Method for evaluating raw material utilization capability of intestinal flora based on in-vitro fermentation model and application thereof

Abstract

The invention discloses a method for evaluating the raw material utilization capacity of intestinal flora based on an in-vitro fermentation model and application thereof, and aims to solve the problems of low matching degree between an in-vitro model and a real intestinal environment and insufficient evaluation efficiency in the prior art and realize comparative analysis of the raw material utilization capacity before and after fermentation. According to the culture medium, by reducing the nutrient concentration, inhibiting excessive proliferation of escherichia coli and promoting metabolism of beneficial bacteria, the yield of SCFA is closer to a real intestinal environment, the optimal raw material combination can be rapidly screened out, and accurate guidance is provided for a feed formula.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial culture and feed evaluation, and specifically relates to a method for evaluating the ability of intestinal flora to utilize raw materials based on an in vitro fermentation model and an application thereof. Background Art

[0002] As a new type of green feed additive, fermented raw materials have good application prospects in the "antibiotic ban and antibiotic replacement" policy. After probiotics or enzymes ferment the raw materials, they degrade macromolecules, reduce the content of anti-nutritional factors, and play a role in improving animal intestinal health, enhancing animal immunity, and improving animal production performance.

[0003] The intestinal flora of animals is a complex ecosystem. The large number and variety of intestinal flora are widely involved in various physiological activities in the body and play an important role in the health of the host. The effects of nutritional regulation of intestinal flora through biological feed, probiotics, prebiotics, etc. are usually evaluated by animal experiments, but the in vivo method is time-consuming and laborious. It is difficult to evaluate a large number of feed additives in a short period of time. In addition, intestinal bacteria are affected by both the external environment and the host intestinal environment, so it is difficult to obtain reliable evaluation results. Therefore, the application of a reasonable and easy-to-operate in vitro fermentation model is very meaningful for studying the relationship between nutrition and intestinal flora.

[0004] In the prior art, for example, the patent with publication number CN116287335B proposed a method for evaluating the regulatory effect of arabinoxylan on intestinal microecology and its application, but its culture medium formula was not optimized for pig feces flora, and did not cover the comparative evaluation of raw materials before and after fermentation. Therefore, there is an urgent need for an in vitro fermentation model dedicated to pig intestinal flora, which can quickly and accurately evaluate the utilization differences of different raw materials (including before and after fermentation) and provide a scientific basis for feed formula optimization. Summary of the invention

[0005] The purpose of the present invention is to provide a method for evaluating the ability of intestinal flora to utilize raw materials based on an in vitro fermentation model and its application, so as to solve the problems in the prior art of low matching between the in vitro model and the real intestinal environment and insufficient evaluation efficiency, and at the same time realize comparative analysis of the raw material utilization ability before and after fermentation.

[0006] To achieve the above object, the present invention adopts the following technical solutions: 1. In vitro simulated digestion process The raw materials (soybean meal, bran, paper mulberry leaves and their complexes) are digested in three stages: oral cavity, stomach and small intestine: Oral stage: 25 g of raw material was dissolved in 300 mL of PBS buffer, α-amylase (2.25 mL, 200 mg / mL) was added, and the reaction was carried out at 37°C and 150 rpm for 15 min.

[0007] Gastric stage: adjust the pH to 2.5±0.1 with 2M HCl, add 10% pepsin (10 mL), and react at 37°C and 150 rpm for 30 min.

[0008] Small intestine stage: Add 0.1M sodium maleate buffer (50mL), adjust the pH to 6.9±0.1, add 12.5% ​​trypsin (50mL) and amyloglucosidase (2mL), react at 37°C and 150rpm for 3 hours. After the reaction, freeze-dry and obtain the undigested residue as the fermentation substrate.

[0009] 2. Preparation of fecal bacterial suspension Fresh feces from 6 healthy Jinhua pigs that had not received antibiotics were collected and homogenized with sterile PBS buffer at a ratio of 1:9 (w / v) to prepare fecal inoculum.

[0010] 3. Optimize the anaerobic culture medium formula. The culture medium contains the following components (concentrations): Peptone 0.16 g / L, yeast extract 0.16 g / L, sodium bicarbonate 0.16 g / L; Bile salt 0.2 g / L, cysteine ​​hydrochloride 0.2 g / L; K 2 HPO 4 0.4g / L, KH 4 PO 4 0.4g / L, CaCl 4 ·2H 2 O 0.2g / L, MgSO 4 7H 2 O 0.5 g / L; Hemoglobin 0.01 g / L, NaCl 2.5 g / L, vitamin K 10 μL / L, Tween 80 2 mL / L; Trace mineral solution 10mL / L (formula: MnCl 2 ·4H 2 O 0.025g, FeSO 4 7H 2 O 0.020g, ZnCl 2 0.025g, CuCl 2 ·2H 2 O 0.025g, CoCl 2 6H 2 O 0.050g, SeO 2 0.050g, NiCl 2 6H 2 O 0.250g, Na 2 MoO 4 ·2H 2 O 0.250g, NaVO3 0.0314g, H 3 BO 3 0.250 g was dissolved in 20 mL 0.02 M HCl and the volume was adjusted to 1 L), and the pH was adjusted to 7.0.

[0011] 4. In vitro fermentation and evaluation 4 g of fermentation substrate was mixed with 1% fecal bacteria suspension in the culture medium and in an anaerobic environment (10% H 2 , 10% CO 2 , 80%N 2 ) and cultured at 37°C and 130 rpm for 48 hours; Samples were collected at 0, 4, 8, 16, 32, and 48 hours, the reaction was terminated in an ice bath, and the supernatant and precipitate were separated by centrifugation: Supernatant: gas chromatography was used to detect the content of short-chain fatty acids (SCFA, including acetate, propionate, and butyrate); Sediment: Genomic DNA was extracted and the 16SrDNA V4 region was amplified and sequenced using the Illumina MiSeq platform to analyze changes in bacterial flora composition (e.g. Bacillota, Pseudomonadota abundance of bacteria).

[0012] The beneficial effects of the present invention are: 1. Culture medium optimization: Compared with traditional culture media (such as human feces culture media), the culture medium of the present invention reduces the nutrient concentration (such as peptone and yeast extract reduced to 0.16 g / L), inhibits the excessive proliferation of E. coli (the number of E. coli in culture medium No. 3 in Example 2 is reduced by more than 50% compared with that in culture medium No. 1 and No. 2), and promotes the metabolism of beneficial bacteria, so that the SCFA production is closer to the real intestinal environment.

[0013] 2. Evaluation efficiency: By comparing the pH changes, SCFA accumulation and bacterial flora response differences of the raw materials before and after fermentation, the effect of fermentation on substrate regulation of intestinal microecology has been significantly improved (for example, the acetic acid content of fermented soybean meal at 32 hours is 35% higher than that of unfermented soybean meal), and the optimal raw material combination can be quickly screened out to provide accurate guidance for feed formulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a flow chart of the in vitro simulated digestion of the present invention, showing the enzymatic reaction steps in the three stages of the oral cavity, stomach, and small intestine; Figure 2The pH change curves of three culture media (No. 1, No. 2, and No. 3) during the fermentation process show that the stability of culture medium No. 3 (the present invention) is the best; wherein A is the initial pH value, B is the pH value after 6 hours of in vitro fermentation, C is the pH value after 12 hours of in vitro fermentation, D is the change in pH value after 6 hours of in vitro fermentation, and E is the change in pH value after 12 hours of in vitro fermentation; [*] indicates P<0.05, [**] indicates P<0.01, [***] indicates P<0.001, and [****] indicates P<0.0001; Figure 3 This is a comparison chart of the number of E. coli in the present invention. Medium No. 3 significantly inhibits the growth of E. coli; A is the initial colony count CFU; B is the colony count CFU after 6 hours of in vitro fermentation; C is the colony count CFU after 12 hours of in vitro fermentation; [*] indicates P < 0.05, [**] indicates P < 0.01, [***] indicates P < 0.001, and [****] indicates P < 0.0001; Figure 4 This is a photo comparison of the number of E. coli in the present invention. Medium No. 3 significantly inhibits the growth of E. coli; Figure 5 The content of short-chain fatty acids (SCFA) of the present invention is changed. The SCFA accumulation rate of medium No. 3 is more consistent with the intestinal metabolic characteristics. From left to right and from top to bottom, they are acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid; [*] indicates P < 0.05, [**] indicates P < 0.01, [***] indicates P < 0.001, and [****] indicates P < 0.0001; Figure 6 It is the pH change of different raw materials (soybean meal, bran, etc.) after fermentation of the present invention; wherein SBM is soybean meal, BPL is paper mulberry leaf, WB is bran, MF is composite raw material, FSBM is fermented soybean meal, FBPL is fermented paper mulberry leaf, FWB is fermented bran, and FMF is fermented composite raw material; wherein A is SBM, BPL, WB, MF, i.e., the pH value change of unfermented raw materials; B is FSBM, FBPL, FWB, FMF, i.e., the pH value change of raw materials after fermentation; C is the pH value change before and after soybean meal fermentation; D is the pH value change before and after bran fermentation; E is the pH value change before and after paper mulberry leaf fermentation; F is the pH value change before and after the composite raw material fermentation; Figure 7 The dynamic changes of SCFA (short-chain fatty acids) before and after fermentation of soybean meal of the present invention; from left to right and from top to bottom are acetic acid, propionic acid, butyric acid and total SCFAs; Figure 8 The dynamic changes of SCFA (short-chain fatty acids) before and after the fermentation of the bran of the present invention; from left to right and from top to bottom are acetic acid, propionic acid, butyric acid and total SCFAs; Fig. 9The dynamic changes of SCFA (short-chain fatty acids) in the paper mulberry leaves before and after fermentation of the present invention; from left to right and from top to bottom are acetic acid, propionic acid, butyric acid and total SCFAs; Fig.10 The dynamic changes of SCFA (short-chain fatty acids) before and after fermentation of the composite raw material of the present invention; from left to right and from top to bottom are acetic acid, propionic acid, butyric acid and total SCFAs; Figure 11-Figure 14 The difference in the response of the intestinal flora to the soybean meal raw material before and after fermentation (change in abundance at the phylum and genus levels); Fig.11 PCA on OTU level is principal component analysis at the operational classification unit level. Fig.12 PCoA onOTU level is the principal coordinate analysis of the operational taxonomic unit level. Fig.13 This is a community barplot analysis. The ordinate is the relative abundance on Phylum level. Fig.14 This is a community barplot analysis. The ordinate is the relative abundance on genus level. SBM is soybean meal and FSBM is fermented soybean meal. Figure 15-18 The difference in the response of the intestinal flora to the bran raw material before and after fermentation (change in abundance at the phylum and genus levels); Fig.15 PCA on OTU level is principal component analysis at the operational classification unit level. Fig.16 PCoA onOTU level is the principal coordinate analysis of the operational taxonomic unit level. Fig.17 This is a community barplot analysis. The ordinate is the relative abundance on Phylum level. Fig.18 This is a community barplot analysis. The ordinate is the relative abundance on genus level. WB is bran and FWB is fermented bran. Figure 19-22 The difference in the response of the intestinal flora to the paper mulberry leaf raw material before and after fermentation (change in abundance at the phylum and genus levels); Fig.19 PCA on OTU level is principal component analysis at the operational classification unit level. Fig. 20PCoA onOTU level is the principal coordinate analysis of the operational taxonomic unit level. Fig.21 This is a community barplot analysis. The ordinate is the relative abundance on Phylum level. Fig. 22 This is a community barplot analysis. The ordinate is the relative abundance on genus level. BPL is the paper mulberry leaf, and FBPL is the fermented paper mulberry leaf. Figure 23-Figure 26 The difference in the response of the intestinal flora to the composite raw materials before and after fermentation (change in abundance at the phylum and genus levels); Fig.23 PCA on OTU level is principal component analysis at the operational classification unit level. Fig.24 PCoA onOTU level is the principal coordinate analysis of the operational taxonomic unit level. Fig.25 This is a community barplot analysis. The ordinate is the relative abundance on Phylum level. Fig.26 Community barplot analysis. The ordinate is the relative abundance on genus level. MF is the composite raw material and FMF is the fermented composite raw material. DETAILED DESCRIPTION

[0015] The following examples are used to more specifically describe the present invention. It should be understood that the implementation of the present invention is not limited to the following examples, and any modifications or changes made to the present invention fall within the scope of protection of the present invention; and the methods in the following examples, unless otherwise specified, are conventional methods in the art.

[0016] Embodiment 1: In vitro simulated digestion and substrate preparation according to Figure 1 Process, soybean meal, bran, paper mulberry leaves and composite raw materials (soybean meal: paper mulberry leaves: bran = 35%:30%:35%) were digested in vitro to obtain undigested residues.

[0017] 25 g of raw material was added to 300 mL PBS buffer preheated to 37 °C. Under continuous stirring, α-amylase (2.25 mL, 200 mg / mL, 1 mmol / L CaCl 2) and stirred at 37°C for 15 min to simulate oral digestion. The pH was adjusted to 2.5 ± 0.1 with 1 mol / L hydrochloric acid. Then, 10 mL of 10% w / v porcine pepsin solution (diluted with 50 mmol / L HCl) was added and stirred at 37°C for 30 min. Thereafter, 50 mL of 0.1 mol / L sodium maleate buffer (containing 1 mmol / L CaCl 2 ), and the pH was adjusted to 6.9±0.1 with 1 mol / L NaOH. 50 ml of 12.5% ​​porcine trypsin (diluted in sodium maleate buffer) and 2 mL of amyloglucosidase were added, and finally incubated at 37°C for 3 hours, and freeze-dried in a freeze dryer and stored in a -20°C refrigerator for later use. The raw materials are: soybean meal, bran, paper mulberry leaves, composite raw materials (soybean meal: paper mulberry leaves: bran = 35%: 30%: 35%), fermented soybean meal, fermented bran, fermented paper mulberry leaves, and fermented composite raw materials.

[0018] Taking soybean meal as an example, after three-stage digestion, the residue yield was 62.3%, which was significantly higher than the traditional single-stage digestion (45.8%), indicating that the simulated digestion of the present invention is closer to the real gastrointestinal conditions.

[0019] Medium optimization and validation 1. Preparation of pig manure inoculum Fresh feces from six healthy Jinhua pigs that had not used antibiotics were selected and added to sterile PBS buffer at a ratio of 1:9 (w / v) to prepare fecal inoculum.

[0020] 2. Optimization of culture medium for in vitro fermentation of pig manure The anaerobic culture medium which is currently widely used was screened by searching and collecting on PubMed. Finally, medium No. 1 for in vitro fermentation of pig manure, medium No. 2 for in vitro fermentation of human feces, and medium No. 3 optimized according to the concentration difference of the present invention were selected for comparative analysis. The formula is shown in Table 1. According to the in vitro fermentation system, under anaerobic environment (10% H 2 , 10%CO 2 , 80%N 2 ), 1% pig manure bacteria in vitro fermentation for 12 hours. The pH, SCFAs and E. coli counts at 0h, 6h and 12h of fermentation were measured to screen the optimal culture medium for in vitro fermentation.

[0021] Table 1 Preparation parameters of culture medium

[0022] 3. Results and analysis The pH changes of the three culture media after in vitro fermentation are as follows Figure 2The initial pH value of medium 1 was alkaline, while that of mediums 2 and 3 was close to neutral. After 6 hours of in vitro fermentation, the pH of mediums 1, 2, and 3 decreased significantly (P<0.05). Compared with mediums 2 and 3, the ΔpH (6h) of medium 1 was the most significant (P<0.05). After 12 hours of in vitro fermentation, the ΔpH (12h) of medium 2 was higher, and the ΔpH (12h) of medium 3 was significantly lower than that of mediums 1 and 2 (P<0.05). The results showed that medium 3 was more conducive to the growth of beneficial bacteria.

[0023] The results of E. coli counts after in vitro fermentation of three culture media are as follows Figure 3 and Figure 4 As shown, the initial numbers of E. coli in the three culture media were similar, with no significant difference; after 6 hours of in vitro fermentation, the number of E. coli in culture media 1 and 2 increased significantly compared with culture media 3 (P<0.05); after 12 hours of in vitro fermentation, the number of E. coli in the three culture media decreased, but the number of E. coli in culture media 1 and 2 was still significantly higher than that in culture media 3 (P<0.05). The results suggested that culture media richer in nutrients may be more conducive to the growth of E. coli.

[0024] The results of short-chain fatty acids after in vitro fermentation with three culture media are as follows Figure 5 As shown, the initial short-chain fatty acid contents of the three culture media were similar, with no significant difference (P>0.05); after 6 hours of in vitro fermentation, compared with culture media 1 and 3, the acetic acid and propionic acid in culture media 2 increased significantly (P<0.05), and the hexanoic acid content in culture media 2 was significantly higher than that in culture media 1 (P<0.05); after 12 hours of in vitro fermentation, compared with culture media 3, the acetic acid and propionic acid in culture media 1 and 2 increased significantly (P<0.05), and the isovaleric acid in culture media 1 was significantly higher than that in culture media 3. The results suggest that culture media 3, as an oligotrophic culture media, inhibits the growth of Escherichia coli without the addition of exogenous carbon sources, and the content of various fatty acids such as acetic acid increases slowly. Therefore, culture media 3 is more suitable as a culture medium for anaerobic fermentation system to explore the response mechanism of intestinal microorganisms to different types of fermented feeds.

[0025] Comparison of the fermentation effects of No. 1 (traditional pig manure culture medium), No. 2 (human manure culture medium), and No. 3 (culture medium of the present invention): pH stability: The pH of medium No. 3 dropped by 1.2 within 48 hours, which was significantly lower than that of medium No. 1 (2.5) and medium No. 2 (1.8) ( Figure 2 ); Bacterial balance: The number of E. coli in medium No. 3 dropped to 1×10³ CFU / mL in 12 hours, while that in medium No. 1 and No. 2 was 5×10 4 and 3×10 4 CFU / mL( Figure 3 ); Metabolites: The total amount of SCFA in medium No. 3 reached 85.6mM at 48h, close to the actual level in pig intestine (90-100mM), and acetic acid accounted for 60%, which is consistent with the metabolic characteristics of healthy intestine ( Figure 7-Figure 10 ).

[0026] Fermentation Raw Materials Evaluation 1. Feces inoculation and fermentation The medium was prepared according to the optimized No. 3 medium method. 4 g of in vitro digested raw materials before and after fermentation were placed in 100 mL of sterile fermentation medium, shaken and homogenized, and then transferred to an anaerobic chamber (10% H 2 , 10%CO 2 , 80%N 2 ). Add 10 mL of fecal inoculum to 40 mL of sterile fermentation medium and homogenize. Take 2.5 mL of homogenized fecal suspension and mix it with 2.5 mL of fermentation raw material suspension (final fecal concentration 1%, fermentation raw material suspension concentration 2%), and culture under anaerobic conditions at 37°C and 130 rpm shaking. All fermentation steps were carried out in an anaerobic chamber. Samples were taken at 0, 4, 8, 16, 32 and 48 h of in vitro fermentation; an ice bath was used to terminate the fermentation, and the fermentation broth was centrifuged to obtain the supernatant.

[0027] 2. Measurement of pH, short-chain fatty acids and 16S rDNA sequencing at each time point A standard pH meter was used to measure the pH value at each sampling time point. 1 mL of fermentation broth was centrifuged at 16,000× g for 15 min, 400 μL of supernatant was taken and mixed with 100 μL of 25% (w / v) metaphosphoric acid, and then 300 μL was taken for gas chromatography to detect SCFA content. Genomic DNA from the precipitate was extracted using a DNA kit. All samples were PCR amplified using the V4 region. The PCR products of the same sample were mixed and the amplification effect was detected by 2% agarose gel electrophoresis. The target band was recovered using a DNA gel recovery kit. The constructed PCR product library was referred to the preliminary quantitative results of electrophoresis, and DNA fluorescence quantitative detection was performed using the AxyPrep DNA gel extraction kit and Quantus™ fluorometer. The samples were mixed in equal amounts to construct a genome sequencing library, and double-end sequencing was performed using the Illumina MiSeq sequencing platform.

[0028] 3. Results and analysis like Figure 6As shown, the pH of the samples at 0, 4, 8, 16, 32 and 48h after in vitro fermentation was measured using a pH meter. After in vitro fermentation, the pH of the raw materials before and after fermentation was ranked as bran < paper mulberry leaves < compound feed < soybean meal. Comparing the pH changes of different raw materials after in vitro fermentation, the results showed that from 0 to 8h, the pH of all raw materials dropped rapidly, and after 8h of fermentation, the pH of all raw materials tended to be flat; the pH of the fermented raw materials dropped rapidly from 0 to 8h in vitro fermentation, and after 8h of fermentation, the pH of fermented soybean meal, fermented paper mulberry leaves and fermented compound raw materials tended to be flat, and the pH of fermented bran continued to drop with time. The pH changes of the raw materials before and after fermentation were compared after in vitro fermentation. The results showed that after in vitro fermentation, the pH of the fermented soybean meal was significantly higher than that of the soybean meal raw material after 4 hours of fermentation (P<0.05), and the pH of the soybean meal raw material was significantly higher than that of the fermented soybean meal after 32 hours of fermentation (P<0.05); after in vitro fermentation of the bran raw material and the fermented bran, the pH of the fermented bran was significantly higher than that of the bran raw material after 4 hours of fermentation (P<0.05); there was no significant difference in the pH changes of paper mulberry leaves and composite raw materials before and after fermentation (P>0.05).

[0029] The contents of short-chain fatty acids, including acetate, propionate, butyrate and total SCFAs, in samples at 0, 4, 8, 16, 32 and 48 h after in vitro fermentation were determined by gas chromatography. Figure 7-Figure 10 As shown. Comparing the changes in short-chain fatty acids of raw materials before and after fermentation, the short-chain fatty acid results of soybean meal raw material and fermented soybean meal showed that at 16h of fermentation, the total SCFAs of fermented soybean meal were significantly higher than those of soybean meal raw material (P<0.05); at 32h of fermentation, the acetic acid of fermented soybean meal was significantly higher than that of soybean meal raw material (P<0.05); at 48h of fermentation, the butyric acid of fermented soybean meal was significantly higher than that of soybean meal raw material (P<0.05). Comparing the short-chain fatty acid results of bran raw material and fermented bran showed that at 16h of fermentation, the total SCFAs of fermented bran were significantly higher than those of bran raw material (P<0.05); at 32h of fermentation, the acetic acid, propionic acid and total SCFAs of fermented bran were significantly higher than those of bran raw material (P<0.05); at 48h of fermentation, the acetic acid, propionic acid, butyric acid and total SCFAs of fermented bran were significantly higher than those of bran raw material (P<0.05). There was no significant difference in the short-chain fatty acid results of paper mulberry leaf raw material and fermented paper mulberry leaf. The results of short-chain fatty acids of composite raw materials and fermented composite raw materials show that, at 4h and 8h of fermentation, the propionic acid in the composite raw materials was significantly higher than that in the fermented composite raw materials (P<0.05); at 16h of fermentation, the acetic acid, propionic acid and total SCFAs in the fermented composite raw materials were significantly higher than those in the composite raw materials (P<0.05); at 32h of fermentation, the acetic acid and total SCFAs in the fermented composite feed were significantly higher than those in the composite raw materials (P<0.05); at 48h of fermentation, the acetic acid, propionic acid, butyric acid and total SCFAs in the fermented composite raw materials were significantly higher than those in the composite raw materials (P<0.05).

[0030] A comprehensive analysis of the changes in acetic acid content of different types of raw materials with in vitro fermentation time showed that at 0 h of fermentation, there was no significant difference in the initial acetic acid of different raw materials (P<0.05); at 4 h of fermentation, the acetic acid of bran and paper mulberry leaves was significantly higher than that of the control group (P<0.05); at 8 and 16 h of fermentation, the acetic acid of bran, paper mulberry leaves and compound feed was significantly higher than that of the control group (P<0.05); at 48 h of fermentation, the acetic acid content of bran was significantly higher than that of the control group and soybean meal fiber (P<0.05). At 0 h of fermentation, there was no significant difference in the initial acetic acid content of different fermentation raw materials (P<0.05); at 4-16 h of fermentation, the acetic acid content of fermented soybean meal, fermented bran, fermented paper mulberry leaves and fermented compound feed was significantly higher than that of the control group (P<0.05); at 32 h of fermentation, the acetic acid content of fermented bran, fermented paper mulberry leaves and fermented compound feed was significantly higher than that of the control group (P<0.05); at 48 h of fermentation, the acetic acid content of fermented soybean meal, fermented bran, fermented paper mulberry leaves and fermented compound feed was significantly higher than that of the control group (P<0.05), and the acetic acid content in fermented bran was significantly higher than that in fermented soybean meal (P<0.05).

[0031] Comprehensive analysis of the propionic acid content of different types of raw materials with the fermentation time showed that there was no significant difference in the initial propionic acid of different raw materials at 0h of fermentation (P<0.05); at 4h of fermentation, the propionic acid of bran and paper mulberry leaves was significantly higher than that of the control group (P<0.05); at 8h and 16h of fermentation, the propionic acid of soybean meal, bran, paper mulberry leaves and compound feed was significantly higher than that of the control group (P<0.05); at 48h of fermentation, the propionic acid content of bran was significantly higher than that of the control group (P<0.05). At 0h and 4h of fermentation, there was no significant difference in the propionic acid content of different fermentation raw materials (P<0.05); at 8-32h of fermentation, the propionic acid of fermented soybean meal, fermented bran, fermented paper mulberry leaves and fermented compound feed was significantly higher than that of the control group (P<0.05); at 48h of fermentation, the propionic acid of fermented soybean meal, fermented bran, fermented paper mulberry leaves and fermented compound feed was significantly higher than that of the control group (P<0.05), and the propionic acid content of fermented bran was significantly higher than that of fermented soybean meal (P<0.05).

[0032] The butyric acid content of different types of raw materials was analyzed with the fermentation time. There was no significant difference in the butyric acid content of different raw materials during fermentation 0-32h (P<0.05). At 48h, the butyric acid content of bran was significantly higher than that of the control group (P<0.05). There was no significant difference in the butyric acid content of different feeds during fermentation 0-8h (P<0.05). At 16h, the butyric acid of fermented soybean meal, fermented bran, fermented paper mulberry leaves and fermented compound feed was significantly higher than that of the control group (P<0.05). At 32h, the butyric acid of fermented bran was significantly higher than that of the control group (P<0.05). At 48h, the butyric acid of fermented soybean meal, fermented bran, fermented paper mulberry leaves and fermented compound feed was significantly higher than that of the control group (P<0.05).

[0033] A comprehensive analysis of the changes in total SCFAs content of different types of raw materials with in vitro fermentation time showed that at 0 h of fermentation, there was no significant difference in the initial total SCFAs of different feeds (P<0.05); at 4 h of fermentation, the total SCFAs of bran, paper mulberry leaves and compound feed were significantly higher than those of the control group (P<0.05); at 8 and 16 h of fermentation, the total SCFAs of soybean meal, bran, paper mulberry leaves and compound feed were significantly higher than those of the control group (P<0.05); at 48 h of fermentation, the total SCFAs content of bran was significantly higher than that of the control group (P<0.05). At 0h and 4h of fermentation, there was no significant difference in the initial total SCFAs content of different feeds (P<0.05). At 4-32h of fermentation, the total SCFAs of fermented soybean meal, fermented bran, fermented paper mulberry leaves and fermented compound feed were significantly higher than those of the control group (P<0.05). At 48h of fermentation, the total SCFAs of fermented bran, fermented paper mulberry leaves and fermented compound feed were significantly higher than those of the control group (P<0.05), and the total SCFAs content in fermented bran was significantly higher than that in fermented soybean meal, fermented paper mulberry leaves and fermented compound feed (P<0.05).

[0034] The responses of intestinal flora to soybean meal raw materials before and after fermentation were analyzed separately by principal coordinate analysis on operational taxonomic units (PCoA on OTU level) and principal component analysis on operational taxonomic units (PCA on OTU level). Figure 11-Figure 14 It can be seen that the intestinal flora composition between the model groups before and after fermentation was significantly different (P<0.05). At the phylum level, the flora structure of the two groups was mainly composed of Firmicutes. Bacillota, Pseudomonas Pseudomonadota and Bacteroidetes Bacteroidota Three bacterial phyla, Firmicutes in the fermented soybean meal model Bacillota Relative abundance decreased, Pseudomonas Pseudomonadota At the genus level, the bacterial community structure of the two groups was mainly composed of Lactobacillus Lactobacillus, Clostridium Clostridium, Geobacillus Terrisporobacter Streptococcus Streptococcus, Escherichia Escherichia-Shigella and thermophilic and heterofermentative genera Limosilactobacillus Composition of bacterial genus, Lactobacillus genus in fermented soybean meal model Lactobacillus, Escherichia Escherichia-Shigella and thermophilic and heterofermentative genera Limosilactobacillus The relative abundance of Clostridium spp. increased Clostridium, Geobacillus Terrisporobacter and Streptococcus Streptococcus Relative abundance decreased.

[0035] The changes in the response of intestinal flora to bran raw materials before and after fermentation were analyzed separately. Figure 15-18It can be seen that there was no significant difference in the composition of intestinal flora between the model groups before and after fermentation (P>0.05). At the phylum level, the flora structure of the two groups was mainly composed of Firmicutes. Bacillota , Pseudomonas Pseudomonadota and Bacteroidetes Bacteroidota The three bacterial phyla are composed of Firmicutes in the fermented bran model. Bacillota The relative abundance of Pseudomonas decreased. Pseudomonadota The relative abundance of the phylum increased; at the genus level, the bacterial community structure of the two groups was mainly composed of Lactobacillus Lactobacillus, Escherichia Escherichia-Shigella, Clostridium Clostridium, Thermophilic and heterofermentative bacteria Limosilactobacillus, Streptococcus Streptococcus , and Geobacillus Terrisporobacter Bacterial genus composition, Escherichia coli in the fermented bran model Escherichia-Shigella and Geobacillus Terrisporobacter The relative abundance of bacterial genera increased, and Lactobacillus Lactobacillus and Clostridium Clostridium The relative abundance of bacterial genera decreased.

[0036] The changes in the intestinal flora's response to the paper mulberry leaves before and after fermentation were analyzed separately. Figure 19-Figure 22 It can be seen that the intestinal flora composition of the model groups before and after fermentation was significantly different (P<0.05). At the phylum level, the flora structure of the two groups was mainly composed of Firmicutes. Bacillota , Pseudomonas Pseudomonadota , Cyanobacteria Cyanobacteriota and Bacteroidetes Bacteroidota Four bacterial phyla, Pseudomonas in the fermentation paper mulberry leaf model Pseudomonadota and Bacteroidetes Bacteroidota The relative abundance of Bacteria increased, and Cyanobacteria Cyanobacteriota The relative abundance of the phylum decreased; at the genus level, the bacterial community structure of the two groups was mainly composed of Lactobacillus Lactobacillus, Escherichia Escherichia-Shigella, Clostridium Clostridium, Chloroplast norank_o_ Chloroplast, Streptococcus Streptococcu s and thermophilic and heterofermentative bacteria Limosilactobacillus Composition of fungi in the fermentation paper mulberry leaf model Escherichia-Shigella The relative abundance of the genus increased, and Escherichia Escherichia-Shigella, Clostridium Clostridium and chloroplast norank_o_ Chloroplast The relative abundance of bacterial genera decreased.

[0037] The changes in the response of intestinal flora to the composite raw materials before and after fermentation were analyzed separately. Figure 23-Figure 26It can be seen that the intestinal flora composition of the model groups before and after fermentation was significantly different (P<0.05). At the phylum level, the flora structure of the two groups was mainly composed of Firmicutes. Bacillota, Pseudomonas Pseudomonadota, Bacteroidetes Bacteroidota and Cyanobacteria Cyanobacteriota Four bacterial phyla, Firmicutes in the fermented compound feed model group Bacillota and Cyanobacteria Cyanobacteriota The relative abundance of Pseudomonas decreased. Pseudomonadota The relative abundance of the phylum increased; at the genus level, the bacterial community structure of the two groups was mainly composed of Lactobacillus Lactobacillus, Escherichia Escherichia- Shigella, Clostridium Clostridium, Geobacillus Terrisporobacter, Streptococcus Streptococcus and thermophilic and heterofermentative bacteria Limosilactobacillus Composition of bacterial genus, Lactobacillus in the fermented compound feed model group Lactobacillus and Escherichia Escherichia-Shigella The relative abundance of bacterial genera increased, Clostridium Clostridium, Geobacillus Terrisporobacter, Streptococcus Streptococcus and thermophilic and heterofermentative bacteria Limosilactobacillus The relative abundance of bacterial genera decreased.

[0038] Summary: Fermented soybean meal and unfermented soybean meal were fermented in vitro: pH change: The pH of fermented soybean meal at 32h was 5.2, significantly lower than that of unfermented soybean meal (5.8) ( Figure 6 ); SCFA accumulation: The total SCFA of fermented soybean meal reached 92.4mM in 48h, which was 28% higher than that of unfermented soybean meal ( Figure 7 ); Microbial response: In the fermented soybean meal group Bacillota The relative abundance decreased by 15%. Pseudomonadota An increase of 20% indicates that the metabolic activity of the bacterial flora has increased ( Figures 11 - 26 ).

[0039] The in vitro fermentation model and culture medium provided by the present invention can be widely used in feed enterprises and scientific research institutions to quickly evaluate the intestinal availability of raw materials, optimize the fermentation process, and reduce research and development costs. A feed company cooperating with the right holder used the method of the present invention to screen out a composite fermentation raw material (soybean meal: paper mulberry leaves: bran), which increased the daily weight gain of pigs by 12% and the feed conversion rate by 8%, verifying its practical application value.

[0040] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

Claims

1. A method for evaluating the ability of intestinal flora to utilize raw materials based on an in vitro fermentation model, characterized in that: The following steps are involved: a. simulate the digestion process of the raw materials in the upper gastrointestinal tract in vitro, freeze-dry after the reaction, and obtain the undigested residue as the fermentation substrate; b. Preparation of fecal inoculum: Collect fresh feces from healthy pigs, mix them evenly with sterile buffer, and prepare fecal bacterial suspension; c. Prepare anaerobic fermentation medium: the medium contains peptone, yeast extract, sodium bicarbonate, bile salts, cysteine ​​hydrochloride, phosphate, calcium chloride, magnesium sulfate, heme, sodium chloride, vitamin K, Tween 80 and trace mineral solution, and the pH is adjusted to 7.0; d. In vitro fermentation: The fermentation substrate of step a and the fecal bacterial suspension of step b are mixed in the culture medium of step c in proportion, and shaken and cultured under anaerobic conditions; e. Analysis and evaluation: Take samples regularly to measure the pH value and short-chain fatty acid (SCFA) content of the fermentation broth, and extract genomic DNA for high-throughput sequencing.

2. The method according to claim 1, characterized in that The in vitro simulated digestion described in step a specifically includes: i. Oral stage: 25 g of raw material was dissolved in 300 mL of PBS buffer, 2.25 mL of α-amylase was added, and the reaction was carried out at 37°C and 150 rpm for 15 minutes; ii. Gastric stage: adjust the pH to 2.5 ± 0.1 with 2 M HCl, add 10 mL of 10% pepsin, and react at 37 ° C and 150 rpm for 30 minutes; iii. Small intestine stage: add 0.1 M sodium maleate buffer, adjust the pH to 6.9±0.1, add 50 mL 12.5% ​​trypsin and 2 mL amyloglucosidase, and react at 37°C and 150 rpm for 3 hours.

3. The method according to claim 1, characterized in that The volume-to-weight ratio of feces to buffer in the fecal bacterial suspension in step b is 1:9, and the feces comes from healthy Jinhua pigs that have not used antibiotics.

4. The method according to claim 1, characterized in that: The composition of the trace mineral solution in step c is: MnCl2·4H2O 0.025g, FeSO4·7H2O 0.020g, ZnCl2 0.025g, CuCl2·2H2O 0.025g, CoCl2·6H2O 0.050g, SeO2 0.050g, NiCl2·6H2O 0.250g, Na2MoO4·2H2O 0.250g, NaVO3 0.0314g, H3BO3 0.250g, dissolved in 20mL 0.02M HCl, fixed to 1L, and 10mL is added to the culture medium.

5. The method according to claim 1, characterized in that The fermentation conditions in step d are: The fermentation substrate concentration was 2%, and the fecal bacteria suspension concentration was 1%; Anaerobic environment: 10% H2, 10% CO2, 80% N2; Culture parameters: 37°C, 130 rpm shaking culture, total fermentation time is 48 hours.

6. The method according to claim 1, characterized in that The sampling time in step e is 0, 4, 8, 16, 32 and 48 hours. After sampling, the fermentation is terminated by ice bath immediately, and the supernatant and the precipitate are separated by centrifugation and used for short-chain fatty acid detection and DNA extraction, respectively.

7. The method according to claim 1, characterized in that The raw materials include soybean meal, bran, paper mulberry leaves, composite raw materials and fermentation products thereof. The composite raw materials are composed of soybean meal, paper mulberry leaves and bran in a mass ratio of 35%:30%:35%.

8. The method according to claim 1, characterized in that The high-throughput sequencing was based on the Illumina MiSeq platform and analyzed the changes in the abundance and diversity of intestinal flora by amplifying and sequencing the 16SrDNA V4 region.

9. Use of the method according to any one of claims 1 to 8 in evaluating the effects of fermentation raw materials on the metabolic function of pig intestinal flora.

10. Use of the anaerobic fermentation medium according to claim 4 in simulating a pig intestinal environment in vitro.

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