Complex probiotic agent for reducing intestinal production of harmful gases and modulating intestinal flora

Abstract

This invention relates to the field of microbial technology, and particularly to a compound probiotic agent for reducing the production of harmful intestinal gases and regulating intestinal flora. The compound probiotic of this invention consists of *Bifidobacterium animalis* subsp. *lactobacter* Bi66, *Lactobacillus plantarum* P16, and *Saccharomyces boulardii* strain Bld-3. This invention combines these three strains to significantly increase the content of short-chain fatty acids in the intestinal flora, increase the production of beneficial gases such as CH4 and H2, and reduce the production of harmful gases such as NH3 and H2S, thereby promoting the growth of beneficial bacteria and inhibiting the growth of harmful bacteria and regulating the intestinal flora. While maintaining a consistent amount of bacteria, compared with intervention methods lacking any one of the strains, the combination of the three strains showed the best effect in improving the intestinal flora, indicating that the three strains can cooperate and promote each other, and can synergistically enhance the effect in regulating the intestinal flora.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound probiotic agent that reduces the production of harmful intestinal gases and regulates intestinal flora. Background Technology

[0002] The human gut is home to a vast and diverse community of microorganisms, collectively known as the gut microbiota. Numerous scientific studies have confirmed that the composition and functional balance of the gut microbiota are closely related to human health. A balanced gut microbiota not only aids in food digestion, nutrient absorption, and vitamin synthesis, but also plays a crucial role in maintaining intestinal barrier function, regulating the immune system, resisting pathogen invasion, and influencing metabolism and neural regulation. Furthermore, harmful gases produced by gut microbiota metabolism can cause discomfort such as bloating, abdominal pain, and bad breath. Excessive amounts can also be toxic to intestinal cells, affecting intestinal barrier function and even being closely related to the development of certain intestinal diseases. Gut microbiota dysbiosis is considered to be associated with the development of various diseases, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), antibiotic-associated diarrhea (AAD), and constipation. Gut microbiota dysbiosis is often accompanied by excessive proliferation of harmful bacteria, reduced beneficial bacteria, and excessive production of harmful metabolites (including the aforementioned harmful gases). Therefore, the dynamic balance of the gut microbiota is crucial for individual health.

[0003] Probiotics, as live microorganisms, can have beneficial effects on the host's health when ingested in sufficient quantities. Currently, regulating and improving the gut microbiota structure through exogenous probiotic supplementation has become an important strategy for the prevention and adjunctive treatment of related diseases.

[0004] Currently, various probiotic products are available on the market, including single-strain or multi-strain combinations (compound probiotics), with dosage forms covering pharmaceuticals, health supplements, and foods (such as yogurt, fermented milk, and probiotic powders). Despite the wide application of probiotics, existing probiotic preparations, especially those involving multi-strain combinations, suffer from arbitrary combinations and a lack of synergistic effects. In particular, research on achieving synergistic reduction of harmful intestinal gases through specific strain combinations is scarce. Therefore, there is an urgent need to develop a novel compound probiotic preparation based on specific strain combinations that have been proven to have synergistic effects, effectively overcoming the shortcomings of existing technologies and achieving a more comprehensive and efficient gut microbiota balance.

[0005] Although the regulatory effects of probiotics on the gut microbiota have been extensively studied, existing probiotic preparations, especially those involving multi-strain combinations, still have significant limitations. For example, single strains have limited efficacy, and multi-strain combinations suffer from arbitrary pairings and a lack of synergistic effects; in particular, there are limited reports on their ability to significantly reduce various harmful intestinal gases.

[0006] Existing technology discloses a compound probiotic capable of alleviating ulcerative colitis, its preparation method, and its application. This compound probiotic includes *Bifidobacterium longum* BL21, *Bifidobacterium animalis subsp. lactis* BLa 80, and *Lactobacillus casei* LC89. Its preparation includes the following steps: separately preparing freeze-dried powders of *Bifidobacterium longum* BL21, *Bifidobacterium animalis subsp. lactis* BLa80, and *Lactobacillus casei* LC89; and mixing the three freeze-dried powders evenly in a specific ratio to prepare the compound probiotic. The compound probiotic provided by this invention can prevent and / or treat ulcerative colitis and has great application potential in the preparation of products for the prevention and / or treatment of ulcerative colitis (such as solid beverages).

[0007] A probiotic agent for enhancing immunity and improving gut microbiota and its application involves compounding five specific, different types of Bifidobacterium strains. These five strains exhibit potential interactions and can synergistically enhance the growth of immune organs and increase the total quantity and diversity of gut microbiota. This agent is used in conjunction with basic fermentation strains for yogurt fermentation, while simultaneously adding compound prebiotics for synergistic fermentation. The resulting yogurt maintains a Bifidobacterium count of at least 1 × 10⁻⁶ at the end of its shelf life after fermentation. 8 CFU / mL or 1×10 8 CFU / g significantly improves the stability of lactic acid bacteria in yogurt, significantly increasing the number of live bacteria that enter the human intestine and exert their effects, resulting in better tolerance in the intestine; the yogurt produced has a delicate texture, rich taste, and strong aroma; and it can also significantly improve the immune decline and intestinal flora disorder caused by cyclophosphamide (CTX), thus having a positive effect on immune enhancement.

[0008] Although the probiotic combination involved in the invention of CN 113430133 B can alleviate ulcerative colitis to a certain extent, the synergistic effect of the three strains used together has not been verified, and the combination may be somewhat arbitrary.

[0009] The probiotic combination involved in the invention CN 119530101 A is used for yogurt fermentation. The actual investigation is on the regulatory effect of fermented yogurt on immunity and gut microbiota. The five strains mentioned have potential interactions and can synergistically promote the growth of immune organs and increase the total number and diversity of gut microbiota. However, no corresponding experimental evidence has been found, which limits the application scope of this compound strain.

[0010] In the existing field of probiotics, especially compound probiotics, there is insufficient discovery and verification of synergistic effects among specific strain combinations (especially three strains). There is a certain degree of randomness in the combination and lack of synergistic effects. Furthermore, there are few reports on the synergistic reduction of harmful intestinal gases through specific strain combinations. Summary of the Invention

[0011] In view of this, the present invention provides a compound probiotic agent that can reduce the production of harmful gases in the intestines and regulate the intestinal flora. The three strains are innovatively combined and have a synergistic effect.

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] In a first aspect, the present invention provides a compound microbial culture, comprising Bifidobacterium animalis subsp. lactis Bi66, Lactobacillus plantarum P16, and Saccharomyces boulardii Bld-3.

[0014] In some specific embodiments of the present invention, the ratio of the viable counts of Bifidobacterium animalis subsp. Lactobacillus Bi66, Lactobacillus plantarum P16 and Saccharomyces boulardii Bld-3 is (1~5):(1~5):(1~5).

[0015] In some specific embodiments of the present invention, the ratio of viable counts of Bifidobacterium animalis subsp. Lactobacillus Bi66, Lactobacillus plantarum P16, and Saccharomyces boulardii Bld-3 is 1:1:1, 5:1:1, or 1:2:5.

[0016] In some specific embodiments of the present invention, the total number of viable bacteria in the compound bacteria is not less than 1×10⁻⁶. 8 CFU / ml or 1×10 8 CFU / g.

[0017] In some specific embodiments of the present invention, the Bifidobacterium lactis subsp. Bi66 is preserved with the accession number CCTCC NO: M 2023769;

[0018] The preservation number of the plant lactobacillus P16 is CCTCC NO: M 2023767;

[0019] The preservation number of the *Blad-3* strain is CCTCC NO: M 2019643.

[0020] Secondly, the present invention also provides the application of the compound bacteria in the preparation of any of the following microecological products;

[0021] (I) Increase the content of short-chain fatty acids (SCFAs) in the gut microbiota;

[0022] (II) Increase the production of beneficial gases;

[0023] (III) Reduce harmful intestinal gases; or

[0024] (IV) Regulate the gut microbiota.

[0025] In some specific embodiments of the present invention, the beneficial gas includes CH4 and / or H2. 2。

[0026] In some specific embodiments of the present invention, the intestinal harmful gases include NH3 and / or H2S.

[0027] In some specific embodiments of the present invention, the regulation of intestinal flora includes promoting the growth of beneficial bacteria and / or inhibiting the growth of harmful bacteria;

[0028] The beneficial bacteria include one or more of Bifidobacterium animalis, Lactobacillus reuteri, or Clostridium capillaris.

[0029] The harmful bacteria include Escherichia coli and / or Salmonella.

[0030] In some specific embodiments of the present invention, the microecological products include compound probiotic products, synbiotic products, or postbiotic products.

[0031] In some specific embodiments of the present invention, the microecological products include food, feed, or feed additives.

[0032] In some specific embodiments of the present invention, the food is a health food, a food for special medical purposes, a functional food, or a pet food.

[0033] In some specific embodiments of the present invention, the dosage form of the microecological product is a powder, granule, capsule, tablet, pill, extract or liquid preparation.

[0034] Thirdly, the present invention also provides microecological products, including the aforementioned compound bacteria.

[0035] In some specific embodiments of the present invention, the microecological products include compound probiotic products, synbiotic products, or postbiotic products.

[0036] In some specific embodiments of the present invention, the microecological products include food, feed, or feed additives.

[0037] In some specific embodiments of the present invention, the food is a health food, a food for special medical purposes, a functional food, or a pet food.

[0038] In some specific embodiments of the present invention, the dosage form of the microecological product is a powder, granule, capsule, tablet, pill, extract or liquid preparation.

[0039] In some specific embodiments of the present invention, the powder includes a lyophilized powder;

[0040] The method for preparing the lyophilized powder includes the following steps:

[0041] Step 1: Activate and culture the Bifidobacterium animalis subsp. lactis Bi66 and the Lactobacillus plantarum P16 respectively to obtain bacterial solutions. Collect the bacterial cells by centrifugation and filtration, mix with a protectant and freeze dry to obtain Bi66 bacterial powder and P16 bacterial powder respectively.

[0042] Step 2: After activating and culturing the Bld-3 yeast strain, the seed fermentation broth is obtained. The cells are collected by centrifugation, mixed with emulsifier, vegetable oil and starch, and then granulated to obtain yeast powder.

[0043] Step 3: The Bi66 bacterial powder, the P16 bacterial powder, and the yeast powder obtained in Step 1 are compounded in a certain proportion to obtain the freeze-dried powder.

[0044] In some specific embodiments of the present invention, the number of live bacteria in the Bi66 bacterial powder is 600 billion CFU / g;

[0045] The P16 bacterial powder contains 200 billion CFU / g of live bacteria;

[0046] The yeast powder contains 20 billion CFU / g of Saccharomyces boulardii.

[0047] In some specific embodiments of the present invention, the method for preparing the lyophilized powder includes the following steps:

[0048] Step 1-1: Inoculate the Bifidobacterium animalis subsp. lactis Bi66 and the Lactobacillus plantarum P16 into MRS liquid medium and activate them by culturing at 37°C for 24 hours. Repeat the activation process twice to obtain the activated solution.

[0049] Steps 1-2: Inoculate the activation solution into MRS liquid medium at an inoculation rate of 3% (v / v) and incubate at 37°C for 22 hours to obtain bacterial culture.

[0050] Steps 1-3: Centrifuge the bacterial solution at 4℃ and 8000r / min for 10min, and filter to obtain bacterial cells;

[0051] Steps 1-4: Mix the bacterial cells with 5 times their weight of the protective agent to obtain a bacterial suspension;

[0052] Steps 1-5: The bacterial suspension was pre-cultured at 37°C for 1 hour and then freeze-dried for 24 hours to obtain Bi66 bacterial powder (600 billion CFU / g) and P16 bacterial powder (200 billion CFU / g).

[0053] Step 2-1: Inoculate the *Blad-3* strain into potato agar liquid medium and incubate at 37°C for 26 hours to activate it and obtain the activated solution;

[0054] Step 2-2: Inoculate the activated solution into the above potato agar liquid culture medium and culture at 30°C for 24 hours to obtain seed fermentation broth;

[0055] Steps 2-3: Centrifuge the seed fermentation broth (8000 r / min, 10 min) to obtain yeast cells;

[0056] Steps 2-4: Add emulsifier and vegetable oil to the yeast cells, add starch and granulate to obtain yeast powder (Saccharomyces boulardii 20 billion CFU / g).

[0057] Step 3: The Bi66 bacterial powder, the P16 bacterial powder, and the yeast powder obtained in Step 1 are compounded in a certain proportion to obtain the freeze-dried powder.

[0058] The compound bacterial agent provided by this invention can significantly increase the content of short-chain fatty acids (SCFAs) in intestinal flora, increase the production of beneficial gases such as CH4 and H2, reduce the production of harmful gases such as NH3 and H2S, promote the growth of beneficial bacteria and inhibit the growth of harmful bacteria, thereby regulating intestinal flora. Furthermore, the three strains have a synergistic effect.

[0059] Biological Preservation Instructions

[0060] Biological material: Saccharomyces boulardii Bld-3, deposited on August 8, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, accession number CCTCC NO: M 20251803.

[0061] Biological material: Bifidobacterium animalis subsp. lactis Bi66, classified and named Bifidobacterium animalis subsp. lactis Bi66, was deposited on May 17, 2023 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2023769.

[0062] Biological material: Lactiplantibacillus plantarum P16, classified and named Lactiplantibacillus plantarum P16, was deposited on May 17, 2023 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2023767. Attached Figure Description

[0063] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0064] Figure 1 Indicates acetic acid production;

[0065] Figure 2 Indicates propionic acid production;

[0066] Figure 3 Butyric acid production;

[0067] Figure 4 Indicates isobutyric acid production;

[0068] Figure 5 Indicates valerate production;

[0069] Figure 6 Indicates isovaleric acid production;

[0070] Figure 7 Indicates CO2 production;

[0071] Figure 8 Indicates H2 production;

[0072] Figure 9 Indicates CH4 production;

[0073] Figure 10 Indicates H2S production;

[0074] Figure 11 This indicates the production of NH3. Detailed Implementation

[0075] This invention discloses a compound probiotic agent that reduces the production of harmful intestinal gases and regulates intestinal flora. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0076] This invention provides a compound probiotic agent that can reduce the production of harmful gases in the intestines and regulate the intestinal flora, including Bifidobacterium animalis subsp. lactis Bi66 with accession number CCTCC NO: M 2023769, Lactobacillus plantarum P16 with accession number CCTCC NO: M 2023767, and Saccharomyces boulardii Bld-3 with accession number CCTCC NO: M 2019643.

[0077] Table 1

[0078]

[0079]

[0080] Preferably, the ratio of viable counts of Bifidobacterium animalis subsp. Lactobacillus Bi66, Lactobacillus plantarum P16, and Saccharomyces boulardii strain Bld-3 is (1~5):(1~5):(1~5).

[0081] Preferably, the total number of live bacteria in the probiotic agent is not less than 1×10⁻⁶. 8 CFU / ml or 1×10 8 CFU / g.

[0082] The probiotic dosage form is not limited, including common lyophilized powders, or further processed dosage forms such as capsules, granules, and tablets. The lyophilized powder can be prepared using the following method:

[0083] Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus plantarum P16 were inoculated into MRS liquid medium and activated by culturing at 37°C for 24 h. This activation was repeated twice to obtain an activated solution. The activated solution was then inoculated into MRS liquid medium at a rate of 3% (v / v) and cultured at 37°C for 22 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 8000 r / min for 10 min at 4°C and filtered to obtain bacterial cells. The bacterial cells were mixed with 5 times their mass of a protective agent to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37°C for 1 h and then freeze-dried for 24 h to obtain Bi66 (600 billion CFU / g) and P16 bacterial powder (200 billion CFU / g).

[0084] Saccharomyces boulardii Bld-3 was inoculated into liquid culture medium and activated by culturing at 37°C for 26 h. The activated solution was then inoculated into liquid culture medium and cultured at 30°C for 24 h to obtain seed fermentation broth. The fermentation broth was centrifuged (8000 r / min, 10 min) to obtain yeast cells. Emulsifier and vegetable oil were added to the cells, and starch was added for granulation to obtain yeast cells (Saccharomyces boulardii 20 billion CFU / g).

[0085] The above-mentioned bacterial powder raw materials were compounded in a certain proportion to obtain probiotic freeze-dried powder.

[0086] The compound probiotic agent involved in this invention can significantly increase the content of short-chain fatty acids (SCFAs) in the intestinal flora, increase the production of beneficial gases such as CH4 and H2, and reduce the production of harmful gases such as NH3 and H2S, thereby promoting the growth of beneficial bacteria and inhibiting the growth of harmful bacteria and regulating the intestinal flora. When maintaining a consistent amount of bacteria, compared with intervention methods that lack any one type of bacteria, the combination of three bacteria is the most effective in reducing the production of harmful intestinal gases and improving the intestinal flora.

[0087] The compound probiotic agent used in this invention to reduce the production of harmful intestinal gases and regulate intestinal flora.

[0088] The present invention will be further illustrated below with reference to the embodiments:

[0089] Preparation method of compound probiotic agent

[0090] The experimental groups are shown below:

[0091] Table 2

[0092]

[0093] Bifidobacterium animalis subsp. lactis Bi66 and Lactobacillus plantarum P16 were inoculated into MRS liquid medium and activated by culturing at 37°C for 24 h. This activation was repeated twice to obtain an activated solution. The activated solution was inoculated into MRS liquid medium at a rate of 3% (v / v) and cultured at 37°C for 22 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 8000 r / min for 10 min at 4°C and filtered to obtain bacterial cells. The bacterial cells were mixed with 5 times their mass of a protective agent (13% skim milk powder) to obtain a bacterial suspension. The bacterial suspension was pre-cultured at 37°C for 1 h and then freeze-dried for 24 h to obtain Bi66 (600 billion CFU / g) and P16 bacterial powder (200 billion CFU / g).

[0094] Saccharomyces boulardii Bld-3 was inoculated into potato agar liquid medium and activated by culturing at 37°C for 26 h. The activated solution was then inoculated into liquid medium and cultured at 30°C for 24 h to obtain seed fermentation broth. The fermentation broth was centrifuged (8000 r / min, 10 min) to obtain yeast cells. Emulsifier (sp60 sorbitan monostearate) and vegetable oil (soybean oil) were added to the cells, followed by the addition of starch for granulation to obtain yeast cells (Saccharomyces boulardii 20 billion CFU / g).

[0095] The above-mentioned bacterial powder raw materials were compounded in a certain proportion to obtain probiotic freeze-dried powder.

[0096] Effects of compound probiotic preparations on the intervention of fecal microbiota fermentation products and the growth of specific probiotics.

[0097] I. Experimental Design

[0098] 1. Preparation of culture medium

[0099] Preparation of YCFA basal medium: To prepare 1L of YCFA basal medium, weigh the following reagents and dissolve them in 1L of purified water: tryptone: 10g; yeast extract: 2.5g; L-cysteine: 1g; heme solution: 2mL; NaCl: 0.9g; calcium chloride hexahydrate: 0.09g; KH2PO4: 0.45g; K2HPO4: 0.45g; magnesium sulfate heptahydrate: 0.09g. After dissolving, boil the solution and immediately purge with nitrogen to maintain an anaerobic environment. Dispense the solution into vials using a peristaltic pump, seal with pressure caps, and autoclave before use.

[0100] Bifidobacterium animalis Bi66, Lactobacillus plantarum P16, Saccharomyces boulardii Bld-3, and Lactobacillus casei Zhang were formulated into a concentration of 10. 8 CFU / mL bacterial suspensions were prepared, and then 250 μL of each of the following bacterial suspensions—Bifidobacterium animalis Bi66, Lactobacillus plantarum P16, Saccharomyces boulardii Bld-3, and Lactobacillus casei Zhang—were co-inoculated into YCFA medium to prepare different culture media (ensuring consistent viable cell counts across different media). Three replicates were prepared for each culture medium. After gentle shaking to mix, the media were incubated at 37°C for 24 h. After incubation, the media were stored at 4°C for later use.

[0101] 2. Collection of fecal samples

[0102] Fresh stool samples were collected from 40 healthy individuals aged 18-28 (20 males and 20 females) and delivered to the laboratory within four hours.

[0103] 3. Pretreatment of feces

[0104] Using an analytical balance in a fume hood, approximately 0.2 g of fresh fecal sample was weighed twice from the fecal sampling box and placed into two 2 mL sterile centrifuge tubes. The original fecal samples were stored at -80°C. Then, 0.6 g of fecal sample was weighed into sterile 15 mL centrifuge tubes, and 6 mL of PBS buffer solution was added. The fecal sample and buffer solution were mixed thoroughly using a shaker. After filtering to remove large particles, a 10% fecal suspension inoculum was prepared. A diluted fecal inoculum solution was prepared according to the required inoculum volume.

[0105] 4. Fermentation experiment

[0106] 4.1 Fecal microbial fermentation

[0107] 250 μL of the prepared fresh fecal suspension was inoculated into the above-mentioned culture media containing different probiotics and YCFA control culture media using a disposable sterile syringe in an anaerobic operating table. Each culture medium was prepared in triplicate. After gentle shaking and mixing, the media was placed in a constant temperature incubator at 37°C for 24 h. After incubation, the media was removed and the total amount and composition of gas were measured using a gas analyzer. Then, the fermentation broth was opened and aliquoted into sterile 2 mL centrifuge tubes. After centrifugation at 12000 r / min for 5 minutes, solid-liquid separation was performed. The precipitate was used to extract DNA, which was then sequenced into 16S rDNA by Shanghai Meiji Company. The supernatant was analyzed for SCFA metabolites by GC.

[0108] 4.2 Gas analysis inside the bottle after fermentation

[0109] Different fermentation bottles inoculated with fecal bacteria were removed after fermentation at 37°C for 24 hours. After cooling to room temperature, the gas was automatically analyzed using a fermentation gas analyzer, and the data was recorded.

[0110] 4.3 Short-chain fatty acids (SCFAs) after fermentation S Determination of )

[0111] (1) Preparation of standard solutions and establishment of standard curves

[0112] Weigh 2.5 g of metaphosphoric acid and add deionized water to a final volume of 100 mL to prepare a 2.5% (w / v) metaphosphoric acid solution. Weigh 0.6464 g of crotonic acid and add it to the metaphosphoric acid solution to a final volume of 100 mL to prepare a crotonic acid / metaphosphoric acid solution. Using the external standard method, prepare six standard solutions of different concentrations with standard samples of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. Then, determine the retention time and peak area of ​​each standard component in the mixed standard solution at the standard concentration. Each sample concentration is tested in triplicate, and the average value is taken. Use the peak area to plot the standard curve for each component concentration.

[0113] (2) Sample processing

[0114] Transfer 500 μL of fermentation broth to a 1.5 mL centrifuge tube, then add 100 μL of crotonic acid metaphosphate solution, vortex to mix, and place in a -80°C freezer for 24 hours to acidify. After acidification, thaw at 4°C, then centrifuge at 12000 r / min for 3 minutes at 4°C. Transfer the supernatant to another centrifuge tube, and use a disposable syringe to draw the supernatant and filter it through a 0.22 μm aqueous microporous membrane into a new centrifuge tube. Transfer 100 μL of the filtrate to a vial, shaking to remove air bubbles from the bottom of the inner tube to prevent air intake during sample loading.

[0115] (3) Gas chromatography determination

[0116] Turn on the instrument, clean the waste liquid, fill with cleaning solution, clean the injection needle with methanol, replace the gaskets regularly, and clean the quartz wool liner. Once ready, load the sample and perform the aging program. Column temperature ramp-up program: Column temperature: 80℃ for 1 min, 10℃ / min, ramp to 190℃, hold for 0.50 min; then reach 240℃ at a rate of 40℃ / min, hold for 5 min; FID detector: 240℃; vaporization chamber: 240℃; carrier gas: nitrogen, flow rate 20 mL / min, hydrogen flow rate 40 mL / min, air flow rate 400 mL / min. Edit the program, start the test, and record the data.

[0117] 5. In vitro growth assay

[0118] Bifidobacterium animalis, Lactobacillus reuteri, Clostridium tenuifolium, Escherichia coli, and Salmonella were activated and cultured overnight at 37°C. Then, 1 mL of saturated O / N culture of these five strains was inoculated into 100 mL of suitable culture media (MRS medium for Bifidobacterium animalis and Lactobacillus reuteri; Clostridium tenuifolium amplification optimized medium (containing 7 g / L glucose, 10 g / L soybean peptone, and 0.7 g / L L-cysteine ​​hydrochloride); and LB medium for Escherichia coli and Salmonella). The probiotic group was simultaneously inoculated with 0.5 mg / mL of compound probiotics at an inoculation volume of 250 μL. After reaching the logarithmic growth phase, the bacterial culture of each strain was serially diluted 10-fold, and viable cells were counted using quantitative plate counting. All experiments were conducted under anaerobic conditions.

[0119] 6. Data Statistics and Analysis

[0120] Gas and SCFAs data were statistically plotted using Excel, and one-way or two-way ANOVA was performed using Minitab software. Compared with the control group, *** represents P < 0.001, ** represents P < 0.01, * represents P < 0.05, and NS represents no significant difference.

[0121] II. Test Results

[0122] 1. Analysis of differences in short-chain fatty acid production among fermentation groups

[0123] By analyzing six SCFAs in the fermentation broth S Production was tested, and the results were as follows: Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 As shown in Table 3. Figure 1 Indicates acetic acid production. Figure 2Indicates propionic acid production. Figure 3 This indicates the yield of butyric acid. Figure 4 Indicates isobutyric acid production. Figure 5 Indicates valerate production. Figure 6 This indicates the production of isovaleric acid. Compared with the control group, the production of acetic acid, propionic acid, butyric acid, and valeric acid increased to varying degrees after intervention in each probiotic group, with the most significant increases observed in the example groups (Bi66+P16+Bld-3 (1:1:1), Bi66+P16+Bld-3 (5:1:1), and Bi66+P16+Bld-3 (1:2:5). There was no significant difference in isovaleric acid and isovaleric acid content among the intervention groups compared to the control group. (From six SCFAs...) S The changes in yield show that the different probiotic intervention groups have an impact on the production of SCFA in fecal microbiota. S It has a good promoting effect, and compared with the example group, the promoting effect is the best. The synergistic effect disappears when any key strain is missing or when the strain combination exceeds the proportion range.

[0124] Table 3

[0125]

[0126] 2. Analysis of differences in gas production among fermentation groups

[0127] The results of gas production during the fermentation process of fecal microbiota are as follows: Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 As shown in Table 4. Figure 7 Indicates CO2 production. Figure 8 Indicates H2 production. Figure 9 Indicates CH4 production. Figure 10 Indicates H2S production. Figure 11 The figure represents NH3 production. Compared with the control group, the production of CH4 and H2 increased to varying degrees after probiotic intervention in each group, with the most significant increases observed in the example groups (Bi66+P16+Bld-3 (1:1:1), Bi66+P16+Bld-3 (5:1:1), and Bi66+P16+Bld-3 (1:2:5). The production of NH3 and H2S showed the opposite trend. Except for the example group, which showed a significant increase in CO2 production compared to the control group, the other probiotic intervention groups showed no significant difference compared to the control group. Based on the differences in gas production, it can be seen that probiotic intervention helps increase the production of CH4 (which has anti-apoptotic effects) and H2 (which has anti-inflammatory effects), while reducing the production of harmful NH3 and H2S. Among all probiotic groups, the example group showed the best effect; the synergistic effect disappeared when any key strain was missing or when the strain combination exceeded the specified ratio.

[0128] Table 4

[0129]

[0130] 3. Analysis of the impact of each fermentation group on specific microorganisms

[0131] In vitro experiments (Table 5) demonstrated that each probiotic intervention group could promote the growth of beneficial bacteria such as Bifidobacterium animalis and Lactobacillus reuteri to varying degrees, while inhibiting the growth of harmful bacteria such as Escherichia coli and Salmonella. Furthermore, the promotion / inhibition effects of the respective example groups (Bi66+P16+Bld-3 (1:1:1), Bi66+P16+Bld-3 (5:1:1), and Bi66+P16+Bld-3 (1:2:5)) were more significant. These results indicate that probiotic intervention regulates the intestinal flora by promoting the growth of beneficial bacteria and inhibiting the growth of harmful bacteria, thereby promoting intestinal health.

[0132] Table 5. Comparison of the effects of each fermentation group on specific microorganisms (LogCFU / ml)

[0133]

[0134] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound bacteria, characterized in that, It is composed of Bifidobacterium animalis subsp. lactis Bi66, Lactobacillus plantarum P16 and Saccharomyces boulardii Bld-3; The ratio of viable counts of Bifidobacterium animalis subsp. Lactobacillus Bi66, Lactobacillus plantarum P16, and Saccharomyces boulardii Bld-3 is 1:1:1, 5:1:1, or 1:2:

5. The preservation number of Bifidobacterium lactis subspecies Bi66 is CCTCC NO: M 2023769; The preservation number of the plant lactobacillus P16 is CCTCC NO: M 2023767; The preservation number of the *Blad-3* strain is CCTCC NO: M 20251803.

2. The compound bacteria as described in claim 1, characterized in that, The total number of viable bacteria in the compound bacteria is not less than 1×10⁻⁶. 8 CFU / ml or 1×10 8 CFU / g.

3. The application of the compound bacteria as described in claim 1 or 2 in the preparation of any of the following microecological products; (I) Increase the content of short-chain fatty acids in the intestinal flora, specifically acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid; (II) Increase the production of beneficial gases CH4 and / or H2; (III) Reduce the production of harmful gases NH3 and / or H2S; or (IV) It promotes the growth of beneficial bacteria such as Bifidobacterium animalis, Lactobacillus reuteri or Clostridium truncatum and regulates the intestinal flora by inhibiting harmful bacteria such as Escherichia coli and / or Salmonella.

4. The application as described in claim 3, characterized in that, The microecological products include compound probiotic products or synbiotic products.

5. The application as described in claim 4, characterized in that, The microecological products include food, feed, or feed additives.

6. The application as described in claim 5, characterized in that, The food products mentioned are health foods, foods for special medical purposes, functional foods, or pet foods.

7. The application as described in claim 5, characterized in that, The dosage forms of the microecological products are powders, granules, capsules, tablets, pills, extracts, or liquid preparations.

8. A microecological product, characterized in that, Includes the compound bacteria as described in claim 1 or 2.

9. The microecological product as described in claim 8, characterized in that, The microecological products include compound probiotic products or synbiotic products.

10. The microecological product as described in claim 8, characterized in that, The microecological products include food, feed, or feed additives.

11. The microecological product as described in claim 10, characterized in that, The food products mentioned are health foods, foods for special medical purposes, functional foods, or pet foods.

12. The microecological product as described in claim 8, characterized in that, The dosage forms of the microecological products are powders, granules, capsules, tablets, pills, extracts, or liquid preparations.

13. The microecological product as described in claim 12, characterized in that, The powder includes lyophilized powder; The method for preparing the lyophilized powder includes the following steps: Step 1: Activate and culture the Bifidobacterium animalis subsp. lactis Bi66 and the Lactobacillus plantarum P16 respectively to obtain bacterial solutions. Collect the bacterial cells by centrifugation and filtration, mix with a protectant and freeze dry to obtain Bi66 bacterial powder and P16 bacterial powder respectively. Step 2: After activating and culturing the Bld-3 yeast strain, the seed fermentation broth is obtained. The cells are collected by centrifugation, mixed with emulsifier, vegetable oil and starch, and then granulated to obtain yeast powder. Step 3: The Bi66 bacterial powder, the P16 bacterial powder, and the yeast powder obtained in Step 1 are compounded in a certain proportion to obtain the freeze-dried powder.

14. The microecological product as described in claim 13, characterized in that, The Bi66 bacterial powder contains 600 billion CFU / g of live bacteria; The P16 bacterial powder contains 200 billion CFU / g of live bacteria; The yeast powder contains 20 billion CFU / g of Saccharomyces boulardii.

15. The microecological product as described in claim 13, characterized in that, The method for preparing the lyophilized powder includes the following steps: Step 1-1: Inoculate the Bifidobacterium animalis subsp. lactis Bi66 and the Lactobacillus plantarum P16 into MRS liquid medium and activate them by culturing at 37°C for 24 hours. Repeat the activation process twice to obtain the activated solution. Steps 1-2: Inoculate the activation solution into MRS liquid medium at an inoculation rate of 3% (v / v) and incubate at 37°C for 22 hours to obtain bacterial culture. Steps 1-3: Centrifuge the bacterial solution at 4℃ and 8000r / min for 10min, and filter to obtain bacterial cells; Steps 1-4: Mix the bacterial cells with 5 times their weight of the protective agent to obtain a bacterial suspension; Steps 1-5: The bacterial suspension was pre-cultured at 37°C for 1 hour and then freeze-dried for 24 hours to obtain Bi66 bacterial powder and P16 bacterial powder. Step 2-1: Inoculate the *Blad-3* strain into liquid culture medium and activate it by culturing at 37°C for 26 hours to obtain the activated solution; Step 2-2: Inoculate the activated solution into a liquid culture medium and culture at 30°C for 24 hours to obtain the seed fermentation broth; Steps 2-3: Centrifuge the seed fermentation broth to obtain yeast cells; Steps 2-4: Add emulsifier and vegetable oil to the yeast cells, add starch and granulate to obtain yeast powder; Step 3: The Bi66 bacterial powder, the P16 bacterial powder, and the yeast powder obtained in Step 1 are compounded in a certain proportion to obtain the freeze-dried powder.

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