A mixed bacterial combination capable of forming a multi-bacteria biofilm on water-insoluble dietary fiber and a method for preparing the multi-bacteria biofilm

Abstract

The present invention discloses a mixed bacterial combination capable of forming a multi-bacteria biofilm on water-insoluble dietary fiber and a method for preparing the multi-bacteria biofilm, and belongs to the fields of microbial technology and medical technology. The present invention provides a mixed bacterial combination composed of 9 types of intestinal bacteria and a dynamic fermentation film-forming method for forming a biofilm on water-insoluble dietary fiber. The multi-bacteria biofilm formed by the M9 mixed bacterial combination and the dynamic fermentation film-forming method provided by the present invention has better film-forming ability and stability than a single-bacteria biofilm, which is specifically reflected in that the M9 multi-bacteria biofilm shows better resistance to environmental changes than a single-bacteria biofilm under different pH environments and bile salt environments, and the M9 multi-bacteria biofilm has a higher amount of viable bacteria; after the M9 mixed bacteria are invaded by human feces, the M9 mixed bacteria can still stably adhere to the water-insoluble dietary fiber and form a biofilm.

Description

Technical Field

[0001] The present invention relates to a mixed bacteria combination capable of forming a multi-bacteria biofilm on water-insoluble dietary fiber and a method for preparing the multi-bacteria biofilm, belonging to the technical fields of microorganisms and medicine. Background Art

[0002] Biofilms are microbial aggregates formed by microorganisms adhering to the surface of a carrier and secreting extracellular polymers such as polysaccharides, proteins, lipid compounds, and nucleic acids. Biofilms can provide microorganisms with a protective ecological niche and a place for material exchange, which can increase the survival rate of microorganisms in harsh environments and enable microorganisms to remain active in environments such as ultraviolet radiation, extreme temperatures, extreme pH values, high salinity, high pressure, malnutrition, and antibiotic interference. Biofilms in natural environments, whether in soil, food surfaces, the mouth, or the intestines, are all multi-bacterial biofilms. Compared with single-bacterial biofilms, multi-bacterial biofilms can more accurately reflect the complex microbial composition in the actual environment and show better resistance to adverse environments.

[0003] Existing biofilm research primarily relies on microplate models, continuous interfaces on agar plates, trickle flow biofilm reactors, and rotating biofilm reactors. The classic method commonly used for biofilm production in the laboratory is the 96-well static fermentation method. However, the small plate area results in low biofilm yields, making continuous sampling for studying biofilm formation mechanisms and the production of high-density biofilms in industrial production impractical. Furthermore, static biofilm formation cannot simulate intestinal motility. While biofilm formation using chemostat, trickle flow, and rotating biofilm reactors allows for continuous sampling and industrial production of biofilms, the equipment is expensive, requires large amounts of culture medium, and is not suitable for laboratory use. Furthermore, these biofilm formation methods lack the anaerobic and temperature conditions necessary for the growth of intestinal bacteria. Currently, commonly used biofilm-forming supports include polystyrene microplates, glass, stainless steel, and plastics. However, these materials are inaccessible to intestinal bacteria in vivo. Currently, intestinal microbial aggregation has been observed to occur primarily in two ecological niches: the mucosa and around undigested food particles. Undigested food particles primarily refer to insoluble dietary fiber that has not been digested by the gastrointestinal tract. As the seventh nutrient, insoluble dietary fiber is consumed in large quantities in our daily diet and has many beneficial effects, such as moisturizing the intestines and promoting bowel movements, improving intestinal flora, enhancing insulin sensitivity, reducing the risk of obesity, cardiovascular disease, and certain cancers, increasing stool volume, promoting bowel movements, and controlling blood sugar levels.

[0004] Developing a method for preparing biofilms using water-insoluble dietary fiber as a film-forming carrier can better simulate the aggregation state of intestinal bacteria in the body. In addition, water-insoluble dietary fiber can be derived from daily diet and is food safe, which is of great significance for the application of biofilms in the food industry. Summary of the Invention

[0005] The invention provides a mixed bacterial combination capable of forming a multi-bacteria biofilm on water-insoluble dietary fiber.

[0006] In one embodiment of the present invention, the mixed bacterial combination consists of 9 intestinal bacteria, including Parabacteroides distichum ( Parabacteroides distasonis ), Escherichia coli ( Escherichia coli ), Bacteroides ovatus ( Bacteroides ovatus ), Bacteroides monomorpha ( Bacteroides uniformis ), Bacteroides cellulolyticus ( Bacteroides cellulolyticus ), Bacteroides fragilis ( Bacteroides fragilis ), Bacteroides doreniae ( Golden seal ), Bacteroides faecalis ( Bacteroides suffocatus ) and Bifidobacterium longum ( Bifidobacterium longum ), called M9 mixed bacteria.

[0007] In one embodiment of the present invention, the ovate Bacteroides includes ovate Bacteroides CCFM1342, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center on September 12, 2023, with a deposit number of GDMCC No: 63798; the Parabacteroides dissimilar includes Parabacteroides dissimilar CCFM1377, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center on February 2, 2024, with a deposit number of GDMCC No: 64368; the single Bacteroides includes single Bacteroides CCFM1358, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center on September 12, 2023, with a deposit number of GDMCC No: 63801; the cellulolytic Bacteroides includes cellulolytic Bacteroides FSDTAELIBHI5; the Escherichia coli includes Escherichia coli CCFM1378, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center on February 2, 2024, with a deposit number of GDMCC No.: 64369; the Bacteroides fragilis includes Bacteroides fragilis FSDTA-HCK-B8; the Bacteroides dorenia includes Bacteroides dorenia FSDLZ62K4; the Bacteroides faecalis includes Bacteroides faecalis FFJLY21K3; the Bifidobacterium longum includes Bifidobacterium longum subspecies infantis FBJCY2M11.

[0008] In one embodiment of the present invention, the Parabacteroides distichum ( Parabacteroides distasonis )CCFM1377 was compared with the nucleic acid sequence of Parabacteroides distichous in NCBI, and the results showed that the nucleic acid sequence similarity with Parabacteroides distichous was as high as 99%. Parabacteroides distasonis CCFM1377 was inoculated into YCFA solid medium and cultured inverted at 37°C for 48 h. The colonies were observed and the bacteria were observed under a microscope. The colonies were milky white, round and convex, and smooth. The bacteria were slightly irregular and round in shape and usually existed alone.

[0009] In one embodiment of the present invention, the Escherichia coli ( Escherichia coli )CCFM1378 was compared with the nucleic acid sequence of Escherichia coli in NCBI, and the results showed that the nucleic acid sequence similarity was as high as 99%. Escherichia coli CCFM1378 was inoculated into YCFA solid medium and cultured inverted at 37°C for 24 h. The colonies were observed and the bacteria were observed under a microscope. The results showed that the colonies were light white, slightly transparent, with round protrusions, smooth, and slightly moist around the colonies. The bacteria were slightly irregular and round in shape and usually existed alone, in pairs, and in small clusters.

[0010] In one embodiment of the present invention, the dietary fiber is insoluble in water, and its main sources are wheat fiber, soybean fiber, soy fiber, apple fiber, oat fiber, grape seed powder, dried bamboo shoot powder, and soy flour.

[0011] The present invention also provides a dynamic fermentation film-forming method for forming a multi-bacteria biofilm on water-insoluble dietary fiber.

[0012] In one embodiment of the present invention, the dynamic fermentation film-forming method of forming a multi-bacteria biofilm on water-insoluble dietary fiber comprises the following steps:

[0013] (1) Add non-water-soluble dietary fiber to the liquid culture medium, mix well, divide into equal volumes, and sterilize under high pressure to prepare dynamic fermentation medium.

[0014] (2) After activation of each intestinal bacteria, the bacterial solution was taken to measure the absorbance value, and the dilution multiple was calculated based on the measured absorbance value. The bacterial solution of each intestinal bacteria was diluted to a uniform absorbance value with fresh liquid culture medium, and the diluted bacterial solutions of the nine single bacteria were mixed.

[0015] (3) Inoculate the mixed bacteria into the dynamic fermentation medium and place it in a constant temperature shaking incubator for dynamic culture.

[0016] Preferably, all liquid culture media in this preparation method are modified YCFA liquid culture media.

[0017] Preferably, the modified YCFA liquid medium comprises: 10 g casein peptone, 10 g tryptone, 5 g yeast extract, 5 g sodium acetate, 5 g lactose monohydrate, 4 g sodium bicarbonate, 0.45 g dipotassium hydrogen phosphate, 1.8 g ammonium sulfate, 0.9 g sodium chloride, 0.045 g magnesium sulfate heptahydrate, 0.09 g anhydrous calcium chloride, 1.445 g L-cysteine ​​hydrochloride, 2 g glucose, 2 g maltose, 2 g cellobiose, 0.01 mg biotin, 0.01 mg VB12, 0.03 mg p-aminobenzoic acid, 0.05 mg folic acid, 0.15 mg VB6, 0.05 mg thiamine hydrochloride, 0.05 mg riboflavin, 5 mg D-pantothenate calcium, 5 mg lipoic acid, 2 mg niacin, 1% (v / v) clarified bovine rumen fluid, and 1% (v / v) hemin solution. Autoclave at 115°C for 20 min. pH 7.5±0.1.

[0018] Preferably, in step (1), the amount of water-insoluble dietary fiber added to the dynamic fermentation medium is 1% to 10% (w / v).

[0019] Preferably, in step (1), the water-insoluble dietary fiber is wheat fiber.

[0020] Preferably, in step (1), the mixture is mixed evenly and then divided into conical flasks according to equal volumes.

[0021] Preferably, in step (2), the diluted bacteria of each single strain are mixed with the same volume of bacterial liquid to form M9 mixed bacteria.

[0022] Preferably, in step (3), the inoculation amount of the mixed bacteria inoculated into the dynamic fermentation medium is 1% to 4% (v / v).

[0023] Preferably, in step (3), the dynamic culture conditions are 35-40°C, 100-180 rpm, and 12-60 h.

[0024] The present invention also provides a product containing an M9 mixed bacterial combination and water-insoluble dietary fiber, which is called an M9 multi-bacteria biofilm.

[0025] In one embodiment of the present invention, the product includes but is not limited to medicine.

[0026] In one embodiment of the present invention, the M9 multi-bacteria biofilm is resistant to changes in pH environment and bile salt environment.

[0027] In one embodiment of the present invention, the M9 multi-bacteria biofilm can still stably adhere to the water-insoluble dietary fiber to form a multi-bacteria biofilm under the invasion of complex intestinal flora.

[0028] Beneficial effects

[0029] (1) The present invention provides a mixed bacterial combination that can form a biofilm on water-insoluble dietary fiber, referred to as the M9 mixed bacterial combination. The film-forming effect of the M9 mixed bacterial combination on water-insoluble dietary fiber is improved by 57% to 321% compared with that of a single bacterial combination.

[0030] (2) The present invention provides a method for preparing a biofilm formed by mixed intestinal bacteria on water-insoluble dietary fiber. The preparation method uses water-insoluble dietary fiber as a film-forming carrier to perform dynamic film formation, which is not only conducive to continuous sampling and convenient for studying the film-forming mechanism, but also can provide the anaerobic and temperature conditions required for the growth of intestinal bacteria and simulate intestinal movement. In addition, this method can be carried out in the laboratory using a smaller capacity instrument or can be expanded and cultured in a larger container, which is feasible for preparing high-density biological cells in industrial production. In addition, water-insoluble dietary fiber can be ingested in daily diet and has a prebiotic effect, which is food safe.

[0031] (3) The M9 multi-bacteria biofilm prepared in the present invention using water-insoluble dietary fiber as a film-forming carrier still has film-forming stability under adverse environmental stress and invasion of complex intestinal flora.

[0032] (4) The M9 multi-bacteria biofilm in the present invention, which uses water-insoluble dietary fiber as a film-forming carrier, has an ecological niche that can be filled and can serve as the basis for mixed-bacteria film formation to construct a more complex multi-bacteria biofilm.

[0033] (5) The present invention provides potential feasibility for intestinal bacteria to utilize water-insoluble dietary fiber to colonize the intestine to form a stable ecological niche and thus regulate intestinal homeostasis, and has very broad research value and application prospects.

[0034] Biomaterial Deposit

[0035] A strain of Bacteroides ovatus ( Bacteroides ovatus )CCFM1342, taxonomically named Bacteroides ovate , was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 12, 2023, with the deposit number GDMCC No: 63798, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0036] A strain of Parabacteroides distichum ( Parabacteroides distasonis )CCFM1377, taxonomically named Parabacteroides distasonis , was deposited in the Guangdong Provincial Microbiological Culture Collection Center on February 2, 2024, with the deposit number GDMCC No: 64368, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0037] A strain of Bacteroides monomorpha ( Bacteroides uniformis)CCFM1358, taxonomically named Bacteroides uniform , was deposited in the Guangdong Provincial Microbiological Culture Collection on September 12, 2023, with the deposit number GDMCC No: 63801, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0038] A strain of Escherichia coli ( Escherichia coli )CCFM1378, taxonomic designation: Escherichia coli , was deposited in the Guangdong Provincial Microbiological Culture Collection Center on February 2, 2024, with the deposit number GDMCC No: 64369, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Comparison of film-forming ability of 18 single bacteria and different mixed bacteria combinations; A is the film-forming ability of 18 single bacteria; B is the film-forming ability of mixed bacteria obtained by combining 6 bacteria that can degrade cellulose and hemicellulose in different ways; C is the film-forming ability of mixed bacteria obtained by adding 12 bacteria that do not have the ability to degrade cellulose and hemicellulose to the basic mixed bacteria combination (M4), and the slashed filled column represents the M9 mixed bacteria combination.

[0040] Figure 2 Field emission scanning electron microscopy (FESEM) observations of wheat fiber and M9 mixed bacterial biofilms. AC shows observations of wheat fiber at different magnifications; DF shows observations of M9 mixed bacterial biofilms at different magnifications. Magnifications are 2500x, 5000x, and 10000x, respectively. White scale bars are 20 μm, 10 μm, and 5 μm, respectively.

[0041] Figure 3 : Particle size distribution of wheat fiber, 9 single bacteria biofilms and M9 mixed bacteria biofilm; A is wheat fiber; BK are Bacteroides ovatus CCFM1342, Parabacteroides distichous CCFM1377, Bacteroides monomorpha CCFM1358, Bacteroides cellulolyticus FSDTAELIBHI5, Escherichia coli CCFM1378, Bacteroides fragilis FSDTA-HCK-B8, Bacteroides doretinoides FSDLZ62K4, Bacteroides faecalis FFJLY21K3, Bifidobacterium longum subsp. infantis FBJCY2M11 and M9 mixed bacteria biofilm; L is the peak size distribution of wheat fiber, 9 single bacteria biofilms and M9 mixed bacteria biofilm.

[0042] Figure 4: Effects of environmental stress on the biofilm-forming ability of single bacteria and M9 mixed bacteria; AD is bile salt stress, and the bile salt concentrations are 0.05%, 0.1%, 0.3% and 0.5% (w / v); EF is pH stress, and the pH values ​​are 6.5, 7.0, 7.5 and 8.0, respectively.

[0043] Figure 5 Figure 3: Changes in bacterial communities after human fecal intrusion with the M9 mixed bacteria; A: Percent relative abundance; B: Circos plot of the relationship between bacterial communities and samples; C: Relative abundance of the top 10 bacterial genera. F: Fecal microbial fluid group; MF: Fecal microbial fluid + M9 mixed bacteria group; FW: Wheat fiber + fecal microbial fluid biofilm group; MFW: Wheat fiber + fecal microbial fluid + M9 mixed bacteria biofilm group; FP: Wheat fiber + fecal microbial fluid planktonic group; MFP: Wheat fiber + fecal microbial fluid + M9 mixed bacteria planktonic group.

[0044] Figure 6 : Lefse diagram of significantly different bacteria in different groups; F: fecal bacteria liquid group; MF: fecal bacteria liquid + M9 mixed bacteria group; FW: wheat fiber + fecal bacteria liquid biofilm group; MFW: wheat fiber + fecal bacteria liquid + M9 mixed bacteria biofilm group; FP: wheat fiber + fecal bacteria liquid planktonic bacteria group; MFP: wheat fiber + fecal bacteria liquid + M9 mixed bacteria planktonic bacteria group.

[0045] Figure 7 : Absolute content of M9 mixed bacteria on water-insoluble dietary fiber after being invaded by human feces; FW: wheat fiber + fecal bacteria liquid biofilm group; MFW: wheat fiber + fecal bacteria liquid + M9 mixed bacteria biofilm group.

[0046] Figure 8 : The film-forming ability of a more complex multi-bacteria biofilm constructed based on M9; M10 is M9+Bacteroides xylanisolvens FGDLZ48K3; M11 is M9+Bacteroides xylanisolvens FGDLZ48K3+Bacteroides faecium FSDTAELIBHI4; M12 is M9+Bacteroides xylanisolvens FGDLZ48K3+Ruminococcus contortus ATCC27756+Faecalibacterium prausnitzii A2-165. DETAILED DESCRIPTION

[0047] The experimental materials involved in the following examples are as follows:

[0048] Casein peptone, tryptone, yeast extract, sodium acetate, lactose monohydrate, sodium bicarbonate, dipotassium hydrogen phosphate, ammonium sulfate, sodium chloride, magnesium sulfate heptahydrate, anhydrous calcium chloride, L-cysteine ​​hydrochloride, glucose, and hemin were purchased from Sinopharm Chemical Reagent Co., Ltd.; maltose, cellobiose, biotin, VB12, p-aminobenzoic acid, folic acid, VB6, thiamine hydrochloride, riboflavin, D-calcium pantothenate, lipoic acid, niacin, XTT sodium salt, menadione, ox bile salt, and VK1 were purchased from McLean Chemical Reagent Co., Ltd.; clarified bovine rumen was purchased from Bofei Meike Reagent Co., Ltd.; the water-insoluble dietary fiber used in the present invention is wheat fiber, which was purchased from Shanghai Songqin Food Co., Ltd.; a bacterial DNA extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.; and a fecal DNA extraction kit was purchased from MP Biotechnology Co., Ltd. BIOMEDICALS; gel recovery and extraction kit, purchased from Hangzhou Beiwo Medical Technology Co., Ltd.; qPCR specific primers, synthesized from Shanghai Shenggong Co., Ltd.; anaerobic bags, purchased from Shanghai Chuangsai Technology Co., Ltd.

[0049] The culture medium involved in the following examples is as follows:

[0050] YCFA liquid medium (L): casein peptone 10 g, tryptone 10 g, yeast extract 5 g, sodium acetate 5 g, lactose monohydrate 5 g, sodium bicarbonate 4 g, potassium hydrogen phosphate dihydrate 0.45 g, ammonium sulfate 1.8 g, sodium chloride 0.9 g, magnesium sulfate heptahydrate 0.045 g, anhydrous calcium chloride 0.09 g, L-cysteine ​​hydrochloride 1.445 g, glucose 2 g, maltose 2 g, cellobiose 2 g, biotin 0.01 mg, VB12 0.01 mg, p-aminobenzoic acid 0.03 mg, folic acid 0.05 mg, VB6 0.15 mg, thiamine hydrochloride 0.05 mg, riboflavin 0.05 mg, D-pantothenate calcium 5 mg, lipoic acid 5 mg, niacin 2 mg, clarified bovine rumen fluid 1% (v / v), hemin solution 1% (v / v). Autoclave at 115°C for 20 min. pH 7.5±0.1.

[0051] YCFA solid medium (L): casein peptone 10 g, tryptone 10 g, yeast extract 5 g, sodium acetate 5 g, lactose monohydrate 5 g, sodium bicarbonate 4 g, potassium dihydrogen phosphate 0.45 g, ammonium sulfate 1.8 g, sodium chloride 0.9 g, magnesium sulfate heptahydrate 0.045 g, anhydrous calcium chloride 0.09 g, L-cysteine ​​hydrochloride 1.445 g, glucose 2 g, maltose 2 g, cellobiose 2 g, biotin 0.01 mg, VB12 0.01 mg, p-aminobenzoic acid 0.03 mg, folic acid 0.05 mg, VB6 0.15 mg, thiamine hydrochloride 0.05 mg, riboflavin 0.05 mg, D-calcium pantothenate 5 mg, lipoic acid 5 mg, niacin 2 mg, clarified bovine rumen fluid 1% (v / v), hemin solution 1% (v / v), agar powder 15 g. Autoclave at 115°C for 20 min. pH 7.5±0.1.

[0052] Dynamic fermentation medium: Weigh wheat fiber at a 4% (w / v) addition rate into an Erlenmeyer flask and add YCFA liquid medium. Seal the flask with parafilm and kraft paper and autoclave at 115°C for 20 min. pH 7.5 ± 0.1.

[0053] The strains involved in the following examples are as follows:

[0054] The deposit number of Bacteroides ovatus CCFM1342 is GDMCC No: 63798; the deposit number of Parabacteroides dienitrosum CCFM1377 is GDMCC No: 64368; the deposit number of Bacteroides monomorpha CCFM1358 is GDMCC No: 63801; the deposit number of Bacteroides cellulolyticus FSDTAELIBHI5 is disclosed in "A key genetic factor governing arabinan utilization in the gut microbiome alleviates constipation"; the deposit number of Escherichia coli CCFM1378 is GDMCC No: 64369; the deposit number of Bacteroides fragilis FSDTA-HCK-B8 is disclosed in "A new Illumina MiSeq high-throughput sequencing-based method for evaluating the composition of the Bacteroides community in the intestine using the rpsD gene sequence"; the deposit number of Bacteroides doretinoides FSDLZ62K4 is disclosed in "A key genetic factor governing arabinan utilization in the gut microbiome alleviates constipation" constipation”; Bacteroides faecalis FFJLY21K3 was published in “A new Illumina MiSeq high-throughput sequencing-based method for evaluating the composition of the Bacteroides community in the intestine using the rpsD genesequence”; Bifidobacterium longum subsp. infantis FBJCY2M11 was published in “Functional genomics of Bifidobacterium longum subsp. infantis and its effect on DSS-induced colitis”.

[0055] Example 1: Film formation experiment of 18 intestinal bacteria on water-insoluble dietary fiber

[0056] The film formation experiment of 18 intestinal bacteria on wheat fiber was investigated. The 18 intestinal bacteria included: Bacteroides ovatus CCFM1342, Parabacteroides distichous CCFM1377, Bacteroides monomorpha CCFM1358, Bacteroides cellulolyticus FSDTAELIBHI5, Bacteroides thetaiotaomicron FTJS1K7, Bacteroides vulgaris FSDTAHCMXY14, Escherichia coli CCFM1378, Bacteroides fragilis FSDTA-HCK-B8, Bacteroides doretii FSDLZ62K4, Bacteroides faecalis FFJLY21K3, Bifidobacterium longum subsp. infantis FBJCY2M11, Bacteroides xylanisolvens FGDLZ48K3, Parabacteroides faecium FSDTAELIBHI4, Bacteroides faecalis FNMHLBE15K4, Streptococcus salivarius B5, Alternaria unshiu DSM19147, Ruminococcus contortus ATCC27756 and Faecalibacterium prausnitzii A2-165. The specific steps are as follows:

[0057] (1) Bacteria activation:

[0058] Eighteen intestinal bacteria were inoculated into YCFA liquid culture medium at a 2% (v / v) inoculation rate and cultured at 37°C under anaerobic conditions for 24-48 h. The activated first-generation bacterial solution was transferred into new YCFA liquid culture medium at a 2% (v / v) inoculation rate and activated at 37°C under anaerobic conditions to the third generation.

[0059] (II) Film formation experiment of 18 single bacteria

[0060] Take 200 μL of the activated 3rd generation single bacterial solution and measure the absorbance at 600 nm. Calculate the dilution factor based on the measured absorbance and adjust the single bacterial solution to a uniform absorbance of 0.1 at 600 nm. Inoculate the 18 diluted single bacteria into the dynamic fermentation medium at a 2% (v / v) inoculation volume. The initial inoculation number of viable bacteria is 10 7 -10 8 CFU / mL, and the culture medium was placed in a sealed box with an anaerobic bag. The sealed box containing the dynamic fermentation medium was placed in a constant temperature shaking incubator at 37°C and 120 rpm for anaerobically incubated for 24 h for the film formation experiment on wheat fiber.

[0061] (III) XTT method for measuring film-forming ability

[0062] In this example, the XTT assay was used to evaluate the film-forming ability of bacteria. A higher XTT value indicates a higher number of viable bacteria adhering to and forming a film on the wheat fiber. 5 mg of XTT was dissolved in 10 mL of 37°C PBS to prepare a 0.5 mg / mL XTT solution. 0.017 g of menadione was dissolved in 10 mL of 100% acetone to prepare a 10 mM menadione solution. Before use, a mixture of the 10 mM menadione solution and the 0.5 mg / mL XTT solution was prepared at a ratio of 1:10 (μL / mL).

[0063] After 24 hours of dynamic fermentation, the biofilm-forming sample was removed from the sealed box. In a clean bench, while gently shaking the conical flask, 350 μL of the sample was pipetted into a 2 mL round-bottom centrifuge tube to ensure a uniform mixture of wheat fiber and fermentation broth. The sample was centrifuged at 100 rpm for 2 minutes to remove floating bacteria. 350 μL of PBS was added to the pellet, and the pellet was centrifuged at 100 rpm for 2 minutes to remove floating bacteria. This washing procedure was repeated once. The washed pellet consisted of wheat fiber and attached biofilm-forming bacteria. 175 μL of a mixture of menadione and XTT solution and 175 μL of PBS were added to the pellet. The reaction was incubated at 37°C in an anaerobic workstation in the dark for 2 hours. 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 492 nm.

[0064] Depend on Figure 1 A shows that the film-forming ability of 18 single bacteria on wheat fiber is significantly different. Alistipes onderdonkii ) DSM19147 has the strongest single bacterial film-forming ability, while Ruminococcus contortus ( Ruminococcus torque ) ATCC27756 had the weakest film-forming ability, with a difference of 5.04 times.

[0065] Example 2: Screening of the best film-forming mixed bacterial combination of intestinal bacteria on water-insoluble dietary fiber

[0066] (1) Determination of basic mixed bacterial combination

[0067] Among the 18 bacteria, 6 bacteria that have the ability to degrade cellulose and hemicellulose were activated to the third generation. The 6 bacteria are: Bacteroides ovatus ( Bacteroides ovatus CCFM1342, A), Parabacteroides dissimilaris ( Parabacteroids discordant CCFM1377, B), Bacteroides monomorpha ( Bacteroides uniformis CCFM1358, C), Bacteroides cellulolyticus ( Bacteroides cellulolyticus FSDTAELIBHI5, D), Bacteroides thetaiotaomicron ( Bacteroides thetaiotaomicronFTJS1K7, E) and Bacteroides vulgaris ( Bacteroides vulgaris FSDTAHCMXY14, F). 200 μL of the three-generation activated bacterial suspension of the six single bacteria was measured at 600 nm. The dilution factor was calculated based on the measured absorbance, and the single bacterial suspension was uniformly adjusted to an absorbance of 0.1 at 600 nm. After dilution, equal volumes of each single bacterial strain were mixed according to the combination method in Table 1. The mixed bacterial combinations in Table 1 were inoculated into the dynamic fermentation medium at a 2% (v / v) inoculation volume. After inoculation, the total viable count of the mixed bacteria was 10 7 -10 8 CFU / mL. The culture medium was placed in a sealed box containing an anaerobic bag and incubated anaerobically for 24 hours in a constant temperature shaking incubator at 37°C and 120 rpm. The sample that had formed a film after 24 hours of dynamic fermentation was removed from the sealed box and the film-forming ability was determined according to the XTT method in Example 1.

[0068] Depend on Figure 1 B shows that after the six groups with the ability to degrade cellulose and hemicellulose were mixed according to Table 1, the basic mixed bacteria combination with the best film-forming ability was composed of four bacteria, namely: Bacteroides ovatus ( Bacteroides ovatus )CCFM1342, Parabacteroides dissimilaris ( Parabacteroides distasonis )CCFM1377, Bacteroides monomorpha ( Bacteroides uniformis )CCFM1358 and Bacteroides cellulolyticus ( Bacteroides cellulolyticus The resulting combination is the basic mixed bacterial combination, referred to in this patent as the M4 multi-bacteria biofilm. The M4 multi-bacteria biofilm exhibited an average 37% higher film-forming capacity than six single bacterial strains capable of degrading cellulose and hemicellulose and the other 23 mixed bacterial combinations listed in Table 1.

[0069] Table 1 Mixed bacterial combinations consisting of six intestinal bacteria capable of degrading cellulose and hemicellulose

[0070]

[0071] (2) Determination of the optimal film-forming combination

[0072] The remaining 12 bacteria that cannot degrade cellulose and hemicellulose were activated to the third generation. The 12 bacteria are: Escherichia coli ( Escherichia coli )CCFM1378, Bacteroides fragilis ( Bacteroides fragilis ) FSDTA-HCK-B8, Bacteroides doreyi ( Golden seal ) FSDLZ62K4, Bacteroides faecalis ( Bacteroides suffocatus )FFJLY21K3, Bifidobacterium longum subsp. infantis ( Bifidobacterium longum )FBJCY2M11, Bacteroides xylanisolvens ( Bacteroides xylanisolvens) FGDLZ48K3, Parabacteroides faecium ( Parabacteroides merdae ) FSDTAELIBHI4, Bacteroides faecalis ( Bacteroides caccae ) FNMHLBE15K4, Streptococcus salivarius ( Streptococcus salivary ) B5, Ang-like bacteria ( Alistipes onderdonkii )DSM19147, Ruminococcus contortus ( Ruminococcus torques )ATCC27756 and Faecalibacterium prausnitzii ( Faecalibacterium prausnitzii )A2-165.

[0073] Take 200 μL of the 12 single bacterial cultures activated for 3 generations and measure the absorbance at 600 nm. Calculate the dilution factor based on the measured absorbance and adjust the single bacterial culture to a uniform absorbance of 0.1 at 600 nm. Add the 12 diluted single bacteria to the basic mixed bacterial combination (M4) and ensure that each bacteria is mixed in equal proportions. Inoculate the mixed bacteria into the dynamic fermentation medium at a 2% (v / v) inoculation volume. After inoculation, the total number of viable bacteria in the mixed bacteria is 10 7 -10 8 CFU / mL. Place the culture medium in a sealed box containing an anaerobic bag and incubate anaerobically for 24 hours in a constant temperature shaking incubator at 37°C and 120 rpm. Remove the sample that has formed a film after 24 hours from the sealed box and measure its film-forming ability using the XTT method described in Example 1. Screen for individual bacteria that promote film formation of the basic mixed bacterial combination (M4).

[0074] Figure 1 C shows that after 12 bacteria without cellulose and hemicellulose degradation ability were added to the basic mixed bacteria combination (M4), 5 bacteria had a promoting effect on the film-forming ability of the basic mixed bacteria combination (M4) and their film-forming ability was stronger than that of all the single bacteria in the combination. The 5 bacteria were: Escherichia coli ( Escherichia coli )CCFM1378, Bacteroides fragilis ( Bacteroides fragilis ) FSDTA-HCK-B8, Bacteroides doreyi ( Phocaeicola dorei ) FSDLZ62K4, Bacteroides faecalis ( Bacteroides stercoris )FFJLY21K3 and Bifidobacterium longum subsp. infantis ( Bifidobacterium longum )FBJCY2M11.

[0075] Furthermore, the above five bacteria were added to the basic mixed bacteria combination (M4) at the same time, and each bacteria was diluted to the same absorbance value and then mixed in equal proportions. The mixed bacteria were inoculated into the dynamic fermentation medium at an inoculum volume of 2% (v / v). After inoculation, the total number of viable bacteria in the mixed bacteria was 10 7 -10 8CFU / mL. The culture medium was placed in a sealed box containing an anaerobic bag and incubated anaerobically for 24 hours in a constant temperature shaking incubator at 37°C and 120 rpm. The sample that had formed a film after 24 hours of dynamic fermentation was removed from the sealed box and the film-forming ability was determined according to the XTT method in Example 1.

[0076] The multi-bacterial biofilm formed by mixing a basic mixed bacterial composition (M4) with five single bacteria with a promoting effect exhibited the best film-forming effect, with an average film-forming capacity increased by 105% compared to each single bacteria composition and a 12% increase compared to the basic mixed bacterial composition (M4). In this patent, the multi-bacterial biofilm formed by this nine-strain mixture on water-insoluble dietary fiber is referred to as the M9 multi-bacterial biofilm. The nine bacteria are: Bacteroides ovatus CCFM1342, Parabacteroides dieldrinii CCFM1377, Bacteroides monomorphis CCFM1358, Bacteroides cellulolyticus FSDTAELIBHI5, Escherichia coli CCFM1378, Bacteroides fragilis FSDTA-HCK-B8, Bacteroides doretinoides FSDLZ62K4, Bacteroides faecalis FFJLY21K3, and Bifidobacterium longum subsp. infantis FBJCY2M11, collectively referred to as the M9 mixed bacterial composition.

[0077] Table 2 Absorbance values ​​at OD492 nm of multi-bacteria biofilms formed by adding 12 bacterial species to the basic mixed bacterial combination (M4)

[0078]

[0079] Example 3: Characterization of the M9 multibacterial biofilm system

[0080] (I) Microscopic observation of M9 multibacterial biofilm

[0081] Take 0.5 mL of the sample obtained by 24 h dynamic fermentation film formation in Example 2 and place it in a centrifuge tube. Centrifuge at 100 rpm for 2 min to remove planktonic bacteria. The resulting precipitate is the M9 mixed bacteria biofilm. Add 1 mL of 2.5% glutaraldehyde solution and fix for 2-4 h. The fixed sample was washed twice with PBS solution and dehydrated with 1 mL of 50%, 70%, 90%, and 100% ethanol solution in sequence, with each dehydration time being 15 min. After the last dehydration, the multi-bacteria biofilm sample was placed on a 0.4 μm filter membrane for natural air drying. Take an appropriate amount of wheat fiber and dried multi-bacteria biofilm sample, stick them on the sample table with conductive adhesive, spray-coat them with gold powder, and observe them in a field emission scanning electron microscope.

[0082] (2) Particle size measurement of M9 multi-bacteria biofilm

[0083] 1 mL of the sample obtained by 24 h of dynamic fermentation biofilm formation in Examples 1 and 2 was placed in a centrifuge tube and centrifuged at 100 rpm for 2 min to remove floating bacteria. The resulting precipitate was the nine single-bacteria biofilms and the M9 mixed-bacteria biofilm. An equal volume of PBS solution was added to the precipitate for resuspending. Wheat fiber was added to a centrifuge tube containing 1 mL of PBS at a concentration of 4% (w / v) and resuspended. Before measurement, the sample in the centrifuge tube was gently pipetted to ensure uniform mixing, and 300 μL of the sample was taken for particle size measurement.

[0084] Depend on Figure 2 It can be seen that the surface structure of the wheat fiber is rough and has small holes. The M9 mixed bacteria are closely connected and regularly stacked on the wheat fiber, forming a three-dimensional structure. Figure 3 The wheat fiber particle size showed a normal distribution, with an average particle size of 45.19 μm. The average particle sizes of the nine single-bacterial biofilms formed by Bacteroides ovatus CCFM1342, Parabacteroides distichous CCFM1377, Bacteroides monomorpha CCFM1358, Bacteroides cellulolyticus FSDTAELIBHI5, Escherichia coli CCFM1378, Bacteroides fragilis FSDTA-HCK-B8, Bacteroides doreyi FSDLZ62K4, Bacteroides faecalis FFJLY21K3, and Bifidobacterium longum subsp. infantis FBJCY2M11 were 50.01 μm, 51.38 μm, 49.76 μm, 49.36 μm, 50.99 μm, 49.19 μm, 49.9 μm, 49.47 μm, and 49.31 μm, respectively. The average particle size of the M9 multi-bacterial biofilm was 96.71 μm. The multi-bacteria biofilm formed by the M9 mixed bacteria had a larger particle size, indicating that the mixed bacteria aggregated more densely on the wheat fibers, forming a thicker film structure. Figure 1 、 Figure 2 and Figure 3 These results indicate that M9 mixed bacteria formed a multi-bacteria biofilm on wheat fiber.

[0085] Example 4: Determination of the content of 9 bacteria in M9 multibacterial biofilm

[0086] To clarify the content of nine bacterial species in the multibacterial biofilm, absolute quantitative qPCR was performed on the M9 multibacterial biofilm in this example.

[0087] The specific steps are as follows:

[0088] (1) Bacterial DNA extraction

[0089] Take 1 mL of the sample obtained by 24 hours of dynamic fermentation and film formation in Example 2 and the sample obtained by 24 hours of dynamic fermentation and film formation of the nine single bacteria in Example 1. For the single-bacterial samples, after sampling, centrifugation was performed at 10,000 rpm for 1 minute, and the supernatant was removed. The resulting precipitate was the biofilm and planktonic bacteria, which was used to establish the qPCR standard curve for each single bacteria. For the mixed-bacterial samples, after sampling, centrifugation was performed at 100 rpm for 2 minutes to remove the planktonic bacteria. The resulting precipitate was the M9 mixed-bacterial biofilm, which was used to measure the specific content of the nine bacterial species in the multi-bacterial biofilm. The total DNA in the precipitate was extracted using the Tiangen Centrifugal Column Bacterial Genomic DNA Extraction Kit (Catalog No.: DP302).

[0090] (II) Absolute quantitative qPCR

[0091] Specific primers were designed for the nine single bacteria based on their specific gene fragments, ensuring that each primer only amplified the corresponding single bacteria and not the remaining eight single bacteria in the M9 multi-bacteria system.

[0092] The qPCR reaction system was 10 μL: 1 μL DNA template, 0.5 μL upstream primer, 0.5 μL downstream primer, 3 μL ddH2O and 5 μL SYBR Green Premix Ex Taq II.

[0093] The qPCR program was as follows: 95°C, 30 s; 95°C, 5 s; 60°C, 30 s, 40 cycles. The melting curve was as follows: 65°C-95°C, increment of 0.5°C every 5 s.

[0094] (III) Establishment of standard curve

[0095] The total bacterial DNA extracted from the 9 single bacterial samples was diluted with 0.9% NaCl solution (10 1 -10 5 ), the diluted DNA was used as a template for qPCR amplification, and the Ct values ​​at different dilutions were obtained.

[0096] Take 1 mL of the sample obtained by 24 h dynamic fermentation of 9 single bacteria in Example 1 and dilute it with 0.9% NaCl solution (10 1 -10 10 Based on the different growth patterns of each bacterial strain, three appropriate dilutions were selected and plated using YCFA solid medium. The plates were then incubated in an anaerobic workstation at 37°C for 48 hours. The CFU / mL value for each bacterial strain was calculated by counting the number of colonies on the plates and converting the dilution factor.

[0097] Log 10The CFU / mL value was used as the horizontal axis and the Ct value was used as the vertical axis for linear fitting to draw the qPCR standard curve of each single bacteria.

[0098] The Ct value of each single bacterium measured in the M9 multibacterial biofilm was substituted into its corresponding single bacterium qPCR standard curve to calculate the absolute content of each single bacterium in the M9 multibacterial biofilm.

[0099] As shown in Table 2, the standard curves of the nine bacteria have a good linear relationship. After the Ct value of each single bacteria measured in the M9 multi-bacteria biofilm was substituted into its corresponding single-bacteria qPCR standard curve, it was found that the absolute contents of Bacteroides ovatus CCFM1342, Parabacteroides dissimilaris CCFM1377, Bacteroides monomorpha CCFM1358, Bacteroides cellulolyticus FSDTAELIBHI5, Escherichia coli CCFM1378, Bacteroides fragilis FSDTA-HCK-B8, Bacteroides doretinoides FSDLZ62K4, Bacteroides faecalis FFJLY21K3 and Bifidobacterium longum subsp. infantis FBJCY2M11 in the M9 multi-bacteria biofilm were 9.375 Log, respectively. 10 CFU / mL, 11.853Log 10 CFU / mL, 10.172 Log 10 CFU / mL, 9.934 Log 10 CFU / mL, 12.879 Log 10 CFU / mL, 11.389Log 10 CFU / mL, 10.084 Log 10 CFU / mL, 9.057 Log 10 CFU / mL and 13.211 Log 10 CFU / mL.

[0100] Table 3 Standard curves of 9 single bacteria and absolute contents of 9 single bacteria in multi-bacteria biofilm (Log 10 CFU / mL)

[0101]

[0102] Example 5: Stress resistance of M9 multibacterial biofilm

[0103] (1) pH stress

[0104] The pH of the dynamic fermentation medium was adjusted to 6.5, 7.0, 7.5, and 8.0 using 1 M HCl and 1 M NaOH solutions. Biofilms were prepared according to the dynamic fermentation film formation method described in Example 2. The film-forming ability of single bacteria and M9 mixed bacteria under different pH stresses was determined according to the XTT method described in Example 1.

[0105] (2) Bile salt stress

[0106] Ox bile salts were added to the dynamic fermentation medium to achieve bile salt concentrations of 0.05%, 0.1%, 0.3%, and 0.5% (w / v). Biofilms were prepared according to the dynamic fermentation film formation method described in Example 2. The film-forming ability of individual bacteria and the M9 mixed strain under different bile salt stresses was determined using the XTT method described in Example 1.

[0107] Depend on Figure 4 AD shows that with the increase of bile salt concentration, the film-forming ability of the remaining eight single bacteria on wheat fiber decreased by an average of 265%, except for M9 multi-bacteria biofilm and Escherichia coli CCFM1378. Although the film-forming ability of M9 multi-bacteria biofilm under bile salt stress decreased by an average of 180% compared with the group without bile salt addition, it still had better film-forming ability than most single bacteria. Figure 4 EH analysis revealed that the M9 multi-bacteria biofilm exhibited superior resistance to changes in environmental pH compared to single-bacteria biofilms. When the pH of the culture medium changed, the biofilm-forming ability of the M9 mixed-bacteria biofilm remained 104% higher than that of each single-bacteria biofilm. However, the ability of the M9 multi-bacteria biofilm to resist changes in environmental pH decreased as the pH changed to slightly acidic (pH 6.5) and alkaline (pH 8.0) environments.

[0108] These results indicate that M9 multi-bacteria biofilm has better ability to resist environmental stress than single-bacteria biofilm.

[0109] Example 6: Stability of M9 multibacterial biofilm

[0110] The M9 mixed bacteria were invaded by human feces to explore whether the M9 mixed bacteria can still form multi-bacterial biofilm on non-water-soluble dietary fiber under the interference of complex bacterial flora.

[0111] The specific steps are as follows:

[0112] (1) Group settings

[0113] The following groups were set up as follows: F: fecal bacteria liquid group; MF: fecal bacteria liquid + M9 mixed bacteria group; FW: wheat fiber + fecal bacteria liquid biofilm group; MFW: wheat fiber + fecal bacteria liquid + M9 mixed bacteria biofilm group; FP: wheat fiber + fecal bacteria liquid planktonic bacteria group; MFP: wheat fiber + fecal bacteria liquid + M9 mixed bacteria planktonic bacteria group.

[0114] The preparation methods of samples in each group are as follows:

[0115] Fresh feces were collected from four voluntary donors (two women and two men, aged 24-29 years) who had no gastrointestinal diseases or antibiotic treatment in the past three months. 1 g of fecal sample was weighed from each donor, mixed, and diluted with PBS solution (pH 7.0) to form a 10% (w / v) fecal homogenate. The fecal homogenate was filtered through two layers of filter cloth to remove the residue in the fecal homogenate to obtain a fecal microbial solution. The M9 mixed microbial solution was prepared according to the method in Example 2. The fecal microbial solution and the M9 mixed microbial solution were also mixed in equal volumes to obtain a fecal microbial solution + M9 mixed microbial solution.

[0116] The dynamic fermentation medium without wheat fiber was placed in an anaerobic workstation at 37°C for 24 hours in advance for gas replacement and oxygen removal. The fecal microbial solution and the fecal microbial solution + M9 mixture were added to the dynamic fermentation medium without wheat fiber at a 2% (v / v) inoculum rate, and the culture medium was placed in a sealed box containing an anaerobic bag. All of these operations were completed in the anaerobic workstation. The sealed anaerobic box was placed in a constant temperature shaking incubator at 120 rpm and 37°C for 24 hours of dynamic fermentation. The resulting samples were designated as the fecal microbial solution group (F) and the fecal microbial solution + M9 mixture group (MF).

[0117] The dynamic fermentation medium containing wheat fiber was placed in a 37°C anaerobic workstation for 24 hours in advance for gas replacement and oxygen removal. The fecal microbial solution and the fecal microbial solution + M9 mixture were added to the dynamic fermentation medium at a 2% (v / v) inoculum rate, and the medium was placed in a sealed box containing an anaerobic bag. All of these operations were performed in the anaerobic workstation. The sealed anaerobic box was placed in a constant temperature shaking incubator at 120 rpm and 37°C for 24 hours of dynamic fermentation.

[0118] The sample of fecal microbial liquid that had been dynamically fermented for 24 h to form a biofilm was centrifuged at 100 rpm for 2 min, and the resulting precipitate was collected as the wheat fiber + fecal microbial liquid biofilm group (FW); the planktonic bacteria were collected as the wheat fiber + fecal microbial liquid planktonic bacteria group (FP).

[0119] The sample of fecal microbial liquid + M9 mixed bacteria that had been dynamically fermented for 24 h to form a biofilm was centrifuged at 100 rpm for 2 min, and the resulting precipitate was collected as the wheat fiber + fecal microbial liquid + M9 mixed bacteria biofilm group (MFW); the planktonic bacteria were collected as the wheat fiber + fecal microbial liquid + M9 mixed bacteria planktonic bacteria group (MFP).

[0120] (2) Sample testing

[0121] Fecal DNA extraction:

[0122] A 1 mL sample was centrifuged at 100 rpm for 2 min to remove floating bacteria. Total DNA was extracted using the FastDNA Spin Kit for Feces (Lot: FD02018).

[0123] 16s rRNA PCR:

[0124] The extracted DNA was used as a template for PCR amplification of the V3-V4 region using primers (341F: CCTAYGGGRBGCASCAG, 806R: GGACTACNNGGGTATCTAAT). The 5' end of the upstream primer contained a 7-base barcode to distinguish between different samples.

[0125] The PCR amplification system was 50 μL: 2 μL DNA template, 1.5 μL 341F, 1.5 μL 806R, 20 μL ddH2O, and 25 μL Taq mix.

[0126] PCR amplification conditions were as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 30 s; and 72°C for 10 min.

[0127] The amplified samples were electrophoresed on 2% agarose gel.

[0128] Rubber recovery and purification:

[0129] The electrophoresis bands were recovered from the gel using a DNA Gel / PCR Purification Miniprep Kit (Lot: B05WD127B).

[0130] 16S rRNA sequencing:

[0131] The purified DNA concentration was determined, and samples were mixed and libraries constructed at equal mass concentrations. Sequencing was performed using the Illumina sequencing platform. Off-line data were processed using the Quantitative Insights IntoMicrobial Ecology2 platform.

[0132] Determination of the absolute content of 9 bacterial species in biofilms under human feces intrusion:

[0133] The DNA extracted in this example was used to measure the Ct values ​​of the nine bacteria according to the absolute quantitative qPCR system and procedure in Example 5, and the measured Ct values ​​were substituted into the standard curve corresponding to each single bacteria to calculate the absolute content of the nine single bacteria on the wheat fiber after fecal intrusion.

[0134] Depend on Figure 5A shows that 32 genera of bacteria can colonize wheat fiber. Escherichia-Shigella, Anaerostipes, Megamonas, Erysipelotrichaceae UCG-003, Prevotella 9, Ruminococcus torques group, Bifidobacterium, Fusicatenibacter, Bacteroides, and Faecalibacterium are the top 10 genera associated with the samples ( Figure 5 B). In the three experimental groups (MF, MFW, and MFP) with the addition of M9 mixed bacteria, Escherichia-Shigella, Megamonas, and Bacteroides were the top three bacterial genera with the highest relative abundance ( Figure 5 C).

[0135] The Lefse analysis method was used to analyze the bacteria with or without exogenous addition of M9 mixed bacteria and the bacteria with significant differences between the biofilm state and the planktonic state. The results are as follows Figure 6 shown. Figure 6 AC showed that the Escherichia-Shigella genera were significantly different in the experimental group with exogenously added M9 mixed bacteria, which indicated that the exogenously added Escherichia coli ( Escherichia coli )CCFM1378 proliferated well. The Ruminococcus gnavus group was also a significantly different genus in the MFW group, indicating that the exogenous addition of M9 mixed bacteria may promote the colonization and formation of Ruminococcus gnavus group on wheat fiber. Analysis of significantly different genus in biofilm state and planktonic state revealed that ( Figure 6(DE), Anaerostipes, Fusicatenibacter, Agathobacter, and Lachnospiraceae UCG_004 genera tended to exist in biofilms and were unaffected by the addition of the M9 mixture. Ruminococcus torques group, Sutterella, and Senegalimassilia genera tended to exist in planktonic states and were unaffected by the addition of the M9 mixture. After the addition of the M9 mixture, Erysipelotrichaceae UCG_003, Faecalibacterium, and Ruminococcus gnavus group genera became significantly differentially expressed in biofilms, indicating that the M9 mixture promoted the colonization of these three genera on wheat fiber. However, Eubacterium hallii group, Lachnoclostridium, and Coprococcus 3 genera were no longer significantly differentially expressed in biofilms after the addition of the M9 mixture, suggesting that the M9 mixture may have inhibited the colonization of these three genera on wheat fiber. In the case of exogenous addition of the M9 mixed bacterial combination, the group with wheat fiber addition (MFW) could promote a significant increase in the genera of Erysipelotrichaceae UCG_003, Bifidobacterium, Fusicatenibacter, Faecalibacterium, Agathobacter, Dialister and Ruminococcus gnavus group.

[0136] The absolute content of M9 mixed bacteria on wheat fiber after being invaded by exogenously added human feces ( Figure 7 ) analysis found that under the influence of human feces, M9 mixed bacteria can still form a film on wheat fiber and the absolute content of all nine bacteria increased significantly. Bacteroides ovatus )CCFM1342, Parabacteroides dissimilaris ( Parabacteroides distasonis )CCFM1377, Bacteroides monomorpha ( Bacteroides uniformis )CCFM1358, Bacteroides cellulolyticus ( Bacteroides cellulosilyticus )FSDTAELIBHI5, Escherichia coli ( Escherichia coli )CCFM1378, Bacteroides fragilis ( Bacteroides fragilis ) FSDTA-HCK-B8, Bacteroides doreyi ( Phocaeicola dorei ) FSDLZ62K4, Bacteroides faecalis ( Bacteroides stercoris )FFJLY21K3 and Bifidobacterium longum subsp. infantis ( Bifidobacterium longum) The absolute content of FBJCY2M11 in the MFW group increased by 2.39%, 4.56%, 2.85%, 11.85%, 14.35%, 8.10%, 3.19%, 2.04% and 7.87% compared with the FW group, respectively.

[0137] These results demonstrate that the M9 mixed bacteria can withstand the complex intestinal flora environment and maintain film-forming stability. Furthermore, the results also show that the multi-bacterial biofilm formed by the M9 mixed bacteria on wheat fiber has a fillable ecological niche, which can accommodate more intestinal bacteria, thereby forming a more complex biofilm with a flora composition closer to that of the human intestinal flora.

[0138] Comparative Example 1: M9 multi-bacteria biofilm as a basis for building a more complex multi-bacteria biofilm

[0139] Using M9 mixed bacteria as the basis, add Bacteroides xylanisolvens ( Bacteroides xylanisolvens FGDLZ48K3), Parabacteroides faecium ( Parabacteroides merdae FSDTAELIBHI4), Ruminococcus contortus ( Ruminococcus torques ATCC27756) and Faecalibacterium prausnitzii ( Faecalibacterium prausnitzii A2-165) constitutes a more complex mixed bacterial system. Biofilms were prepared according to the mixed bacterial dynamic fermentation film formation method in Example 2. Film-forming ability was determined according to the XTT method in Example 1.

[0140] from Figure 8 It can be seen that using the M9 mixed bacteria as the basis, multi-bacterial biofilms with more complex bacterial compositions can be constructed, such as the M10 multi-bacterial biofilm (M9 + Bacteroides xylanisolvens, OD value 0.355), the M11 multi-bacterial biofilm (M9 + Bacteroides xylanisolvens + Parabacteroides faecium, OD value 0.388), and the M12 multi-bacterial biofilm (M9 + Bacteroides xylanisolvens + Ruminococcus contortus + Faecalibacterium prausnitzii, OD value 0.353). However, the addition of other strains to the M9 multi-bacterial biofilm significantly reduced its film-forming ability.

[0141] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, they can also use other strains of the same species but different strains as the 9 bacteria in the present invention, and add other strains to the M9 mixed bacteria, and other improvements. These improvements should also be considered within the scope of protection of the present invention.

Claims

1. An intestinal mixed bacteria combination M9 mixed bacteria capable of forming a multi-bacteria biofilm on water-insoluble dietary fiber, characterized in that: The M9 mixed bacteria consist of Bacteroides ovatus ( Bacteroides ovatus ), Parabacteroides dielinii ( Parabacteroides distasonis ), Bacteroides monomorpha ( Bacteroides uniformis ), Bacteroides cellulolyticus ( Bacteroides cellulosilyticus ), Escherichia coli ( Escherichia coli ), Bacteroides fragilis ( Bacteroides fragilis ), Bacteroides doreniae ( Phocaeicola dorei ), Bacteroides faecalis ( Bacteroides stercoris ) and Bifidobacterium longum ( Bifidobacterium longum )composition; The Bacteroides ovatus is Bacteroides ovatus CCFM1342, which was deposited in Guangdong Provincial Microbial Culture Collection on September 12, 2023, with the deposit number GDMCC No: 63798; The Parabacteroides distichum is Parabacteroides distichum CCFM1377, which was deposited in Guangdong Provincial Microbial Culture Collection on February 2, 2024, with the deposit number GDMCC No: 64368; The Bacteroides monomorpha is Bacteroides monomorpha CCFM1358, which was deposited in Guangdong Provincial Microbial Culture Collection on September 12, 2023, with the deposit number GDMCC No: 63801; The Bacteroides cellulolyticus is Bacteroides cellulolyticus FSDTAELIBHI5; The Escherichia coli is Escherichia coli CCFM1378, which was deposited in Guangdong Provincial Microbiological Culture Collection on February 2, 2024, with the deposit number GDMCC No: 64369; The Bacteroides fragilis is Bacteroides fragilis FSDTA-HCK-B8; The Bacteroides doreniae is Bacteroides doreniae FSDLZ62K4; The faecal Bacteroides is faecal Bacteroides FFJLY21K3; The Bifidobacterium longum is Bifidobacterium longum subspecies infantis FBJCY2M11.

2. The M9 mixed bacteria according to claim 1, characterized in that Bacteroides ovatus, Parabacteroides distichous, Bacteroides monomorpha, Bacteroides cellulolyticus, Escherichia coli, Bacteroides fragilis, Bacteroides doretii, Bacteroides faecalis and Bifidobacterium longum subsp. infantis were mixed at equal viable counts.

3. The M9 mixed bacteria according to claim 1, characterized in that The water-insoluble dietary fiber includes wheat fiber, soybean fiber, soy fiber, apple fiber, oat fiber, grape seed powder, dried bamboo shoot powder or soy flour.

4. A dynamic fermentation film-forming method for forming a multi-bacteria biofilm on water-insoluble dietary fiber, characterized in that: The M9 mixed bacteria described in any one of claims 1 to 3 are inoculated into a dynamic fermentation medium for dynamic fermentation film formation; the dynamic fermentation medium contains water-insoluble dietary fiber, and the water-insoluble dietary fiber includes wheat fiber, soybean fiber, soybean fiber, apple fiber, oat fiber, grape seed powder, dried bamboo shoot powder or soybean powder.

5. The dynamic fermentation film-forming method according to claim 4, characterized in that: The amount of water-insoluble dietary fiber added to the dynamic fermentation medium is 1% to 10% w / v.

6. The dynamic fermentation film-forming method according to claim 4, characterized in that: The amount of M9 mixed bacteria inoculated into the dynamic fermentation medium is 1%~4% v / v.

7. The dynamic fermentation film-forming method according to claim 4, characterized in that: The dynamic fermentation conditions are 35-40° C., 100-180 rpm, and anaerobic.

8. A multi-bacteria biofilm product, characterized in that: The product contains the M9 mixed bacteria and water-insoluble dietary fiber according to any one of claims 1 to 3.

9. The product according to claim 8, characterized in that The products include pharmaceuticals.

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