A postbiotic with the function of regulating intestinal flora and its application

Abstract

The present invention provides a postbiotic having the function of regulating intestinal flora and its application, wherein the postbiotic comprises an inactivated Saccharomyces boulardii cell and its fermentation metabolites, wherein the Saccharomyces boulardii is Saccharomyces cerevisiae An-15 strain, with a deposit number of CCTCC NO: M2019643, which is deposited in China Center for Type Culture Collection (CCTCC). The postbiotic of the present invention can significantly increase the content of short-chain fatty acids (SCFAs) of intestinal flora, increase the production of beneficial gases such as H2 and CH4, reduce the gas production of NH3 and H2S that are harmful to the human body, promote the growth of specific probiotics and promote them to produce more short-chain fatty acids, and regulate the intestinal flora well to make it in dynamic balance.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to a postbiotic capable of regulating intestinal flora and application thereof. Background Art

[0002] There are more than 10,000 kinds of microorganisms in the human intestine. In recent years, as people's understanding of intestinal microorganisms continues to deepen, the relationship between intestinal flora and human health has received increasing attention. In a healthy state, the intestinal flora is in a dynamic balance. Beneficial microorganisms can form a protective barrier in the intestine to inhibit the growth of pathogens (such as Salmonella, Campylobacter, Clostridium and Escherichia coli). Once the balance of intestinal microorganisms in the human body is destroyed, dysbiosis will eventually lead to the occurrence or aggravation of various diseases. Therefore, the dynamic balance of intestinal flora is crucial to personal health.

[0003] At present, the methods for regulating intestinal flora mainly include diet, drugs, fecal transplantation, probiotics or prebiotics, etc. Dietary intervention can affect the composition and structure of intestinal microorganisms, but this effect lacks long-term effect. Once the eating habits are restored, the composition and structure of intestinal microorganisms will return to the state before the intervention. Although fecal transplantation is an effective means of intestinal flora reconstruction, the process is complicated and has certain ethical controversies, and there is a risk of transmitting viruses or pathogens in the donor's body. In contrast, probiotic intervention is a safe and effective means of regulating intestinal flora, and its research and application are increasingly concerned by the industry. Currently, the commonly used probiotics are mainly bacterial probiotics such as Lactobacillus and Bifidobacterium, while fungal probiotics are rarely studied.

[0004] As a type of fungal probiotic, Saccharomyces boulardii is non-pathogenic and non-diffusible. It usually grows well at a high temperature of 37°C and has special biological activity. After Saccharomyces boulardii enters the intestines of humans and animals, it can not only degrade toxins produced by pathogenic bacteria, but also stimulate the intestinal mucosa to enhance immune function, while regulating the balance of intestinal microecology and preventing the invasion of pathogens. Although yeast has obvious advantages over bacterial probiotics, such as tolerance to antibiotics, greater resistance to high temperatures, acids and bile salts, and higher survival rate in the body, it is still sensitive to the environment and temperature as a live bacteria. Its activity will be lost during processing, storage and transportation, which will affect the effect of the product to a certain extent and greatly limit its scope of application. Summary of the invention

[0005] The problems existing in the current existing technologies are that there is a lack of effective methods for regulating intestinal flora, active probiotics are sensitive to the environment and temperature, which affects the effect of the product to a certain extent, and bacterial probiotics are pathogenic and diffusible.

[0006] The boulardii yeast of the present invention is a kind of fungal probiotic, which has no pathogenicity and diffusibility, and usually grows well at a high temperature of 37°C and has special biological activity. The postbiotics prepared therefrom have functions similar to those of live bacteria. Specifically, the present invention proposes the following technical solutions:

[0007] In a first aspect, the present invention provides a postbiotic, characterized in that the postbiotic comprises inactivated Saccharomyces boulardii cells and fermentation metabolites thereof.

[0008] Preferably, the postbiotic is characterized in that the Saccharomyces boulardii is Saccharomyces cerevisiae An-15 strain, with a preservation number of CCTCC NO: M2019643, which is preserved in China Center for Type Culture Collection (CCTCC).

[0009] Preferably, the postbiotic is characterized in that the mass percentage of polyphenols contained in the postbiotic is ≥3%.

[0010] In a second aspect, the present invention provides a method for preparing the postbiotics, characterized in that the bacteria are inactivated after fermentation and cultivation.

[0011] Preferably, the preparation method of the postbiotics is characterized in that the preparation method further comprises a drying step after inactivation; preferably, the drying is selected from spray drying or freeze drying.

[0012] In a third aspect, the present invention provides a postbiotic prepared by the method for preparing postbiotics.

[0013] In a fourth aspect, the present invention provides an application of the postbiotics in the preparation of intestinal flora regulating foods, health foods or medicines.

[0014] Preferably, the application is characterized in that the postbiotics increase the production of short-chain fatty acids in the intestinal flora. Preferably, the short-chain fatty acids are selected from one or more of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.

[0015] Preferably, the application is characterized in that the postbiotics increase the production of beneficial gases by intestinal flora. Preferably, the beneficial gases include one or both of H2 or CH4.

[0016] Preferably, the application is characterized in that the postbiotics reduce the production of harmful gases by intestinal flora. Preferably, the harmful gases include one or both of NH3 or H2S.

[0017] Preferably, the application is characterized in that the postbiotics promote the growth of intestinal probiotics. Preferably, the intestinal probiotics are selected from one or more of Bifidobacterium animalis, Lactobacillus reuteri and Clostridium tenuissima.

[0018] Preferably, the application is characterized in that the postbiotics inhibit the growth of harmful intestinal bacteria. Preferably, the harmful intestinal bacteria are selected from one or both of Escherichia coli and Salmonella.

[0019] In a fifth aspect, the present invention provides the use of the postbiotics in food, medicine and health products.

[0020] Preferably, the use of postbiotics in food, medicine and health products is characterized in that the use in food includes use in bread, beverages and candies.

[0021] Preferably, the postbiotics are used in food, medicine and health food, wherein the food is bread, and the postbiotics are preferably added to the raw materials for bread preparation in an amount of ≥0.01% by mass relative to the flour.

[0022] The beneficial effects of the present invention include:

[0023] The postbiotics of the present invention can quickly enter the body through the intestinal mucosa, saving the time for live bacteria to colonize in the intestine and quickly play a role. In addition, the inactivated bacteria are stable, not only resistant to high temperature, gastric acid, and bile salts, but also not limited in the later processing process, and can be applied to a variety of fields and product types.

[0024] The postbiotics of the present invention contain rich nutrients such as high-quality protein, polysaccharides, multiple vitamins, minerals, etc., which not only play an intestinal protective role but also provide rich nutrients for the host.

[0025] The postbiotics of the present invention can significantly increase the content of short-chain fatty acids (SCFAs) in the intestinal flora, increase the production of beneficial gases such as H2 and CH4, reduce the production of NH3 and H2S that are harmful to the human body, promote the growth of specific probiotics and prompt them to produce more short-chain fatty acids, and well regulate the intestinal flora to keep it in a dynamic balance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A Shown is the total production of short-chain fatty acids (SCFAs) produced by the fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0027] Figure 1B Shown is the acetic acid production produced by the fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0028] Figure 1C Shown is the propionic acid production produced by the fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0029] Figure 1D Shown is the butyrate production produced by the fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0030] Figure 1E Shown is the isobutyric acid production produced by the fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0031] Figure 1F Shown is the valeric acid production produced by the fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0032] Figure 1G Shown is the isovaleric acid production produced by the fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0033] Figure 2A Shown is the total gas production of fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0034] Figure 2B Shown is the CO2 production of fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0035] Figure 2C Shown is the H2 production of fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0036] Figure 2D Shown is the CH4 production of fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0037] Figure 2E Shown is the H2S production of fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0038] Figure 2F Shown is the NH3 production of fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group in Example 2.

[0039] Figure 3 Shown is the effect of the postbiotics in Example 2 on the metabolism of Bifidobacterium animalis, Lactobacillus reuteri, Clostridium perfringens, Escherichia coli and Salmonella.

[0040] Strain deposit information

[0041] The Saccharomyces cerevisiae An-15 strain provided by the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on August 19, 2019, with a deposit number of CCTCC NO: M 2019643, a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052; and it is disclosed in a patent application with patent publication number CN113881583A. DETAILED DESCRIPTION

[0042] The breeding method and identification of the Saccharomyces cerevisiae An-15 strain provided by the present invention have been disclosed in a patent application with patent publication number CN113881583A. A Saccharomyces cerevisiae An-1.27 strain in the Angel strain library is continuously evolved to obtain an evolved culture strain, and the evolved culture strain is subjected to atmospheric pressure and room temperature plasma mutagenesis breeding to obtain a mutant strain. The mutant strain is sequentially subjected to high-throughput screening and multiple iterative mutagenesis to obtain the Saccharomyces cerevisiae An-15 strain, which has better acid resistance, bile salt resistance, artificial gastric juice resistance and artificial intestinal juice resistance.

[0043] Unless otherwise specified, the various reagents and instruments used in the examples and comparative examples of the present invention are conventional commercially available products. The source information of the instruments and reagents used in the examples and comparative examples of the present invention is shown in Table 1 below.

[0044] Table 1

[0045]

[0046]

[0047]

[0048] The formula of the liquid culture medium used for strain activation and shake flask culture in the embodiment of the present invention is: sucrose 80-120 g / L, yeast extract 15-30 g / L, KH2PO4 0.5-1 g / L, MgSO4 0-1 g / L.

[0049] The formula of the liquid culture medium used for seed culture in the embodiment of the present invention is: molasses (reducing sugar) 80-150 g / L, magnesium sulfate 0.5-2.0 g / L, ammonium sulfate 10-30 g / L, NH4H2PO4 1-5 g / L, copper sulfate pentahydrate 0.001-0.02 g / L.

[0050] The formula of the liquid culture medium used for commercial fermentation in the embodiment of the present invention is: molasses 80-200g / L, mass concentration of 20% ammonia water 5-30ml / L, NH4H2PO4 1-5g / L, copper sulfate pentahydrate 0.001-0.02g / L.

[0051] Example 1 Preparation method of postbiotics

[0052] 1. Bacteria activation

[0053] A loopful of Saccharomyces boulardii An-15 colony was selected and inoculated into 100 ml of liquid culture medium for activation. The specific composition of the liquid culture medium was: sucrose 100 g / L, yeast extract 20 g / L, KH2PO4 1 g / L, MgSO4 0.5 g / L. The culture time was 26 h at 200 rpm and at 30 °C.

[0054] 2. Shake flask culture

[0055] 1 mL of the activated fermentation broth was inoculated into a shake flask containing 1 L of liquid culture medium, wherein the specific composition of the liquid culture medium was: sucrose 100 g / L, yeast extract 20 g / L g / L, KH2PO4 1.0 g / L, MgSO4 0.5 g / L, pH 5.0-5.5. The culture conditions were: 30°C, 200 rpm, and cultured for 24 h.

[0056] 4. Seed fermentation

[0057] Take 1L of the liquid culture medium after shake flask culture and inoculate it into a fermenter containing 9L of seed culture medium to continue fermentation culture. The specific composition of the liquid culture medium used for seed culture is: molasses (reducing sugar) 120g / L, magnesium sulfate 1.2g / L, ammonium sulfate 24g / L, NH4H2PO4 3.0g / L, copper sulfate pentahydrate 0.004g / L.

[0058] The initial air volume is 9L / min and the speed is 200rpm. Molasses, ammonium sulfate, and NH4H2PO4 are added. As the seed fermentation proceeds, the air volume eventually reaches 30L / min and the speed reaches 600rpm. The fermentation time is 24h, and the temperature of the entire fermentation process is 30℃. After the fermentation is completed, the seed fermentation liquid is separated by a centrifuge until the yeast concentration in the heavy phase reaches more than 500g / L. The heavy phase solution at this time is the seed liquid.

[0059] Yeast concentration measurement method: take 10mL of solution and put it into a centrifuge tube, measure the weight as m0 (g), centrifuge at 5000rpm for 4min, take out the centrifuge tube, pour out the light phase solution, turn the centrifuge tube mouth downward, shake it three times to remove the light phase solution, and measure the weight of the centrifuge tube as m1 (g). The yeast concentration in the solution (g / L) is: (m0-m1) / L.

[0060] 5. Product fermentation

[0061] The seed liquid was inoculated into a fermentation tank containing 9L of commercial fermentation medium, and the yeast concentration of the fermentation liquid after inoculation was 50g / L. The specific composition of the liquid culture medium used for commercial fermentation is: molasses 150g / L, 20% ammonia water 15mL / L, NH4H2PO4 2.0g / L, copper sulfate pentahydrate 0.004g / L. The initial air volume is 15L / min, the initial speed is 400rpm, and the temperature is 31°C. Molasses, ammonia water, and NH4H2PO4 are added, and as the commercial fermentation time progresses, the final air volume reaches 30L / min and the speed reaches 600rpm. The fermentation time is 15h, and the final fermentation temperature is 34°C. After the fermentation is completed, the fermentation liquid is heated to 90°C and sterilized for 60min.

[0062] 6. Spray drying

[0063] The inactivated fermentation product was spray dried with an air inlet temperature of 160°C, an air outlet temperature of 85°C, and a feed rate of 1 L / h. After drying, the dried powder was collected to obtain yeast postbiotics.

[0064] Example 2 Study on the intervention of postbiotics on fecal flora fermentation products and the growth of specific probiotics

[0065] 1. Collection and storage of stool samples

[0066] The volunteers in this embodiment are from internal employees of the company. A total of 40 volunteers who meet the requirements have collected fresh feces, including 20 volunteers without intestinal diseases and 20 volunteers with intestinal diarrhea diseases. All volunteers have eaten normally in the past month and have not received antibiotics, prebiotics or probiotics and other drug treatments. In addition, healthy volunteers did not show gastrointestinal diseases such as constipation, flatulence and diarrhea. The basic characteristics of all volunteers are shown in Table 2. The recruitment of all volunteers and the collection of fecal samples were carried out in cooperation with Angel Yeast Co., Ltd. and Yichang Center for Disease Control and Prevention. All volunteers should quickly pick at least 3g of feces in the middle area with less food residue and less oxygen contact during defecation, and place it in a disposable sterile feces sampling box, and record the name, gender and age of the donor. The collected fecal samples are stored at 4°C and need to be fermented within 4 hours.

[0067] Table 2

[0068]

[0069] 2. Sample pretreatment

[0070] A ready-to-use PBS tablet was dissolved in ultrapure water and sterilized by high pressure steam at 121°C for 15 min, and then cooled to obtain a sterile PBS buffer solution.

[0071] Take 0.8 g of fresh feces sample and put it into a 10 mL sterile centrifuge tube. Then add 8 mL of sterile PBS buffer, seal and shake to mix. After removing large particles of impurities with sterile gauze, a 10% feces suspension inoculum can be obtained.

[0072] The postbiotic powder was suspended in sterile PBS buffer to prepare a yeast suspension with a concentration of 50 mg / ml, which was used for the fermentation samples described in "4. In vitro batch simulated fermentation of intestinal flora" below.

[0073] 3. Preparation of fermentation medium

[0074] The in vitro simulated fermentation medium for intestinal flora used in this example has the following composition: 10 g / L tryptone, 2.5 g / L yeast extract, 1 g / L L-cysteine, 2 ml / L heme solution with a concentration of 5 mg / mL, 0.9 g / L sodium chloride, 0.09 g / L calcium chloride hexahydrate, 0.45 g / L potassium dihydrogen phosphate, 0.45 g / L potassium hydrogen phosphate, and 0.09 g / L magnesium sulfate heptahydrate. The blank control medium can be obtained by weighing the above ingredients in proportion and dissolving them with ultrapure water.

[0075] After the liquid fermentation medium is prepared, it is heated with a magnetic heating stirrer to keep boiling to reduce the gas contained in the medium solution. During this period, it is dispensed into vials with a peristaltic pump, and each bottle is dispensed with 4.5 ml. At the same time, nitrogen should be continuously filled into the vials during the dispensing process to maintain an anaerobic environment. After the dispensing is completed, it is quickly sealed with a cap and marked. Finally, all vials filled with the medium are sterilized at 115°C with high-pressure steam for 30 minutes, cooled and stored for later use.

[0076] 4. In vitro batch fermentation of intestinal flora

[0077] Four different fermentation samples were set up: healthy control group, diarrhea control group, healthy postbiotic group, and diarrhea postbiotic group.

[0078] The healthy control group was prepared by mixing 250 μL of fecal suspension inoculum from volunteers without intestinal diseases and 250 μL of sterile PBS buffer in an anaerobic workstation, aspirating all the bubbles in the syringe with a disposable sterile syringe, and injecting it into a sterile blank culture medium; the diarrhea control group was prepared by mixing 250 μL of fecal suspension inoculum from volunteers with intestinal diarrhea diseases and 250 μL of sterile PBS buffer in an anaerobic workstation, aspirating all the bubbles in the syringe with a disposable sterile syringe, and injecting it into a sterile blank culture medium; the healthy postbiotic group was prepared by taking 250 μL of fecal suspension inoculum from volunteers without intestinal diseases and 250 μL of yeast suspension (such as the "50 mg / ml yeast suspension" prepared in "2. Sample pretreatment") was placed in a sterile centrifuge tube, and 500 μL of mixed bacterial suspension was obtained after shaking and mixing, which was injected into a sterile blank culture medium using a disposable sterile syringe; the diarrhea postbiotic group was prepared by taking 250 μL of fecal suspension inoculum of volunteers with intestinal diarrhea and 250 μL of yeast suspension (such as the "50 mg / ml yeast suspension" prepared in "2. Sample pretreatment") in a sterile centrifuge tube, shaking and mixing to obtain 500 μL of mixed bacterial suspension, which was injected into a sterile blank culture medium using a disposable sterile syringe.

[0079] Make three parallels for each culture medium. After gently shaking to mix, place all the culture media in a water-tight incubator and culture them at 37°C for 24 hours. After the culture is completed, store them in a 4°C refrigerator for later use.

[0080] 5. In vitro simulated fermentation gas production detection of intestinal flora

[0081] Take out the vial that has completed fermentation, wait for it to return to room temperature, and then use the intestinal microbial gas detector to detect gas. After the gas detector is turned on and preheated, press the record button, and then connect its air inlet and outlet to the vial through a rubber tube and a disposable syringe needle. During the measurement, the needle is prohibited from touching the culture medium liquid surface to prevent water from entering the machine. The machine can detect the concentrations of CH4, H2S, NH3, CO2, and H2. The concentrations of the five gases will increase and then decrease over time, and the highest value is the volume concentration of the gas produced by the fermentation of fecal flora. After the values ​​of the five gases have dropped to zero, the detection of the next sample bottle can be started.

[0082] 6. Detection of short-chain fatty acids (SCFAs) produced by in vitro simulated fermentation of intestinal flora

[0083] (1) Drawing of standard curve

[0084] Dissolve 2.5g of metaphosphoric acid in deionized water and dilute to 100mL to obtain a 2.5% (w / v) metaphosphoric acid solution, then weigh 0.64g of crotonic acid and dissolve it in the metaphosphoric acid solution and dilute to 100mL to obtain a crotonic acid / metaphosphoric acid solution. The area internal standard method was used to establish the standard curve of each component. Chromatographically pure acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid were diluted with ultrapure water to 6 standard solutions with different concentration gradients. The peak time and peak area of ​​the six SCFAs and crotonic acid at different standard concentrations were recorded. The peak area ratio and concentration ratio of each SCFA to crotonic acid were used to make the corresponding standard curve at different concentrations. Three parallels were made for each standard sample concentration and the average value was taken.

[0085] (2) Fermentation sample processing

[0086] After the gas detection is completed, open the vial in the clean bench, shake and mix the fermentation liquid, then take 1.8mL of the fermentation liquid into a 2mL sterile centrifuge tube, centrifuge at 12000rpm for 6min, take 500μL of the supernatant into a 1.5mL sterile centrifuge tube, add 100μL of crotonic acid / metaphosphoric acid solution, mix well, and acidify in a -80℃ refrigerator for at least 24h. The fermentation liquid precipitate is also stored in a -80℃ refrigerator.

[0087] After acidification, thaw in a 4°C refrigerator, then centrifuge at 4°C and 12,000 rpm for 6 min, take the supernatant, filter with a 0.22 μm water-based microporous filter membrane, add 150 μL of the filtrate to the inner tube of the gas phase injection bottle, shake the injection bottle to expel the bubbles at the bottom of the inner tube to prevent air suction during gas chromatograph injection.

[0088] (3) Gas chromatography method

[0089] Install the gas chromatograph column, remove the waste liquid, add the cleaning liquid, clean or replace the injection pad, liner and injection needle. After preparation, turn on the machine, open the gas valve and gas purifier, and perform the aging procedure. The column temperature rise procedure is: the column temperature is maintained at 80℃ for 1 minute, then increased to 190℃ at a rate of 10℃ / min, maintained for 0.50min, and then increased to 240℃ at a rate of 40℃ / min, maintained for 5min. The FID detector and vaporization chamber are maintained at 240℃. The three carrier gas flow rates are 20mL / min (nitrogen), 40mL / min (hydrogen) and 400mL / min (air).

[0090] 7. In vitro growth assay

[0091] Bifidobacterium animalis, Lactobacillus reuteri, Clostridium perfringens, Escherichia coli, and Salmonella were activated and cultured overnight at 37°C. Then, 1 mL of the saturated O / N culture of these five strains was inoculated into 100 mL of appropriate culture medium as a control group. The postbiotic group was inoculated with 0.5 mg / mL yeast suspension at the same time. After culturing to the logarithmic phase, the bacterial solution of each strain was serially diluted 10 times, and the viable cells were counted by quantitative plate counting. All experiments were performed under anaerobic conditions.

[0092] 8. Determination of total polyphenol content of strains (Folin-Ciocalteu (FC) colorimetric method)

[0093] (1) Preparation of sample solution

[0094] The fermentation broth of Saccharomyces boulardii An-15 was vacuum filtered with 200-mesh filter paper, and the filtrate was ultrafiltered through a 100 μm molecular weight ultrafiltration column to obtain a clarified filtrate, which was the sample solution.

[0095] (2) Determination of standard curve

[0096] Accurately weigh 10 mg of gallic acid, place it in a 100 ml volumetric flask, dilute to the mark with deionized water, shake well for later use, and this is the gallic acid standard solution. Accurately pipette 0, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60 and 0.70 ml of the standard solution into a 10 ml colorimetric tube, add 1.0 ml of FC reagent (0.2 mol / L) and 2 ml of 15% Na2CO3 solution in sequence, dilute to 10 ml with deionized water, mix thoroughly, place at room temperature for 1 hour, measure the absorbance at a wavelength of 700 nm, and establish a standard curve.

[0097] (3) Determination of total polyphenol content in sample solution

[0098] Use a pipette to accurately transfer 1 mL of the sample solution diluted 30 times (i.e., the concentration percentage is 3.34%) into a test tube, and repeat 5 times in total. The other operating conditions are the same as those for establishing the standard curve. The total polyphenol content in the sample solution is calculated according to the regression equation of the standard curve.

[0099] 9. Data statistics and analysis

[0100] The data of gas and SCFAs were statistically plotted using Excel, and one-way or two-way ANOVA was performed using Minitab software. All test results are expressed as mean ± SD. The significance level was set as follows: *, #: P < 0.05 indicates a significant difference; **, ##: P < 0.01 indicates an extremely significant difference. * indicates the comparison of the fermentation results of fecal flora of healthy or diarrheal volunteers in two culture media, and # indicates the comparison of the fermentation results of fecal flora of healthy volunteers and diarrheal volunteers in the same culture medium.

[0101] 10. Test results

[0102] (1) Analysis of differences in the production of short-chain fatty acids in fecal flora of healthy volunteers and diarrhea volunteers in the control fermentation group and the postbiotic fermentation group

[0103] The production of six SCFAs after fecal flora fermentation in the healthy control group and diarrhea control group, as well as the healthy postbiotic group and diarrhea postbiotic group set up in “4. In vitro batch simulated fermentation of intestinal flora” was tested. The results are as follows: Figure 1A , 1B , 1C, 1D, 1E, 1F, 1G and Table 3, where Figure 1A is the total production of SCFAs, Figure 1B is the acetic acid yield, Figure 1C is the propionic acid yield, Figure 1D is the butyrate production, Figure 1E is the isobutyric acid yield, Figure 1F The production of valeric acid and Figure 1G is the production of isovaleric acid. Among the six SCFAs, the production of acetic acid was the highest, followed by propionic acid and butyric acid, while the production of isobutyric acid, valeric acid and isovaleric acid was the lowest. Whether it was the fecal flora of healthy or diarrheal volunteers, the total production of SCFAs, acetic acid, propionic acid, butyric acid and valeric acid in the postbiotic group were significantly higher than those in the control group, while there was no significant difference in the production of isovaleric acid and isobutyric acid. The postbiotic group of healthy volunteers had an average increase of 59% in total SCFAs production, 75% in acetic acid production, 51% in propionic acid production, 40% in butyric acid production, 100% in valeric acid production, 10% in isovaleric acid production and 25% in isobutyric acid production compared with the control group; the postbiotic group of diarrheal volunteers had an average increase of 36% in total SCFAs production, 43% in acetic acid production, 140% in propionic acid production, 50% in butyric acid production, 350% in valeric acid production, 20% in isovaleric acid production and 30% in isobutyric acid production compared with the control group.

[0104] In addition, in the control group, the production of propionic acid and valeric acid in the fecal flora of healthy volunteers was significantly higher than that of diarrhea volunteers. In the control group, the production of propionic acid in the fecal flora of healthy volunteers was 75% higher than that of diarrhea volunteers, and the production of valeric acid in the fecal flora of healthy volunteers was 150% higher than that of diarrhea volunteers. In the postbiotic group, there were significant differences in the total production of SCFAs and acetic acid between healthy and diarrhea volunteers. In the postbiotic group, the total production of SCFAs in the fecal flora of healthy volunteers was 33% higher than that of diarrhea volunteers, and the total production of acetic acid in the fecal flora of healthy volunteers was 40% higher than that of diarrhea volunteers. From the changes in the production of the six SCFAs, it can be seen that the postbiotic has a good promoting effect on the production of SCFAs in the fecal flora of healthy and diarrhea volunteers, and the intervention of postbiotics can help the intestinal flora of diarrhea people to return to normal.

[0105] Table 3

[0106]

[0107]

[0108] (2) Analysis of gas production differences among fermentation groups

[0109] The production of six SCFAs and the total production of SCFAs during the fecal flora fermentation of the healthy control group and diarrhea control group set up in "4. In vitro batch simulated fermentation of intestinal flora" and the healthy postbiotic group and diarrhea postbiotic group were compared. Figure 2A , 2B , 2C, 2D, 2E, 2F and Table 4, where Figure 2A is the total gas production, Figure 2B is the CO2 production, Figure 2C is the H2 production, Figure 2D is the CH4 production, Figure 2E The H2S production and Figure 2Fis the production of NH3. Among the five gases, CO2 has the largest gas production, followed by H2, and the production of CH4, H2S, and NH3 is relatively small. Whether it is the fecal flora of healthy or diarrheal volunteers, there are significant differences in the production of the five gases in the two groups. Among them, the production of CO2, H2, and CH4 in the postbiotic group is significantly higher than that in the control group, while the production of H2S and NH3 shows the opposite trend. Compared with the control group, the production of CO2 in healthy volunteers in the postbiotic group increased by an average of 165%, the production of CH4 increased by an average of 114%, the production of H2 increased by an average of 50%, the production of H2S decreased by an average of 290%, and the production of NH3 decreased by an average of 550%; compared with the control group, the production of CO2 in diarrheal volunteers in the postbiotic group increased by an average of 170%, the production of CH4 increased by an average of 220%, the production of H2 increased by an average of 67%, the production of H2S decreased by an average of 240%, and the production of NH3 decreased by an average of 471%.

[0110] In addition, in both groups, the production of H2 and CH4 in healthy volunteers was significantly higher than that in diarrhea volunteers, while the production of NH3 was the opposite, but there was no significant difference in the production of CO2 and H2S. In the control group, the H2 production of healthy volunteers increased by an average of 67%, the CH4 production increased by an average of 133%, and the NH3 production decreased by an average of 54% compared with that of diarrhea volunteers; in the postbiotic group, the H2 production of healthy volunteers increased by an average of 50%, the CH4 production increased by an average of 56%, and the NH3 production decreased by an average of 43% compared with that of diarrhea volunteers. According to the difference in gas production, it can be seen that after the intervention of this postbiotic, it helps to increase the production of H2 with anti-inflammatory effect and CH4 with anti-apoptotic effect, reduce the production of NH3 and H2S that are harmful to the human body, and reduce the production of harmful gases such as NH3 in the fecal flora of diarrhea volunteers, indicating that this postbiotic may play a good role in regulating intestinal flora, inhibiting and alleviating the occurrence and development of some intestinal diseases.

[0111] Table 4

[0112]

[0113]

[0114] (3) This postbiotic can significantly promote the growth of specific probiotics and the production of short-chain fatty acids (SCFAs), while also inhibiting the proliferation of certain harmful bacteria.

[0115] In vitro tests have shown that this postbiotic can significantly promote the growth of animal Bifidobacterium, Lactobacillus reuteri and Clostridium perfringens, and promote them to produce more short-chain fatty acids. In addition, this postbiotic has a significant inhibitory effect on harmful bacteria such as Escherichia coli and Salmonella. The results are as follows Figure 3The above results indicate that this postbiotic can maintain the dynamic balance of intestinal flora by promoting the growth of specific probiotics and the production of SCFAs, as well as inhibiting the growth of certain specific harmful bacteria.

[0116] (4) Analysis of total polyphenol content of postbiotics

[0117] The total polyphenol content percentage of the postbiotics is shown in Table 5. The mass percentage of polyphenols contained in the postbiotics of the present invention is ≥3%.

[0118] Table 5

[0119]

[0120] Polyphenols have many biological activities such as antioxidant, anti-inflammatory, and antibacterial. The high polyphenol content means that the postbiotics formed during the entire fermentation process have significant health benefits.

[0121] Example 3 Application of postbiotics in bread

[0122] This embodiment discloses a method for preparing freshly baked bread containing the postbiotic, and the bread formula is as described in Table 6.

[0123] Table 6 Bread recipe

[0124]

[0125] After accurately weighing all kinds of raw materials according to the formula, they are placed in a bread machine for automatic kneading for 30 minutes, and then 50g of butter is added to the bread machine to continue kneading. After 15 minutes, the bread machine cover is covered for the first fermentation. When the dough is fermented to twice its original volume, 50mg of the postbiotics obtained in Example 1 is added to the bread machine, the dough is evenly divided into 5 parts after exhausting and kneading the postbiotic powder, and rolled into a tongue cake shape, rolled up and placed in a bread mold. Set the bread machine temperature to 30°C and continue fermenting for 30 minutes, then brush a little egg liquid on the surface, place the bread in the oven and bake for 20 minutes, and the baking temperature is 200-230°C to obtain a functional bread that can regulate intestinal flora.

[0126] The method for making bread has simple operation steps, soft texture, mellow taste, sweet but not greasy. The content of postbiotic powder in freshly baked bread is 50 mg (about 1 billion CFU), which effectively avoids the influence of baking temperature of freshly baked bread on the activity of live bacteria, while maintaining the same effect of regulating intestinal flora as live bacteria, expanding the application range of probiotics.

[0127] Example 4 Application of postbiotics in flavored beverages

[0128] The present embodiment discloses a method for preparing a postbiotic flavored beverage: first, water, maltitol liquid, skim milk powder, white granulated sugar, polydextrose, glucose, and soluble soybean polysaccharide are proportioned and stirred to obtain a mixture A; then, sucralose, food flavoring, and postbiotic powder are added while stirring; after all materials are added, they are further homogenized to fully mix all materials to obtain a mixture B; finally, the mixture B is filled and sterilized to obtain a flavored beverage containing postbiotics.

[0129] The postbiotic beverage described in the method contains the following components by weight per 100g: 5g maltitol liquid, 45g skim milk powder, 5g white sugar, 3g polydextrose, 1g glucose, 1g soluble soybean polysaccharide, 0.1g sucralose, 0.05g food flavoring, 0.5g postbiotics, and water is added to 100g.

[0130] Example 5 Application of postbiotics in candy

[0131] This embodiment discloses a method for preparing a compressed candy containing postbiotics: the formula is as follows (weight components contained per 100g): 60g of sorbitol, 25g of isomalt, 8g of resistant dextrin, 5g of strawberry powder, 0.5g of postbiotics, 1g of citric acid, and 0.5g of magnesium stearate. The above materials are accurately weighed and fully mixed, and the tablets are compressed using a rotary tablet press to obtain a compressed candy containing postbiotics.

[0132] The postbiotic compressed candy is not affected by factors such as environmental temperature and humidity, tablet press pressure, etc. during the tableting process, thereby ensuring the probiotic effect of the product after tableting.

[0133] Comparative Example 1 Study on the Intervention of Live Saccharomyces boulardii on Fermentation Products of Intestinal Flora and the Growth of Specific Probiotics

[0134] The research method in Example 2 was used to study the intervention of live bacteria on the fermentation products of intestinal flora and the effect on the growth of specific probiotics. The comparison results of live bacteria and postbiotics are shown in Tables 7, 8, 9 and 10.

[0135] Table 7 Production of six SCFAs and total SCFAs by intestinal flora of volunteers in each fermentation group

[0136]

[0137]

[0138] Table 8 Five gas production and total gas production of intestinal flora of volunteers in each fermentation group

[0139]

[0140] Table 9 Comparison of the effects of each fermentation group on the metabolism of specific microorganisms

[0141]

[0142]

[0143] Table 10 Comparison of the effects of each fermentation group on the production of SCFAs (mM) by specific microorganisms

[0144]

[0145] From the comparison results, live Saccharomyces boulardii can also significantly increase the production of SCFAs in intestinal fecal flora, promote the growth of specific probiotics and the production of SCFAs, and inhibit the proliferation of certain harmful bacteria. In addition, in terms of gas production, live bacteria significantly promote the production of beneficial gases such as H2 and CH4, and reduce the production of NH3 and H2S that are harmful to the human body. However, compared with the postbiotic group, there is no significant advantage. Therefore, the postbiotic group and the live bacteria group have equivalent effects on regulating intestinal flora.

[0146] Comparative Example 2 Application of live Saccharomyces boulardii in bread, flavored beverages and candies and the number of live bacteria

[0147] Bread, flavored beverages and candies containing live bacterial powder of Saccharomyces boulardii strain An-15 were prepared according to the methods of Examples 3, 4 and 5. The amount of live bacteria of Saccharomyces boulardii strain An-15 added was 1×10 9 CFU / portion, the addition amount of other components in bread, flavored beverages and candies is the same as that in Example 3, Example 4 and Example 5. The determination method of live yeast in bread, flavored beverages and candies is determined by referring to the determination method in the National Standard of the People's Republic of China GB 4789.15-2016 "National Food Safety Standard Food Microbiological Examination Mold and Yeast Count". The specific test results are shown in Table 11 below:

[0148] Table 11 Saccharomyces boulardii viable bacteria content (CFU / portion)

[0149]

[0150] From the results of comparative example 1, live Saccharomyces boulardii can also significantly increase the production of SCFAs in intestinal fecal flora, promote the growth of specific probiotics and the production of SCFAs, and inhibit the proliferation of certain harmful bacteria. In addition, in terms of gas production, live bacteria significantly promote the production of beneficial gases such as H2 and CH4, and reduce the production of NH3 and H2S that are harmful to the human body. However, compared with the postbiotic group, there is no significant advantage. As can be seen from comparative example 2, live Saccharomyces boulardii is completely lost in products that require high-temperature processing procedures such as bread and flavored beverages, so its efficacy in regulating intestinal flora in such products is also damaged and lost. In products such as compressed candies, although they are still active after processing, the activity loss rate is as high as more than 90%, and their activity is completely lost after 7 days. It is speculated that the pressure may cause damage to the yeast living cells, causing them to rapidly inactivate. Therefore, compared with live bacteria, postbiotics have a wider range of applications and can be made into functional products that regulate intestinal flora in different forms and types.

[0151] The above description is only a preferred embodiment adopted by the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. An application of a postbiotic in the preparation of a food or medicine for regulating intestinal flora, characterized in that: The postbiotics include inactivated Saccharomyces boulardii bacteria and fermentation metabolites thereof; the Saccharomyces boulardii is Saccharomyces cerevisiae An-15 strain, with a preservation number of CCTCC NO: M 2019643, which is preserved in the China Center for Type Culture Collection (CCTCC).

2. The use according to claim 1, characterized in that: The mass percentage of polyphenols contained in the postbiotics is ≥3%.

3. The use according to claim 1, characterized in that: The postbiotics are prepared by the following method: Saccharomyces boulardii An-15 strain is fermented and then inactivated.

4. The use according to claim 2, characterized in that: The postbiotics are prepared by the following method: Saccharomyces boulardii An-15 strain is fermented and then inactivated.

5. The use according to claim 3, characterized in that: The method further comprises a drying step after inactivation.

6. The use according to claim 4, characterized in that: The method further comprises a drying step after inactivation.

7. The use according to claim 5, characterized in that: The drying is selected from spray drying or freeze drying.

8. The use according to claim 6, characterized in that: The drying is selected from spray drying or freeze drying.

9. The use according to any one of claims 1 to 8, characterized in that: The postbiotics increase the production of short-chain fatty acids by the intestinal flora.

10. The use according to claim 9, characterized in that: The short-chain fatty acid is selected from one or more of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.

11. The use according to any one of claims 1 to 8, characterized in that: The postbiotics increase the production of beneficial gases by the intestinal flora; and / or, the postbiotics reduce the production of harmful gases by the intestinal flora.

12. The use according to claim 11, characterized in that: The beneficial gas includes one or both of H2 or CH4; and / or, the harmful gas includes one or both of NH3 or H2S.

13. The use according to any one of claims 1 to 8, characterized in that: The postbiotics promote the growth of intestinal probiotics; and / or, the postbiotics inhibit the growth of intestinal harmful bacteria.

14. The use according to claim 13, characterized in that The intestinal probiotics are selected from one or more of Bifidobacterium animalis, Lactobacillus reuteri and Clostridium tenella; and / or, the intestinal harmful bacteria are selected from one or more of Escherichia coli and Salmonella.

15. The use according to any one of claims 1 to 8, characterized in that: The food includes health products.

16. The use according to any one of claims 1 to 8, characterized in that: Foods that regulate intestinal flora include bread, beverages and candies that regulate intestinal flora.

17. The use according to claim 9, characterized in that: Foods that regulate intestinal flora include bread, beverages and candies that regulate intestinal flora.

18. The use according to claim 11, characterized in that: Foods that regulate intestinal flora include bread, beverages and candies that regulate intestinal flora.

19. The use according to claim 13, characterized in that: Foods that regulate intestinal flora include bread, beverages and candies that regulate intestinal flora.

20. The use according to claim 16, characterized in that In the preparation of bread for regulating intestinal flora, the added amount of postbiotics is ≥ 0.01% based on the mass of raw flour.

21. The use according to claim 17, characterized in that In the preparation of bread for regulating intestinal flora, the added amount of postbiotics is ≥ 0.01% based on the mass of raw flour.

22. The use according to claim 18, characterized in that In the preparation of bread for regulating intestinal flora, the added amount of postbiotics is ≥ 0.01% based on the mass of raw flour.

23. The use according to claim 19, characterized in that In the preparation of bread for regulating intestinal flora, the added amount of postbiotics is ≥ 0.01% based on the mass of raw flour.

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