In vitro simulation of folate-deficient intestinal environment and in vitro functional evaluation method of different folates

Abstract

The application belongs to the field of food biotechnology, and discloses application of a folic acid determination culture medium in in-vitro function evaluation of folic acid; intestinal flora or fecal suspension liquid containing intestinal flora is added into the folic acid determination culture medium, and then reduced folic acid or non-reduced folic acid to be measured is added, so that the regulation function of reduced folic acid and non-reduced folic acid on intestinal flora is determined. The application also simultaneously provides a method for establishing an in-vitro intestinal simulation system lacking folic acid and a method for measuring regulation effects of different types of folic acid on human intestinal flora. The application develops a new use of the folic acid determination culture medium, and uses the culture medium to replace an existing in-vitro intestinal simulation culture medium, so as to establish an in-vitro intestinal simulation system lacking folic acid (a folic acid deficiency model).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of food biotechnology, and specifically relates to the establishment of a folate-deficient in vitro intestinal simulation system and a method for regulating human intestinal flora by different types of folate. BACKGROUND

[0002] Folic acid, also known as VB9 or pteroylglutamic acid, is an essential water-soluble vitamin for the human body, and the European Union has approved the recommended daily intake of adults as 400 μg / day. Folic acid is involved in important metabolic activities such as the biosynthesis of nucleic acids, DNA methylation, and homocysteine regeneration methionine in the human body. Since humans cannot synthesize folic acid by themselves, the sources of folic acid for humans are: first, through dietary intake. Green vegetables, animal liver, egg yolk, and folic acid fortified foods are good sources of folic acid for the human body. However, whether it is naturally derived or artificially generated folic acid, it will eventually be converted into 5-methyltetrahydrofolic acid, which is the main form of folic acid in serum. Second, from intestinal microorganisms. The colon is the main storage of folic acid, and colon symbiotic microorganisms can synthesize folic acid. Genes related to folic acid biosynthesis have been found in 512 gastrointestinal microbial genomes of the Bacteroidetes, Fusobacteria, Proteobacteria, and Actinobacteria phyla. Folic acid synthesis gene clusters have been proven to exist in various lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus plantarum), which can produce 5-methyltetrahydrofolic acid in the presence of p-aminobenzoic acid. However, so far, the folic acid production of the reported LAB strains cannot meet the recommended standard of daily folic acid intake. Folic acid deficiency is still the most common vitamin deficiency worldwide.

[0003] With the in-depth study of folic acid, it is found that it not only has the function of preventing and treating cardiovascular and cerebrovascular diseases and gastrointestinal cancer, but also plays an important role in human intestinal health. Studies have found that inflammatory bowel disease (IBD) as one of the diseases related to folic acid deficiency, patients show specific intestinal microecological imbalance. Colon microorganisms can use folic acid production pathways to regulate host metabolism and folic acid seems to be able to improve the health status of the body by regulating the intestinal microecology. The existing research on the regulatory role of folic acid on intestinal microorganisms lacks key evidence, and most of them are limited to animal experiments. Mammals (mice or pigs) have been widely used to test drug metabolism, toxicity, and efficacy. However, due to poor reproducibility and individual differences, the influence of experimental ethics and economic factors, their application is greatly limited. In addition, it is very important that due to the differences in physiological structure and flora structure between animals and humans, the results of animal experiments are difficult to accurately extrapolate to humans. Cell culture models as a typical in vitro model have been widely used in the field of food and drug bioavailability and toxicology research. The cell culture model currently used is carried out in two-dimensional (2D) conditions, one or more cell lines are placed in (co-) culture, and an insert system consisting of a rigid porous membrane is often used. This model can study the molecular mechanisms of the gastrointestinal tract in a simple and reproducible way, such as using Caco-2, T84 cells, which are often used to study nutrient transport, intestinal absorption, cell differentiation, and human diseases including carcinogenesis. However, the disadvantages of cell culture models are also obvious, they only contain a single intestinal cell type and cannot represent the complexity and diversity of the human intestinal epithelial environment. In addition, by using Caco-2 as a human colorectal adenocarcinoma cell, the intestinal epithelial cell line established by it may cause problems with model stability due to multiple gene mutations. Therefore, the timely development of various types of in vitro intestinal models is crucial for the development of the field of intestinal microecology.

[0004] In order to explore the effect of folic acid on the intestinal microecology, the in-vitro model of human colon ecology is a key element of the research. The existing technology informs that the optimization of the fermentation medium is the premise of the optimization of the whole in-vitro intestinal fermentation system, but there is no uniform standard for the medium used in the in-vitro simulation fermentation. Inorganic salt, bile salt, pectin, starch, glucose, trypsin, mucin are the medium components of the in-vitro intestinal simulation fermentation, through the orthogonal experiment of 7 factors and 2 levels, the medium components are adjusted for static fermentation, the OD600 absorbance value is measured once every 24 h, and the bacterial DNA is extracted after each group is fermented for 48 h, and the 16S rDNA sequencing is carried out. The experimental results are analyzed by variance analysis, and it is found that trypsin is the main factor affecting the bacterial phylum, and inorganic salt and mucin are the secondary factors. However, the effect of vitamin components on the in-vitro intestinal simulation fermentation has not been considered in the medium. In summary, different research purposes need to match different in-vitro simulation media. At present, there is no report on the experimental model of folic acid (Vb9) on the in-vitro intestinal flora of human. Therefore, the regulatory function of reduced folic acid (5-MTHF) on the flora and the regulatory function of non-reduced folic acid (synthetic folic acid, FA) on the flora are not known. SUMMARY

[0005] The technical problem to be solved by the present application is to provide an in-vitro simulation method of folic acid deficiency intestinal environment and different folic acid in-vitro function evaluation

[0006] To solve the above technical problems, the present application provides an application of a folic acid determination medium in folic acid in-vitro function evaluation, and the preparation method of the folic acid determination medium is as follows:

[0007] Acid hydrolysis casein 10.0 g, glucose 40.0 g, sodium acetate 40.0 g, DL-tryptophan 0.2 g, L-cysteine hydrochloride 0.5 g, adenine 0.01 g, guanine hydrochloride 0.01 g, uracil 0.01 g, xanthine 0.02 g, KH2PO4 1.0 g, K2HPO4 1.0 g, Tween 80 1 mL, reduced glutathione 0.005 g, MgCl2 0.2 g, NaCl 0.02 g, FeSO4 0.02 g, MnSO4 0.015 g, riboflavin 0.001 g, p-aminobenzoic acid 0.002 g, pyridoxine hydrochloride 0.004 g, thiamine hydrochloride 0.0004 g, calcium pantothenate 0.0008 g, biotin 0.00002 g, dissolved in 1 L of water, after dissolution, 1 mL of 0.1 g / L resazurin solution (anaerobic indicator) is added, and boiled until the color changes (red to yellow);

[0008] Then bottle, immediately flush the bottle with nitrogen, keep the surface of the medium in an anaerobic state, then cap, sterilize (high pressure sterilization at 121℃ for 15 minutes), and cool down for standby use.

[0009] Improvement of the application of the folate assay medium of the present application in the in vitro functional evaluation of folate:

[0010] The water is the second grade water stipulated in GB / T 6682-2008.

[0011] Further improvement of the application of the folate assay medium of the present application in the in vitro functional evaluation of folate:

[0012] The enteric flora or the fecal suspension containing the enteric flora is added into the folate assay medium, and then the reduced folate (5-MTHF) or the non-reduced folate (synthetic folate, FA) to be detected is added, so as to determine the regulation function of the reduced folate (5-MTHF) and the non-reduced folate (synthetic folate, FA) on the enteric flora.

[0013] The present application also simultaneously provides a method for establishing an in vitro intestinal simulation system (folate deficiency model) and determining the regulation effect of different types of folate on human intestinal flora, comprising the following steps:

[0014] The fecal suspension is inoculated into the folate assay medium at an inoculation amount of (10±1) % (v / v), so as to obtain the in vitro intestinal simulation system (folate deficiency model);

[0015] The reduced folate (5-MTHF) or the non-reduced folate (synthetic folate, FA) to be detected is added into the above-mentioned in vitro intestinal simulation system (folate deficiency model) to form a reaction system, so as to determine the regulation function of the reduced folate (5-MTHF) and the non-reduced folate (synthetic folate, FA) on the enteric flora.

[0016] Improvement of the method of the present application:

[0017] The preparation method of the fecal suspension is as follows: (0.8±0.1) g of fresh morning feces of a donor (volunteer) per person is added into 8 mL of 0.1 mM PBS buffer (pH=6.8) after sterilization, and then vortexed and mixed, and large particles are filtered out by using a 0.24 mm filter screen, so as to obtain the fecal suspension.

[0018] Further improvement of the method of the present application: the reaction system is incubated at 37°C for 24 h; and the obtained culture is used for 16S rDNA identification and detection of short-chain fatty acids.

[0019] As a further improvement of the method of the application: Illumina platform is used for 16S rDNA gene V3-V4 region double-end sequencing of the sequencing sample; the sequencing platform is MiSeq PE250, and the sequencing primer is a universal primer; the amplification primer is 341F: 5'-CCTAYGGGRBGCASCAG-3' and 806R: 5'-GGACTACHVGGGTWTCTAAT-3', and the thermal cycle condition is as follows: 95 DEG C for 5 minutes; 30 cycles of 95 DEG C for 30 seconds, 50 DEG C for 30 seconds, 72 DEG C for 40 seconds, and 72 DEG C for 7 minutes.

[0020] As a further improvement of the method of the application: GC-MS is used for detecting short-chain fatty acids in the fecal suspension fermentation sample.

[0021] The chromatographic system is Agilent DB-WAX capillary column (30mm*0.25mm*0.25um); the carrier gas is high-purity nitrogen (the purity is not less than 99.999%), the flow rate is 1.0mL / min; the injection port temperature is 220 DEG C, the injection amount is 1ul, the splitless injection is used, and the solvent delay time is 2.5min;

[0022] The mass spectrometry system is an electron impact ion source (EI), the ion source temperature is 230 DEG C, and the interface temperature is 220 DEG C.

[0023] So that the content of the short-chain fatty acid in the fermentation liquor is determined.

[0024] The Vis culture medium used in the prior art often contains folic acid pollution, and HPLC results show that the content of folic acid in the Vis culture medium is about 69.42ng / mL. The folic acid determination culture medium of the application does not contain folic acid, and other nutrients are complete, so that the intestinal flora grows under the condition of the simulated folic acid deficiency in later life, and a folic acid deficiency model can be constructed.

[0025] The application provides a new use of folic acid, that is, 5-methyltetrahydrofolic acid and non-reduced folic acid have a regulating effect on the microecology of an in-vitro simulated intestinal flora of a folic acid deficiency population.

[0026] The application uses an in-vitro intestinal simulation experiment to explore the effect of 5-methyltetrahydrofolic acid (active folic acid, purity > 99%) and non-reduced folic acid (synthetic folic acid, purity > 98%) on intestinal flora, that is, the effect of different types of Vb9 on intestinal flora.

[0027] The application uses an improved in-vitro intestinal simulation model to perform an in-vitro intestinal simulation experiment, proves that folic acid has the function of regulating intestinal flora, and then compares different folic acids and finds that they all have the function of improving intestinal flora (that is, beneficial to the regulation of human intestinal flora).

[0028] That is, since reduced folate (5-methyltetrahydrofolate, MTHF) and synthetic folate (FA) are two different folate forms, they have some differences in structure, source and bioavailability, etc. The present application carries out in vitro functional evaluation by analyzing the composition of intestinal flora and the level of short-chain fatty acids.

[0029] 5-methyltetrahydrofolate promotes the growth of Bifidobacterium and Lactobacillus in the intestine, thereby promoting the intestinal flora to produce short-chain fatty acids. Non-reduced folate promotes the growth of Bifidobacterium, Lactobacillus and Pediococcus, thereby promoting the intestinal flora to produce short-chain fatty acids.

[0030] The present application provides an in vitro folate-deficient environment, and different types of folate have very significant effects on intestinal regulation (intestinal flora and flora metabolites). It has a very obvious promoting effect on the growth of Bifidobacterium and Lactobacillus in the intestine. After treatment with non-reduced folate, the abundance of Pediococcus is significantly increased, which has a certain promoting effect on inhibiting the growth of pathogenic bacteria and promoting the intestinal flora of the human body to produce short-chain fatty acids (SCFAs).

[0031] It should be emphasized that: the present application adopts an in vitro intestinal simulation system (folate-deficient model), while the prior art adopts an animal model (mouse model) in clinical research, and there is no in vitro functional evaluation of folate; the experimental model of the present application has high repeatability, high safety and low experimental cost. The use of folate determination medium avoids the interference of background folate from the source. Compared with the postnatal modeling of mouse model, the difference between groups is small, the biological difference is ignored, the operation is convenient and the data reliability is high.

[0032] The present application has the following technical advantages:

[0033] First, the present application uses a new method to realize in vitro functional evaluation of folate (intestinal microecological regulation) for the first time:

[0034] Some of the existing culture media in the field of in vitro simulation technology are mainly based on VIs (veal infusion broth), VL (Viande Levure) medium, and YCFA (yeast extract, casein hydrolysate, fatty acids) medium. HPLC results show that the existing in vitro intestinal simulation medium VIs has sufficient folate content (i.e., there is folate contamination), which cannot exclude the influence of background folate on intestinal flora.

[0035] The application develops a new use of a folic acid determination medium, which replaces the existing in vitro intestinal simulation medium.

[0036] The application first emphasizes the pollution of water to the medium.

[0037] The scheme of the application removes the interference of background folic acid while ensuring the intestinal microbial nutritional conditions, uses the folic acid determination medium to replace the known medium, and enables the intestinal flora to grow in the folic acid deficiency condition created by the afterlife.

[0038] Therefore, the application first realizes the in vitro functional evaluation of folic acid, and the prior art cannot guarantee the interference of background folic acid in the medium.

[0039] Secondly, the application finds that folic acid has intestinal flora regulation function, mainly promoting Lactobacillus.

[0040] The application constructs an in vitro intestinal flora model to verify the regulation effect of folic acid on intestinal flora, and compared with the previous clinical animal model technology, has the advantages of high repeatability, high safety and low experimental cost, and compared with the known animal model technology, the application is more easy to exclude the interference of self conditions and external conditions, and the experimental results show that folic acid has intestinal flora regulation function, mainly promoting the abundance of Lactobacillus genus, and the intestinal flora of the control group is significantly different.

[0041] In summary, the application first finds that folic acid supplementation has a positive regulation effect on the intestinal specific genus Lactobacillus.

[0042] Thirdly, the application finds that reduced folic acid (5-MTHF) and non-reduced folic acid (synthetic folic acid, FA) have different regulation functions on flora.

[0043] Reduced folic acid (5-MTHF) is the main bioactive form of folic acid metabolism, and it does not need an activation process, and after supplementing reduced folic acid, the abundance of Bifidobacterium genus is significantly increased; and non-reduced folic acid (synthetic folic acid, FA) is unactivated folic acid, which is the main form of folic acid in drugs and part of health foods, and needs to be converted into 5-MTHF through multiple folic acid metabolism steps, and in the experiment, the abundance of Pediococcus genus is significantly increased after supplementing non-reduced folic acid.

[0044] In summary, the application first finds that reduced folic acid (5-MTHF) and non-reduced folic acid (synthetic folic acid, FA) have different regulation functions on flora, which provides an idea for dietary intervention treatment of the folic acid-intestinal microbial pathway in practice. Attached Figure Description

[0045] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0046] Figure 1 HPLC identification of folic acid content in VIS culture medium:

[0047] A: HPLC analysis of in vitro intestinal simulated culture medium VIS (10×);

[0048] B: HPLC quantitative standard curve of folic acid standard;

[0049] Specifically:

[0050] The folic acid was calibrated and analyzed using Vis, a commonly used in vitro intestinal simulant culture medium. Vis medium was concentrated tenfold, and folic acid was quantified based on peak area using an external standard method. Figure 1 A shows a characteristic peak at 11.831 min. Figure 1 B calculated that the folic acid concentration was 694.21 ng / mL, meaning that synthetic folic acid was present in the original Vis medium, and the concentration was approximately 69.4 ng / mL.

[0051] Figure 2 In the middle: A is the petal plot analysis based on the OUT level; B is the PCoA analysis of 4 groups (Ctrl, FA, MTHF, Ori).

[0052] Ori represents the average level of the 8 replicates in the original fecal sample group;

[0053] Ctrl represents the average level of 8 replicates fermented on folic acid assay medium.

[0054] FA represents the average value of 8 replicates in the fermentation group inoculated with non-reduced folic acid at a concentration of 2.0 ng / mL;

[0055] MTHF represents the average value of 8 replicates inoculated with 2.0 ng / mL of 5-methyltetrahydrofolate;

[0056] Specifically:

[0057] Figure 2 The differences in gut microbiota can be indicated by operational taxonomic units (OTUs) and beta diversity. Figure 2 A found that there were 488 OTUs in common across the four groups, 2326 unique OTUs in the FA group, and 1631 unique OTUs in the MTHF group, which were significantly different from the Ctrl group. Figure 2B By PCoA analysis, the eigenvalues and eigenvectors were sorted, and the eigenvalues mainly in the top few were selected, which showed the PCoA scores among the 4 groups, respectively. On the level of OTU, Axis.1 and Axis.2 explained 10.87% and 6.37% of the variation at this level, respectively. Folic acid was closely related to the changed intestinal microbial ecology, showing a tendency of the fitting circle transferring from the negative half of Axis 1 to the positive half from the control group to the FA group, and then to the MTHF group. The sampling points of the Ctrl group always surrounded (0, 0), indicating that the difference among groups was small, and the microbial community composition of this group remained stable.

[0058] Figure 3 Medium: A~B: The average level of 8 replicates of the group at the level of phylum and genus;

[0059] Ori represents the average level of 8 replicates of the fecal original group;

[0060] Ctrl represents the average level of 8 replicates of the fermentation group inoculated with folic acid;

[0061] FA represents the average level of 8 replicates of the fermentation group inoculated with non-reduced folic acid at a concentration of 2.0 ng / mL;

[0062] MTHF represents the average level of 8 replicates of the fermentation group inoculated with 5-methyltetrahydrofolic acid at a concentration of 2.0 ng / mL;

[0063] Specifically:

[0064] Figure 3is the relative abundance of "door" and "genus" flora after fermentation of the in vitro intestinal model. It can be clearly seen that the dominant bacterial phylum of the four groups is Firmicutes, but compared with the Ctrl group, the abundance of Firmicutes in the MTHF group increases, and the abundance of Firmicutes in the FA group decreases. At the genus level, Lactobacillus becomes the new dominant genus after fermentation. Compared with the Ctrl group, the Bacteroides genus in the experimental groups (FA and MTHF) is down-regulated to different degrees, accounting for 17% and 18%, respectively. In terms of the relative abundance of Bifidobacterium, the folic acid supplement groups (MTHF and FA) are higher than the control group (p<0.05). This shows that folic acid supplements can be used by folic acid-producing bacteria, and the beneficial effects of Bifidobacterium are exerted to regulate the intestinal flora. In addition, in terms of the relative abundance of Pediococcus, the abundance of the control group is 0.06%, and after supplementing non-reduced folic acid, the relative abundance reaches 0.81%. Some Pediococcus show folic acid dependence, and their growth can be used as a marker for folic acid absorption and utilization. Among them, the relative abundance of the Ctrl group is lower than that of the Ori group, which can prove that the folic acid deficiency model is successfully constructed.

[0065] Figure 4 is the change of Lactobacillus, Bifidobacterium, Bacteroides and Pediococcus after fermentation of the in vitro intestinal model.

[0066] Figure 4

[0067] A is the growth of Lactobacillus;

[0068] B is the growth of Bifidobacterium;

[0069] C is the growth of Bacteroides;

[0070] D is the growth of Pediococcus;

[0071] Ori represents the average level of 8 replicates of the fecal sample group;

[0072] Ctrl represents the average level of 8 replicates of the fecal sample group fermented in folic acid assay medium;

[0073] FA represents the average level of 8 replicates of the fermentation group inoculated with non-reduced folic acid with a concentration of 2.0 ng / mL;

[0074] MTHF represents the average level of 8 replicates inoculated with 5-methyltetrahydrofolic acid with a concentration of 2.0 ng / mL.

[0075] Figure 5 is the effect of folic acid on the metabolism of intestinal flora to generate short-chain fatty acids in the in vitro intestinal model system; ​

[0076] Figure 5 middle:

[0077] A represents the total acid concentration after fermentation in the in vitro intestinal model; B represents the acetic acid concentration after fermentation in the in vitro intestinal model; C represents the propionic acid concentration after fermentation in the in vitro intestinal model; D represents the butyric acid concentration after fermentation in the in vitro intestinal model; E represents the isobutyric acid concentration after fermentation in the in vitro intestinal model; F represents the valerate concentration after fermentation in the in vitro intestinal model; G represents the isovaleric acid concentration after fermentation in the in vitro intestinal model; H represents the hexanoic acid concentration after fermentation in the in vitro intestinal model.

[0078] Fermentation time was 24 hours. The horizontal axis represents different culture media, and the vertical axis represents concentration.

[0079] Ctrl represents the average level of 8 replicates fermented on folic acid assay medium.

[0080] FA represents the average value of 8 replicates in the fermentation group inoculated with non-reduced folic acid at a concentration of 2.0 ng / mL;

[0081] MTHF represents the average value of 8 replicates inoculated with 2.0 ng / mL of 5-methyltetrahydrofolate;

[0082] Specifically:

[0083] SCFAs are the main fermentation products of intestinal anaerobic bacteria, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, which have positive effects on the human body. Figure 5 As shown, after 24 hours of fermentation, the content of short-chain fatty acids in the intestine did not accumulate with the extension of fermentation time. There was no significant difference in SCFAs between the MTHF and FA groups. Compared with the control group, the acetic acid content decreased to varying degrees after fermentation, and the acetic acid content in the FA group was lower than that in the MTHF group. In terms of isovaleric acid content, both folic acid supplementation groups showed varying degrees of upregulation compared with the control group.

[0084] Figure 6 This is a heatmap showing the correlation between gut microbiota and short-chain fatty acids.

[0085] The horizontal axis represents total acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid after fermentation, while the vertical axis represents the genus of intestinal microorganisms.

[0086] Specifically:

[0087] Figure 6 The correlation between gut microbial genera and SCFAs was analyzed and evaluated. The Spearman correlation coefficient was used as a measure to study the relationship between short-chain fatty acids and microbial species richness (alpha diversity), and the pairwise correlations and significance P-values ​​were obtained. Figure 6As shown, the correlation coefficients between the concentrations of most short-chain fatty acids and the species composition varied greatly. Overall, hexanoic acid was strongly positively correlated with Ralstonia and Serratia (p<0.001), and the total acid content was strongly positively correlated with Parasutterella (p<0.001). The isovaleric acid level was strongly negatively correlated with the abundance of Bacteroides, Lachnospiraceae_UCG_010, and Lachnospira (p<0.01). DETAILED DESCRIPTION

[0088] The application will be further described below in conjunction with specific examples, but the scope of protection of the application is not limited to this:

[0089] The components in the culture medium of the application can be obtained through conventional commercial forms; for example:

[0090] The reagents such as Tween 80 were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.; the reagents of hemin and CoSO4·7H2O were purchased from Shanghai Macklin Biochemical Technology Co., Ltd.; the reagents of tryptone, proteose peptone, and yeast extract were purchased from Oxoid; the starch was purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd.; the reagents of 3# cholate (3 cholate), mucin, and L-cysteine hydrochloride were purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.; NaCl was purchased from Xilong Scientific Co., Ltd.; KCl was purchased from Guangdong Guanghua Science and Technology Co., Ltd.; CaCl2·6H2O was purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.; KH2PO4, K2HPO4, and MnCl2·4H2O were purchased from Hangzhou Changqing Chemical Co., Ltd.; MgSO4·7H2O, ZnSO4·7H2O, CuSO4·5H2O, NiCl2·6H2O, and FeSO4·7H2O were purchased from Xilong Scientific Co., Ltd.; and the folic acid determination medium was purchased from Beijing Luqiao Company.

[0091] I. Setting up the experimental system

[0092] Vis medium: Accurately weigh 3 g of tryptone, 4.5 g of yeast extract, 5 g of starch, 3 g of peptone, 0.5 g of mucin, 0.8 g of L-cysteine hydrochloride, 0.4 g of bile salt No. 3, 4.5 g of NaCl, 2.5 g of KCl, 4.5 g of MgCl2-6H2O, 0.2 g of CaCl2-6H2O, 0.4 g of KH2PO4, 0.45 g of K2HPO4, 1 mL of Tween 80, and dissolve in 1 L of deionized water. After complete dissolution, add 10 mL of a hemin solution (0.1 g / L) and 2 mL of a trace element solution (see Table 1). Autoclave at 121 °C for 15 minutes. When the medium cools to about 50 °C, add 10 mL of a hemin solution. Divide into 10 mL of a test tube. Add 5 mL of the medium to each test tube. Transfer to an anaerobic workstation for anaerobic pretreatment (i.e., anaerobic prevention at 37 °C for 12 hours. The purpose of this anaerobic pretreatment is to remove residual air in the test tube). Then, seal with a cap. Use as a VIS medium.

[0093] Hemin solution: Weigh 0.5 g of hemin powder, dissolve with 1 mL (1 M) of sodium hydroxide solution, and dilute to 100 mL with water. Autoclave at 121 °C. Store at 4 °C in a refrigerator.

[0094] Table 1 Trace element solution Tab.1Trace element solution

[0095]

[0096]

[0097] Folic acid assay medium: acid hydrolysis casein 10.0 g, glucose 40.0 g, sodium acetate 40.0 g, DL-tryptophan 0.2 g, L-cysteine hydrochloride 0.5 g, adenine 0.01 g, guanine hydrochloride 0.01 g, uracil 0.01 g, xanthine 0.02 g, KH2PO4 1.0 g, K2HPO4 1.0 g, Tween 80 1 mL, reduced glutathione 0.005 g, MgCl2 0.2 g, NaCl 0.02 g, FeSO4 0.02 g, MnSO4 0.015 g, riboflavin 0.001 g, p-aminobenzoic acid 0.002 g, pyridoxine hydrochloride 0.004 g, thiamine hydrochloride 0.0004 g, calcium pantothenate 0.0008 g, biotin 0.00002 g, dissolved in 1 L of GB / T 6682-2008 specified secondary water, after complete dissolution, 1 mL of resazurin solution (0.1 g / L) was added, boiled until the color changed (red to yellow), the medium was added to the prepared 10 mL of a test tube, 5 mL per tube. Then immediately flush in nitrogen, keep the surface of the medium anaerobic, press the cap with the instrument, autoclave at 121 ℃ for 15 min, then cool to room temperature and reserve.

[0098] Folic acid sample: 5-methyltetrahydrofolic acid and folic acid standard powder (Sigma) were taken, and 5-methyltetrahydrofolic acid solution with a concentration of 2.0 ng / mL and non-reduced folic acid solution with a concentration of 2.0 ng / mL were respectively configured according to the daily intake standard of human folic acid.

[0099] Fecal bacteria suspension: fecal samples were provided by 8 volunteers (4 men and 4 women), who were recruited in Hangzhou, Zhejiang. The volunteer standard: aged between 22-35 years old, regular work and rest, normal diet, no disease, no antibiotics, prebiotics, and no gastrointestinal disease in the past six months, and all volunteers obtained informed consent. Each volunteer was operated as follows: 0.80 g of fresh feces per person was taken from the volunteer in the morning, 8 mL of sterilized (sterilized in a high-pressure steam sterilization pot at 121 ℃ for 15 min) 0.1 mM PBS (pH = 6.8) was added, vortexed and mixed, and large particles were filtered out with a 0.24 mm filter screen to obtain a fecal suspension.

[0100] II.1: Determination of folic acid content in Vis medium

[0101] HPLC e2695 system (Waters, USA) was used. After VIS medium was concentrated ten times, Vis (10x) medium was obtained. Then the filtrate was filtered at 4 ℃, and loaded onto the column.

[0102] VIS medium was concentrated ten times by using a rotary evaporator (BUCHI, Switzerland) at 37°C, 30 rpm, 20 mbar.

[0103] The HPLC assay conditions were as follows:

[0104] Column: Sun Fire™ C18 column (5 μm, 4.6 x 150 mm)

[0105] Wavelength: 280 nm,

[0106] Column temperature: 25°C

[0107] Injection volume: 20 μL

[0108] The solvents consisted of 0.1% (v / v) HCOOH in water (solvent A) and acetonitrile (solvent B);

[0109] Elution conditions: Gradient elution from 0-3 min, 10% B; 3-5 min, 10-15% B; 5-8 min, 15% B; 8-13 min 15-50% B; 13-16 min, 50-10% B, then isocratic elution at 10% B for 9 min.

[0110] For example, "3-5 min, 10-15% B" means that the volume concentration of solvent B in the solvent is uniformly increased from 10% to 15% in the time period of 3-5 min.

[0111] The results obtained are shown in the chromatogram Figure 1 A) shows the presence of folic acid in the form of non-reduced folic acid in the VIS medium.

[0112] The folic acid concentration-peak area standard curve Figure 1 B) shows the standard curve established with different concentrations of synthetic folic acid (non-reduced folic acid) as the abscissa (10 μg / mL, 30 μg / mL, 50 μg / mL, 80 μg / mL, 100 μg / mL) and the peak area (μAU) as the ordinate:

[0113] The formula is: y = 32035x - 116743;

[0114] The peak area of the filtrate of the Vis (10x) medium is brought into the formula to calculate, and the folate content in the Vis (10x) medium is 694.21 ng / mL, that is, the folate content in the Vis medium is about 69.4 ng / mL. The reason is that there is folate pollution in the medium formula, so that the folate concentration is in the super high range (the normal value of folate in the human body is 4.85-35.69 ng / mL), and therefore the medium has defects for the in vitro functional evaluation research of folate. It is crucial to create a folate-deficient environment for exploring the function of folate in regulating the intestinal microecology.

[0115] In order to eliminate the interference of background folate, the medium is improved through designed research, so as to obtain the folate determination medium specially used in the application.

[0116] II.2: The folate determination medium is detected according to the method of II.1, and the characteristic peaks of reduced folate (5-methyltetrahydrofolate, MTHF) and synthetic folate (FA) do not appear, so it can be known that the folate determination medium does not contain reduced folate (5-methyltetrahydrofolate, MTHF) and synthetic folate (FA).

[0117] III: Group fermentation of human fecal suspension

[0118] Take 0.5 mL of the fecal suspension obtained in the above step one, place it in a new 2 mL sterilized centrifuge tube, mix uniformly, and store in a-80°C refrigerator as a pre-fermentation analysis control sample of human feces. Each volunteer sets up a corresponding pre-fermentation analysis control sample of human feces, which is marked as Ori.

[0119] In an anaerobic workstation (Do Whitley Scientific), inoculate the fecal suspension in the folate determination medium at a 10% (V / V) inoculation amount. That is,

[0120] The experimental group is divided into Ctrl, MTHF, and FA groups, each group has 8 repeated samples, that is, 8 samples provided by 8 volunteers, and each sample has 3 parallel groups.

[0121] Ori represents the original fecal group: 500 μL of fecal suspension;

[0122] Ctrl represents the control group: 0.5 mL of fecal suspension, 5 mL of folate determination medium

[0123] MTHF represents the 5-methyltetrahydrofolate supplement group: 0.5 mL of fecal suspension, 5 mL of folate determination medium, and 100 μL of 2.0 ng / mL 5-methyltetrahydrofolate;

[0124] FA represents non-reduced folate supplement group: 0.5 mL of fecal suspension, 5 mL of folate assay medium, 100 μL of folate standard with a concentration of 2.0 ng / mL;

[0125] The above-mentioned 3 groups of corresponding fermentation samples were placed in a constant temperature incubator (SPX-150B-Z, Shanghai Boxun Industry Co., Ltd.) at 37°C and incubated anaerobically for 24 h. After incubation, the samples were shaken uniformly, the bottle cap was opened with a bottle opener, and 500 μL of the fermentation suspension was taken for sub-packaging. The samples were stored in a -80°C refrigerator as post-fermentation analysis samples of human feces. Therefore, a total of 8*3=24 post-fermentation analysis samples of human feces were obtained, and each sample had 3 replicates.

[0126] Four: DNA extraction and 16S rDNA sequencing analysis of fecal suspension fermentation samples

[0127] Specifically as follows:

[0128] The Omega kit (M5635-02, Omega Bio-Tek, USA) was used for DNA extraction of the 4 groups of fecal fermentation before and after fermentation (post-fermentation samples Ctrl, FA, MTHF and pre-fermentation sample Ori).

[0129] The pre- and post-fermentation samples were sent to Shenzhen Microskol Technology Group Co., Ltd. for high-throughput sequencing of the V3-V4 region of the 16S rDNA gene. The Illumina platform was used for double-end sequencing of the sequencing samples. The sequencing platform was MiSeq PE250, and the sequencing primer was a universal primer. The amplification primer was 341F: 5'-CCTAYGGGRBGCASCAG-3' and 806R: 5'-GGACTACHVGGGTWTCTAAT-3', and the thermal cycling conditions were as follows: 95°C for 5 minutes; 30 cycles of 95°C for 30 seconds, 50°C for 30 seconds, 72°C for 40 seconds, and 72°C for 7 minutes. The obtained data was screened, optimized and spliced, and the specific composition of each sample (group) at different classification levels was analyzed (and whether there was a statistically significant difference between groups was tested). Through various multivariate statistical analysis tools, the differences in the microbial community structure between different samples (groups) and the species related to the differences were further measured.

[0130] The results are shown in Figure 2 As shown in Figure A, the changes in intestinal microorganisms after inoculation of different folates (FA, MTHF) were evaluated. It was found that there were 2326 unique operational taxonomic units (OTUs) in the FA group, and 1631 unique OTUs in the MTHF group were significantly different in number from the Ctrl group, and 488 OTUs were common to the three groups. Figure 2B By principal coordinate analysis (PCoA) to clarify the overall composition of in vitro intestinal simulated microbial community at OTU level, Axis.1 and Axis.2 explained 10.87% and 6.37% of the variation at the level, respectively. As Figure 2 B As shown from Ctrl group to FA group and finally to MTHF group, the fitting circle has a trend of moving from the negative half of Axis 1 to the positive half, which proves that folate is closely related to the ecological changes of intestinal microorganisms. The sampling points of the Ctrl group are always gathered near (0, 0), indicating that the difference between groups is small, indicating that the composition of the microbial community in this group is stable. After fermentation after supplementing FA and MTHF, the difference between samples increased significantly.

[0131] Figure 3 The microbial community at the door, genus and level was analyzed to analyze the differences in microbial composition between different types of folate. At the door level ( Figure 3 A), Firmicutes, Bacteroidetes and Proteobacteria are the main bacterial phyla, and the proportion of the three bacterial phyla accounts for more than 90% of the total. After fermentation, compared with the relative abundance of Firmicutes (60.41%) in the control group Ctrl, the relative abundance of Firmicutes in the test group FA is slightly lower (56.7%), while the increase in the MTHF group (64.9%) is more obvious. This result shows that compared with the addition of ordinary folate (non-reduced folate), 5-methyltetrahydrofolate (reduced folate) or can promote the growth of Firmicutes in fecal samples. At the genus level ( Figure 3 B), after fermentation, Lactobacillus became the new dominant genus. Compared with the Ctrl group, the Bacteroides genus in the experimental group (FA, MTHF) was down-regulated to different degrees. In terms of the relative abundance of Bifidobacterium, the folate supplement group (MTHF and FA) was higher than the control group (p<0.05). This shows that folate supplements can be used by folate-producing bacteria, and the beneficial effects of Bifidobacterium are exerted to regulate the intestinal flora. In addition, the relative abundance of Pediococcus in the control group is 0.06%, and after supplementing non-reduced folate, the relative abundance reaches 0.81%. Some Pediococcus show folate dependence, and their growth can be used as a marker for folate absorption and utilization. The relative abundance of the Ctrl group is lower than that of the Ori group, which can prove that the folate-deficient model is successfully constructed.

[0132] Figure 4 The results show that in the FA group, the abundance of Lactobacillus genus decreased slightly (22.75%), but in the MTHF group, the abundance of Lactobacillus genus increased to 30.76% ( Figure 4 A). AsFigure 4 B shows that the Bacteroides genus in the experimental group (FA, MTHF) is down-regulated to different degrees compared with the Ctrl group, accounting for 17% and 18%, respectively. In terms of the relative abundance of the Bifidobacterium genus, the MTHF group (1.06%) shows a significant difference (p < 0.05) compared with the control group (0.24%), and the FA group (0.70%) also shows similar results Figure 4 C). Figure 4 D shows that the relative abundance of the Pediococcus genus reaches 0.81% after the addition of FA, which is higher than that of the control group.

[0133] Five: GC-MS detection of short-chain fatty acids in fecal suspension fermentation samples

[0134] Short-chain fatty acids are an important indicator for measuring the growth and metabolism of intestinal flora, including acetic acid, propionic acid, butyric acid, isovaleric acid, isobutyric acid, etc. The three sample groups (FA, MTHF, Ctrl) after fermentation were taken out from the -80°C refrigerator, thawed at room temperature, and then 300 μL of 50% sulfuric acid was added, followed by 100 μL of 500 mg / L internal standard (cyclohexanone) solution and 2 mL of diethyl ether homogenate for 1 min, 4°C centrifugation (12000 rpm, 10 min) to take the supernatant, and then filtered with a 0.22 μm filter. Take 100 μL of the filtrate and add it to the gas phase sample bottle, tightly cover it with a lid to exclude air bubbles, and then it can be analyzed, and the rest of the filtrate is stored at -30°C for future use. Gas chromatography conditions:

[0135] Chromatographic column: Agilent DB-WAX capillary column 30 mm x 0.25 mm x 0.25 μm;

[0136] Column temperature: 60°C, 30°C / min to 210°C for 3 min;

[0137] Inlet temperature: 220°C;

[0138] Injection volume: 1.0 μL;

[0139] Non-split injection; solvent delay time 2.5 min

[0140] Carrier gas: high-purity nitrogen (purity not less than 99.999%);

[0141] Flow rate 1.0 mL / min;

[0142] Mass spectrometry system: electron impact ion source (EI), ion source temperature 230°C, interface temperature 220°C.

[0143] The determination results can be automatically calculated by the workstation according to the standard curve equation internal standard method, and the sample determination results are automatically converted to the amount of target short-chain fatty acid in the sample.

[0144] The results are shown in Figure 5 After 24 hours of fermentation, the content of intestinal short-chain fatty acids did not accumulate with the extension of fermentation time. There was no significant difference in SCFAs between the MTHF group and the FA group. Compared with the control group, the content of acetic acid decreased to different degrees after fermentation, and the content of acetic acid in the FA group was lower than that in the MTHF group. In the content of isovaleric acid, the two groups of folic acid supplement groups had different degrees of up-regulation compared with the control group.

[0145] The results show that the two different types of folic acid show consistent trends in regulating human intestinal flora, but the two different types of folic acid supplements, MTHF and FA, have a regulating effect on specific intestinal flora. After supplementing non-reduced folic acid (synthetic folic acid, FA) under folic acid deficiency conditions, the abundance of Pediococcus genus significantly increased (0.81%). It is known in the prior art that members of the Pediococcus genus can produce short-chain fatty acids in the metabolic process in the intestine. They produce short-chain fatty acids such as propionic acid and butyric acid by fermenting carbohydrates such as dietary fiber Figure 5 D-E). These produced short-chain fatty acids can be absorbed and utilized by intestinal epithelial cells to provide energy for cells and have a positive effect on intestinal health.

[0146] 5-methyltetrahydrofolic acid is similar to natural folic acid and can be directly absorbed and utilized by the human body. Therefore, its bioavailability is relatively high. After supplementing reduced folic acid (5-MTHF), the abundance of Bifidobacterium genus in the intestinal flora significantly increased (1.06%), and Bifidobacterium genus is an important member of the intestinal flora and widely exists in the human intestine. It is known in the prior art that Bifidobacterium genus produces short-chain fatty acids such as acetic acid, propionic acid and butyric acid by fermenting dietary fiber, oligosaccharides and other carbohydrates, and the trend of short-chain fatty acid determination results Figure 5 A,C-E) is consistent.

[0147] According to the correlation analysis between the abundance level of intestinal flora and SCFAs, Figure 6), the correlation coefficients between most of the short-chain fatty acids and the species composition were significantly different. According to the results, at the genus level, Lahnospiraceae_ND3004, Roseburia, Lachnospira were positively correlated with acetic acid. There were 15 genera that showed positive correlation with propionic acid content, among which the genus Oscillospira UCG_005 showed stronger correlation (p<0.01). Ralstonia and Nocardioides showed positive correlation with butyric acid content, while Lachnospiraceae_UCG_010 showed a significant negative correlation. It was observed that isobutyric acid content was positively correlated with Nocardioides, Serratia. The content of valeric acid was positively correlated with Phocea, Desulfovibrio, and negatively correlated with Megamonas and Bamesiella. And most of the bacteria with negative correlation with isovaleric acid content were from the phylum Firmicutes, among which Lachnospiraceae_UCG_010, Lachnispira and Roseburia showed strong negative correlation with isovaleric acid. The content of caproic acid was strongly positively correlated with Ralstonia, Serratia, Parasutterella in the phylum Proteobacteria. Finally, at the total acid level, the abundance of 7 genera (Parasutterella, Olsenella, Alcanivorax, Eubacterium_ruminantium, Bifidobacterium, Incertae_Sedis, Coprococcus) was significantly positively correlated with the total acid content.

[0148] Experiment 1, the present application found that folic acid has the function of regulating intestinal flora, mainly promoting the genus Lactobacillus.

[0149] Specifically as follows:

[0150] The fermentation samples before and after fermentation are sent to Shenzhen Micro-Science Alliance Technology Group Co., Ltd. for 16S rDNA high-throughput sequencing. Illumina NovaSeq platform is used for double-end sequencing of the sequencing samples. The DADA2 plug-in in Qiime2 software is used for quality control (filtered), denoising (correcting sequencing error sequences, denoised), splicing (merged), and non-chimeric of all sample input sequences, and OTU is formed. Based on the absolute abundance and annotation information of OTU, the species composition, inter-group OTU difference significance, Alpha diversity and Beta diversity of each sample are analyzed. The experimental results show that compared with the control group, the Lactobacillus genus abundance in the folic acid supplement group (FA, MTHF) is 22.75% and 30.76% respectively, and the difference in flora abundance is significant compared with the control group.

[0151] However, the prior art only indicates that the folic acid producing lactic acid bacteria has a significant regulatory effect on the Akkermansia flora in the intestinal tract, and the effect of the strain itself is greater than that of folic acid production, so the regulatory function of folic acid on intestinal microecology research cannot be fully proved.

[0152] The above-mentioned discovery of the present application can be applied to the efficient screening of Lactobacillus genus in human intestinal tract.

[0153] Experiment 2, the present application finds that reduced folic acid (5-MTHF) and non-reduced folic acid (synthetic folic acid, FA) have different regulatory functions on flora.

[0154] Specifically as follows:

[0155] After supplementing reduced folic acid (5-MTHF), the abundance of Bifidobacterium genus is significantly increased (1.06%); after supplementing non-reduced folic acid (synthetic folic acid, FA), the abundance of Pediococcus genus is significantly increased (0.81%).

[0156] The above-mentioned discovery of the present application is beneficial to refining the classification of folic acid fortified foods in practice, and increasing the diet intervention-intestinal microbial imbalance therapy.

[0157] Comparative experiment 1, replace the "second grade water" specified in GB / T 6682-2008 in the "folic acid determination medium" with conventional "deionized water", and the rest is the same as the folic acid determination medium; the obtained is named folic acid determination medium A.

[0158] Because the deionized water cannot effectively remove the organic matter in water, including folic acid; therefore, the folic acid determination medium A appears the characteristic peak of reduced folic acid (5-methyltetrahydrofolic acid, MTHF) and synthetic folic acid (FA) through detection.

[0159] Finally, it should be noted that the above list is only a few embodiments of the present application. Obviously, the present application is not limited to the above embodiments, but can have many variations. All variations that can be directly derived or inferred from the disclosure of the present application by those of ordinary skill in the art should be considered as falling within the scope of the present application.

Claims

1. Use of a folic acid assay medium in the in vitro evaluation of folic acid functions, characterized in that: The preparation method of the folic acid assay medium is as follows: casein hydrochloride 10.0 g, glucose 40.0 g, sodium acetate 40.0 g, DL-tryptophan 0.2 g, L-cysteine hydrochloride 0.5 g, adenine 0.01 g, guanine hydrochloride 0.01 g, uracil 0.01 g, xanthine 0.02 g, KH2PO4 1.0 g, K2HPO4 1.0 g, Tween 80 1 mL, reduced glutathione 0.005 g, MgCl2 0.2 g, NaCl 0.02 g, FeSO4 0.02 g, MnSO4 0.015 g, riboflavin 0.001 g, p-aminobenzoic acid 0.002 g, pyridoxine hydrochloride 0.004 g, thiamine hydrochloride 0.0004 g, calcium pantothenate 0.0008 g, biotin 0.00002 g, dissolved in 1 L of water, after dissolution, 1 mL of 0.1 g / L resazurin solution is added, boiled until the color changes; Then bottle, immediately flush nitrogen into the bottle, keep the surface of the medium in an anaerobic state, then cap, sterilize and cool; The water is the second grade water specified in GB / T 6682-2008; Add enteric flora or fecal suspension containing enteric flora to the folic acid assay medium, and then add the reduced folic acid or non-reduced folic acid to be tested, so as to determine the regulation function of the reduced folic acid and non-reduced folic acid on the enteric flora. Comprise the following steps:

2. A method for establishing an in vitro intestinal simulation system of folate deficiency and determining the regulatory effects of different types of folate on human intestinal flora, characterized in that Add the fecal suspension to the folic acid assay medium in claim 1 at a volume ratio of (10±1)%, so as to obtain an in vitro intestinal simulation system lacking folic acid; Add the reduced folic acid or non-reduced folic acid to be tested to the above-mentioned in vitro intestinal simulation system lacking folic acid to form a reaction system, so as to determine the regulation function of the reduced folic acid and non-reduced folic acid on the enteric flora.

3. The method of claim 2, characterized in that: The preparation method of the fecal suspension is as follows: take fresh morning feces of the donor (0.8±0.1) g / person, add 8 mL of 0.1 mM PBS after sterilization, vortex and mix uniformly, and filter out large particles in the fecal suspension with a 0.24 mm filter screen.

4. The method of claim 3, characterized in that: Culture the reaction system at 37℃ for 24 hours; the obtained culture is used for 16S rDNA identification and detection of short-chain fatty acids.

5. The method of claim 4, characterized in that: Illumina platform is used for double-end sequencing of the 16S rDNA gene V3-V4 region of the sequencing sample; the sequencing platform is MiSeq PE250, the sequencing primer is a universal primer, the amplification primer is 341F: 5'-CCTAYGGGRBGCASCAG-3' and 806R: 5'-GGACTACHVGGGTWTCTAAT-3', and the thermal cycling conditions are as follows: 95℃ for 5 minutes; 30 cycles of 95℃ for 30 seconds, 50℃ for 30 seconds, 72℃ for 40 seconds, and 72℃ for 7 minutes. ​ 6. The method of claim 5, wherein: GC-MS is used to detect short-chain fatty acids in the fecal suspension fermentation sample; Chromatographic system: Agilent DB-WAX capillary column; carrier gas is high-purity nitrogen, flow rate is 1.0 mL / min; injection port temperature is 220°C, injection amount is 1 μL, splitless injection, solvent delay time is 2.5 min; Mass spectrometry system: electron impact ion source, ion source temperature is 230°C, interface temperature is 220°C; The content of short-chain fatty acids in the fermentation broth is determined.

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