; and then Lactobacillus bulgaricus, The Bacillus subtilis seed liquid is respectively inoculated into the culture medium in the fermentor for further fermentation to obtain the fermentation liquid. The fermentation temperature is, for example, 28-30°C, the fermentation pH is, for example, 4-8, preferably 5-7, and the amount of dissolved oxygen is ≥20%, for example, 50-70%.
[0087] In a more preferred embodiment of the present invention, the steps of activation and expansion of the strain of Astrodon cristata are: inoculating the strain of Astrodon cristata on a PDA culture slant, and culture it at 28°C Activate, wait for the hyphae to grow over the slope, wash the hyphae with 5ml sterile saline, inoculate 0.2ml into a 500mL Erlenmeyer flask containing 200mL PDA liquid medium, and place it on a shaker at 28°C and 160rpm for 120h ; Then transfer the seeds from the secondary shaker flask at a 10% inoculum amount and place them on a shaker at 28°C and 160 rpm for 72 hours;
[0088] The steps for the activation and expansion of Lactobacillus bulgaricus strains are as follows: inoculate the Lactobacillus bulgaricus strain on the modified MRS agar medium plate, place it at 37°C for activation and culture for 48 hours, scrape 1 loop with an inoculation loop and inoculate it with 80mL MRS Place the liquid culture medium in a 250mL Erlenmeyer flask and place it on a shaker at 30°C and 160r/min for 48 hours.
[0089] The steps of activation and expansion of Bacillus subtilis strains are as follows: inoculate Bacillus subtilis strains on agar medium (the composition of the medium is: 1L distilled water+20g glucose+15g peptone+0.5g beef extract+20g agar) slant, Place it at 30°C for 48h activation and culture, use an inoculating loop to scrape 1 loop and inoculate it into a 250mL Erlenmeyer flask containing 80mL liquid medium (the composition of the medium: 1L distilled water + 20g glucose + 15g peptone + 0.5g beef extract). Place it on a shaker at 30°C and 160r/min for 36 hours.
[0090] In a preferred embodiment of the present invention, wherein, in the fermentation step, the inoculation amount of P. cristatus is 5-15%, preferably, the fermentation temperature is 28-30°C, and more preferably, The fermentation pH is 4-7, and more preferably, the fermentation time is 66-78h.
[0091] In a preferred embodiment of the present invention, wherein, in the fermentation step, the inoculation amount of Lactobacillus bulgaricus is 2-5%, preferably, the fermentation temperature is 28-30°C, and more preferably, the fermentation pH For 5-8.
[0092] In a preferred embodiment of the present invention, wherein, in the fermentation step, the inoculation amount of Bacillus subtilis is 2-5%, preferably, the fermentation temperature is 28-30°C, and more preferably, the fermentation pH For 5-8.
[0093] In a more preferred embodiment of the present invention, in the fermentation step, the fermentation medium (1% sugar and milk powder) is put into the fermenter for high-pressure steam sterilization at a volume of about 60%. After cooling the fermentation medium, insert 10% of the expanded saccharomycetes, and add active raw materials throughout the fermentation process, control DO not less than 20%, temperature 28-30℃, pH5-6, culture for 66-78h, and then The expanded Lactobacillus bulgaricus and Bacillus subtilis strains were connected at the inoculum of 3% respectively, and the temperature of the fermentation culture was controlled at 28-30℃, pH 6-7, rotation speed controlled at 140-160r/min, and controlled The dissolved oxygen is not less than 20%, and the culture is continued for 60-80 hours to obtain a composite fermentation broth.
[0094] Wherein, the DO is the amount of oxygen dissolved in water (unit: mg/L). The percentage of air saturation is often used in fermentation to indicate the dissolved oxygen concentration (the 100% air saturation concentration of oxygen in the fermentation broth at 28°C is only about 0.25 mmol/L).
[0095] In a preferred embodiment of the present invention, in the solid-liquid separation step, the supernatant contains rare ginsenosides. In the solid-liquid separation step, solid-liquid separation can be achieved by plate and frame, ultrafiltration, ceramic membrane and other equipment, and then the solids are collected to obtain probiotics, and the supernatant is concentrated, separated and purified to obtain rare saponin.
[0096] In a preferred embodiment of the present invention, the active raw materials are carried out in a flow-feeding manner, by mixing the active raw materials and ultrapure water into a suitable mother liquor, wherein the flow-through amount of the active raw materials is based on the solid active material. Judging by the mass and the volume after the addition, the final ratio of active raw materials is 5%-50% (w/v), preferably 10%-30%, more preferably 25%, added through the entire fermentation process Into the mixed culture medium.
[0097] The present invention provides rare ginsenosides prepared by the above-mentioned method.
[0098] The present invention provides probiotic powder prepared by the method described above.
[0099] The present invention provides a probiotic powder, wherein the probiotic powder contains P. cristatus, Bacillus subtilis and Lactobacillus bulgaricus; preferably, in a unit volume, the live bacteria of P. cristatus The ratio of the total number to the total number of live bacteria of Bacillus subtilis and Lactobacillus bulgaricus is 1:1-10, preferably 1:1-5, and more preferably 1:1-2.
[0100] In a unit volume, the ratio of the total number of viable bacteria of the saccharomyces cristatum to the total number of viable bacteria of the sum of Bacillus subtilis and Lactobacillus bulgaricus can be, for example, 1:1, 1:2, 1:3, 1: 4. 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any range in between.
[0101] The saccharomyces cristatum is a selenium-enriched strain of sclerotium cristatum (Eurotium Cristatum XD-01), which was deposited in the General Microbiology Center of the China Microbial Culture Collection and Management Committee on April 2, 2018, and the deposit number is CGMCCNo. The preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The strain first appeared in a public document with Chinese Patent Application No. CN201911381242.9.
[0102] The Bacillus subtilis is commercially purchased from the China Industrial Microbial Culture Collection and Management Center (CICC), and the preservation number is CICC10071, and its preservation address is Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing.
[0103] The Lactobacillus bulgaricus is commercially purchased from the China Agricultural Microbial Culture Collection and Management Center (CICC), the preservation number is ACCC03598, and its preservation address is the Institute of Agricultural Resources and Agricultural Regional Planning, Chinese Academy of Agricultural Sciences, No. 12, Zhongguancun South Street, Haidian District, Beijing Resource Building 206.
[0104] In a preferred embodiment of the present invention, the ratio of the total number of live bacteria of Bacillus subtilis to the total number of live bacteria of Lactobacillus bulgaricus is 1-5:1, preferably 1-2:1.
[0105] For example, the ratio of the total number of live bacteria of Bacillus subtilis to the total number of live bacteria of Lactobacillus bulgaricus can be, for example, 1:1, 2:1, 3:1, 4:1, 5:1, or any range therebetween.
[0106] The present invention provides a composition comprising rare ginsenosides and the probiotics described above, the mass ratio of the rare ginsenosides to the probiotics is 1:1-10, preferably 1:1-5, More preferably, it is 1:2-3.
[0107] The mass ratio of the rare ginsenosides to the probiotics can be, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1: 9. 1:10 or any range in between.
[0108] In a preferred embodiment of the present invention, the rare ginsenoside is Rh4.
[0109] The present invention provides the use of the above-mentioned composition in food, health products or medicine, preferably in medicine, and further preferably in preparation for preventing or treating diabetes, hyperlipidemia or complications caused by diabetes and hyperlipidemia Use in medicine.
[0110] The present invention provides the use of the above-mentioned composition in reducing blood glucose concentration, total cholesterol, triglyceride or lipoprotein load.
[0111] The present invention provides a health product composition, which comprises an effective dose of the above-mentioned composition and additives.
[0112] Among them, the "effective dose" refers to the dose corresponding to the desired function. The effective dose is the dose that can be obtained by a person skilled in the art in a limited number of experiments according to actual needs. The additives are those skilled in the art. Well-known by the person, for example, it may be magnesium stearate, sodium carboxymethyl cellulose, microcrystalline cellulose or sodium starch glycolate.
[0113] The present invention provides a food composition comprising an effective dose of the above-mentioned composition and additives.
[0114] Among them, the "effective dose" refers to the dose corresponding to the desired function. The effective dose is the dose that can be obtained by a person skilled in the art in a limited number of experiments according to actual needs. The additives are those skilled in the art. Well-known by the person, for example, it may be magnesium stearate, sodium carboxymethyl cellulose, microcrystalline cellulose or sodium starch glycolate.
[0115] The present invention provides a pharmaceutical composition comprising an effective dose of the above-mentioned composition and pharmaceutically acceptable excipients.
[0116] Among them, "effective dose" refers to the dose used when it is used as a drug to exert its pharmaceutical function.
[0117] The pharmaceutically acceptable excipients include pharmaceutically acceptable carriers, excipients, diluents, etc., which are compatible with the active ingredient. The pharmaceutically acceptable excipients are well known to those skilled in the art, such as magnesium stearate, sodium carboxymethyl cellulose, microcrystalline cellulose or sodium starch glycolate.
[0118] The present invention provides a preparation for preventing or treating diabetes, hyperlipidemia or complications caused by diabetes and hyperlipidemia, which is prepared by including the raw materials of the above-mentioned pharmaceutical composition.
[0119] In a preferred embodiment of the present invention, the dosage form is a solid preparation or a liquid preparation, and the solid preparation is a tablet, pill, capsule (including sustained release or delayed release form), powder or granule. The liquid preparations are suspensions, tinctures, syrups, emulsions or suspensions.
[0120] In a more preferred embodiment of the present invention, the dosage form is a solid preparation.
[0121] The present invention provides the use of the above-mentioned health product composition in reducing blood glucose concentration, total cholesterol, triglyceride or lipoprotein load.
[0122] The present invention provides the use of the above-mentioned food composition in reducing blood glucose concentration, total cholesterol, triglyceride or lipoprotein load.
[0123] The present invention provides the use of the above-mentioned pharmaceutical composition in reducing blood glucose concentration, total cholesterol, triglyceride or lipoprotein load.
[0124] The present invention provides the use of the above-mentioned preparation in reducing blood glucose concentration, total cholesterol, triglyceride or lipoprotein load.
Example Embodiment
[0126] Example 1 Preparation of rare ginsenosides and probiotics
Example Embodiment
[0127] Example 1.1
[0128] a. Strain activation and expansion
[0129] 1) S. cristatus: inoculate the strain of S. cristatus on the PDA medium slant, culture at 28°C for activation, wait for the hyphae to grow over the slant, wash the hypha with 5ml sterile saline , Inoculate 0.2ml into a 500mL Erlenmeyer flask containing 200mL PDA liquid medium, place it on a shaker at 28°C and 160rpm for 120h; then transfer the seeds in a secondary shake flask at 28°C, 160rpm at a 10% inoculum Cultured on the shaker for 72 hours, the number of viable bacteria measured was 2.0×10 2 cfu/g;
[0130] 2) Lactobacillus bulgaricus: Inoculate the Lactobacillus bulgaricus strain on the modified MRS agar medium plate, place it at 37°C for 48h activation and culture, scrape 1 loop with the inoculating loop and inoculate it into a 250mL Erlenmeyer flask containing 80mL MRS liquid medium Cultured on a shaker at 30℃ and 160r/min for 48h, the number of viable bacteria was 1.8×10 3 cfu/g;
[0131] 3) Bacillus subtilis: inoculate Bacillus subtilis strains on a solid medium (medium composition: 1L distilled water+20g glucose+15g peptone+0.5g beef extract+20g agar) slant, place it at 30℃ for activation and culture for 48h Use an inoculating loop to scrape 1 loop and inoculate it into a 250mL Erlenmeyer flask containing 80mL liquid culture medium (medium composition: 1L distilled water+20g glucose+15g peptone+0.5g beef extract), and place it at 30℃, 160r/min Cultured on the shaker for 36 hours, the number of viable bacteria measured was 2.4×10 3 cfu/g;
[0132] b. Fermentation
[0133] Put the fermentation medium (1% sugar and milk powder) into the fermentor for high-pressure steam sterilization according to the amount of about 60% of the liquid. After the fermentation medium is cooled, it is connected to the 10% coronatus sp. During the whole fermentation process, feed active raw materials at a rate of 3-12mL/L/H, control dissolved oxygen to 50%, temperature 28°C, pH 5, culture for 66 hours, and then expand the seed solution of Lactobacillus bulgaricus and Bacillus subtilis respectively according to 3% of the inoculum was connected, and the culture was continued for 60 hours, the temperature of the fermentation culture was controlled at 28°C, the rotation speed was controlled at 140r/min, and the dissolved oxygen was controlled at 60% to obtain a composite fermentation broth.
[0134] c. Fermentation product collection: After solid-liquid separation is achieved by plate and frame filtration, the solids are collected and lyophilized to obtain probiotic powder, the supernatant is collected and then concentrated to obtain ginsenoside Rh4.
[0135] d. Detection of related indicators:
[0136] ① Active rare ginsenosides: The purity of ginsenoside Rh4 is 95% and the content is 118mg/mL as determined by the American SSI liquid chromatography.
[0137] ② Detection of the number of viable bacteria: prepare to weigh 1g of freeze-dried powder, and after dilution, spread PDA plates, modified MRS agar medium plates, and Bacillus subtilis solid medium plates on an ultra-clean workbench. The count of cultured colonies shows that, The three strains have symbiosis and mutual promotion during the fermentation process.
[0138] Table 1: Colony count
[0139] S. cristatum Lactobacillus bulgaricus Bacillus subtilis Colony number before fermentation cfu/g 2.0×10 2 cfu/g
PUM
| Property | Measurement | Unit |
| Viable count | 200.0 | colony_forming_unit/g |
| Viable count | 250.0 | colony_forming_unit/g |
| Viable count | 230.0 | colony_forming_unit/g |
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