CN114908019B - Enterococcus faecium compound microbial inoculum, compounding method and application thereof - Google Patents
Enterococcus faecium compound microbial inoculum, compounding method and application thereof Download PDFInfo
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Abstract
The invention discloses an enterococcus faecium compound microbial inoculum, a compounding method and application thereof. The invention adopts a mode of compounding the enterococcus faecium B13 and the saccharomyces cerevisiae Y21 obtained by screening, and utilizes the mutual synergistic effect of the enterococcus faecium B13 and the saccharomyces cerevisiae Y21 to form the high-efficiency stable enterococcus faecium compound microbial inoculum. The enterococcus faecium compound microbial agent and the compounding method thereof can regulate the population composition of intestinal microorganisms, reduce the abundance of harmful bacteria in the intestinal microorganisms, promote the abundance of beneficial bacteria, strengthen the intestinal immunity and maintain the integrity of intestinal barriers of mice. In animal feeding test, the contents of constant bile acid Taurocholate (TCA), taurochenodeoxycholic acid (TCDCA), taurochenodeoxycholic acid (TDCA) and taurochenodeoxycholic acid (TUDCA) in the compound group of enterococcus faecium B13 and Saccharomyces cerevisiae Y21 are obviously improved (P < 0.05).
Description
Technical Field
The invention relates to the field of application of microbial technology, in particular to an enterococcus faecium compound bacterial agent, a compounding method and application thereof.
Background
Enterococcus faecium (Enterococcus Faecium) belongs to the genus enterococcus and is part of the normal flora in the human and animal intestinal tract. The enterococcus faecium is round or oval, is gram-positive cocci arranged singly, in pairs or in short chains, has no spores and flagella, is aerobic or facultative anaerobic bacteria, and has fewer pairs and chains when observed under an oil microscope.
A great deal of experimental evidence suggests that ingestion of probiotics can alleviate or prevent various diseases such as lactose intolerance, diarrhea, and atopic diseases. In animal production, the probiotic preparation can improve animal production performance and ensure animal health. Enterococcus faecium and saccharomycetes are used as common inner flora in food, and have the effects of improving intestinal flora, inhibiting growth of harmful bacteria, enhancing immunity and maintaining bile acid metabolism balance. Currently, the products of the microecological preparations sold on the market can have a certain probiotic effect in actual use, but a plurality of products have a plurality of defects: the probiotic effect exerted by a single probiotic preparation has limitations; the viable count of the product is unstable, so that the actual efficacy of the product is greatly reduced; the organism gastrointestinal tract environment has complexity and variability, and the probiotics composite strain has unstable effect in actual use. Therefore, the probiotic composite microbial inoculum with low cost, high yield and long-term activity maintenance is developed, and has important significance for practical production and application of the probiotic composite microbial inoculum.
Disclosure of Invention
Aiming at the defects, the invention aims to provide an enterococcus faecium compound bacterial agent, a compounding method and application thereof. The enterococcus faecium B13 and the saccharomyces cerevisiae Y21 with better safety are obtained by adopting the early-stage experimental screening method, and the enterococcus faecium B13 and the saccharomyces cerevisiae Y21 are mutually synergistic to form the high-efficiency stable enterococcus faecium compound microbial inoculum, so that the compound microbial inoculum can adapt to the complexity and variability of the gastrointestinal tract environment of the organism. The enterococcus faecium compound bacterial agent can reduce the abundance of harmful bacteria in intestinal microorganisms, promote the abundance of beneficial bacteria, strengthen intestinal immunity, maintain the integrity of intestinal barrier, and has important significance for improving intestinal flora and bile acid metabolic disorder.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides enterococcus faecium (Enterococcus faecium), which is named enterococcus faecium B13 and is preserved in China general microbiological culture Collection center (CGMCC No. 23693) with the preservation address of North Chen Xiyu No. 1, no. 3 in the Korean region of Beijing city, and the preservation date of 2021, 10 months and 29 days.
The invention provides a saccharomyces cerevisiae (Saccharomyces cerevisiae), which is named as saccharomyces cerevisiae Y21 and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, wherein the preservation center is No. 1, 3 of North Chen Xili in the area of Beijing, the ward, and the preservation date is 2021, 10 months and 29 days, and the preservation number is CGMCC No.23692.
The invention provides an enterococcus faecium composite microbial agent, which consists of the following strains: enterococcus faecium B13 and Saccharomyces cerevisiae Y21.
Further, the compounding ratio of enterococcus faecium B13 to saccharomyces cerevisiae Y21 is 10-100:1; preferably 100:1.
Further, the enterococcus faecium compound bacterial agent is in the form of freeze-dried bacterial powder or liquid aqua.
The preparation form of the enterococcus faecium composite bacterial agent is not limited to the freeze-dried bacterial powder or the liquid water agent, and can be selected according to actual needs, and auxiliary materials commonly used in the field can be added.
The invention also provides a compounding method of the enterococcus faecium composite microbial inoculum, which comprises the following steps: enterococcus faecium B13 and saccharomyces cerevisiae Y21 are subjected to compound feeding.
It should be noted that the basic ration may be prepared according to conventional methods in the art or purchased directly from commercial products, and is not a critical and necessary component of the enterococcus faecium complex inoculant of the present invention.
The invention also provides application of the enterococcus faecium composite microbial agent in animal cultivation.
A microbial additive for animal cultivation comprises the enterococcus faecium compound microbial agent.
The invention also provides application of the enterococcus faecium composite microbial inoculum in preparing medicines for inhibiting inflammation caused by Proteus infection.
Further, in the above application, the Proteus includes Klebsiella and Vibrio desulphus.
The invention also provides application of the enterococcus faecium compound microbial inoculum in preparing medicines for regulating bile acid metabolism in ileum.
The invention also provides application of the enterococcus faecium composite microbial inoculum in preparation of a microbial inoculum for repairing or enhancing intestinal functions.
The invention also provides application of the enterococcus faecium composite microbial inoculum in preparing an organism inflammation microbial inoculum for preventing and/or regulating food stress.
An intestinal probiotic preparation comprises the enterococcus faecium compound bacterial agent.
In summary, the invention has the following advantages:
1. the invention provides an enterococcus faecium compound microbial inoculum and a compounding method thereof, wherein the enterococcus faecium B13 and the saccharomycete Y12 screened by the interaction and co-culture interaction of metabolites are compounded, and the enterococcus faecium compound microbial inoculum is formed by utilizing the interaction synergistic effect of the enterococcus faecium B13 and the saccharomycete Y21; the feeding test of mice proves that compared with a single-bacteria preparation and normal feed feeding control group, the compounding method of enterococcus faecium B13 and saccharomyces cerevisiae Y21 can reduce the proportion of harmful flora proteobacteria in intestinal flora at the portal level, increase the proportion of the firmicutes, and the research shows that the rising of the firmicutes can improve the fat metabolism function of organisms, so that the intestinal barrier and the immune function of the organisms of the mice are more perfect. The ratio of harmful bacteria Klebsiella and Vibrio desulphus can be reduced at the genus level, the ratio of beneficial bacteria lactobacillus and Chaetomium can be increased, common lactobacillus in the intestinal tract comprises lactobacillus reuteri, lactobacillus acidophilus, lactobacillus bulgaricus and the like, and the lactobacillus has various probiotics such as promoting intestinal development, enhancing intestinal immunity, reducing diarrhea rate and the like; the genus chaetomium is a class of butyrate-producing bacteria that maintain the intestinal barrier integrity of mice. The method for compounding enterococcus faecium and Saccharomyces cerevisiae can improve intestinal microbial flora and protect intestinal tract. The invention has important influence on the reduction of harmful bacteria and the increase of beneficial bacteria in the intestinal flora, and the combination mode of enterococcus faecium and saccharomyces cerevisiae can be developed into a probiotic preparation for improving the imbalance of the intestinal flora and maintaining the healthy balance of the intestinal flora.
2. According to the invention, only enterococcus faecium B13 and saccharomyces cerevisiae Y21 are used for compounding for the first time, and the oral enterococcus faecium and saccharomyces cerevisiae compound liquid reagent is fed, so that the contents of constant bile acid Taurocholate (TCA), taurochenodeoxycholic acid (TCDCA), taurochenodeoxycholic acid (TDCA) and taurochenodeoxycholic acid (TUDCA) can be obviously improved (P is less than 0.05). The TCA can reduce the permeability of inflammatory tissue capillaries, inhibit inflammatory swelling, inhibit the generation of inflammatory mediators such as NO, PGE2, histamine and the like, improve the content of the inflammatory mediators in ileum and effectively increase the anti-inflammatory effect of the intestinal tract; TCDA can normalize clinical inflammation parameters and reduce intestinal inflammation; the elevation of bile acids TCDCA, TUDCA can activate the activity of liver bile acid synthase, promote the metabolic process of cholesterol synthesis bile acid, thereby reducing cholesterol level, and TCDCA can normalize clinical inflammation parameters, and reduce intestinal inflammation. Therefore, the complex microbial inoculum can reduce inflammatory reaction of intestinal tracts and prevent lipid metabolism disorder.
Drawings
Fig. 1-3: the compounding method of enterococcus faecium B13 and saccharomyces cerevisiae Y21 has important influence on the reduction of harmful bacteria and the increase of beneficial bacteria in the intestinal flora of mice, and can improve the imbalance of the intestinal flora and maintain the healthy balance of the intestinal flora.
Wherein, fig. 1: enterococcus faecium B13 and saccharomyces cerevisiae Y21 are compounded and fed into a mouse experimental route;
fig. 2: the compounding method of enterococcus faecium B13 and saccharomyces cerevisiae Y21 proves that the compounding method of enterococcus faecium B13 and saccharomyces cerevisiae Y21 can reduce the proportion of harmful flora and Proteus in intestinal flora at the portal level and increase the proportion of thick-walled bacteria portal;
fig. 3: the compounding method of enterococcus faecium B13 and Saccharomyces cerevisiae Y21 can reduce the proportion of harmful bacteria Klebsiella and Vibrio desulphus at the genus level and increase the proportion of beneficial bacteria Lactobacillus and Chairosporum.
Fig. 4-7: the compounding method of enterococcus faecium B13 and saccharomyces cerevisiae Y21 can promote the intestinal barrier function of mice and promote the metabolic balance of intestinal bile acid.
Wherein, fig. 4-5: the invention adopts UPLC-ESI-MS/MS to measure the bile acid content of the ileum content of the mice, and the total ion flow diagram reflects that the instrument has good repeatability of metabolite detection, and the detection result of the sample is good, so that the sample can be used as the subsequent result analysis.
Fig. 6-7: the invention adopts UPLC-ESI-MS/MS to measure 42 bile acid contents in the ileum content of the mice, and the detected bile acid content is 27.
Wherein, fig. 6: as a result of quantitative analysis by UPLC-ESI-MS/MS, the concentration of 18 kinds of constant bile acid is more than 1 mug/g. The major bile acids Taurocholate (TCA), taurochenodeoxycholate (TCDCA), taurochenodeoxycholate (TDCA) and Taurochenodeoxycholate (TUDCA) were found to be significantly elevated in content (P < 0.05) in the constant results.
Fig. 7: as a result of quantitative analysis of UPLC-ESI-MS/MS, 9 kinds of trace bile acids are obtained, the content is low and is less than 1 mug/g, and no obvious difference exists between groups.
FIGS. 8-10 are, respectively, enterococcus faecium B13 (Enterococcus Faecium B13) 16S rRNA gene sequence phylogenetic tree, enterococcus faecium B13 (Enterococcus Faecium B) rpoA gene sequence phylogenetic tree and Saccharomyces cerevisiae Y21 (Saccharomyces cerevisiae Y21) 26S rRNA gene sequence phylogenetic tree of the present invention.
FIGS. 11-12 are strain morphology diagrams of enterococcus faecium B13 and Saccharomyces cerevisiae Y21, respectively, in the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention.
Thus, the following detailed description of the embodiments of the invention, as provided, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.
EXAMPLE 1A method for compounding enterococcus faecium B13 and Saccharomyces cerevisiae Y21 can improve intestinal microbiota and protect intestinal tract
1.1 interaction relationship between enterococcus faecium (Enterococcus faecium) and Saccharomyces cerevisiae (Saccharomyces cerevisiae) and compound proportion determination
1.1.1 separation, purification, screen and identification of enterococcus faecium B13 and Saccharomyces cerevisiae Y21
The separation and purification of enterococcus faecium B13 comprises the following steps:
grinding 10g of millet pickled peppers, adding into a 10mL sterilized EP tube, sucking 0.1mL of supernatant by a gun head, performing gradient dilution by a 10-time dilution method with sterile water, and selecting 100uL 10 -3 ～10 -5 The diluted samples were plated on sterilized modified MC medium and incubated at 30℃for 24-48h. And (3) picking colonies generating calcium circles, repeatedly streaking and culturing in an MRS solid culture medium until the colonies are single, carrying out gram staining and thixotropic enzyme test on the separated strains, and preserving for later use, wherein all strains which are gram positive and thixotropic enzyme negative are used as starting strains for subsequent screening.
The separation and purification of the saccharomyces cerevisiae Y21 comprises the following steps:
under aseptic condition, weighing 25mL of yogurt sample into conical flask containing 225mL of aseptic water, shaking thoroughly, and preparing 10 simultaneously -2 ，10 -3 ，10 -4 ，10 -5 ，10 -6 The diluted sample homogenate is multiplied, 0.1mL of the diluted solution is sucked and coated into YPD enrichment medium, and the culture is carried out at the constant temperature of 30 ℃ for 48 hours. Single colony strains conforming to the yeast morphology are selected for plate streaking for 3 times, and pure strain is obtained. Purified strains were inoculated into YPD medium, colony morphology was observed, cell structure was determined by microscopic observation, and preliminary classification was performed.
Morphological observation and physiological and biochemical identification of enterococcus faecium B13
Morphological observation and physiological and biochemical experiments are carried out on enterococcus faecium B13. The strain was allowed to stand still in MRS solid medium at 30℃for 24 hours, and the colony morphology was observed. The growth experiment of the milk of the meran, the hydrolysis experiment of the gelatin, the indole experiment and the sugar fermentation experiment refer to the "Berger's bacterial System Classification Manual" and the "lactic acid bacteria Classification and experiment method".
Morphological observation and physiological biochemical identification of Saccharomyces cerevisiae
Morphological observation and physiological and biochemical experiments are carried out on the saccharomyces cerevisiae Y21, the strain is subjected to static culture at the temperature of 30 ℃ in a YPD solid culture medium for 24 hours, and colony morphology is observed. The physiological and biochemical analysis of the strain refers to the handbook of characteristics and identification of yeast, including sugar fermentation experiments, carbon source assimilation experiments, indole experiments, gelatin hydrolysis experiments, urea experiments, and v.p. experiments.
Identification results of enterococcus faecium B13 and Saccharomyces cerevisiae Y21:
(1) Enterococcus faecium B13 strain morphology and culture characteristic observation and physiological and biochemical characteristic measurement
As shown in FIG. 11, the bacterial strain forms colony with diameter of 0.2-0.3 mm, smooth surface, uniform texture and milky white when growing in MRS solid culture medium. The strain is cultured in the methylene blue milk for 48 hours, the methylene blue milk is discolored, and the sugar fermentation experimental results are shown in the table below.
Physiological and biochemical characteristics of enterococcus faecium B13
Enterococcus Faecium B13 Physiological and biochemical characteristics
Note that: positive reaction; negative reaction
(2) Saccharomyces cerevisiae Y21 strain morphology and culture characteristic observation and physiological and biochemical characteristic measurement
As can be seen from the experimental results, as shown in fig. 12: the cultured saccharomycete has circular, oval and cylindrical shape, and its colony is milky white. The results of the sugar fermentation experiment, carbon source assimilation experiment, indole experiment, gelatin hydrolysis experiment, urea experiment, and v.p. experiment are shown in the following table.
Physiological and biochemical characteristics of Saccharomyces cerevisiae Y21
Saccharomvces cerevisiae Y21 Physiological and biochemical characteristics
Note that: positive reaction; negative reaction
(3) Molecular biological identification of strain and construction of phylogenetic tree thereof
According to the invention, through molecular biological identification of the strain and construction of phylogenetic tree, the following identification results are obtained: the enterococcus faecium B13 (Enterococcus Faecium B13) 16S rRNA gene sequence phylogenetic tree is shown in FIG. 8; the enterococcus faecium B13 (Enterococcus Faecium B13) rpoA gene sequence phylogenetic tree is shown in figure 9; the phylogenetic tree of Saccharomyces cerevisiae Y21 (Saccharomyces cerevisiae Y21) 26S rRNA gene sequences is shown in FIG. 10.
1.1.2 determination of the mixture ratio of enterococcus faecium B13 and Saccharomyces cerevisiae Y21
On the basis, the in-vitro probiotics characteristics and the safety of enterococcus faecium B13 are evaluated, the probiotics characteristics are strong, the safety is high, the enterococcus faecium B13 is an excellent strain, and then a subsequent feeding experiment is carried out.
Through the interaction analysis and co-culture interaction analysis of fermentation products, a pair of strains with stable interaction effect, namely saccharomyces cerevisiae Y21 and enterococcus faecium B13, are screened. Since enterococcus faecium B13 shows a difference in co-culture and the change of yeast is not obvious, enterococcus faecium B13 is the object of investigation when two strains are subjected to an inoculation ratio experiment. Enterococcus faecium B13 and Saccharomyces cerevisiae Y21 were subjected to 10:1, 20:1, 30:1, 40:1, 50:1, 100:1, and from the overall result, the inoculation ratio of enterococcus faecium to Saccharomyces cerevisiae is 10:1 to 100:1, the interaction effect is not obviously different, which indicates that the strain does not show different species relations along with the increase of the inoculation proportion. Can be used as recommended inter-fit ratio. The ratio of 100:1 was chosen as the addition ratio for the subsequent animal experiments. The number of viable bacteria of enterococcus faecium B13 co-cultured with enterococcus faecium B13 and Saccharomyces cerevisiae Y21 in different ratios is shown in Table 1 below.
TABLE 1
2. Test animals and test design
24 SPF grade ICR mice (purchased from Chengdu laboratory animal Co.) with good growth at 21 days of age were selected as test animals and randomly divided into 3 treatment groups of 8 replicates each, 1 mouse each:
control group: basic ration + 0.2mL physiological saline for gastric lavage every day;
group A: basic diet + 0.2mL enterococcus faecium per day of gastric lavage (viable count 4×10) 7 CFU)；
Group B: basic ration + 0.2mL of composite bacteria (enterococcus faecium viable count is 4 multiplied by 10) per day of gastric lavage 7 CFU, saccharomyces cerevisiae viable count is 4×10 5 CFU)；
During the feeding period, all ICR mice were free to ingest and drink water, feed was changed daily and feed intake was recorded, with litter changed every 3 d. The whole feeding period is 28d, the room temperature is 25+/-1 ℃, the air humidity is 55+/-1%, the daily illumination time is 12 hours, and daily ration purchase is achieved from experimental animal company in experiments. At test 29d, all ICR mice were weaned for 12h, weighed, anesthetized with carbon dioxide, and then immediately sacrificed at neck-breaking. The ileal content of the mice was collected and the like for subsequent analysis (fig. 1).
3. Intestinal microbiologic diversity
The ileal contents of 24 mice in total in the control group, the A group and the B group of the animal experiment group were taken as samples.
(1) Genomic DNA extraction and library construction
Genomic DNA of the sample was extracted using a DNA extraction kit, and then the concentration of DNA was detected using agarose gel electrophoresis and NanoDrop 2000. PCR was performed using genomic DNA as a template and specific primers with barcode, takara Corp. Tks Gflex DNA Polymerase, to ensure amplification efficiency and accuracy.
The PCR products are detected by agarose electrophoresis, purified by magnetic beads, purified to be used as a two-round PCR template, amplified by two-round PCR, detected again by electrophoresis, purified by magnetic beads, and quantitated by Qubit. Equal amount of mixing is carried out according to the concentration of the PCR product, and sequencing is carried out on a machine.
16S RdnaV 3-V4 region primer:
343F:5’-TACGGRAGGCAGCAG-3’
798R:5’-AGGGTATCTAATCCT-3’。
(2) Credit analysis
After the sequencing result was obtained, the following analysis was performed
Sample OTUs statistics and distribution.
Statistical analysis of the multivariate variables of microorganisms, calculation of differential species between different groups (based on portal and genus levels) by statistical algorithm (ANOVA/Kruskal Wallis/T test/Wilcoxon), and plotting of differential foreign species heatmaps etc.
The experimental result shows that the compounding method of the enterococcus faecium B13 and the saccharomyces cerevisiae Y21 has important influence on the reduction of harmful bacteria and the increase of beneficial bacteria in the intestinal flora of the mice, can improve the imbalance of the intestinal flora and maintain the healthy balance of the intestinal flora (shown in figures 2 and 3, wherein English marks at the lower left part in figure 2 are bacteria-pseudobacillus, fusobacterium-fusobacterium, firmicutes-thick-walled bacteria, gemtimonades-budomonas, proteus-deformable bacteria, spirochets-spirochete, actionbacteria-actinomycota, nitroirae-nitrifying spiral bacteria, epsilon bacteria-epsilon deformable bacteria, pateibacillus, deferbacteria-deironium, fibriform bacteria, bacteria-wall bacteria, bacteria-sterile bacteria, bacteria-Acidobacteria-other bacteria, and other bacteria-green bacteria.
Example 2-enterococcus faecium B13 and saccharomyces cerevisiae Y21 compounding method can significantly improve the content (P < 0.05) of constant bile acid Taurocholate (TCA), taurochenodeoxycholic acid (TCDCA), taurocholate (TDCA) and taurochenodeoxycholic acid (TUDCA), promote intestinal barrier function of mice, promote intestinal bile acid metabolism balance (fig. 6 and 7), promote intestinal barrier function of mice, and promote intestinal bile acid metabolism balance.
1. Analysis flow
After sample collection, the samples were first pre-treated (ground, purified, enriched, purified) and qualitatively and quantitatively analyzed using ultra-high performance liquid chromatography and mass spectrometry.
2. Sample pretreatment
The ileal contents of 3 groups of 24 mice in total were taken as samples in Control group, a group and B group. 50mg of ileal content was weighed and 200 μl of pre-chilled methanolic water (v (methanol): v (water) =1:1, isotopically containing internal standards CDCA-d4 and CA-d 4) solution was added; adding 2 steel balls, and grinding by a grinder (60 Hz,2 min); adding 1mL of ice acetonitrile, and carrying out ultrasonic extraction for 20min in an ice water bath; standing at-20deg.C for 30min; centrifuging for 10min (4deg.C, 13000 rpm), volatilizing the supernatant, re-dissolving with 200mL methanol water (V (methanol): V (water) =1:1, containing 100ng/mL Lyso PC17 and 20ng/mL GCA-C13), swirling for 30s, and performing ultrasonic treatment for 5min; centrifuging for 10min (13000 rpm, 4deg.C), sucking 200mL supernatant with a syringe, filtering with a 0.22 μm organic phase pinhole filter, transferring to a brown sample bottle, and preserving at-80deg.C until the supernatant is analyzed on the machine; the quality control sample (QC) is prepared by mixing all the sample extracting solutions in equal volumes, and the volume of the QC sample is the same as that of the experimental sample. Remarks: all extraction reagents were pre-chilled at-20 ℃.
3. Chromatographic mass spectrometry method
The experiment adopts a UPLC-ESI-MS/MS analysis method to carry out qualitative and quantitative detection on the target metabolite, and the specific analysis conditions and the analysis method are as follows:
chromatographic conditions:
sample injection amount: 5mL. Flow rate: 0.4mL/min. Mobile phase: a (0.1% formic acid-water solution), B (methanol: acetonitrile: isopropanol=1:1:1, containing 0.1% formic acid). Gradient elution method (Gradient Elution Procedures): 1min A/B (70:30, V/V), 5min A/B (60:40, V/V), 13min A/B (45:55, V/V), 17min A/B (20:80, V/V), 18min A/B (10:90, V/V), 19min A/B (10:90, V/V), 19.1min A/B (70:30, V/V), 20min A/B (70:30, V/V).
Mass spectrometry conditions:
air curtain gas: 45psi;
collision-induced ionization (CAD) parameters: medium;
negative ion spray voltage: -4500V;
ion source temperature: 450 ℃;
column temperature: 45 DEG C
Ion source: gas1:55psi; gas2:55psi.
4. Principle of qualitative and quantitative determination
Metabolite quantification was analyzed using the multiple reaction detection (MRM) mode of triple quadrupole mass spectrometry. In the MRM mode, the quaternary rod firstly screens precursor ions (parent ions) of target substances, and eliminates ions corresponding to other molecular weight substances to primarily eliminate interference; the precursor ions are ionized by the collision chamber in an induction way to be broken to form a plurality of fragment ions, and the fragment ions are filtered by the triple quaternary rod to select a required characteristic fragment ion, so that the interference of non-target ions is eliminated, the quantification is more accurate, and the repeatability is better. Mass spectrometry data of different samples were obtained, peak area integration was performed on all the chromatographic peaks, and integral correction was performed on chromatographic peaks in which the same substance was in different samples (fig. 4 to 5).
Experimental results show that the enterococcus faecium B13 and saccharomyces cerevisiae Y21 compounding method reagent can remarkably improve the contents (P < 0.05) of the constant bile acid Taurocholate (TCA), taurochenodeoxycholic acid (TCDCA), taurocholate (TDCA) and taurochenodeoxycholic acid (TUDCA), promote the intestinal barrier function of mice, promote the metabolism balance of intestinal bile acid, and have no remarkable difference among groups in trace bile acid (figures 6 and 7).
Comparative example 1
Based on example 2, the compound formulation of enterococcus faecium B13 and Saccharomyces cerevisiae Y21 was adjusted to the compound formulation of commercially available Lactobacillus plantarum, enterococcus faecium, bacillus subtilis strain and Saccharomyces cerevisiae (wherein, each 1kg of the compound microbial inoculum contains 4.9X10 7 94.8g of Saccharomyces cerevisiae per gram and 6.9X10 g of Saccharomyces cerevisiae 7 131.6g of enterococcus faecium per gram, and the rest steps and parameters are the same.
Compared with the compounding method of enterococcus faecium B13 and saccharomyces cerevisiae Y21 in the embodiment 2 of the invention, the improvement amount of Taurocholate (TCA) and tauchenodeoxycholic acid (TCDCA) in the ileum of the mice in the embodiment is respectively reduced by 5.2 percent and 6.1 percent in the same ratio, and the improvement amount of taurochenodeoxycholic acid (TDCA) is reduced by 4.1 percent in the same ratio; the intestinal barrier function and the intestinal bile acid metabolism balance performance of the mice are weakened to a certain extent.
In conclusion, the enterococcus faecium compound microbial agent and the compounding method thereof can regulate the population composition of intestinal microorganisms, reduce the abundance of harmful bacteria in the intestinal microorganisms, promote the abundance of beneficial bacteria, strengthen intestinal immunity and maintain the integrity of intestinal barriers of mice. In animal feeding experiments, the contents of the constant bile acids Taurocholate (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA) and taurochenodeoxycholic acid (TUDCA) in the complex group of enterococcus faecium B13 and saccharomyces cerevisiae Y21 were significantly increased (P < 0.05).
The foregoing is merely illustrative and explanatory of the invention as it is claimed, as modifications and additions may be made to, or similar to, the particular embodiments described, without the benefit of the inventors' inventive effort, and as alternatives to those of skill in the art, which remain within the scope of this patent.
Claims (7)
1. Enterococcus faeciumEnterococcus faecium) The enterococcus faecium B13 is named and preserved in China general microbiological culture collection center, the preservation center address is North Xielu No. 1, no. 3 in the Korean region of Beijing, the preservation date is 2021, 10 months and 29 days, and the preservation number is CGMCC No.23693.
2. Saccharomyces cerevisiae @ sSaccharomyces cerevisiae) The strain is named as saccharomyces cerevisiae Y21 and is preserved in China general microbiological culture collection center (CGMCC) with the preservation address of 1 # 3 of North west road of the Korean area of Beijing, the preservation date of 2021, 10 months and 29 days and the preservation number of 23692.
3. The enterococcus faecium composite bacterial agent is characterized by comprising the following bacterial species: the enterococcus faecium B13 of claim 1 and the saccharomyces cerevisiae Y21 of claim 2 are mixed in a ratio of 10-100:1.
4. The method for compounding the enterococcus faecium composite microbial inoculum according to claim 3, which is characterized by comprising the following steps: enterococcus faecium B13 according to claim 1 and Saccharomyces cerevisiae Y21 according to claim 2.
5. Use of the enterococcus faecium complex microbial agent of claim 3 in animal cultivation.
6. A microbial additive for animal cultivation, comprising the enterococcus faecium complex microbial agent according to claim 3.
7. The use of the enterococcus faecium complex microbial agent of claim 3 in the preparation of a microbial agent for repairing or enhancing intestinal function.
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