Culture medium for improving activity of cellulose-degrading bacteria outside intestinal flora as well as preparation method and application of culture medium

Abstract

The invention discloses a culture medium for improving the activity of cellulolytic bacteria outside intestinal flora as well as a preparation method and application of the culture medium, and belongs to the technical field of microbial culture mediums. The culture medium provided by the invention can effectively improve the activity of fiber-degrading bacteria in intestinal flora and promote the growth and metabolism of the fiber-degrading bacteria, so that the fiber degradation capability of in-vitro flora is greatly improved, and compared with the conventional common culture medium, the total short-chain fatty acid concentration in a fiber fermentation product can be maximally improved by about 50%; compared with a common culture medium at present, the culture medium provided by the invention can change the flora composition of cellulolytic bacteria in intestinal flora, and the fact that the abundance of nine genus bacteria is significantly different is detected, which may be a reason for different fermentation efficiencies; the culture medium provided by the invention is helpful to more fully evaluate the fermentability of the feed fiber, further researches the metabolic pathway, product synthesis, enzyme activity and biological catalysis mechanism in the feed fiber, and has a great value for optimization of a bionic digestion system in the field of animal nutrition.

Description

Technical Field

[0001] The invention relates to the technical field of microbial culture media, and in particular to a culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora, and a preparation method and application thereof. Background Art

[0002] In recent years, dietary fiber has received extensive attention as a functional substance. Short-chain fatty acids (SCFA) are the main products of dietary fiber fermentation, which not only provide energy for intestinal epithelial cells, but also promote intestinal health and immune function. However, there are many types of dietary fiber with different properties, which are subject to many restrictions in production and application.

[0003] The evaluation of fiber fermentation characteristics is an important basis for the precise application of fiber in production, which can be divided into two methods: in vivo evaluation and in vitro evaluation. The fermentability of fiber in the intestine of monogastric animals is mainly evaluated based on the digestibility and the concentration of short-chain fatty acids in the intestinal contents. However, the in vivo method has the disadvantages of high test cost, long test cycle, and large individual differences. Compared with the in vivo test method, in vitro digestion simulation technology is also an effective method to evaluate the nutritional value of pig feed, which can overcome 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 fiber degradation rate and fermentation characteristics of rumen microorganisms in ruminant nutrition research, but there are still many problems when it is introduced into intestinal microbial research. The traditional in vitro fiber fermentation model is mainly the culture medium reported by Menke et al. or its simplified version, which can provide nitrogen sources, inorganic salts and trace elements for the growth of microorganisms. By adding fiber substrate as a carbon source, it provides basic conditions for the growth of bacterial flora. However, there are some shortcomings in traditional culture media: 1) The reducing agent Na in the components 2 S is corrosive and toxic, which is not conducive to bacterial growth; 2) lack of intestinal bacteria, especially anaerobic growth factors. Therefore, further optimization of the fermentation system culture medium is particularly important. Summary of the invention

[0005] The purpose of the present invention is to provide a culture medium for improving the activity of in vitro fiber-degrading bacteria in the intestinal flora, and a preparation method and application thereof, so as to solve the problem that the culture medium used in the current in vitro fermentation technology is not conducive to bacterial growth, lacks intestinal bacteria such as anaerobic growth factors, and leads to an imperfect in vitro fermentation model.

[0006] To achieve the above object, the present invention provides a culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora, comprising the following components: 0.19-0.21 g / L peptone, 0.95-1.05 g / L cysteine ​​hydrochloride, NH 4 HCO 30.2~0.5g / 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, hemin chloride 4~5.5mg / L, vitamin K10.8~1.5mg / L, mucin content is 9~15mg / L, reducing agent 250~750mg / L, oligosaccharides 35~50mg / L, bile acid 150~350mg / L, acetic acid.

[0007] Preferably, the following components are included: peptone 0.2 g / L, cysteine ​​hydrochloride 1.0 g / L, NH 4 HCO 3 0.4 g / L, sodium bicarbonate 3.5g / L, disodium hydrogen phosphate 9.45g / L, potassium dihydrogen phosphate 6.2g / L, magnesium sulfate heptahydrate 0.6g / L, calcium chloride dihydrate 13.2mg / L, manganese chloride tetrahydrate 10mg / L, cobalt chloride hexahydrate 1mg / L, ferric chloride hexahydrate 8mg / L, sodium hydroxide 200mg / L, hemin chloride 5mg / L, vitamin K11mg / L, mucin content is 10mg / L, reducing agent 500mg / L, oligosaccharides 40mg / L, bile acid 200mg / L, acetic acid.

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

[0009] Preferably, the oligosaccharide is an oligosaccharide, including arabinoxylan, isomaltooligosaccharide, mannooligosaccharide, fructo-oligosaccharide and xylo-oligosaccharide.

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

[0011] Preferably, the bile acid includes bile acid and hyodolic acid; the concentration of the bile acid is 0 to 50 mg / L, and the concentration of the hyodolic acid is 150 mg / L to 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 for improving the activity of in vitro fiber-degrading bacteria in intestinal flora as described above comprises the following steps:

[0014] S1. Mix the required amount of fiber substrate, peptone, cysteine ​​hydrochloride, and NH 4 HCO 3 、NaHCO 3 、Na 2 HPO 4 12H 2 O.K 2 HPO 4 MgSO 4 7H 2 O, CaCl 2 ·2H 2 O、MnCl 2 ·4H 2 O、CoCl 2 6H 2 O, FeCl 3 6H 2 Dissolve O, hemin, vitamin K1, and cholic acid in distilled water to the required volume, and sterilize in a high-temperature sterilizer at 121°C for 15 min;

[0015] S2. Prepare vitamin C and reduced glutathione mother solutions with ultrapure water, filter with a membrane, add the solutions to the sterilized solution obtained in step S1 in an ultraclean workbench according to the required volume of final concentration and mix well to obtain Buffer A;

[0016] S3. Dissolve the required amount of arabinoxylan, isomaltooligosaccharide, mannooligosaccharide, fructooligosaccharide, xylo-oligosaccharide and mucin in distilled water and make up to the required volume, and sterilize in a high temperature sterilizer at 121°C for 15 min;

[0017] S4. Cool the sterilization solution obtained in step S4 to 30-40° C., add it to the 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 caliber of the filter membrane in step S2 is 45 μm.

[0020] A culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora as described above is used in evaluating an in vitro fermentation model of fiber raw materials.

[0021] Therefore, the present invention provides a culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora, and a preparation method and application thereof, and the specific technical effects thereof are as follows:

[0022] (2) 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-degrading ability of the in vitro flora. Compared with the commonly used culture medium, the total short-chain fatty acid concentration in the fiber fermentation product can be increased by up to about 50%;

[0023] (2) Compared with the commonly used culture medium, the culture medium provided by the present invention can change the composition of fiber-degrading bacteria in the intestinal flora. A total of 9 genera were detected with significant differences in the abundance of bacteria, which is different from the dominant flora in the fiber fermentation products of the commonly used culture medium, which 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 to the optimization of bionic digestion systems in the field of animal nutrition. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0026] Figure 1 is the test result of the effect of the reducing agent on the microbial alpha diversity (A) and principal component analysis (B) in the culture solution in Example 4 of the present invention;

[0027] Figure 2 The results of investigating the effect of the optimized culture medium in Example 6 of the present invention 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;

[0028] Figure 3 This is the result of investigating the effect of the 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 investigating the effect of the optimized culture medium on the abundance of fiber-degrading bacteria in the fermentation broth in Example 6 of the present invention;

[0030] Figure 5 These are the results of investigating the effect of the initial pH of the culture medium on the concentrations of acetate (A), propionate (B), butyrate (C), and SCFAs (D) in the fiber fermentation broth in Example 7 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0032] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

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

[0034] Embodiment 1

[0035] Prepare the basal medium as follows:

[0036] Accurately weigh 5 g of fiber substrate, 0.2 g of peptone, 1.0 g of cysteine ​​hydrochloride, and NH 4 HCO 3 0.4 g, NaHCO 3 3.5g, Na 2 HPO 4 12H 2 O 9.45g, K 2 HPO 4 6.2 g MgSO 4 7H 2 O 0.6g, CaCl 2 ·2H 2 O 13.2mg, MnCl 2 ·4H 2 O 10mg, CoCl 2 6H 2 O 1mg, FeCl 3 6H 2 O 8mg, hemin 5mg, vitamin K11mg, Na 2 S300mg, add to the fermentation bottle and stir with 800mL distilled water until dissolved, then dilute to 1L with distilled water, seal the fermentation bottle, fill with sterile nitrogen to close to atmospheric pressure, and sterilize in a high-temperature sterilizer at 121℃ for 15min to obtain the basal culture medium.

[0037] Embodiment 2

[0038] Prepare organic reducing agent modified medium, the specific steps are as follows:

[0039] S21, accurately weigh 5g of fiber substrate, 0.2g of peptone, 1.0g of cysteine ​​hydrochloride, and NH 4 HCO3 0.4 g, NaHCO 3 3.5g, Na 2 HPO 4 12H 2 O 9.45g, K 2 HPO 4 6.2 g MgSO 4 7H 2 O 0.6g, CaCl 2 ·2H 2 O 13.2mg, MnCl 2 ·4H 2 O 10mg, CoCl 2 6H 2 O 1mg, FeCl 3 6H 2 O 8mg, hemin 5mg, vitamin K11mg, add to the fermentation bottle and stir with 800mL distilled water until dissolved, then dilute to 1L with distilled water, seal the fermentation bottle, fill with sterile nitrogen to near atmospheric pressure, and sterilize in a high temperature sterilizer at 121℃ for 15min. Sodium hydroxide needs to be added when hemin is dissolved, so that the final concentration of sodium hydroxide is 200mg / L.

[0040] S22. Dissolve 1.0 g of vitamin C and 1.0 g of reduced glutathione in 100 mL of ultrapure water, filter using a 45 μm water-soluble filter membrane to obtain vitamin C and reduced glutathione solutions with a concentration of 10.0 g / L, respectively. Pipette 12.5 mL, 25.0 mL, and 37.5 mL into the clean bench and add them to the solution that was sterilized in step S21 and cooled to room temperature, stir and mix, so that the final concentrations of vitamin C are 125 mg / L, 250 mg / L, and 375 mg / L, respectively, and the final concentrations of reduced glutathione are 125 mg / L, 250 mg / L, and 375 mg / L, respectively, to obtain different organic reducing agent improved culture media.

[0041] Embodiment 3

[0042] Prepare the optimized culture medium. The specific steps are as follows:

[0043] S31, accurately weigh 0.2 g of peptone, 1.0 g of cysteine ​​hydrochloride, and NH 4 HCO 3 0.4 g, NaHCO 3 3.5g, Na 2 HPO 4 12H 2 O 9.45g, K 2 HPO 4 6.2 g MgSO4 7H 2 O 0.6g, CaCl 2 ·2H 2 O 13.2mg, MnCl 2 ·4H 2 O10mg, CoCl 2 6H 2 O 1mg, FeCl 3 6H 2 Add 8 mg of 1,2-dichlorobenzene, 5 mg of hemin, 11 mg of vitamin K, and 200 mg of cholic acid to the fermentation bottle, stir with 800 mL of distilled water until dissolved, and then dilute to 1 L with distilled water. Seal the fermentation bottle, fill it with sterile nitrogen to near atmospheric pressure, and sterilize it in a high-temperature autoclave at 121°C for 15 minutes. After cooling, obtain Buffer A.

[0044] S32. Dissolve 1.0 g of vitamin C and 1.0 g of reduced glutathione in 100 mL of ultrapure water, filter using a 45 μm-diameter water-soluble filter membrane to obtain vitamin C and reduced glutathione solutions with a concentration of 10.0 g / L, respectively. Mix the two 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 mannooligosaccharide, 4.0 g of fructooligosaccharide, 6.0 g of xylo-oligosaccharide, and 10.0 g of mucin, add to the fermentation bottle, stir with 800 mL of distilled water until dissolved, then dilute to 1 L with distilled water, sterilize in a high temperature sterilizer at 121°C for 15 min. After cooling, obtain Buffer C.

[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] Embodiment 4

[0048] Screening the optimal organic reducing agent combination concentration, the specific steps are as follows:

[0049] Resistant starch was used as the fiber substrate, and the basal culture medium prepared in Example 1 was used as the control group; experimental culture medium 1 to culture medium 9 were configured according to the orthogonal experimental design, and the preparation method was the same as that described in Example 2, which was recorded as the experimental group. The components of the basal culture medium, culture medium 1 to culture medium 9 are shown in Table 1, and other unspecified components are the same as those in Example 2. There are 2 parallels for each culture medium, and the volume of each parallel culture medium is 100 mL. 1 mL of pig manure suspension was added to the culture medium of the experimental group and the control group, and 1 mL of sterilized 0.1% resazurin solution was added to indicate anaerobic conditions. Two parallels were set for each culture medium, and the amount of resistant starch added to each parallel was 0.5 g. Place in a constant temperature shaker at 38°C and ferment for 96 hours. At the end of the 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] <![CDATA[Na 2 Smg / L]]> VCmg / L GSHmg / L Basal 300 0 0 Medium 1 0 125 125 Medium 2 0 125 250 Medium 3 0 125 375 Medium 4 0 250 125 Medium 5 0 250 250 Medium 6 0 250 375 Medium 7 0 375 125 Medium 8 0 375 250 Medium 9 0 375 375

[0052] The results are as follows Figure 1 As shown in Figure 2, the effect of reducing agents on the α diversity of the fermentation broth flora is as follows: Figure 1 As shown in A. The results showed that the Shannon index and the number of observable species of the basal culture medium were 1.782 and 91 on average, respectively. Among all the culture media tested, the species richness of medium 4, medium 5, medium 6, medium 8, and medium 9 was relatively high, and the average number of observable species was 128, 129, 148, 136, and 146, respectively, which was higher than that of the basal culture medium. Among them, medium 4 and medium 5 had the highest Shannon index, which were 1.765 and 1.908, respectively, which were similar to the basal culture medium. Therefore, 250 mg / L vitamin C and 125 mg / L or 250 mg / L reduced glutathione can effectively improve the microbial diversity of intestinal flora cultured in vitro.

[0053] The results of RDA analysis showed that the two samples of the same culture medium were close in position in RDA analysis, indicating good parallelism; the variance explained by the two principal components for the changes in bacterial community structure reached 87.21%, and PC1 reflected Na 2 S affects the structure of the bacterial community, and PC2 reflects the influence of VC and GSH on the structure of the bacterial community ( Figure 1 B).

[0054] Embodiment 5

[0055] The effects of bile acid and oligosaccharide on the concentration of short-chain fatty acids and the composition of bacterial flora in the in vitro fermentation model / experiment of Duroc pigs and Ningxiang pigs were investigated. The specific steps are as follows:

[0056] The rice bran meal was pretreated by simulating gastric-small intestinal digestion (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 to the digestibility of total energy and crude protein in the diet during porcine bionic digestion. Journal of Animal Nutrition), centrifuged at 4000rpm / s for 15min to separate the insoluble residue and the supernatant; 4 times the volume of anhydrous ethanol was added to the supernatant, and the mixture was centrifuged at 6000rpm / s for 15min to collect the flocculent precipitate; the insoluble residue and the flocculent precipitate were mixed, dried, ground or crushed, and sieved through a 60-mesh sieve, and the obtained rice bran meal fiber was put into a ziplock bag for storage.

[0057] The prepared rice bran meal fiber was used as a fiber substrate, and the culture medium 5 in Example 4 was used as a control group (Basal). The test culture medium 1 to culture medium 18 were configured according to the orthogonal experimental design, and the preparation method was the same as that in Example 3. The components in the basal medium and culture medium 1 to culture medium 18 are shown in Table 2, and other unspecified components are exactly the same as those in Example 3, each culture medium has 3 parallels, each parallel culture medium volume is 100 mL, and the amount of rice bran meal fiber added in each parallel is 0.5 g.

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

[0059] Table 2 Orthogonal test design table

[0060]

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

[0062] As shown in Table 3, HCA (hyodeoxycholic acid) as a covariate had a significant effect on acetic acid concentration (P<0.05), and had an influencing trend on the concentrations of total 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 had a trend of increasing the concentrations of SCFAs and TVFAs (0.05<P<0.10). The HCA concentration significantly affected the acetic acid concentration in the fermentation broth (P<0.05), and had an influencing trend on the TVFAs concentration (0.05<P<0.10). There was an interactive influencing trend between OS and HCA on the propionic acid concentration in the fermentation broth (0.05<P<0.10). Among them, when the oligosaccharide solution and medium-dose hyodeoxycholic acid (OS*HCA ++ ) were added, the concentrations of propionic acid, SCFAs and TVFAs in the fermentation broth were the highest. The results showed that the combination of the oligosaccharide solution and 200 mg / L HCA could effectively improve the fiber fermentation ability of swine manure bacterial liquid.

[0063] Table 3 Influence of orthogonal factors as covariates on VFAs concentration in fermentation broth

[0064]

[0065] Note: *In the GLM model, the source of the inoculum as the main effect on the VFAs concentration.

[0066] Influence of orthogonal factors as covariates on VFAs concentration; OS is the oligosaccharide solution, HCA is hyodeoxycholic acid, CA is cholic acid, the same below.

[0067] Table 4 Influence of oligosaccharides and hyodeoxycholic acid on VFAs concentration (μg / mL) in fermentation broth

[0068]

[0069] Note: The superscript letters in the same column a、b represent the homogeneous subsets in Duncan multiple comparison. OS- means the oligosaccharide solution was not added, OS means the oligosaccharide solution was added. HCA- - means no hyodeoxycholic acid was added, HCA + means low dose (0.1 g / L), HCA ++ means medium dose (0.2 g / L), HCA +++ means high dose (0.4 g / L).

[0070] Example 6

[0071] Investigate the influence of the optimized medium on the composition of the in vitro flora and the fiber fermentation ability of Duroc×Landrace×Yorkshire pigs and Ningxiang pigs. The specific steps are as follows:

[0072] The rice bran fiber prepared in Example 5 was selected as the substrate, the organic reducing agent improved culture medium prepared in Example 2 was used as the control group, and the optimized culture medium prepared in Example 3 was used as the test group, with 3 parallels in each group, and the volume of each parallel culture medium was 100 mL.

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

[0074] Basal medium and optimized medium (OS*HCA ++ The results of the detection of VFAs concentration in the fermentation broth of Figure 2 As shown, compared with Basal medium, OS*HCA ++ The culture medium significantly increased the concentrations of acetate, propionate, butyrate, SCFAs and TVFAs in the fermentation broth of NX pigs (P<0.05), significantly increased the concentration of propionate in the fermentation broth of DLY pigs (P<0.05), and significantly reduced the concentration of butyrate in the fermentation broth of DLY pigs (P<0.05). The concentrations of acetate, propionate and butyrate in the fermentation broth of the two pig breeds also differed. The concentrations of propionate and butyrate in the fermentation broth of NX pigs were significantly higher than those of DLY pigs (P<0.05), while the concentration of acetate in the fermentation broth was significantly lower than that of DLY pigs (P<0.05).

[0075] Microbiome sequencing results Figure 3 As shown in the figure, in Basal medium, the top 5 dominant bacteria with the average abundance at the genus level in the in vitro microbiota of NX pigs were Enterococcus, Clostridi um_sensu_stricto_1, Amphibacillus, Colidextribacter and Flavonifractor, and the top 5 dominant bacteria with the average abundance at the genus level in the in vitro microbiota of DLY pigs were Enterococcus, Bacillus, Eubacterium, Lactobacillus and Paludicola. ++In the culture medium, the top 5 dominant bacteria with the average abundance at the genus level in the in vitro microbiota of NX pigs were Enterococcus, Bacteroides, Bilophila, Cloacibacillus and Desulfo vibrio, and the top 5 dominant bacteria with the average abundance at the genus level in the in vitro microbiota of DLY pigs were Bacillus, Enterococcus, Eubacterium, Lactobacillus and Paludicola. ++ The statistical results of the differences in the microbial genus level in the culture medium fermentation broth are as follows Figure 4 As shown in Figure 2, a total of 9 genera were found to be abundant in OS*HCA. ++ There were differences in the culture medium and Basal culture medium. Compared with the basal culture medium, the optimized culture medium significantly increased the relative abundance of anaerobic fiber-degrading bacteria Bacteroides, Lactobacillus, Paludicola, Ruminococcus, Parabacteroides, NK4A214_group, UCG-005 and Christensenellaceae_R.7_group in the in vitro microbiota of NX pigs (P<0.05), significantly increased the relative abundance of anaerobic fiber-degrading bacteria Lactobacillus, Ruminococcus and Parabacteroides in the in vitro microbiota of DLY pigs (P<0.05), and significantly decreased the relative abundance of facultative anaerobic bacteria Enterococcus in NX pigs and DLY pigs (P<0.05).

[0076] Embodiment 7

[0077] The effect of initial pH on the fiber fermentation capacity of in vitro bacterial flora was investigated. The specific steps are as follows:

[0078] The rice bran fiber prepared in Example 5 was selected as the substrate and the optimized culture medium described in Example 3 to establish an in vitro fermentation model, with 4 parallels in each group and a culture medium volume of 100 mL for each parallel. After dripping acetic acid to make the initial pH value of the culture medium reach 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, 1 mL of Ningxiang pig manure suspension was inoculated, and 1 mL of sterilized 0.1% resazurin solution was added to indicate anaerobic conditions. Place in a 38°C constant temperature shaker and ferment for 96 hours. At the end of the fermentation, 2 mL of fermentation broth was extracted from each bottle and stored at -80°C for detection of short-chain fatty acids (SCFAs) in the fermentation broth.

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

[0080] Therefore, 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-degrading ability of the in vitro flora. Compared with the currently commonly used culture medium, the total short-chain fatty acid concentration in the fiber fermentation product can be increased by up to about 50%; compared with the currently commonly used culture medium, the culture medium provided by the present invention can change the composition of the fiber-degrading bacteria in the intestinal flora, and a total of 9 genera were detected to have significant differences in the abundance of bacteria, which may be the reason for the different fermentation efficiencies; the culture medium provided by the present invention is helpful to more fully evaluate the fermentability of feed fiber, and then further study the metabolic pathways, product synthesis, enzyme activity and biocatalytic mechanism therein, which is of great value to the optimization of bionic digestion systems in the field of animal nutrition.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora, characterized in that: The invention comprises the following components: 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 dihydrogen 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 .5~10.5mg / L, cobalt chloride hexahydrate 0.95~1.05mg / L, ferric chloride hexahydrate 6.0~8.2mg / L, sodium hydroxide 200mg / L, hemin chloride 4~5.5mg / L, vitamin K10.8~1.5mg / L, mucin content is 9~15mg / L, reducing agent 250~750mg / L, oligosaccharides 35~50mg / L, bile acid 150~350mg / L, acetic acid.

2. A culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora according to claim 1, characterized in that: It includes the following components: peptone 0.2g / L, cysteine ​​hydrochloride 1.0g / L, NH4HCO3 0.4g / L, sodium bicarbonate 3.5g / L, disodium hydrogen phosphate 9.45g / L, potassium dihydrogen phosphate 6.2g / L, magnesium sulfate heptahydrate 0.6g / L, calcium chloride dihydrate 13.2mg / L, manganese chloride tetrahydrate 10mg / L, cobalt chloride hexahydrate 1mg / L, ferric chloride hexahydrate 8mg / L, sodium hydroxide 200mg / L, hemin chloride 5mg / L, vitamin K11mg / L, mucin content of 10mg / L, reducing agent 500mg / L, oligosaccharides 40mg / L, bile acid 200mg / L, and acetic acid.

3. A culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora according to claim 1, characterized in that: The reducing agent is an organic reducing agent, including 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.

4. A culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora according to claim 1, characterized in that: The oligosaccharides are oligosaccharides, including arabinoxylan, isomaltooligosaccharide, mannooligosaccharide, fructo-oligosaccharide and xylo-oligosaccharide; the concentration of the arabinoxylan is 25-45 mg / L, the concentration of the isomaltooligosaccharide is 10-20 mg / L, the concentration of the manno-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.

5. A culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora according to claim 1, characterized in that: The bile acid includes bile acid and hyobolic acid; the concentration of the bile acid is 0-50 mg / L, and the concentration of the hyobolic acid is 150 mg / L-300 mg / L.

6. A culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora according to claim 1, characterized in that: The acetic acid is used to adjust the pH of the culture medium to 6.9-7.

5.

7. A method for preparing a culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora according to any one of claims 1 to 6, characterized in that: Here are the steps: S1. Dissolve the required amount of fiber substrate, peptone, cysteine ​​hydrochloride, NH4HCO3, NaHCO3, Na2HPO4·12H2O, K2HPO4, MgSO4·7H2O, Ca Cl2·2H2O, MnCl2·4H2O, CoCl2·6H2O, FeCl3·6H2O, hemin chloride, vitamin K1, and hyoric acid in distilled water and make up to the required volume, and sterilize in a high temperature sterilizer at 121°C for 15 min; S2. Prepare vitamin C and reduced glutathione mother solutions with ultrapure water, filter with a membrane, add the solutions to the sterilized solution obtained in step S1 in an ultraclean workbench according to the required volume of final concentration and mix well to obtain Buffer A; S3. Dissolve the required amount of arabinoxylan, isomaltooligosaccharide, mannooligosaccharide, fructooligosaccharide, xylo-oligosaccharide and mucin in distilled water and make up to the required volume, and sterilize in a high temperature sterilizer at 121°C for 15 min; S4. Cool the sterilization solution obtained in step S4 to 30-40° C., add it to the Buffer A obtained in step S2 in a clean bench, and mix well.

8. The method for preparing a culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora according to claim 7, characterized in that: In the step S1, the system is sealed and filled with sterile nitrogen to a pressure close to atmospheric pressure before sterilization.

9. The method for preparing a culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora according to claim 7, characterized in that: The caliber of the filter membrane in step S2 is 45 μm.

10. Use of a culture medium for improving the activity of in vitro fiber-degrading bacteria in intestinal flora as claimed in any one of claims 1 to 6 in evaluating an in vitro fermentation model of fiber raw materials.

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