Composite probiotic agent for reducing generation of harmful gases in intestinal tract and regulating intestinal flora

Abstract

The invention relates to the technical field of microorganisms, in particular to a compound probiotic agent for reducing harmful gases generated in intestinal tracts and regulating intestinal flora. The compound probiotics disclosed by the invention are prepared from bifidobacterium animalis subsp. Lactis Bi66, plant lactobacillus P16 and a saccharomyces boulardii Bld-3 strain. The three strains are compounded and combined for use, so that the content of short-chain fatty acid in intestinal flora can be remarkably increased, the yield of beneficial gases such as CH4 and H2 can be increased, the yield of NH3 and H2S which are harmful to a human body can be reduced, beneficial bacteria can be promoted, and the growth of harmful bacteria can be inhibited, so that the intestinal flora can be regulated. Under the condition of keeping the same amount of the used bacteria, compared with a strain intervention mode without any bacteria, the compounding of the three bacteria has the best effect in the aspect of improving the intestinal flora, which indicates that the three bacteria can be mutually matched and promoted, and can have synergistic interaction in the aspect of regulating the intestinal flora.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology, in particular to a complex probiotic agent for reducing the production of harmful intestinal gases and regulating intestinal flora. BACKGROUND

[0002] The human intestinal tract is inhabited by a large number of microorganisms of various species, collectively known as intestinal flora. A large number of scientific studies have confirmed that the composition and functional balance of intestinal flora are closely related to human health. A balanced intestinal flora not only helps 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 affecting metabolism, neural regulation, etc. In addition, harmful gases produced by intestinal flora metabolism not only cause discomfort symptoms such as abdominal distension, abdominal pain, and bad breath, but also may be toxic to intestinal cells when excessive, affecting intestinal barrier function, and even closely related to the occurrence and development of some intestinal diseases. Intestinal flora imbalance is considered to be related to the occurrence and development of many diseases, such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), antibiotic-associated diarrhea (AAD), constipation, etc. Intestinal flora imbalance is often accompanied by excessive proliferation of harmful bacteria, reduction of beneficial bacteria, and excessive production of harmful metabolites (including the above harmful gases). Therefore, the dynamic balance of intestinal flora is crucial to personal health.

[0003] Probiotics, as a kind of living microorganism, have beneficial effects on host health when ingested in sufficient quantities. Currently, exogenous supplementation of probiotics to regulate and improve intestinal flora structure has become an important strategy for preventing and adjuvant treatment of related diseases.

[0004] There are a variety of probiotic products on the market, including single strains or combinations of multiple strains (complex bacterial agents), and dosage forms covering drugs, health products, foods (such as yogurt, fermented milk, probiotic powders), etc. Although probiotics are widely used, the probiotic preparations in the prior art, especially involving multiple strain combinations, have random combinations and lack of synergistic effects, especially there are few reports on the synergistic reduction of intestinal harmful gases by specific strain combinations. Therefore, there is an urgent need to develop a new type of complex probiotic agent, which is based on specific strain combinations and has been proven to have synergistic effects, can effectively overcome the shortcomings of the prior art, and achieve more comprehensive and efficient flora balance.

[0005] Although the regulatory effect of probiotics on intestinal flora has been extensively studied, the probiotic agents in the prior art, especially involving multiple strain combinations, still have obvious limitations. For example, single strain has limited efficacy, multiple strain combinations have random combinations and lack of synergistic effects, and there are limited reports on the significant reduction of multiple intestinal harmful gases.

[0006] The prior art discloses a composite probiotic capable of relieving ulcerative colitis, a preparation method and application thereof, the composite probiotic comprising Bifidobacterium longum BL21, Bifidobacterium animalis subs p. lactis BLa 80 and Lactobacillus casei LC89; the preparation method comprises the following steps: respectively preparing Bifidobacterium longum BL21, Bifidobacterium animalis subs p. lactis BLa 80 and Lactobacillus casei LC89 freeze-dried powder; and the three kinds of freeze-dried powders are mixed in proportion to prepare the composite probiotic.

[0007] A probiotic agent for increasing immunity and improving intestinal flora and application thereof, five specific different types of Bifidobacterium strains are compounded, there is potential interaction between the five strains, which can cooperate with each other, synergistically promote the growth of immune organs and increase the total number and diversity of intestinal flora, and the compounded basic fermentation strain is used for yogurt fermentation, and a composite prebiotic is added for synergistic fermentation of yogurt, the number of Bifidobacterium at the end of the shelf life after the yogurt fermentation is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g, which significantly improves the stability of lactic acid bacteria in yogurt, significantly increases the number of live bacteria entering the human intestinal tract and playing a role, has good tolerance in the intestinal tract, and the prepared yogurt has delicate texture, rich taste and rich aroma; and can significantly improve the immune decline and intestinal flora disorder caused by cyclophosphamide (CTX) and has a positive effect on immune enhancement.

[0008] Although the probiotic combination involved in CN 113430133 B invention can relieve ulcerative colitis to a certain extent, the synergistic effect of the combination of the three strains has not been verified, and the combination may have certain randomness.

[0009] The probiotic combination involved in CN 119530101 A invention is used for yogurt fermentation, and the actual exploration is the regulation effect of the fermented yogurt on immunity and intestinal flora, the five strains mentioned have potential interaction, which can synergistically promote the growth of immune organs and increase the total number and diversity of intestinal flora, and no corresponding test evidence is found, and the application range of the composite strain is limited.

[0010] The existing probiotics, especially the complex bacterial agent field, lacks discovery and verification of synergistic effect between specific strain combinations (especially three strains), has certain combination randomness and synergistic effect loss, and there are few reports on the research of synergistically reducing intestinal harmful gas through specific strain combinations. SUMMARY

[0011] Therefore, the present application provides a complex probiotic agent which can reduce the production of intestinal harmful gas and regulate intestinal flora, and the three strains are innovatively combined and have synergistic effect.

[0012] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0013] In a first aspect, the present application provides a complex bacteria, which comprises animal Bifidobacterium lactis Bi66, plant Lactiplantibacillus P16 and Saccharomyces boulardii Bld-3.

[0014] In some specific embodiments of the present application, the ratio of the viable bacterial number of the animal Bifidobacterium lactis Bi66, the plant Lactiplantibacillus P16 and the Saccharomyces boulardii Bld-3 is (1-5):(1-5):(1-5).

[0015] In some specific embodiments of the present application, the ratio of the viable bacterial number of the animal Bifidobacterium lactis Bi66, the plant Lactiplantibacillus P16 and the Saccharomyces boulardii Bld-3 is 1:1:1, 5:1:1 or 1:2:5.

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

[0017] In some specific embodiments of the present application, the preservation number of the animal Bifidobacterium lactis Bi66 is CCTCC NO: M 2023769.

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

[0019] The preservation number of the Saccharomyces boulardii Bld-3 is CCTCC NO: M 2019643.

[0020] In a second aspect, the present application also provides the use of the complex bacteria in the preparation of a microecological product for any of the following purposes:

[0021] (I) increasing the content of intestinal flora short-chain fatty acids (SCFAs);

[0022] (II) increasing the production of beneficial gas;

[0023] (III) reducing the production of intestinal harmful gas; or

[0024] (IV) regulating intestinal flora.

[0025] In some embodiments of the present application, the beneficial gas comprises CH4 and / or H 2。

[0026] In some embodiments of the present application, the intestinal harmful gas comprises NH3 and / or H2S.

[0027] In some embodiments of the present application, the regulating intestinal flora comprises promoting the growth of beneficial bacteria and / or inhibiting the growth of harmful bacteria;

[0028] The beneficial bacteria comprise one or more of animal bifidobacterium, lactobacillus reuteri or clostridium leptum.

[0029] The harmful bacteria comprise escherichia coli and / or salmonella.

[0030] In some embodiments of the present application, the microecological preparation comprises a complex probiotic preparation, a synbiotic preparation or a postbiotic preparation.

[0031] In some embodiments of the present application, the microecological preparation comprises a food, a feed or a feed additive.

[0032] In some embodiments of the present application, the food is a health food, a special medical purpose food, a functional food or a pet food.

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

[0034] In a third aspect, the present application further provides a microecological preparation comprising the complex bacteria.

[0035] In some embodiments of the present application, the microecological preparation comprises a complex probiotic preparation, a synbiotic preparation or a postbiotic preparation.

[0036] In some embodiments of the present application, the microecological preparation comprises a food, a feed or a feed additive.

[0037] In some embodiments of the present application, the food is a health food, a special medical purpose food, a functional food or a pet food.

[0038] In some embodiments of the present application, the dosage form of the microecological preparation is powder, granules, capsules, tablets, pills, extract or liquid preparation.

[0039] In some embodiments of the present application, the powder comprises lyophilized powder.

[0040] The preparation method of the lyophilized powder comprises the following steps:

[0041] Step 1: respectively activate and culture the Bifidobacterium animalis lactis Bi66 and the Plantarum P16 to obtain bacterial liquid, centrifuge and filter to collect bacterial bodies, mix with protective agent, and then lyophilize to obtain Bi66 bacterial powder and P16 bacterial powder respectively;

[0042] Step 2: activate and culture the Saccharomyces boulardii Bld-3 to obtain seed fermentation liquid, centrifuge to collect bacterial bodies, mix with emulsifier, vegetable oil and starch, and then granulate to obtain yeast powder;

[0043] Step 3: compound the Bi66 bacterial powder, the P16 bacterial powder and the yeast powder obtained in step 2 according to a proportion to obtain the lyophilized powder.

[0044] In some embodiments of the present application, the number of viable bacteria in the Bi66 bacterial powder is 600 billion CFU / g.

[0045] The number of viable bacteria in the P16 bacterial powder is 200 billion CFU / g.

[0046] The number of Saccharomyces boulardii in the yeast powder is 200 billion CFU / g.

[0047] In some embodiments of the present application, the preparation method of the lyophilized powder comprises the following steps:

[0048] Step 1-1: inoculate the Bifidobacterium animalis lactis Bi66 and the Plantarum P16 into MRS liquid medium respectively, activate at 37℃ for 24h, and continuously activate for 2 times to obtain activation liquid;

[0049] Step 1-2: inoculate the activation liquid into MRS liquid medium at an inoculation amount of 3%(v / v), and culture at 37℃ for 22h to obtain bacterial liquid;

[0050] Step 1-3: centrifuge the bacterial liquid at 8000r / min at 4℃ for 10min, and filter to obtain bacterial bodies;

[0051] Step 1-4: mix the bacterial bodies with 5 times of protective agent to obtain bacterial suspension;

[0052] Step 1-5: After pre-culturing the bacterial suspension at 37 DEG C for 1 h, freeze-drying is performed for 24 h to obtain Bi66 bacterial powder (6000 billion CFU / g) and P16 bacterial powder (2000 billion CFU / g);

[0053] Step 2-1: The Saccharomyces boulardii Bld-3 is inoculated into a potato agar liquid culture medium, activated at 37 DEG C for 26 h, and an activation liquid is obtained;

[0054] Step 2-2: The activation liquid is inoculated into the above-mentioned potato agar liquid culture medium, and is cultured at 30 DEG C for 24 h to obtain a seed fermentation liquid;

[0055] Step 2-3: The seed fermentation liquid is centrifuged (8000 r / min, 10 min) to obtain a yeast cell body;

[0056] Step 2-4: The yeast cell body is added with an emulsifier and vegetable oil, and starch is added for granulation to obtain a yeast bacterial powder (Saccharomyces boulardii 200 billion CFU / g);

[0057] Step 3: The Bi66 bacterial powder prepared in step 1, the P16 bacterial powder and the yeast bacterial powder prepared in step 2 are compounded in proportion to obtain the freeze-dried powder.

[0058] The composite bacterial agent provided by the application can significantly increase the content of intestinal flora short-chain fatty acids (SCFAs), increase the yield of beneficial gases such as CH4 and H2, reduce the yield of harmful gases such as NH3 and H2S to the human body, promote the growth of beneficial bacteria and inhibit the growth of harmful bacteria to regulate intestinal flora, and the three strains have a synergistic effect.

[0059] Biological preservation instructions

[0060] Biological material: Saccharomyces boulardii Bld-3, classified and named as Saccharomyces boulardii Bld-3, preserved in the China Center for Type Culture Collection on August 8, 2025, located at Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20251803.

[0061] Biological material: animal Bifidobacterium lactis subsp. Bi66, classified and named as Bifidobacterium animalis subsp. lactis Bi66 (Bifidobacterium animalis subsp. lactis Bi66), preserved in the China Center for Type Culture Collection on May 17, 2023, located at Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2023769.

[0062] Biological material: Lactiplantibacillus plantarum P16, classification name: Lactiplantibacillus plantarum P16, preserved in China Center for Type Culture Collection on May 17, 2023, address: China, Wuhan, Wuhan University, preservation number: CCTCC NO: M 2023767. BRIEF DESCRIPTION OF DRAWINGS

[0063] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below.

[0064] Figure 1 Example 1: Acetic acid production;

[0065] Figure 2 Example 1: Acetic acid production;

[0066] Figure 3 Example 1: Acetic acid production;

[0067] Figure 4 Example 1: Acetic acid production;

[0068] Figure 5 Example 1: Acetic acid production;

[0069] Figure 6 Example 1: Acetic acid production;

[0070] Figure 7 Example 1: Acetic acid production;

[0071] Figure 8 Example 1: Acetic acid production;

[0072] Figure 9 Example 1: Acetic acid production;

[0073] Figure 10 Example 1: Acetic acid production;

[0074] Figure 11 Example 1: Acetic acid production; DETAILED DESCRIPTION

[0075] The present application discloses a complex probiotic agent for reducing intestinal harmful gas production and regulating intestinal flora. Those skilled in the art can refer to the content herein and appropriately improve the process parameters for implementation. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0076] The present application provides a kind of complex probiotic agent that can reduce intestinal harmful gas production and regulate intestinal flora, which includes animal Bifidobacterium lactis Bi66 with preservation number CCTCC NO: M 2023769, plant Lactobacillus plantarum P16 with preservation number CCTCC NO: M 2023767 and Saccharomyces boulardii Bld-3 with preservation number CCTCC NO: M 2019643.

[0077] Table 1

[0078]

[0079]

[0080] Preferably, the ratio of viable cell number of the animal Bifidobacterium lactis Bi66, plant Lactobacillus plantarum P16 and Saccharomyces boulardii Bld-3 strains is (1-5):(1-5):(1-5).

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

[0082] The probiotic agent form is not limited, including common freeze-dried powder, or further prepared capsule, granule, tablet and other dosage forms. The freeze-dried powder can be prepared by the following method:

[0083] Animal Bifidobacterium lactis Bi66 and plant Lactobacillus plantarum P16 are inoculated into MRS liquid medium respectively, activated at 37°C for 24h, continuously activated for 2 times to obtain the activation liquid; the activation liquid is inoculated into MRS liquid medium at an inoculation amount of 3%(v / v), cultured at 37°C for 22h to obtain the bacterial liquid; the bacterial liquid is centrifuged at 8000r / min for 10min at 4°C, filtered to obtain the bacterial body; the bacterial body is mixed with 5 times the mass of the protective agent to obtain the bacterial suspension; the bacterial suspension is pre-cultured at 37°C for 1h, then freeze-dried, the freeze-drying time is 24h, to obtain Bi66 (6000 billion CFU / g) and P16 bacterial powder (2000 billion CFU / g).

[0084] Saccharomyces boulardii Bld-3 is inoculated into liquid medium, activated at 37°C for 26h, then the activation liquid is inoculated into liquid medium and cultured at 30°C for 24h to obtain the seed fermentation liquid, the fermentation liquid is centrifuged (8000r / min, 10min) to obtain the yeast body, then emulsifier and vegetable oil are added to the yeast body, and starch is added for granulation to obtain the yeast body (Saccharomyces boulardii 200 billion CFU / g).

[0085] The above bacterial powder raw materials are compounded according to a proportion to obtain the probiotic freeze-dried powder.

[0086] The composite probiotic agent can significantly increase the content of intestinal flora short-chain fatty acids (SCFAs), increase the production of beneficial gases such as CH4 and H2, reduce the production of harmful gases such as NH3 and H2S to the human body, promote the growth of beneficial bacteria and inhibit the growth of harmful bacteria, and thus regulate the intestinal flora. Compared with the strain intervention mode in which any one of the strains is missing, the compounding of the three strains is the best in reducing the production of harmful gases in the intestinal tract and improving the intestinal flora under the condition that the amount of the used strains is consistent.

[0087] The composite probiotic agent for reducing the production of harmful gases in the intestinal tract and regulating the intestinal flora provided by the application comprises

[0088] The application will be further described below in combination with examples:

[0089] Preparation method of the composite probiotic agent

[0090] The experimental groups are shown as follows:

[0091] Table 2

[0092]

[0093] Bi66 and P16 were inoculated into MRS liquid medium respectively, activated at 37℃ for 24h, continuously activated for 2 times, and activated liquid was obtained; the activated liquid was inoculated into MRS liquid medium at an inoculation amount of 3%(v / v), and cultured at 37℃ for 22h to obtain bacterial liquid; the bacterial liquid was centrifuged at 8000r / min for 10min at 4℃, and the bacterial body was obtained by filtration; the bacterial body was mixed with 5 times of protective agent (13% defatted milk powder) to obtain bacterial suspension; the bacterial suspension was pre-cultured at 37℃ for 1h, and then freeze-dried for 24h to obtain Bi66 (6000 billion CFU / g) and P16 bacterial powder (2000 billion CFU / g).

[0094] Bld-3 was inoculated into potato agar liquid medium, activated at 37℃ for 26h, and then the activated liquid was inoculated into liquid medium and cultured at 30℃ for 24h to obtain seed fermentation liquid; the fermentation liquid was centrifuged (8000r / min, 10min) to obtain yeast body, and then emulsifier (sp60 sorbitan monostearate) and vegetable oil (soybean oil) were added to the yeast body, and starch was added for granulation to obtain yeast body (Buller's yeast 200 billion CFU / g).

[0095] The above bacterial powder raw materials are compounded according to a proportion to obtain the probiotic freeze-dried powder.

[0096] Effect Example Intervention of composite probiotic agent on fecal bacterial flora fermentation products and growth of specific probiotics

[0097] I. Experimental design

[0098] 1. Preparation of culture medium

[0099] YCFA base medium preparation: To prepare 1 L of YCFA base medium, the following reagents were weighed and dissolved in 1 L of purified water: tryptone, 10 g; yeast extract, 2.5 g; L-cysteine, 1 g; hematin solution, 2 mL; NaCl, 0.9 g; calcium chloride hexahydrate, 0.09 g; KH2PO4, 0.45 g; K2HPO4, 0.45 g; magnesium sulfate heptahydrate, 0.09 g. After dissolution, boil immediately and maintain anaerobic conditions by nitrogen flushing. Use a peristaltic pump to dispense the medium into a flask, seal with a cap, and sterilize with high-pressure steam before use.

[0100] The animal Bifidobacterium Bi66, Lactobacillus plantarum P16, Saccharomyces boulardii Bld-3, and Lactobacillus casei Zhang were prepared into bacterial suspensions with a concentration of 10 8 CFU / mL, and then 250 μL of each bacterial suspension was inoculated into YCFA medium, resulting in different culture media (ensuring consistent viable cell counts in different culture media). Each medium was prepared in triplicate, mixed gently, and incubated at 37°C in a constant temperature incubator for 24 h. After incubation, the cultures were stored at 4°C for future use.

[0101] 2. Collection of fecal samples

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

[0103] 3. Pretreatment of fecal samples

[0104] Using an analytical balance in a fume hood, 0.2 g of fresh fecal sample was weighed from the fecal sampling box and placed in two 2 mL sterile centrifuge tubes. The original fecal samples were stored in a -80°C freezer. Then, 0.6 g of fecal sample was weighed into a sterile 15 mL centrifuge tube, and 6 mL of PBS buffer solution was added. The fecal sample and buffer solution were mixed using a vortex mixer, and the large particles were removed by filtration to prepare a 10% fecal suspension inoculum. The fecal dilution inoculum was prepared according to the inoculum size.

[0105] 4. Fermentation experiment

[0106] 4.1 Fecal bacterial flora fermentation

[0107] 250 μL of the treated fresh fecal suspension was inoculated into the different probiotic culture media and YCFA control medium described above using a sterile disposable syringe in an anaerobic workstation, with three replicates for each medium. After mixing gently, the culture was incubated at 37°C in a constant temperature incubator. After 24 h of incubation, the total gas and gas composition were determined using a gas analyzer. Then, the fermentation broth was taken out, and the supernatant was separated from the precipitate by centrifugation at 12000 r / min for 5 min. The precipitate was used to extract DNA for 16S rDNA sequencing by Shanghai Meiji Company. The supernatant was analyzed for SCFAs metabolites by GC.

[0108] 4.2 Analysis of the gas in the bottle after fermentation

[0109] After 24 h of fermentation at 37°C, the different fermentation bottles inoculated with fecal bacteria were taken out and cooled to room temperature. The gas in the bottles was then automatically analyzed using a fermentation gas analyzer, and the data were recorded.

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

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

[0112] 2.5 g of metaphosphoric acid was weighed and dissolved in deionized water to make a 100 mL metaphosphoric acid solution with a mass / volume ratio (w / v) of 2.5%. 0.6464 g of crotonic acid was weighed and dissolved in the metaphosphoric acid solution to make a 100 mL crotonic acid / metaphosphoric acid solution. Area external standard method was used to prepare six standard solutions with different concentrations of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid standard samples. Then, the retention time and peak area of each component in the mixed standard were determined at the standard concentration. Each sample concentration had three replicates, and the average value was taken. The standard curve of the concentration of each component was drawn using the peak area.

[0113] (2) Sample treatment

[0114] 500 μL of the fermentation broth was taken into a 1.5 mL centrifuge tube, and then 100 μL of the crotonic acid / metaphosphoric acid solution was added. After shaking and mixing, the mixture was placed in a -80°C refrigerator for acidification for 24 hours. After acidification, the mixture was thawed at 4°C and then centrifuged at 12000 r / min for 3 minutes at 4°C. The supernatant was transferred to another centrifuge tube, and the supernatant was filtered through a 0.22 μm water-based microporous filter membrane using a disposable syringe. The filtered liquid was then transferred to a new centrifuge tube, and 100 μL of the filtered liquid was taken into a sample vial. The vial was shaken to remove the air bubbles at the bottom of the inner cannula to prevent air suction during sample injection. ​

[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 different 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, there were no significant differences between the other probiotic intervention groups and 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 influence of each fermentation group on specific microorganisms

[0131] In vitro tests (Table 5) show that each probiotic intervention group can promote the growth of beneficial bacteria such as bifidobacterium and lactobacillus reuteri and inhibit the growth of harmful bacteria such as escherichia coli and salmonella to varying degrees, and the promotion / inhibition effect of each example group (Bi66+P16+Bld-3 (1:1:1), Bi66+P16+Bld-3 (5:1:1), Bi66+P16+Bld-3 (1:2:5)) is more significant. The above results show that the probiotic group adjusts the intestinal flora by promoting the growth of beneficial bacteria and inhibiting the growth of harmful bacteria after intervention, thereby achieving the effect of promoting intestinal health.

[0132] Table 5 Comparison of the influence of each fermentation group on specific microorganisms (Log CFU / ml)

[0133]

[0134] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. A composite bacterium, characterized in that, The animal Bifidobacterium lactis Bi66, the plant Lactobacillus plantarum P16 and the yeast Saccharomyces boulardii Bld-3.

2. The complex bacteria as claimed in claim 1, wherein, The ratio of the viable cell numbers of the animal Bifidobacterium lactis Bi66, the plant Lactobacillus plantarum P16 and the yeast Saccharomyces boulardii Bld-3 is (1-5):(1-5):(1-5).

3. The complex bacteria as claimed in claim 1, wherein, The ratio of the viable cell numbers of the animal Bifidobacterium lactis Bi66, the plant Lactobacillus plantarum P16 and the yeast Saccharomyces boulardii Bld-3 is 1:1:1, 5:1:1 or 1:2:

5.

4. The complex bacteria as claimed in claim 1, wherein, The total number of viable bacteria in the complex bacteria is not less than 1 x 10 8 CFU / ml or 1 x 10 8 CFU / g.

5. The complex bacteria as claimed in claim 1, wherein, The preservation number of the animal Bifidobacterium lactis Bi66 is CCTCC NO: M 2023769; The preservation number of the plant Lactobacillus plantarum P16 is CCTCC NO: M 2023767; The preservation number of the yeast Saccharomyces boulardii Bld-3 is CCTCC NO: M 20251803.

6. Use of the complex bacteria according to any one of claims 1 to 5 in the preparation of a microecological preparation for any one of the following purposes: (I) increasing the content of short-chain fatty acids in the intestinal flora; (II) increasing the production of beneficial gas; (III) reducing harmful gas in the intestine; or (IV) regulating the intestinal flora.

7. Use according to claim 6, wherein The beneficial gas includes CH4 and / or H2.

8. The use according to claim 6, wherein The harmful gas in the intestine includes NH3 and / or H2S.

9. The use according to claim 6, wherein The regulation of the intestinal flora includes promoting the growth of beneficial bacteria and / or inhibiting the growth of harmful bacteria. The beneficial bacteria include one or more of Bifidobacterium animalis, Lactobacillus reuteri or Clostridium leptum. The harmful bacteria include Escherichia coli and / or Salmonella.

10. The use according to claim 6, wherein The microecological preparation includes a complex probiotic preparation, a synbiotic preparation or a postbiotic preparation.

11. Use according to claim 10, wherein the compound is ###0002### The microecological preparation includes a food, a feed or a feed additive.

12. The use according to claim 11, wherein the compound is ###0002### The food is a health food, a special medical purpose food, a functional food or a pet food.

13. The use according to claim 11, wherein the compound is ###0002### The dosage form of the microecological preparation is a powder, a granule, a capsule, a tablet, a pill, an extract or a liquid preparation.

14. Microecological preparation, characterized in that, The microecological preparation includes a complex probiotic preparation, a synbiotic preparation or a postbiotic preparation.

15. The microecological preparation according to claim 14, characterized in that, The microecological preparation includes a food, a feed or a feed additive.

16. The microecological preparation according to claim 14, characterized in that, The food is a health food, a special medical purpose food, a functional food or a pet food.

17. The microecological preparation according to claim 16, characterized in that, The dosage form of the microecological preparation is a powder, a granule, a capsule, a tablet, a pill, an extract or a liquid preparation.

18. The microecological preparation according to claim 14, characterized in that, The powder includes a freeze-dried powder.

19. The microecological preparation according to claim 18, characterized in that, The preparation method of the freeze-dried powder includes the following steps: Step 1: respectively activate and culture the animal Bifidobacterium lactis Bi66 and the plant Lactobacillus plantarum P16 to obtain bacterial liquid, centrifuge and filter to collect bacterial cells, mix with a protective agent and freeze-dry to respectively obtain Bi66 bacterial powder and P16 bacterial powder; Step 2: activate and culture the yeast Saccharomyces boulardii Bld-3 to obtain seed fermentation liquid, centrifuge to collect bacterial cells, mix with an emulsifying agent, vegetable oil and starch and granulate to obtain yeast bacterial powder; Step 3: compound the Bi66 bacterial powder, the P16 bacterial powder and the yeast bacterial powder prepared in step 2 in a certain proportion to obtain the freeze-dried powder. ​ 20. The microecological preparation according to claim 19, characterized in that, The viable cell number of the Bi66 bacterial powder is 600 billion CFU / g; The viable cell number of the P16 bacterial powder is 200 billion CFU / g; The viable cell number of the Saccharomyces boulardii in the yeast bacterial powder is 20 billion CFU / g.

21. The microecological preparation as claimed in claim 19, characterized in that, The preparation method of the freeze-dried powder comprises the following steps: Step 1-1: inoculate the animal Bifidobacterium lactis Bi66 and the plant Bifidobacterium lactis P16 into MRS liquid medium respectively, activate at 37℃ for 24h, continuously activate for 2 times, and obtain an activation liquid; Step 1-2: inoculate the activation liquid into MRS liquid medium at an inoculation amount of 3%(v / v), culture at 37℃ for 22h, and obtain a bacterial liquid; Step 1-3: centrifuge the bacterial liquid at 8000r / min for 10min at 4℃, and filter to obtain bacterial bodies; Step 1-4: mix the bacterial bodies with 5 times the mass of a protective agent to obtain a bacterial suspension; Step 1-5: pre-culture the bacterial suspension at 37℃ for 1h, freeze-dry, and freeze-dry for 24h to obtain Bi66 bacterial powder and P16 bacterial powder; Step 2-1: inoculate the Saccharomyces boulardii Bld-3 into liquid medium, activate at 37℃ for 26h, and obtain an activation liquid; Step 2-2: inoculate the activation liquid into liquid medium, culture at 30℃ for 24h to obtain a seed fermentation liquid; Step 2-3: centrifuge the seed fermentation liquid to obtain yeast bacterial bodies; Step 2-4: add an emulsifier and vegetable oil to the yeast bacterial bodies, add starch for granulation, and obtain a yeast bacterial powder; Step 3: proportionally compound the Bi66 bacterial powder prepared in step 1, the P16 bacterial powder, and the yeast bacterial powder prepared in step 2 to obtain the freeze-dried powder.

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