A culture medium for enhancing the activity of extracellular fiber-degrading bacteria in the gut microbiota, its preparation method, and its application.

Abstract

This invention discloses a culture medium for enhancing the activity of in vitro fiber-degrading bacteria in the gut microbiota, its preparation method, and its application, belonging to the field of microbial culture medium technology. The culture medium provided by this invention can effectively enhance the activity of fiber-degrading bacteria in the gut microbiota, promote their growth and metabolism, and thus significantly improve the fiber degradation capacity of in vitro microbiota. Compared with commonly used culture media, the concentration of total short-chain fatty acids in fiber fermentation products can be increased by up to approximately 50%. Compared with commonly used culture media, the culture medium provided by this invention can alter the composition of the fiber-degrading bacterial community in the gut microbiota, with significant differences detected in the abundance of nine genera, which may be the reason for the different fermentation efficiencies. The culture medium provided by this invention helps to more fully evaluate the fermentability of dietary fiber, and further study its metabolic pathways, product synthesis, enzyme activity, and biocatalytic mechanisms, which has significant value for the optimization of biomimetic digestive systems in the field of animal nutrition.

Description

Technical Field

[0001] This invention relates to the field of microbial culture medium technology, and in particular to a culture medium for improving the activity of extracellular fiber-degrading bacteria in intestinal flora, its preparation method, and its application. Background Technology

[0002] In recent years, dietary fiber has received widespread attention as a functional substance. Short-chain fatty acids (SCFAs) are the main products of dietary fiber fermentation, providing energy not only for intestinal epithelial cells but also promoting intestinal health and immune function. However, the diverse types and properties of dietary fiber limit its production and application.

[0003] The assessment of fiber fermentation characteristics is crucial for the precise application of fiber in production and can be divided into in vivo and in vitro assessment methods. The fermentability of fiber in the intestines of monogastric animals is primarily assessed based on digestibility and the concentration of short-chain fatty acids in the intestinal contents. However, in vivo methods suffer from drawbacks such as high testing costs, long testing cycles, and significant individual variability. Compared to in vivo methods, in vitro digestion simulation technology is also an effective method for assessing the nutritional value of pig feed, overcoming the limitations of traditional biological methods such as low efficiency and poor reproducibility.

[0004] In vitro fermentation technology is an effective method for evaluating the degradation rate and fermentation characteristics of rumen microbial fiber in ruminant nutrition research, but its introduction into gut microbiota research still faces many challenges. Traditional in vitro fiber fermentation models mainly rely on the culture media reported by Menke et al. or simplified versions thereof, which provide nitrogen, inorganic salts, and trace elements for microbial growth. The addition of fiber substrates as a carbon source provides the basic conditions for bacterial community growth. However, traditional culture media have some shortcomings: 1) the reducing agent Na₂S in the components is corrosive and toxic, which is detrimental to bacterial growth; 2) they lack growth factors for gut bacteria, especially anaerobic bacteria. Therefore, further optimization of the fermentation system culture medium is particularly important. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium for improving the activity of in vitro fiber-degrading bacteria in the gut microbiota, its preparation method, and its application, in order to solve the problem that the culture media used in current in vitro fermentation technology are not conducive to bacterial growth and lack anaerobic growth factors for gut bacteria, resulting in an imperfect in vitro fermentation model.

[0006] To achieve the above objectives, the present invention provides a culture medium for enhancing the activity of extracellular fiber-degrading bacteria in the gut microbiota, comprising the following components: peptone 0.19~0.21 g / L, cysteine ​​hydrochloride 0.95~1.05 g / L, and NH4HCO3. 0.2~0.5g / L, sodium bicarbonate 2~5.5g / L, disodium hydrogen phosphate 7.35~1.0g / L, potassium dihydrogen phosphate 4.45~7.5g / L, magnesium sulfate heptahydrate 0.5~0.75g / L, calcium chloride dihydrate 12.5~14.0mg / L, manganese chloride tetrahydrate 9.5~10.5mg / L, cobalt chloride hexahydrate 0.95~1.05mg / L, ferric chloride hexahydrate 6.0~8.2mg / L, sodium hydroxide 200mg / L, heme chloride 4~5.5mg / L, vitamin K1 0.8~1.5mg / L, mucin content 9~15mg / L, reducing agent 250~750mg / L, oligosaccharide 35~50mg / L, bile acid 150~350mg / L, acetic acid.

[0007] Preferably, the composition includes the following components: peptone 0.2 g / L, cysteine ​​hydrochloride 1.0 g / L, NH4HCO3 0.4 g / L, sodium bicarbonate 3.5 g / L, disodium hydrogen phosphate 9.45 g / L, potassium dihydrogen phosphate 6.2 g / L, magnesium sulfate heptahydrate 0.6 g / L, calcium chloride dihydrate 13.2 mg / L, manganese chloride tetrahydrate 10 mg / L, cobalt chloride hexahydrate 1 mg / L, ferric chloride hexahydrate 8 mg / L, sodium hydroxide 200 mg / L, heme chloride 5 mg / L, vitamin K1 1 mg / L, mucin content 10 mg / L, reducing agent 500 mg / L, oligosaccharide 40 mg / L, bile acid 200 mg / L, and acetic acid.

[0008] Preferably, the reducing agent is an organic reducing agent, including vitamin C and reduced glutathione; the concentration of vitamin C is 125~375 mg / L, and the concentration of reduced glutathione is 125~375 mg / L.

[0009] Preferably, the oligosaccharide is a oligosaccharide, including arabinoxylan, isomaltooligosaccharide, mannose, fructooligosaccharide, and xylooligosaccharide.

[0010] Preferably, the concentration of arabinoxylan is 25-45 mg / L, the concentration of isomaltooligosaccharide is 10-20 mg / L, the concentration of mannose oligosaccharide is 10-45 mg / L, the concentration of fructooligosaccharide is 10-20 mg / L, and the concentration of xylooligosaccharide is 10-20 mg / L.

[0011] Preferably, the bile acids include cholic acid and porcine cholic acid; the concentration of cholic acid is 0~50 mg / L, and the concentration of porcine cholic acid is 150 mg / L~300 mg / L.

[0012] Preferably, the acetic acid is used to adjust the pH of the culture medium to 6.9-7.5.

[0013] A method for preparing a culture medium that enhances the activity of extracellular fiber-degrading bacteria in the gut microbiota, as described above, comprises the following steps:

[0014] S1. Dissolve the required amounts of fiber substrate, peptone, cysteine ​​hydrochloride, NH4HCO3, NaHCO3, Na2HPO4·12H2O, K2HPO4, MgSO4·7H2O, CaCl2·2H2O, MnCl2·4H2O, CoCl2·6H2O, FeCl3·6H2O, heme chloride, vitamin K1, and porcine cholic acid in distilled water and bring the volume to the required level. Sterilize in a high-temperature sterilizer at 121°C for 15 minutes.

[0015] S2. Prepare a mother solution of vitamin C and reduced glutathione using ultrapure water, filter it through a filter membrane, and add it to the sterilized solution obtained in step S1 in an ultra-clean workbench according to the volume required for the final concentration. Mix well to obtain Buffer A.

[0016] S3. Dissolve the required amounts of arabinoxylan, isomaltooligosaccharide, mannose, fructooligosaccharide, xylooligosaccharide, and mucin in distilled water and bring the volume to the required level. Sterilize in a high-temperature sterilizer at 121°C for 15 minutes.

[0017] S4. Cool the sterilization solution obtained in step S4 to 30~40℃, add it to Buffer A obtained in step S2 in a clean bench, and mix well.

[0018] Preferably, in step S1, the container is sealed and filled with sterile nitrogen to atmospheric pressure before sterilization.

[0019] Preferably, the diameter of the filter membrane in step S2 is 45 μm.

[0020] The application of a culture medium, as described above, that enhances the activity of in vitro fiber-degrading bacteria in the gut microbiota in evaluating in vitro fermentation models of fiber raw materials.

[0021] Therefore, the present invention provides a culture medium for improving the activity of extracellular fiber-degrading bacteria in the gut microbiota, its preparation method, and its application, the specific technical effects of which are as follows:

[0022] (1) The culture medium provided by the present invention can effectively improve the activity of fiber-degrading bacteria in the intestinal flora, promote their growth and metabolism, and thus greatly improve the fiber degradation ability of the in vitro flora. Compared with the commonly used culture medium, the concentration of total short-chain fatty acids in the fiber fermentation product can be increased by up to about 50%;

[0023] (2) Compared with the commonly used culture media, the culture media provided by the present invention can change the composition of the fiber-degrading bacteria in the intestinal flora. A total of 9 genera of bacteria were detected to have significant differences in abundance, which are different from the dominant bacteria in the fiber fermentation products of commonly used culture media. This may be the reason for the different fermentation efficiency.

[0024] (3) The culture medium provided by the present invention helps to more fully evaluate the fermentability of dietary fiber, and further study the metabolic pathways, product synthesis, enzyme activity and biocatalytic mechanism therein, which is of great value for the optimization of biomimetic digestive systems in the field of animal nutrition. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The results of the detection of the effect of the reducing agent on the α-diversity (A) and principal component analysis (B) of microorganisms in the culture medium in Example 4 of the present invention are as follows:

[0027] Figure 2 This is the result of the investigation into the effects of the optimized culture medium on the concentrations of acetic acid (A), propionic acid (B), butyric acid (C), SCFAs (D), BCFAs (E), and VFAs (F) in the fiber fermentation broth in Example 6 of the present invention;

[0028] Figure 3 This is the result of the investigation into the effect of optimized culture medium on the abundance of dominant species at the genus level in the fiber fermentation broth in Example 6 of the present invention;

[0029] Figure 4 This is the result of the investigation into the effect of optimized culture medium on the abundance of fiber-degrading bacteria in the fermentation broth in Example 6 of the present invention;

[0030] Figure 5 This is the result of the study on the effect of the initial pH of the culture medium on the concentrations of acetic acid (A), propionic acid (B), butyric acid (C), and SCFAs (D) in the fiber fermentation broth in Example 7 of the present invention. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0033] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0034] Example 1

[0035] The specific steps for preparing the basal culture medium are as follows:

[0036] Accurately weigh 5g of fiber substrate, 0.2g of peptone, 1.0g of cysteine ​​hydrochloride, 0.4g of NH4HCO3, 3.5g of NaHCO3, 9.45g of Na2HPO4·12H2O, 6.2g of K2HPO4, 0.6g of MgSO4·7H2O, 13.2mg of CaCl2·2H2O, 10mg of MnCl2·4H2O, 1mg of CoCl2·6H2O, 8mg of FeCl3·6H2O, 5mg of heme chloride, 1mg of vitamin K1, and 300mg of Na2S. Add these to a fermentation flask and stir with 800mL of distilled water until dissolved. Then, bring the volume to 1L with distilled water. Seal the fermentation flask, purge it with sterile nitrogen to near atmospheric pressure, and sterilize it in an autoclave at 121℃ for 15 minutes to obtain the basal culture medium.

[0037] Example 2

[0038] The specific steps for preparing an organic reducing agent-modified culture medium are as follows:

[0039] S21. Accurately weigh 5g of fiber substrate, 0.2g of peptone, 1.0g of cysteine ​​hydrochloride, 0.4g of NH4HCO3, 3.5g of NaHCO3, 9.45g of Na2HPO4·12H2O, 6.2g of K2HPO4, 0.6g of MgSO4·7H2O, 13.2mg of CaCl2·2H2O, 10mg of MnCl2·4H2O, 1mg of CoCl2·6H2O, 8mg of FeCl3·6H2O, 5mg of heme chloride, and 1mg of vitamin K1. Add these to a fermentation flask and stir with 800mL of distilled water until dissolved. Then, bring the volume to 1L with distilled water. Seal the fermentation flask, purge it with sterile nitrogen to near atmospheric pressure, and sterilize it in an autoclave at 121℃ for 15 minutes. Sodium hydroxide needs to be added when dissolving heme chloride to achieve a final sodium hydroxide concentration of 200mg / L.

[0040] S22. Dissolve 1.0g of vitamin C and 1.0g of reduced glutathione in 100mL of ultrapure water, respectively, and filter using a 45μm diameter water-soluble filter membrane to obtain vitamin C and reduced glutathione solutions with a concentration of 10.0g / L. In a clean bench, add 12.5mL, 25.0mL, and 37.5mL of the solutions, respectively, to the solutions sterilized in step S21 and cooled to room temperature. Stir and mix well to achieve final concentrations of vitamin C of 125mg / L, 250mg / L, and 375mg / L, and final concentrations of reduced glutathione of 125mg / L, 250mg / L, and 375mg / L, respectively, thus obtaining different organic reducing agent modified culture media.

[0041] Example 3

[0042] The specific steps for preparing the optimized culture medium are as follows:

[0043] S31. Accurately weigh 0.2g peptone, 1.0g cysteine ​​hydrochloride, 0.4g NH4HCO3, 3.5g NaHCO3, 9.45g Na2HPO4·12H2O, 6.2g K2HPO4, 0.6g MgSO4·7H2O, 13.2mg CaCl2·2H2O, 10mg MnCl2·4H2O, 1mg CoCl2·6H2O, 8mg FeCl3·6H2O, 5mg heme chloride, 1mg vitamin K1, and 200mg cholic acid. Add these to a fermentation flask and stir with 800mL distilled water until dissolved. Then, bring the volume to 1L with distilled water. Seal the fermentation flask, purge with sterile nitrogen to near atmospheric pressure, and sterilize in an autoclave at 121℃ for 15min. After cooling, Buffer A is obtained.

[0044] S32. Dissolve 1.0g of vitamin C and 1.0g of reduced glutathione in 100mL of ultrapure water, respectively, and filter using a 45μm diameter water-soluble filter membrane to obtain vitamin C and reduced glutathione solutions with a concentration of 10.0g / L. Mix the two solutions in equal proportions to obtain Buffer B.

[0045] S33. Accurately weigh 10.0 g of arabinoxylan, 4.0 g of isomaltooligosaccharide, 16.0 g of mannose oligosaccharide, 4.0 g of fructooligosaccharide, 6.0 g of xylooligosaccharide, and 10.0 g of mucin. Add them to a fermentation flask and stir with 800 mL of distilled water until dissolved. Then, bring the volume to 1 L with distilled water and sterilize in an autoclave at 121 °C for 15 min. After cooling, Buffer C is obtained.

[0046] S34. Take 25 mL of Buffer B and 10 mL of Buffer C in sequence and add them to Buffer A (1.0 L) prepared in step S31 to obtain the optimized culture medium.

[0047] Example 4

[0048] The specific steps for screening the optimal combination concentration of organic reducing agents are as follows:

[0049] Using resistant starch as the fiber substrate and the basal culture medium prepared in Example 1 as the control group, experimental culture media 1 to 9 were prepared according to an orthogonal experimental design, using the same method as described in Example 2, and are referred to as the experimental groups. The components of the basal culture medium and culture media 1 to 9 are shown in Table 1, and other unspecified components are the same as in Example 2. Each culture medium was tested in duplicate, with each duplicate culture medium having a volume of 100 mL. 1 mL of pig manure bacteria suspension was added to both the experimental and control culture media, along with 1 mL of sterile 0.1% resazurin solution to indicate anaerobic conditions. Each culture medium was tested in duplicate, with 0.5 g of resistant starch added in each duplicate. The mixture was placed in a 38°C constant temperature shaker and fermented for 96 h. At the end of fermentation, 2 mL of fermentation broth was extracted from each bottle and stored at -80°C for microbiome sequencing.

[0050] Table 1 Experimental Design

[0051]

[0052] The results are as follows Figure 1 As shown, the effect of reducing agents on the α-diversity of the fermentation broth microbial community is as follows: Figure 1 As shown in Figure A. The results showed that the average Shannon index and the average number of observable species in the basal medium were 1.782 and 91, respectively. Among all the tested media, media 4, 5, 6, 8, and 9 had higher species richness, with average numbers of observable species of 128, 129, 148, 136, and 146, respectively, which were higher than those in the basal medium. Media 4 and 5 had the highest Shannon indices, at 1.765 and 1.908, respectively, similar to those in the basal medium. Therefore, 250 mg / L vitamin C combined with 125 mg / L or 250 mg / L reduced glutathione can effectively improve the microbial diversity of in vitro cultured gut microbiota.

[0053] RDA analysis results showed that the two samples from the same culture medium were located close to each other in the RDA analysis, indicating good parallelism; the two principal components explained 87.21% of the variance in changes in bacterial community structure, with PC1 reflecting the effect of Na2S on bacterial community structure and PC2 reflecting the effects of VC and GSH on bacterial community structure. Figure 1 B).

[0054] Example 5

[0055] The effects of bile acids and oligosaccharides on the concentration of short-chain fatty acids and microbial community composition in Duroc, Landrace, and Ningxiang pig in vitro fermentation models / experiments were investigated. The specific steps are as follows:

[0056] Rice bran meal was pretreated using a simulated gastrointestinal digestion method (see Wang Ya, Zhao Feng, Zhang Hu, Dang Fangkun, Gao Qingtao, & Yu Yao et al. (2019). Contribution of digestive enzymes in the small intestine stage of pig biomimetic digestion to the digestibility of total dietary energy and crude protein. Journal of Animal Nutrition). The meal was centrifuged at 4000 rpm / s for 15 min to separate the insoluble residue and supernatant. Four volumes of anhydrous ethanol were added to the supernatant, and the mixture was centrifuged at 6000 rpm / s for 15 min to collect the flocculent precipitate. The insoluble residue and flocculent precipitate were mixed, dried, ground or pulverized, and passed through a 60-mesh sieve. The obtained rice bran meal fiber was then stored in a resealable bag for later use.

[0057] Using the prepared rice bran fiber as the fiber substrate, and with culture medium 5 from Example 4 as the control group (Basal), experimental culture media 1 to 18 were prepared according to an orthogonal experimental design, using the same preparation method as in Example 3. The components of the basal culture medium and culture media 1 to 18 are shown in Table 2. Other unspecified components are exactly the same as in Example 3. Each culture medium was tested in triplicate, with each parallel culture medium having a volume of 100 mL and each parallel containing 0.5 g of rice bran fiber.

[0058] Fecal microbial suspensions were prepared from fresh feces of Duroc, Landrace, Large White, and Ningxiang pigs (DLY) and NX, respectively. 1 mL of each of these suspensions was added to each culture medium, along with 1 mL of sterile 0.1% resazurin solution to indicate anaerobic conditions. The mixtures were placed in a shaker at 38°C and fermented for 96 hours. At the end of fermentation, 2 mL of the fermentation broth was extracted from each bottle, divided into two portions, and stored at -80°C for VFAs detection and microbiome sequencing, respectively.

[0059] Table 2 Orthogonal Experimental Design Table

[0060]

[0061] Note: OS: 0 indicates no addition, 1 indicates an addition concentration of 2.5 ml / L; CA, HCA: 0 indicates no addition, 1 indicates an addition concentration of 0.1 g / L, 2 indicates an addition concentration of 0.2 g / L, 3 indicates an addition concentration of 0.4 g / L.

[0062] As shown in Table 3, HCA (porcine bile acid) has a significant effect on acetic acid concentration as a covariate. P <0.05, showing a trend of affecting the concentrations of total total fatty acids (TVFAs) and total short-chain fatty acids (SCFAs) (0.05 < P<0.10). As shown in Table 4, the oligosaccharide solution (OS) significantly increased the acetic acid concentration in the fermentation broth ( P <0.05), and there is a trend to increase the concentrations of SCFAs and TVFAs (0.05 < P <0.10. HCA concentration significantly affects acetic acid concentration in fermentation broth ( P <0.05), and there is a trend that it affects TVFA concentration (0.05 < P <0.10). OS and HCA showed an interactive effect on propionic acid concentration in the fermentation broth (<0.05). P <0.10), wherein the oligosaccharide solution and a medium dose of porcine cholic acid were added. The concentrations of propionic acid, SCFAs, and TVFAs in the fermentation broth were highest at this time. The results indicate that the combination of oligosaccharide solution and 200 mg / L HCA can effectively improve the fiber fermentation capacity of pig manure bacterial broth.

[0063] Table 3. Effects of orthogonal factors as covariates on VFA concentration in fermentation broth.

[0064]

[0065] Note: The source of the inoculum in the model is considered as the main effect on the concentration of VFAs.

[0066] † The effect of orthogonal factors as covariates on VFA concentration; OS is the oligosaccharide solution, HCA is porcine cholic acid, CA is cholic acid, and so on.

[0067] Table 4. Effects of oligosaccharides and porcine cholic acid on the concentration of VFAs (μg / mL) in the fermentation broth.

[0068]

[0069] Note: The letters on the same column are above the headings. a、b This indicates a subset of the same type during Ducan multiple comparisons. OS- indicates the oligosaccharide solution was not added, and OS indicates the oligosaccharide solution was added. HCA - This indicates that no porcine bile acid or HCA is added. + This indicates a low dose (0.1 g / L) of HCA. ++ This indicates a medium dose (0.2 g / L) of HCA. +++ This indicates a high dose (0.4 g / L).

[0070] Example 6

[0071] The effects of optimized culture medium on the composition of in vitro microbiota and fiber fermentation capacity of Duroc, Landrace, and Greenhouse pigs were investigated. The specific steps are as follows:

[0072] The rice bran fiber prepared in Example 5 was selected as the substrate, the organic reducing agent modified culture medium prepared in Example 2 was selected as the control group, and the optimized culture medium prepared in Example 3 was selected as the experimental group. Each group had 3 replicates, and the volume of each replicate culture medium was 100 mL.

[0073] Fecal microbial suspensions were prepared from fresh feces of Duroc Landrace-Large White (DLY) and Ningxiang pigs (NX), respectively. 1 mL of the prepared fecal microbial suspension was added to the culture medium of both the control and experimental groups, along with 1 mL of sterile 0.1% resazurin solution to indicate anaerobic conditions. The mixture was placed in a shaker at 38°C and fermented for 96 h. At the end of fermentation, 2 mL of fermentation broth was extracted from each bottle, divided into two portions, and stored at -80°C for VFAs detection and microbiome sequencing, respectively.

[0074] Basal and Optimized Media The detection results of VFAs concentration in the fermentation broth of ) are as follows Figure 2 As shown, compared to Basal medium, The culture medium significantly increased the concentrations of acetic acid, propionic acid, butyric acid, SCFAs, and TVFAs in the NX pig fermentation broth. P <0.05%, significantly increasing the propionic acid concentration in DLY pig fermentation broth ( P <0.05%, significantly reducing the butyric acid concentration in DLY pig fermentation broth ( P <0.05). There were also differences in the concentrations of acetic acid, propionic acid, and butyric acid in the fermentation broth of the basal culture medium between the two pig breeds; the concentrations of propionic acid and butyric acid in the fermentation broth of NX pigs were significantly higher than those of DLY pigs. P <0.05), while the acetic acid concentration in the fermentation broth was significantly lower than that in DLY pigs ( P <0.05).

[0075] Microbiome sequencing results as follows Figure 3 As shown, in Basal medium, the top 5 dominant bacteria in terms of average abundance at the genus level of the NX porcine in vitro flora are, in descending order: Enterococcus , Clostridium_sensu_stricto_1 , Amphibacillus , Colidextribacter and Flavonifractor The top 5 dominant bacteria in the DLY porcine in vitro microbiota at the genus level, in order of average abundance, are: Enterococcus , Bacillus , Eubacterium , Lactobacillus and Paludicola .exist In the culture medium, the top 5 dominant bacteria in terms of average abundance at the genus level of the NX porcine in vitro flora are, in descending order: Enterococcus , Bacteroides , Bilophila , Cloacibacillus andDesulfovibrio The top 5 dominant bacteria in the DLY porcine in vitro microbiota at the genus level, in order of average abundance, are: Bacillus , Enterococcus , Eubacterium , Lactobacillus and Paludicola Basal medium and Statistical results of differences in microbial genus level in fermentation broth of culture medium as follows: Figure 4 As shown, the abundance of 9 genera was found to be in the range of [missing information]. There were differences between the culture medium and the basal medium. Compared with the basal medium, the optimized medium significantly increased the number of anaerobic fiber-degrading bacteria in the NX porcine in vitro microbiota. Bacteroides , Lactobacillus , Paludicola , Ruminococcus , Parabacteroides , NK4A214_ group , UCG-005 and Christensenellaceae_R.7_group Relative abundance ( P <0.05), significantly increased the anaerobic fiber-degrading bacteria in the DLY porcine in vitro microbiota. Lactobacillus , Ruminococcus and Parabacteroides Relative abundance ( P <0.05), significantly reduced facultative anaerobic bacteria in NX and DLY pigs. Enterococcus Relative abundance ( P <0.05).

[0076] Example 7

[0077] The effect of initial pH on the in vitro cellulosic fermentation capacity of microbial communities was investigated, and the specific steps are as follows:

[0078] Using rice bran fiber prepared in Example 5 as the substrate and the optimized culture medium described in Example 3, an in vitro fermentation model was established, with four replicates per group and each replicate containing 100 mL of culture medium. Acetic acid was added dropwise to adjust the initial pH of the culture medium to 6.00±0.05, 6.40±0.05, 6.80±0.05, 7.20±0.05, 7.60±0.05, 8.00±0.05, and 8.40±0.05, respectively. Then, 1 mL of Ningxiang pig manure bacteria suspension was inoculated, and 1 mL of sterile 0.1% resazu solution was added to indicate anaerobic conditions. The mixture was placed in a 38℃ constant temperature shaker and fermented for 96 h. At the end of fermentation, 2 mL of fermentation broth was extracted from each bottle and stored at -80℃ for the detection of short-chain fatty acids (SCFAs) in the fermentation broth.

[0079] The results are as follows Figure 5As shown, the concentrations of acetic acid, propionic acid, butyric acid, and total short-chain fatty acids (SCFAs) in the fermentation end product were highest when the initial pH of the culture medium was 6.8 and 7.2. Therefore, it was found that using acetic acid to adjust the pH to 6.8 to 7.2 was beneficial for the in vitro fermentation of fiber.

[0080] Therefore, the culture medium provided by this invention can effectively enhance the activity of fiber-degrading bacteria in the gut microbiota, promote their growth and metabolism, and thus significantly improve the fiber degradation capacity of in vitro microbiota. Compared with commonly used culture media, the concentration of total short-chain fatty acids in fiber fermentation products can be increased by up to about 50%. Compared with commonly used culture media, the culture medium provided by this invention can change the composition of fiber-degrading bacteria in the gut microbiota, with significant differences in the abundance of 9 genera detected, which may be the reason for the different fermentation efficiencies. The culture medium provided by this invention helps to more fully evaluate the fermentability of dietary fiber, and further study its metabolic pathways, product synthesis, enzyme activity, and biocatalytic mechanisms, which has great value for the optimization of biomimetic digestive systems in the field of animal nutrition.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A culture medium for improving the in vitro fiber-degrading bacterial activity of swine intestinal flora, characterized by, The culture medium comprises the following components: proteose peptone 0.19-0.21 g / L, cysteine hydrochloride 0.95-1.05 g / L, NH4HCO3 0.2-0.5 g / L, sodium bicarbonate 2-5.5 g / L, disodium hydrogen phosphate 7.35-1.0 g / L, potassium hydrogen phosphate 4.45-7.5 g / L, magnesium sulfate heptahydrate 0.5-0.75 g / L, calcium chloride dihydrate 12.5-14.0 mg / L, manganese chloride tetrahydrate 9.5-10.5 mg / L, cobalt chloride hexahydrate 0.95-1.05 mg / L, ferric chloride hexahydrate 6.0-8.2 mg / L, sodium hydroxide 200 mg / L, hematin chloride 4-5.5 mg / L, vitamin k1 0.8-1.5 mg / L, mucin content 9-15 mg / L, reducing agent 250-750 mg / L, oligosaccharide 35-50 mg / L, bile acid 150-350 mg / L, and acetic acid. The reducing agent is an organic reducing agent, comprising vitamin C and reduced glutathione; the concentration of the vitamin C is 125-375 mg / L, and the concentration of the reduced glutathione is 125-375 mg / L. The oligosaccharide is a low oligosaccharide, comprising arabinoxylan, isomalto-oligosaccharide, mannan-oligosaccharide, fructo-oligosaccharide, and xylo-oligosaccharide; the concentration of the arabinoxylan is 25-45 mg / L, the concentration of the isomalto-oligosaccharide is 10-20 mg / L, the concentration of the mannan-oligosaccharide is 10-45 mg / L, the concentration of the fructo-oligosaccharide is 10-20 mg / L, and the concentration of the xylo-oligosaccharide is 10-20 mg / L. The bile acid is pig gall acid; the concentration of the pig gall acid is 150 mg / L-300 mg / L. The acetic acid is used to adjust the pH of the culture medium to 6.9-7.

5. The in-vitro fiber-degrading bacteria of the pig intestinal flora are Bacteroides, Lactobacillus, Paludicola, Ruminococcus, NK4A214_group, UCG-005, Christensenellaceae_R.7_group, and Parabacteroides.

2. The culture medium for improving the activity of the fiber-degrading bacteria of the swine intestinal flora in vitro according to claim 1, characterized in that, The culture medium comprises the following components: proteose peptone 0.2 g / L, cysteine hydrochloride 1.0 g / L, NH4HCO3 0.4 g / L, sodium bicarbonate 3.5 g / L, disodium hydrogen phosphate 9.45 g / L, potassium hydrogen phosphate 6.2 g / L, magnesium sulfate heptahydrate 0.6 g / L, calcium chloride dihydrate 13.2 mg / L, manganese chloride tetrahydrate 10 mg / L, cobalt chloride hexahydrate 1 mg / L, ferric chloride hexahydrate 8 mg / L, sodium hydroxide 200 mg / L, hematin chloride 5 mg / L, vitamin K1 1 mg / L, mucin content 10 mg / L, reducing agent 500 mg / L, oligosaccharide 40 mg / L, pig gall acid 200 mg / L, and acetic acid.

3. A method for preparing a culture medium for improving the activity of in vitro fiber-degrading bacteria of swine intestinal flora according to claim 1 or 2, characterized by, The steps are as follows: S1, the required amount of fiber substrate, proteose peptone, cysteine hydrochloride, NH4HCO3, NaHCO3, Na2HPO4·12H2O, K2HPO4, MgSO4·7H2O, CaCl2·2H2O, MnCl2·4H2O, CoCl2·6H2O, FeCl3·6H2O, hematin chloride, vitamin K1, pig gallate were dissolved with distilled water, and then the volume was adjusted to the required volume, and sterilized in a high-temperature sterilization pot at 121℃ for 15min; S2, prepare a vitamin C and reduced glutathione stock solution with ultrapure water, filter with a filter membrane, and mix with the sterilized solution obtained in step S1 in the required volume of the final concentration in an ultraclean bench to obtain Buffer A; S3, dissolve the required amount of arabinoxylan, isomaltooligosaccharide, mannooligosaccharide, fructooligosaccharide, xylooligosaccharide, mucin in distilled water, and then adjust the volume to the required volume, and sterilize in a high-temperature sterilization pot at 121℃ for 15min; S4, cool the sterilized solution obtained in step S4 to 30-40℃, and then add Buffer A obtained in step S2 in an ultraclean bench, and mix well.

4. The method for preparing a culture medium for improving the activity of in vitro fiber-degrading bacteria of swine intestinal flora according to claim 3, characterized in that: The step S1 is sealed before sterilization and filled with sterile nitrogen gas to close to atmospheric pressure.

5. The method for preparing a culture medium for improving the activity of in vitro fiber-degrading bacteria of swine intestinal flora according to claim 3, characterized in that: The pore size of the filter membrane in step S2 is 45μm.

6. Use of the culture medium for improving the activity of in vitro fiber-degrading bacteria of pig intestinal flora according to claim 1 or 2 in evaluating the in vitro pig feces fermentation model of fiber raw materials.

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