Method for in-vitro culture of human intestinal microflora

Abstract

The invention relates to a method for in-vitro culture of human intestinal microflora. The method comprises the following steps: S1, adding an intestinal flora culture solution and simulated gastric juice into a first fermentation tank, adjusting pH and temperature, and reacting for 0.5-3.5 hours to obtain a culture solution digested by the simulated gastric juice; s2, pumping the culture solution digested by the simulated gastric juice in the first fermentation tank and the simulated pancreatic juice into a second fermentation tank, adjusting the pH value and the temperature, and reacting for 0.5-4.5 hours to obtain the culture solution digested by the simulated pancreatic juice, namely digestive juice; pumping the digestive juice into a third fermentation tank, and adjusting the temperature and the pH value; s3, preparing a human intestinal microflora inoculation solution, and inoculating the human intestinal microflora inoculation solution into the digestive juice obtained in the step S2; after inoculation is completed, fermentation is performed for 0-120 h in an anaerobic environment and is not 0, and a fermentation product contains rich human intestinal microflora. According to the method, diversified nutrient substances can be provided for human intestinal flora, stable pH is maintained in the culture process, the diversity and the structure of the obtained flora are similar to those of inoculation liquid, and the culture effect is good.

Description

Technical Field

[0001] The present application belongs to the technical field of intestinal microorganism cultivation, and relates to a method for in vitro cultivation of human intestinal microbial communities. Background Art

[0002] The human intestine hosts a unique and complex microbial ecosystem, with trillions of bacteria, fungi, archaea and viruses, forming a mutually beneficial symbiotic relationship with the host. [1] , each individual's intestinal flora is unique. The intestinal flora of healthy adults is generally dominated by Firmicutes and Bacteroidetes, with Actinobacteria and Proteobacteria accounting for a smaller proportion.

[0003] At present, some studies on human intestinal flora are carried out through animal models, but the diversity and structure of animal intestinal flora are different from those of human intestinal flora. Comparing the diversity of intestinal flora in animal models and humans, 7758 strains of bacteria have been isolated and identified in human intestinal flora, from 11 classes, 16 orders, 40 families and 133 genera. [2] Only 244 bacterial strains from 5 phyla and 28 families were isolated and identified in the mouse intestinal flora. [3] ; 1100 bacterial strains were isolated from the intestinal flora of pigs, of which 117 strains were identified, from 9 phyla and 40 families [4] The intestinal flora of adult chickens is very similar to that of mammals at the higher taxonomic level, but at the genetic level, it is only about 10% similar to the genes of human intestinal flora. [5] .

[0004] Comparing the structure of the intestinal flora of animal models and humans, 98% of the intestinal bacteria in the human intestinal flora can be classified as Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria, of which Bacteroidetes and Firmicutes account for 90%. The zebrafish intestinal flora is mainly composed of bacteria from the Proteobacteria (36.87%), Bacteroidetes (36.11%) and Actinobacteria (26.53%). [6] The intestinal flora of carp is mainly composed of Bacteroidetes (18.55%), Fusobacteria (76.27%) and Proteobacteria (4.58%). [7] The intestinal flora of chickens is mainly composed of Firmicutes (80%) and Ascomycota (5%). [5] The main flora in the earthworm gut are Proteobacteria (50%), Firmicutes (30%), Bacteroidetes (6%), and Verrucomicrobia (3%). [8]The intestinal flora of experimental mice and rats was mainly composed of Bacteroidetes (50%), Firmicutes (35%) and Campylobacter (5%) [9] Therefore, there is a great deal of uncertainty in extrapolating the results of animal model studies to humans.

[10] , and considering the scientific ethics of animal experiment disciplines - the 3R principle (Reduction, Replacement, Refinement), it is very necessary to find animal alternatives in human intestinal bacteria experiments that are closer to the real situation of the human body. Animal alternatives can not only reduce or avoid the use of experimental animals, but also better conduct research from the perspective of the human body, so that the research results can be more in line with the situation under real conditions.

[0005] In vitro cultivation of human intestinal flora is one of the alternatives to animal model experiments, but there are currently many difficulties that need to be solved in the in vitro cultivation of human intestinal flora. One of them is that the culture method used is difficult to achieve community cultivation of intestinal microorganisms close to the human body and cannot reflect the true role of the human intestinal flora. This greatly limits the development of in vitro simulation cultivation of human intestinal flora.

[0006] For example, Sivieri used a culture medium containing high sugar and high mucin to culture only dozens of bacterial species, and in the colon fermenter, they were mainly Clostridium spp., Enterococcus spp., and Enterobacterium spp. The cultured communities were significantly different from the human intestinal flora in terms of diversity and structural composition.

[11] Barros used a medium containing high sugar, high protein, and high fat, and the bacterial community he cultivated was mainly composed of Firmicutes (50%), Bacteroidetes (30%), and Actinobacteria (15%).

[12] Liu used MSHIEM medium composed of high sugar and high inorganic salt components, and the bacterial community he cultivated was mainly composed of Firmicutes (70%) and Proteobacteria (20%) bacteria.

[13] De et al. used a medium composed of high protein, high sugar and low vitamins, and the bacterial flora obtained were mainly Clostridium sp. and Enterobacteriaceae.

[14] Wu et al. used a medium composed of high sugar and obtained mainly Actinobacteria (70%) and Firmicutes (10%) bacteria.

[15] .

[0007] In some patent documents, for example, comparative patent 1 (CN 111500499 B), although the intestinal flora of mice can be well cultured in vitro using Sab and Hab culture media supplemented with OPN, the diversity and structure of the intestinal flora of mice are significantly different from those of the human intestinal flora, and the complexity of the human intestinal flora is higher than that of mice. This comparative patent may not be able to achieve in vitro culture of the human intestinal flora; comparative patent 2 (CN 113474448A) can combine a complex community containing 40 to 500 intestinal microbial strains in vitro, but comparative patent 2 uses a purified single microbial strain for community assembly, and the assembled complex microbial community is transplanted into mice to observe the effect of the complex microbial community on Clostridium difficile, but the human intestinal flora as a whole is a highly complex mixed flora, and the use of a single microbial strain for community assembly cannot restore the composition structure of the real human intestinal flora; comparative patent 3 (CN 118460323A) uses a dual-chamber simulated intestinal reactor device to culture human intestinal flora in vitro. Although the structure and diversity of the human intestinal flora cultured by the device are good, when using the device to culture human intestinal flora in vitro, the human fecal inoculum used is prepared from human fecal samples that have been frozen and preserved with glycerol. In actual operation, it is found that the human intestinal flora is easily affected by external factors (such as temperature, pH, light and oxygen content, etc.) during the in vitro culture process. The culture results of fresh human fecal inoculum (not treated with freezing preservation and other measures) in the experiment are closer to the human intestinal flora in diversity and structure. The use of frozen human fecal samples to prepare human fecal inoculum will cause some human intestinal flora to be inactivated, which may not represent the true intestinal flora composition of the human body.

[0008] In summary, the strains obtained in previous studies are relatively limited and of low complexity. The diversity and structure of the flora cultivated in these studies are significantly different from the human intestinal flora. The comparative patent may not be able to cultivate the human intestinal flora in vitro or the intestinal flora cultivated in vitro may not represent the true composition of the human intestinal flora.

[0009] References involved in the background technology:

[0010] [1]LV M, LIU Y, SHI C, et al. A Missing Puzzle Piece of Pollutant Metabolism in Humans: The Gut Microbiota[J]. Environmental Science & Technology Letters, 2024, 11(2): 48-50.

[0011] [2]POYET M, GROUSSIN M, GIBBONS SM, et al. A library of human gutbacterial isolates paired with longitudinal multiomics data enables mechanistic microbiome research[J]. Nature Medicine, 2019, 25(9):1442-1452.

[0012] [3] LIU C, ZHOU N, DU MX, et al. The Mouse Gut Microbial Biobank expands the coverage of cultured bacteria[J]. Nature Communications, 2020, 11(1):79.

[0013] [4]WYLENSEK D, HITCH TCA, RIEDEL T, et al. A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity [J]. Nature Communications, 2020, 11(1): 6389.

[0014] [5] Zhou Xueyan, Li Qiongyi, Ding Gongtao, et al. Establishment, development, distribution and physiological significance of chicken intestinal microbial flora[J]. Acta Microbiologica Sinica, 2020, 60: 641-652.

[0015] [6] Sun Wen, Wang Yongjie, Bao Junjie, et al. Aeromonas hydrophila causes intestinal physiological damage and intestinal flora imbalance in zebrafish[J]. Oceanology and Lakes, 2023, 54(04): 1191-1198.

[0016] [7]JUNG-SCHROERS V,ADAMEK M,HARRIS S,et al.Response of the intestinalmucosal barrier of carp(Cyprinus carpio)to a bacterial challenge by Aeromonashydrophila intubation after feeding withβ-1,3 / 1,6-glucan[J].Journal of FishDiseases,2018,41(7):1077-1092.

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[0019]

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[0020]

[11] SIVIERI K,MORALES M L V,SAAD S M I,et al.Prebiotic Effect ofFructooligosaccharide in the Simulator of the Human Intestinal MicrobialEcosystem( Model)[J].Journal of Medicinal Food,2014,17(8):894-901.

[0021]

[12] BARROSD M V P,SALGACO M K,PIMENTEL T C,et al.Spirulina platensisbiomass enhances the proliferation rate of Lactobacillus acidophilus 5(La-5)and combined with La-5impact the gut microbiota of medium-age healthyindividuals through an in vitro gut microbiome model[J].Food ResearchInternational,2022,154:110880.

[0022]

[13] LIU L,MAHALAK K K,BOBOKALONOV J T,et al.Impact of Ivermectin onthe Gut Microbial Ecosystem[J].International Journal of Molecular Sciences,2023,24(22).

[0023]

[14] DE B P,VERSTRAETE W,DEPLANCKE B.Fermentation by Gut MicrobiotaCultured in a Simulator of the Human Intestinal Microbial Ecosystem IsImproved by Supplementing a Soygerm Powder[J].The Journal of Nutrition,2000,130(10):2599-2606.

[0024]

[15] WU T, GROOTAERT C, PITART J, et al.Aronia(Aronia melanocarpa)Polyphenols Modulate the Microbial Community in a Simulator of the HumanIntestinal Microbial Ecosystem(SHIME)and Decrease Secretion ofProinflammatory Markers in aCaco-2 / endothelial Cell Coculture Model[J].Molecular Nutrition&Food Research,2018,62(22):1800607. Summary of the invention

[0025] The in vitro culture schemes for human intestinal flora existing in the prior art obtain relatively limited strains of bacteria, have low complexity and are significantly different from the diversity and structure of human intestinal flora. Based on the above problems and shortcomings, the purpose of this application is to provide a method for in vitro culture of human intestinal microbial communities.

[0026] The purpose of this application can be achieved through the following technical solutions:

[0027] The technical solution of the present application provides a method for culturing a human intestinal microbial community in vitro, comprising the following steps:

[0028] S1. Add the intestinal flora culture fluid and simulated gastric fluid to the first fermentation tank, adjust the pH to 1.8-2.2, adjust the temperature to 36-37.5°C, react for 0.5-3.5h, and obtain the culture fluid after being digested by simulated gastric fluid;

[0029] S2. The culture fluid digested with simulated gastric juice and the simulated pancreatic juice in the first fermentation tank are pumped into the second fermentation tank, the pH is adjusted to 6.8-7.2, the experimental temperature is set to 36-37.5°C, and the reaction is carried out for 0.5-4.5 hours to obtain the culture fluid digested with simulated pancreatic juice, i.e., the digestion fluid; the digestion fluid is pumped into the third fermentation tank, the experimental temperature is adjusted to 36-37.5°C, and the pH is adjusted to 5.5-7.5;

[0030] S3. Prepare a human intestinal microbial community inoculum and inoculate it into the digestive fluid obtained in step S2; after the inoculation is completed, fermentation is carried out under anaerobic conditions, and the obtained fermentation product contains rich human intestinal microbial communities.

[0031] Furthermore, in step S1, the added volume ratio of the intestinal flora culture solution and the simulated gastric fluid is (1-3):(0.5-1.5); in step S1, the first fermentation tank is a simulated stomach fermentation tank.

[0032] Furthermore, in step S2, the added volume ratio of the culture medium after simulated gastric juice digestion and the simulated pancreatic juice is (3-9):(1-3); in step S2, the second fermentation tank is a simulated small intestine fermentation tank; and the third fermentation tank is a simulated colon fermentation tank.

[0033] Furthermore, in step S3, the inoculation ratio of the human intestinal microbial community inoculum in the digestive fluid is 1% to 2% v / v.

[0034] Furthermore, the fermentation condition in step S3 is 120-180 rpm, and the fermentation time is 0-120 h and not 0, preferably 0-24 h and not 0; the anaerobic condition is achieved by introducing nitrogen or other inert gases.

[0035] Furthermore, the composition of the intestinal flora culture solution in step S1 includes the following components according to the final concentration:

[0036] Peptone, 2-5 g / L, yeast extract, 1-4 g / L, CaCl 2 , 7~10g / L, D-cellobiose, 0.5~1.2g / L, casein peptone, 2~5g / L, K 2 HPO 4 , 65~80g / L, D-maltose, 0.5~1.2g / L, sodium lactate, 0.2~0.6g / L, NaHCO 3 , 0.1~0.5g / L, MgSO 4 7H 2 O, 1~4g / L, D-glucose, 0.4~0.8g / L, propionic acid, 1~4mL / L, D-fructose, 0.5~1.2g / L, beef extract, 3~6g / L, L-cysteine, 1~4g / L, NaCl, 0.05~0.09g / L, KH 2 PO 4 , 22~36g / L, FeSO 4 , 0.5~1.0g / L, acetic acid 1~4mL / L, butyric acid 1~4mL / L, Tween 80, 0.5~1.0mL / L, vitamin K, 0.05~2.5mL / L, isovaleric acid, 0.05~0.25mL / L, hemoglobin 0.05~2.5mL / L, trace element solution, 0.8~1.3mL / L, vitamin solution, 0.8~1.3mL / L;

[0037] The ingredients of the vitamin solution are: biotin (vitamin B7 ), 10~35mg / L, pyridoxine hydrochloride (vitamin B 6 ), 80~120mg / L, riboflavin (vitamin B 2 ), 35~65mg / L, thiamine hydrochloride (vitamin B 1 ), 30~60mg / L, folic acid (vitamin B 9 )10~35mg / L, lipoic acid, 35~55mg / L, calcium pantothenate (vitamin B 5 ), 35~65mg / L, niacin (vitamin B 3 ), 35~65mg / L, p-aminobenzoic acid (vitamin B x ), 35~55mg / L, cyanocobalamin (vitamin B 12 ), 0.5~2.5mg / L;

[0038] The components of the trace element solution are: NaCl, 0.5-2.5 g / L, MgSO 4 7H 2 O, 2.5-4.5 g / L, nitrilotriacetic acid, 0.5-2.5 g / L, CuSO 4 ·5H 2 O, 0.01~0.04g / L, Na 2 MoO 4 ·2H 2 O, 0.005~0.025g / L, ZnSO 4 7H 2 O, 0.05~0.25g / L, CoSO 4 7H 2 O, 0.05~0.25g / L, FeSO 4 7H 2 O, 0.05-0.25 g / L, CaCl 2 ·2H 2 O, 0.05~0.25g / L, AlK(SO 4 ) 2 , 0.005~0.025g / L, CuSO 4 ·5H 2 O, 0.005~0.025g / L, H 3 BO 3 , 0.005~0.025g / L, NiCl 2 6H 2 O, 0.005~0.025g / L.

[0039] In some specific embodiments of the present application, the preparation of the intestinal flora culture solution in step S1 is: after the culture solution is prepared according to the above-mentioned composition, the culture solution is sterilized at 121°C for 15 to 30 minutes. After sterilization, it is taken out and placed in a clean bench, and cooled to room temperature for use.

[0040] Furthermore, the components of the simulated gastric fluid in step S1 are: NaCl, 1-4 g / L, pepsin, 2-5 g / L, 36.5% concentrated hydrochloric acid, 5-15 mL / L;

[0041] In some specific embodiments of the present application, the preparation steps of the simulated gastric fluid are: first add NaCl, dissolve it with ultrapure water, sterilize it at 121°C for 15 to 30 minutes, then place it in an ultraclean bench, cool it to room temperature, add 36.5% concentrated hydrochloric acid and pepsin, dissolve it and set it aside.

[0042] Furthermore, the composition of the simulated pancreatic juice in step S2 is: NaHCO 3 , 9~18g / L, trypsin, 0.7~1.2g / L, bile salt, 1~5g / L;

[0043] In some specific embodiments of the present application, the preparation steps of the simulated pancreatic juice are: first adding NaHCO 3 After dissolving with ultrapure water, sterilize at 121°C for 15 to 30 minutes. After sterilization, take out and place in an ultra-clean bench. After cooling to room temperature, add bile salts and trypsin, dissolve and set aside.

[0044] In some specific embodiments of the present application, the reagents used to adjust the pH are hydrochloric acid solution and sodium hydroxide solution, and the concentrations of the acid and alkali solutions are both 0.5 to 2 mol / L;

[0045] In some specific embodiments of the present application, the steps for preparing the acid and base solutions are: take a certain amount of ultrapure water and place it in two covered glass bottles, sterilize it at 121°C for 15 to 30 minutes, take it out after sterilization and place it in an ultra-clean bench, cool it to room temperature and then add concentrated hydrochloric acid and NaOH respectively, the concentrations of the prepared hydrochloric acid solution and sodium hydroxide solution are 0.5 to 2 mol / L, and set aside.

[0046] Furthermore, the human intestinal microbial community inoculum described in step S3 includes the following steps: collecting a fresh human intestinal microbial community sample, adding sterile phosphate buffer and sodium thioglycolate; shaking well, then centrifuging, and saving the supernatant for later use, and the supernatant is the human intestinal microbial community inoculum.

[0047] Furthermore, the ratio of the fresh human intestinal microbial community sample, sterile phosphate buffer and sodium thioglycolate is (10-30) g: (50-150) mL: (1.5-2.5) g, the concentration of the sterile phosphate buffer is 0.1 mol / L; the shaking condition is 7000-9000 rpm, 5-15 min; the centrifugation condition is 6000-8000 rpm, 5-15 min.

[0048] In some specific embodiments of the present application, the human intestinal microbial community inoculum is a fecal inoculum, and the fresh human intestinal microbial community sample refers to a fresh fecal sample, and the fresh fecal sample comes from a healthy volunteer who has not taken antibiotics within 3 months before sampling.

[0049] In some specific embodiments of the present application, the operation of preparing the feces inoculum comprises the following steps:

[0050] Collect fresh fecal samples from volunteers, weigh 10-30 g of the fecal samples, add 50-150 mL of 0.1 mol / L sterile phosphate buffer and 1.5-2.5 g of sodium thioglycolate; place it in a homogenizer and shake it at 7000-9000 rpm for 5-15 min, then centrifuge it at 6000-8000 rpm for 5-15 min, and save the supernatant for later use. The supernatant is the fecal inoculum.

[0051] In addition, 0.5 to 1.5 mL of fecal inoculum was stored at -20°C as the sample at fermentation time 0 for testing.

[0052] In some specific embodiments of the present application, a human intestinal flora in vitro culture device (such as Figure 1 As shown in the figure, the human intestinal flora in vitro culture device is used to simulate the digestion of the culture fluid by the human intestine, which is closer to the actual situation of the human body. The human intestinal flora in vitro culture device includes three fermentation tanks connected in sequence, which are respectively recorded as the first fermentation tank, the second fermentation tank, and the third fermentation tank; peristaltic pumps are arranged at the inlets of the three fermentation tanks, the outlets of the third fermentation tank, between the first fermentation tank and the second fermentation tank, and between the second fermentation tank and the third fermentation tank, for the inlet and outlet and / or transfer of intestinal flora culture fluid, simulated gastric juice, simulated pancreatic juice, acid-base solution and digestive fluid / fermentation fluid between the fermentation tanks; the first fermentation tank is used to simulate the human stomach environment, the second fermentation tank is used to simulate the human small intestine environment, and the third fermentation tank is used to simulate the human colon environment.

[0053] In some specific embodiments of the present application, all fermentation tank bodies are equipped with conduits and probes, which can be added or removed as needed. Each tank body is equipped with a stirring device, which is controlled by a micro-integrated servo small stirring motor. The stirring speed can be adjusted within a control range of 50 to 800 rpm and a control accuracy of ±5 rpm.

[0054] In some specific embodiments of the present application, the human intestinal flora in vitro culture device is controlled by a software system, and during the in vitro culture process, the pH and temperature in the fermentation tank can be monitored in real time and self-regulated according to the set values.

[0055] In some specific embodiments of the present application, the diversity and structure of the sample are analyzed by sampling at regular intervals, which specifically includes the following steps:

[0056] After the experimental timing of step S4 begins, sampling is performed according to the experimental plan within 0 (feces inoculum sample) to 12 hours. The samples after each sampling are centrifuged at 4°C and 11000-13000 rpm for 3-7 minutes, and then the supernatant is transferred to a clean centrifuge tube for storage, and then 450-550 μL of 0.1 mol / L sterile phosphate buffer is added to the precipitate for washing. After sufficient shaking, the precipitate is centrifuged again, the washing solution is discarded, and the washing is repeated 3 times to obtain a precipitate sample; the supernatant and the precipitate sample are quickly frozen with liquid nitrogen and stored at -20°C for later use.

[0057] The diversity and structure analysis of the fecal inoculum sample, supernatant and precipitate sample specifically comprises the following steps:

[0058] The prepared fecal inoculum and the collected supernatant and precipitate samples are subjected to gene sequencing analysis, and the results obtained from the gene sequencing analysis are subjected to diversity analysis (e.g., α diversity analysis, β diversity analysis) and flora structure analysis, and the diversity and structure of the intestinal flora in the collected samples and fecal inoculum are compared. The α diversity analysis can reflect the abundance and diversity of the microbial community; the β diversity analysis is used to compare the diversity between different samples, that is, the differences between samples.

[0059] 1. Alpha diversity: It was finally found that the Pielou index of alpha diversity in the collected samples and fecal inoculum was greater than or equal to 0.30, and the difference in the average Pielou index of the two did not exceed 0.01;

[0060] 2. For β diversity, the difference between the Jaccrad distance analysis results of β diversity between the sample and the fecal inoculum should not exceed 0.05;

[0061] 3. For the bacterial community structure, the difference in the relative abundance of each bacterium in the sample and the fecal inoculum should not exceed 10%.

[0062] Compared with the prior art, the beneficial effects of this application are:

[0063] (1) The in vitro culture method of the human intestinal flora of the present invention aims to cultivate the human intestinal microbial community. The culture medium contains a variety of nutrients such as carbohydrates, peptones, vitamins, fatty acids, trace elements, electrolytes and mixed nutrients. During the in vitro culture process, it can provide diversified nutrients for the human intestinal flora to meet the needs of life activities.

[0064] (2) The in vitro culture method of the human intestinal flora of the present invention can maintain a stable pH during the entire in vitro culture process of the human intestinal flora, thereby avoiding the interference of pH changes on the in vitro culture of the intestinal flora.

[0065] (3) In the in vitro culture method of the human intestinal flora of the present invention, the culture medium before inoculation with the human feces inoculation liquid must first undergo two digestion processes in the stomach and small intestine fermentation tank before inoculation, which is closer to the digestion conditions of the human intestine.

[0066] (4) The diversity and structure of the human intestinal flora cultured by the in vitro culture method of the human intestinal flora of the present invention are similar to the diversity and structure of the intestinal flora in the inoculation solution, and the culture effect is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is a schematic diagram of an in vitro culture device for human intestinal flora;

[0068] Figure 2 Alpha diversity analysis results of fecal inocula and samples collected for in vitro culture of human intestinal flora;

[0069] Figure 3 Results of flora structure analysis of fecal inocula and samples collected for in vitro culture of human intestinal flora. DETAILED DESCRIPTION

[0070] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0071] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0072] Example 1

[0073] The human stool sample was obtained from volunteer 1, who was healthy and had not taken antibiotics within 3 months before sampling.

[0074] In vitro culture of human intestinal flora includes the following steps:

[0075] (1) Preparation of intestinal flora culture medium: peptone, 2 g / L, casein peptone, 2 g / L, yeast extract, 1 g / L, D-glucose, 0.4 g / L, D-cellobiose, 1.0 g / L, D-maltose, 1.0 g / L, D-fructose, 1.0 g / L, beef extract, 5 g / L, NaHCO 3 ,0.4g / L,NaCl,0.08g / L,K 2 HPO 4 , 70g / L, KH 2 PO 4 , 28g / L, L-cysteine, 2g / L, sodium lactate, 0.45g / L, MgSO 4 7H 2 O, 2.5 g / L, CaCl 2 , 8.0g / L, FeSO 4 , 0.75g / L, Tween 80, 0.7g / L, trace element solution, 1mL / L, vitamin solution, 1.0mL / L, acetic acid 1.5mL / L, isovaleric acid, 0.1mL / L, propionic acid 2mL / L, butyric acid 2mL / L, vitamin K, 1.0mL / L, hemoglobin 1.0mL / L;

[0076] The ingredients of the vitamin solution are: biotin (vitamin B 7 ), 20mg / L, folic acid (vitamin B 9 ), 20mg / L, pyridoxine hydrochloride (vitamin B 6 ), 100mg / L, Thiamine Hydrochloride (Vitamin B 1 ), 50mg / L, riboflavin (vitamin B 2 ), 50mg / L, niacin (vitamin B 3 ), 50mg / L, calcium pantothenate (vitamin B 5 ), 50mg / L, cyanocobalamin (vitamin B 12 ), 1mg / L, p-aminobenzoic acid (vitamin B x ), 50mg / L, lipoic acid, 50mg / L;

[0077] The components of the trace element solution are: nitrilotriacetic acid, 1.5 g / L, MgSO 4 7H 2 O, 3.0g / L, NaCl, 0.5g / L, FeSO 4 7H 2O, 0.1 g / L, CoSO 4 7H 2 O, 0.1 g / L, CaCl 2 ·2H 2 O, 0.1 g / L, ZnSO 4 7H 2 O, 0.1 g / L, CuSO 4 ·5H 2 O, 0.01 g / L, AlK(SO 4 ) 2 , 0.01g / L, H 3 BO 3 , 0.01g / L, Na 2 MoO 4 ·2H 2 O, 0.01 g / L, CuSO 4 ·5H 2 O, 0.02 g / L, NiCl 2 6H 2 O, 0.01g / L.

[0078] After the culture medium is prepared, sterilize it at 121°C for 15 minutes. After sterilization, take it out and place it in a clean bench, cool it to room temperature and set it aside.

[0079] (2) Preparation of gastric juice, pancreatic juice and acid-base solutions

[0080] The composition of gastric juice is: NaCl, 2.0 g / L, pepsin, 3 g / L, 36.5% concentrated hydrochloric acid, 7 mL / L;

[0081] The components of pancreatic juice are: trypsin, 0.9 g / L, bile salts, 3 g / L, NaHCO 3 , 12g / L.

[0082] Preparation of gastric juice: first add NaCl, dissolve it with ultrapure water, sterilize it at 121°C for 15 minutes, then place it in a clean bench, wait for it to cool to room temperature, add concentrated hydrochloric acid and pepsin, dissolve it and set aside;

[0083] Preparation of pancreatic juice: First add NaHCO 3 After dissolving in ultrapure water, sterilize at 121°C for 15 minutes. After sterilization, take out and place on a clean bench. After cooling to room temperature, add bile salts and trypsin and dissolve for later use.

[0084] Preparation of acid and alkali solutions: Take a certain amount of ultrapure water and place it in a glass bottle with a cover. Sterilize it at 121°C for 15 to 30 minutes. After sterilization, take it out and place it in an ultra-clean bench. After cooling to room temperature, add concentrated hydrochloric acid and NaOH. The prepared hydrochloric acid solution and sodium hydroxide solution have a concentration of 1 mol / L respectively and are set aside.

[0085] (3) Preparation of in vitro culture device for human intestinal flora

[0086] The in vitro culture device for human intestinal flora is controlled by a software system. During the in vitro culture process, the pH and temperature in the fermentation tank can be monitored in real time and self-adjusted according to the set values. All fermentation tanks are equipped with catheters and probes, which can be added or removed as needed. Each tank is equipped with a stirring device, which is controlled by a micro-integrated servo small stirring motor. The stirring speed can be adjusted within a control range of 50 to 800 rpm and a control accuracy of ±5 rpm.

[0087] (4) Digestion process

[0088] 1) 200 mL of culture medium and 100 mL of gastric juice were added to the gastric fermentation tank by a peristaltic pump, the pH was adjusted to 2.00 using the prepared hydrochloric acid solution, the experimental temperature was adjusted to 37°C, and the reaction was carried out for 3 h.

[0089] 2) The culture fluid digested by gastric juice and 100 mL of pancreatic juice in the stomach fermenter were pumped to the small intestine fermenter, and the pH was adjusted to 7.00 using the prepared sodium hydroxide solution. The experimental temperature was set to 37°C and the reaction was continued for 4 hours. The culture fluid digested by pancreatic juice in the small intestine fermenter was then pumped to the colon fermenter, and the experimental temperature was adjusted to 37°C.

[0090] (5) Preparation of fecal inoculum

[0091] Collect fresh fecal samples from volunteers, weigh 25g of fecal sample, add 100mL sterile phosphate buffer (0.1mol / L) and 2g sodium thioglycolate. Place it in a homogenizer (8000rpm, 8min) and centrifuge (7000rpm, 8min), and save the supernatant for later use. In addition, take 1.0mL of fecal inoculation solution and store it at -20℃ for testing.

[0092] (6) Inoculation of fecal inoculum

[0093] After the fecal inoculum was prepared, the pH in the colon fermenter to be inoculated was adjusted to 6.0 using the prepared hydrochloric acid solution and sodium hydroxide solution and recorded, the stirring device speed was adjusted to 150 rpm, the fecal suspension was injected into the fermenter, nitrogen was introduced into the fermenter for 15 minutes to create an anaerobic environment, and the experiment started timing when the nitrogen was stopped.

[0094] (7) Sampling and storage

[0095] After the experiment started, samples were taken at 0 (fecal inoculum sample), 4, 8 and 12h, and the samples were centrifuged at 4°C, 12000rpm for 5min, and then the supernatant was transferred to a clean centrifuge tube for storage. Then 500μL of 0.1mol / L sterile phosphate buffer was added to the precipitate for washing, and the precipitate was centrifuged again after being shaken thoroughly. The washing solution was discarded, and the washing was repeated 3 times to obtain the precipitate sample. The supernatant and precipitate samples were quickly frozen with liquid nitrogen and stored at -20°C.

[0096] (8) Sample diversity and structure analysis

[0097] The prepared fecal inoculum and the collected samples were subjected to gene sequencing analysis, and the results obtained from the gene sequencing analysis were subjected to diversity analysis and flora structure analysis, etc., to compare the diversity and structure of the intestinal flora in the collected samples and the fecal inoculum.

[0098] Example 2

[0099] The human stool sample was obtained from volunteer 2, who was healthy and had not taken antibiotics within 3 months before sampling.

[0100] In vitro culture of human intestinal flora includes the following steps:

[0101] (1) Preparation of intestinal flora culture medium: peptone, 2 g / L, casein peptone, 2 g / L, yeast extract, 1 g / L, D-glucose, 0.4 g / L, D-cellobiose, 1.0 g / L, D-maltose, 1.0 g / L, D-fructose, 1.0 g / L, beef extract, 5 g / L, NaHCO 3 ,0.4g / L,NaCl,0.08g / L,K 2 HPO 4 , 70g / L, KH 2 PO 4 , 28g / L, L-cysteine, 2g / L, sodium lactate, 0.45g / L, MgSO 4 7H 2 O, 2.5 g / L, CaCl 2 , 8.0g / L, FeSO 4 , 0.75g / L, Tween 80, 0.7g / L, trace element solution, 1mL / L, vitamin solution, 1.0mL / L, acetic acid 1.5mL / L, isovaleric acid, 0.1mL / L, propionic acid 2mL / L, butyric acid 2mL / L, vitamin K, 1.0mL / L, hemoglobin 1.0mL / L;

[0102] The ingredients of the vitamin solution are: biotin (vitamin B 7 ), 20mg / L, folic acid (vitamin B9 ), 20mg / L, pyridoxine hydrochloride (vitamin B 6 ), 100mg / L, Thiamine Hydrochloride (Vitamin B 1 ), 50mg / L, riboflavin (vitamin B 2 ), 50mg / L, niacin (vitamin B 3 ), 50mg / L, calcium pantothenate (vitamin B 5 ), 50mg / L, cyanocobalamin (vitamin B 12 ), 1mg / L, p-aminobenzoic acid (vitamin B x ), 50mg / L, lipoic acid, 50mg / L;

[0103] The components of the trace element solution are: nitrilotriacetic acid, 1.5 g / L, MgSO 4 7H 2 O, 3.0g / L, NaCl, 0.5g / L, FeSO 4 7H 2 O, 0.1 g / L, CoSO 4 7H 2 O, 0.1 g / L, CaCl 2 ·2H 2 O, 0.1 g / L, ZnSO 4 7H 2 O, 0.1 g / L, CuSO 4 ·5H 2 O, 0.01 g / L, AlK(SO 4 ) 2 , 0.01g / L, H 3 BO 3 , 0.01g / L, Na 2 MoO 4 ·2H 2 O, 0.01 g / L, CuSO 4 ·5H 2 O, 0.02 g / L, NiCl 2 6H 2 O, 0.01g / L.

[0104] After the culture medium is prepared, sterilize it at 121°C for 15 minutes. After sterilization, take it out and place it in a clean bench, cool it to room temperature and set it aside.

[0105] (2) Preparation of gastric juice, pancreatic juice and acid-base solutions

[0106] The composition of gastric juice is: NaCl, 2.0 g / L, pepsin, 3 g / L, 36.5% concentrated hydrochloric acid, 7 mL / L;

[0107] The components of pancreatic juice are: trypsin, 0.9 g / L, bile salts, 3 g / L, NaHCO 3 , 12g / L.

[0108] Preparation of gastric juice: first add NaCl, dissolve it with ultrapure water, sterilize it at 121°C for 15 minutes, then place it in a clean bench, wait for it to cool to room temperature, add concentrated hydrochloric acid and pepsin, dissolve it and set aside;

[0109] Preparation of pancreatic juice: First add NaHCO 3 After dissolving in ultrapure water, sterilize at 121°C for 15 minutes. After sterilization, take out and place on a clean bench. After cooling to room temperature, add bile salts and trypsin and dissolve for later use.

[0110] Preparation of acid and alkali solutions: Take a certain amount of ultrapure water and place it in a glass bottle with a cover. Sterilize it at 121°C for 15 to 30 minutes. After sterilization, take it out and place it in an ultra-clean bench. After cooling to room temperature, add concentrated hydrochloric acid and NaOH. The prepared hydrochloric acid solution and sodium hydroxide solution have a concentration of 1 mol / L respectively and are set aside.

[0111] (3) Preparation of in vitro culture device for human intestinal flora

[0112] The in vitro culture device for human intestinal flora is controlled by a software system. During the in vitro culture process, the pH and temperature in the fermentation tank can be monitored in real time and self-adjusted according to the set values. All fermentation tanks are equipped with catheters and probes, which can be added or removed as needed. Each tank is equipped with a stirring device, which is controlled by a micro-integrated servo small stirring motor. The stirring speed can be adjusted within a control range of 50 to 800 rpm and a control accuracy of ±5 rpm.

[0113] (4) Digestion process

[0114] 1) 200 mL of culture medium and 100 mL of gastric juice were added to the gastric fermentation tank by a peristaltic pump, the pH was adjusted to 2.00 using the prepared hydrochloric acid solution, the experimental temperature was adjusted to 37°C, and the reaction was carried out for 3 h.

[0115] 2) The culture fluid digested by gastric juice and 100 mL of pancreatic juice in the stomach fermenter were pumped to the small intestine fermenter, and the pH was adjusted to 7.00 using the prepared sodium hydroxide solution. The experimental temperature was set to 37°C and the reaction was continued for 4 hours. The culture fluid digested by pancreatic juice in the small intestine fermenter was then pumped to the colon fermenter, and the experimental temperature was adjusted to 37°C.

[0116] (5) Preparation of fecal inoculum

[0117] Collect fresh fecal samples from volunteers, weigh 25g of fecal sample, add 100mL sterile phosphate buffer (0.1mol / L) and 2g sodium thioglycolate. Place it in a homogenizer (8000rpm, 8min) and centrifuge (7000rpm, 8min), and save the supernatant for later use. In addition, take 1.0mL of fecal inoculation solution and store it at -20℃ for testing.

[0118] (6) Inoculation of fecal inoculum

[0119] After the fecal inoculum was prepared, the pH in the colon fermenter to be inoculated was adjusted to 6.0 using the prepared hydrochloric acid solution and sodium hydroxide solution and recorded, the stirring device speed was adjusted to 150 rpm, the fecal suspension was injected into the fermenter, nitrogen was introduced into the fermenter for 15 minutes to create an anaerobic environment, and the experiment started timing when the nitrogen was stopped.

[0120] (7) Sampling and storage

[0121] After the experiment started, samples were taken at 0 (fecal inoculum sample), 4, 8 and 12h, and the samples were centrifuged at 4°C, 12000rpm for 5min, and then the supernatant was transferred to a clean centrifuge tube for storage. Then 500μL of 0.1mol / L sterile phosphate buffer was added to the precipitate for washing, and the precipitate was centrifuged again after being shaken thoroughly. The washing solution was discarded, and the washing was repeated 3 times to obtain the precipitate sample. The supernatant and precipitate samples were quickly frozen with liquid nitrogen and stored at -20°C.

[0122] (8) Sample diversity and structure analysis

[0123] The prepared fecal inoculum and the collected samples were subjected to gene sequencing analysis, and the results obtained from the gene sequencing analysis were subjected to diversity analysis and flora structure analysis, etc., to compare the diversity and structure of the intestinal flora in the collected samples and the fecal inoculum.

[0124] Example 3

[0125] The human stool sample was obtained from volunteer 3, who was healthy and had not taken antibiotics within 3 months before sampling.

[0126] In vitro culture of human intestinal flora includes the following steps:

[0127] (1) Preparation of intestinal flora culture medium: peptone, 2 g / L, casein peptone, 2 g / L, yeast extract, 1 g / L, D-glucose, 0.4 g / L, D-cellobiose, 1.0 g / L, D-maltose, 1.0 g / L, D-fructose, 1.0 g / L, beef extract, 5 g / L, NaHCO 3 ,0.4g / L,NaCl,0.08g / L,K 2 HPO4 , 70g / L, KH 2 PO 4 , 28g / L, L-cysteine, 2g / L, sodium lactate, 0.45g / L, MgSO 4 7H 2 O, 2.5 g / L, CaCl 2 , 8.0g / L, FeSO 4 , 0.75g / L, Tween 80, 0.7g / L, trace element solution, 1mL / L, vitamin solution, 1.0mL / L, acetic acid 1.5mL / L, isovaleric acid, 0.1mL / L, propionic acid 2mL / L, butyric acid 2mL / L, vitamin K, 1.0mL / L, hemoglobin 1.0mL / L;

[0128] The ingredients of the vitamin solution are: biotin (vitamin B 7 ), 20mg / L, folic acid (vitamin B 9 ), 20mg / L, pyridoxine hydrochloride (vitamin B 6 ), 100mg / L, Thiamine Hydrochloride (Vitamin B 1 ), 50mg / L, riboflavin (vitamin B 2 ), 50mg / L, niacin (vitamin B 3 ), 50mg / L, calcium pantothenate (vitamin B 5 ), 50mg / L, cyanocobalamin (vitamin B 12 ), 1mg / L, p-aminobenzoic acid (vitamin B x ), 50mg / L, lipoic acid, 50mg / L;

[0129] The components of the trace element solution are: nitrilotriacetic acid, 1.5 g / L, MgSO 4 7H 2 O, 3.0g / L, NaCl, 0.5g / L, FeSO 4 7H 2 O, 0.1 g / L, CoSO 4 7H 2 O, 0.1 g / L, CaCl 2 ·2H 2 O, 0.1 g / L, ZnSO 4 7H 2 O, 0.1 g / L, CuSO 4 ·5H 2 O, 0.01 g / L, AlK(SO 4 ) 2 , 0.01g / L, H 3 BO 3 , 0.01g / L, Na2 MoO 4 ·2H 2 O, 0.01 g / L, CuSO 4 ·5H 2 O, 0.02 g / L, NiCl 2 6H 2 O, 0.01g / L.

[0130] After the culture medium is prepared, sterilize it at 121°C for 15 minutes. After sterilization, take it out and place it in a clean bench, cool it to room temperature and set it aside.

[0131] (2) Preparation of gastric juice, pancreatic juice and acid-base solutions

[0132] The composition of gastric juice is: NaCl, 2.0 g / L, pepsin, 3 g / L, 36.5% concentrated hydrochloric acid, 7 mL / L;

[0133] The components of pancreatic juice are: trypsin, 0.9 g / L, bile salts, 3 g / L, NaHCO 3 , 12g / L.

[0134] Preparation of gastric juice: first add NaCl, dissolve it with ultrapure water, sterilize it at 121°C for 15 minutes, then place it in a clean bench, wait for it to cool to room temperature, add concentrated hydrochloric acid and pepsin, dissolve it and set aside;

[0135] Preparation of pancreatic juice: First add NaHCO 3 After dissolving in ultrapure water, sterilize at 121°C for 15 minutes. After sterilization, take out and place on a clean bench. After cooling to room temperature, add bile salts and trypsin and dissolve for later use.

[0136] Preparation of acid and alkali solutions: Take a certain amount of ultrapure water and place it in a glass bottle with a cover. Sterilize it at 121°C for 15 to 30 minutes. After sterilization, take it out and place it in an ultra-clean bench. After cooling to room temperature, add concentrated hydrochloric acid and NaOH. The prepared hydrochloric acid solution and sodium hydroxide solution have a concentration of 1 mol / L respectively and are set aside.

[0137] (3) Preparation of in vitro culture device for human intestinal flora

[0138] The in vitro culture device for human intestinal flora is controlled by a software system. During the in vitro culture process, the pH and temperature in the fermentation tank can be monitored in real time and self-adjusted according to the set values. All fermentation tanks are equipped with catheters and probes, which can be added or removed as needed. Each tank is equipped with a stirring device, which is controlled by a micro-integrated servo small stirring motor. The stirring speed can be adjusted within a control range of 50 to 800 rpm and a control accuracy of ±5 rpm.

[0139] (4) Digestion process

[0140] 1) 200 mL of culture medium and 100 mL of gastric juice were added to the gastric fermentation tank by a peristaltic pump, the pH was adjusted to 2.00 using the prepared hydrochloric acid solution, the experimental temperature was adjusted to 37°C, and the reaction was carried out for 3 h.

[0141] 2) The culture fluid digested by gastric juice and 100 mL of pancreatic juice in the stomach fermenter were pumped to the small intestine fermenter, and the pH was adjusted to 7.00 using the prepared sodium hydroxide solution. The experimental temperature was set to 37°C and the reaction was continued for 4 hours. The culture fluid digested by pancreatic juice in the small intestine fermenter was then pumped to the colon fermenter, and the experimental temperature was adjusted to 37°C.

[0142] (5) Preparation of fecal inoculum

[0143] Collect fresh fecal samples from volunteers, weigh 25g of fecal sample, add 100mL sterile phosphate buffer (0.1mol / L) and 2g sodium thioglycolate. Place it in a homogenizer (8000rpm, 8min) and centrifuge (7000rpm, 8min), and save the supernatant for later use. In addition, take 1.0mL of fecal inoculation solution and store it at -20℃ for testing.

[0144] (6) Inoculation of fecal inoculum

[0145] After the fecal inoculum was prepared, the pH in the colon fermenter to be inoculated was adjusted to 6.0 using the prepared hydrochloric acid solution and sodium hydroxide solution and recorded, the stirring device speed was adjusted to 150 rpm, the fecal suspension was injected into the fermenter, nitrogen was introduced into the fermenter for 15 minutes to create an anaerobic environment, and the experiment started timing when the nitrogen was stopped.

[0146] (7) Sampling and storage

[0147] After the experiment started, samples were taken at 0 (fecal inoculum sample), 4, 8 and 12h, and the samples were centrifuged at 4°C, 12000rpm for 5min, and then the supernatant was transferred to a clean centrifuge tube for storage. Then 500μL of 0.1mol / L sterile phosphate buffer was added to the precipitate for washing, and the precipitate was centrifuged again after being shaken thoroughly. The washing solution was discarded, and the washing was repeated 3 times to obtain the precipitate sample. The supernatant and precipitate samples were quickly frozen with liquid nitrogen and stored at -20°C.

[0148] (8) Sample diversity and structure analysis

[0149] The prepared fecal inoculum and the collected samples were subjected to gene sequencing analysis, and the results obtained from the gene sequencing analysis were subjected to diversity analysis and flora structure analysis, etc., to compare the diversity and structure of the intestinal flora in the collected samples and the fecal inoculum.

[0150] The experimental results of Examples 1 to 3 are shown in Figure 2 and Figure 3 , the inoculum is the sample at time 0, and the culture result is the integrated result of the above three times 4, 8, and 12:

[0151] Figure 2 The Pielou's Evenness Index is a method for measuring species diversity. It is used to measure the uniformity of species distribution in a community, also known as the evenness index. The index ranges from 0 to 1, and the larger the value, the more uniform the vegetation distribution. The calculation formula of the Pielou index is:

[0152]

[0153] Where k is the number of OTUs or ASVs obtained by sequencing, n i is the number of OTUs or ASVs containing i sequences, N is the total number of bacterial species sequences detected, and S is the total abundance of bacterial species detected in the intestinal flora of the collected samples. The Pielou index was analyzed, calculated, and plotted using R language packages such as vegan, ggplot2, tidyverse, agricolae, and aplot.

[0154] Figure 2 The Pielou indexes of inoculum 1 and culture result 1 were 0.51±0.004 and 0.53±0.004, respectively; the Pielou indexes of inoculum 2 and culture result 2 were 0.54±0.006 and 0.55±0.006, respectively; the Pielou indexes of inoculum 3 and culture result 3 were 0.55±0.010 and 0.54±0.009, respectively; the differences in the average values ​​of the Pielou indexes of the three experimental groups were 0.02, 0.01 and 0.01, respectively. This shows that the microbial community in the fermentation broth obtained by the in vitro culture method provided in this application can maintain the diversity of the microbial community in the inoculum.

[0155] In addition, the present application characterized the β diversity between the fermentation broth and the inoculation broth by calculation, and the calculation expression of the Jaccard distance of β diversity is:

[0156]

[0157] Among them, A and B are all bacteria obtained by sequencing samples and fecal inoculum, respectively. The differences in Jaccard distances of the three experimental groups were 0.01, 0.03 and 0.02, respectively.

[0158] Figure 3The results of the flora structure analysis of the fecal inoculum and samples collected from in vitro culture of the human intestinal flora. Each legend in the figure represents a microorganism, and the calculation formula for the relative abundance is:

[0159]

[0160] Among them, m is the number of each OTU or ASV obtained by sequencing, and M is the total number of OTUs or ASVs obtained by sequencing; the experimental results are calculated by using R language packages such as ggplot2 to calculate the relative abundance of each OTU or ASV obtained by sequencing in the total number of sequences, and the bacterial species with a total relative abundance greater than or equal to 80% are selected for graphical display. The correlation coefficient between each group of inoculation solution and culture results is less than 0.05, which also shows that the microbial community in the fermentation broth obtained by the in vitro culture method provided in this application can maintain the diversity and richness of the microbial community in the inoculation solution.

[0161] The above OTU means OTU cluster (Operational Taxonomic Unit). Each OTU obtained by sequencing is a sequence from a strain. Usually, the representative sequence with the highest abundance or center is selected after 97% clustering.

[0162] The above ASV means Amplicon Sequence Variant. Each ASV is a sequence from a strain, and is usually clustered at 100%. ASV is a method for simulating and identifying DNA sequence changes. It can more accurately identify microbial species, distinguish closely related species, and subspecies level microorganisms, and increase the accuracy and sensitivity of microbial diversity analysis.

[0163] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for culturing human intestinal microbial communities in vitro, characterized in that: The method comprises the following steps: S1. Add the intestinal flora culture fluid and simulated gastric fluid to the first fermentation tank, adjust the pH to 1.8-2.2, adjust the temperature to 36-37.5°C, react for 0.5-3.5h, and obtain the culture fluid after being digested by simulated gastric fluid; S2. The culture fluid digested with simulated gastric juice and the simulated pancreatic juice in the first fermentation tank are pumped into the second fermentation tank, the pH is adjusted to 6.8-7.2, the experimental temperature is set to 36-37.5°C, and the reaction is carried out for 0.5-4.5 hours to obtain the culture fluid digested with simulated pancreatic juice, i.e., the digestion fluid; the digestion fluid is pumped into the third fermentation tank, the experimental temperature is adjusted to 36-37.5°C, and the pH is adjusted to 5.5-7.5; S3. Prepare a human intestinal microbial community inoculum and inoculate it into the digestive fluid obtained in step S2; after the inoculation is completed, fermentation is carried out under anaerobic conditions, and the obtained fermentation product contains rich human intestinal microbial communities.

2. The method for in vitro cultivation of human intestinal microbial communities according to claim 1, characterized in that: In step S1, the volume ratio of the intestinal flora culture solution and the simulated gastric fluid is (1-3): (0.5-1.5); in step S1, the first fermentation tank is a simulated stomach fermentation tank.

3. The method for in vitro cultivation of human intestinal microbial communities according to claim 1, characterized in that: In step S2, the volume ratio of the culture medium after simulated gastric juice digestion and the simulated pancreatic juice is (3-9): (1-3); in step S2, the second fermentation tank is a simulated small intestine fermentation tank; and the third fermentation tank is a simulated colon fermentation tank.

4. The method for in vitro cultivation of human intestinal microbial communities according to claim 1, characterized in that: In step S3, the inoculation ratio of the human intestinal microbial community inoculum in the digestive fluid is 1% to 2% v / v.

5. The method for in vitro cultivation of human intestinal microbial communities according to claim 1, characterized in that: The fermentation condition in step S3 is 120-180 rpm, and the fermentation time is 0-120 h and is not 0; the anaerobic condition is achieved by introducing nitrogen or other inert gases.

6. The method for in vitro cultivation of human intestinal microbial communities according to claim 1, characterized in that: The composition of the intestinal flora culture solution in step S1, measured according to the final concentration, includes the following components: peptone, 2-5 g / L, yeast extract, 1-4 g / L, CaCl2, 7-10 g / L, D-cellobiose, 0.5-1.2 g / L, casein peptone, 2-5 g / L, K2HPO4, 65-80 g / L, D-maltose, 0.5-1.2 g / L, sodium lactate, 0.2-0.6 g / L, NaHCO3, 0.1-0.5 g / L, MgSO4·7H2O, 1-4 g / L, D-glucose, 0.4-0.8 g / L, propionic acid, 1-4 mL / L, D- Sugar, 0.5-1.2 g / L, beef extract, 3-6 g / L, L-cysteine, 1-4 g / L, NaCl, 0.05-0.09 g / L, KH2PO4, 22-36 g / L, FeSO4, 0.5-1.0 g / L, acetic acid 1-4 mL / L, butyric acid 1-4 mL / L, Tween 80, 0.5-1.0 mL / L, vitamin K, 0.05-2.5 mL / L, isovaleric acid, 0.05-0.25 mL / L, hemoglobin 0.05-2.5 mL / L, trace element solution, 0.8-1.3 mL / L, vitamin solution, 0.8-1.3 mL / L; The ingredients of the vitamin solution are: biotin, 10-35 mg / L, pyridoxine hydrochloride, 80-120 mg / L, riboflavin, 35-65 mg / L, thiamine hydrochloride, 30-60 mg / L, folic acid, 10-35 mg / L, lipoic acid, 35-55 mg / L, calcium pantothenate, 35-65 mg / L, niacin, 35-65 mg / L, p-aminobenzoic acid, 35-55 mg / L, cyanocobalamin, 0.5-2.5 mg / L; The components of the trace element solution are: NaCl, 0.5-2.5 g / L, MgSO4·7H2O, 2.5-4.5 g / L, nitrilotriacetic acid, 0.5-2.5 g / L, CuSO4·5H2O, 0.01-0.04 g / L, Na2MoO4·2H2O, 0.005-0.025 g / L, ZnSO4·7H2O, 0.05-0.25 g / L, CoSO4·7H2O, 0.05-0.25 g / L, FeSO4·7H2O, 0.05-0.25 g / L, CaCl2·2H2O, 0.05-0.25 g / L, AlK(SO4)2, 0.005-0.025 g / L, CuSO4·5H2O, 0.005-0.025 g / L, H3BO 3， 0.005~0.025g / L, NiCl2·6H2O, 0.005~0.025g / L.

7. The method for in vitro cultivation of human intestinal microbial communities according to claim 1, characterized in that: The composition of the simulated gastric fluid in step S1 includes the following components, measured according to the final concentration: NaCl, 1-4 g / L, pepsin, 2-5 g / L, and 36.5% concentrated hydrochloric acid, 5-15 mL / L.

8. The method for in vitro cultivation of human intestinal microbial communities according to claim 1, characterized in that: The composition of the simulated pancreatic juice in step S2 includes the following components, measured according to the final concentration: NaHCO3, 9-18 g / L, trypsin, 0.7-1.2 g / L, and bile salts, 1-5 g / L.

9. The method for in vitro cultivation of human intestinal microbial communities according to claim 1, characterized in that: The human intestinal microbial community inoculation liquid described in step S3 comprises the following steps: collecting a fresh human intestinal microbial community sample, adding sterile phosphate buffer and sodium thioglycolate; shaking well, then centrifuging, and saving the supernatant for later use, and the supernatant is the human intestinal microbial community inoculation liquid.

10. The method for in vitro cultivation of human intestinal microbial communities according to claim 9, characterized in that: The ratio of the fresh human intestinal microbial community sample, sterile phosphate buffer and sodium thioglycolate is (10-30) g: (50-150) mL: (1.5-2.5) g, and the concentration of the sterile phosphate buffer is 0.1 mol / L; the shaking condition is 7000-9000 rpm, 5-15 min; and the centrifugation condition is 6000-8000 rpm, 5-15 min.

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