Preparation method and application of postbiotics capable of inhibiting bacterial diarrhea pathogens

Abstract

The present invention relates to the technical field of postbiotic fermentation, and a preparation method and application of a postbiotic with inhibition of bacterial diarrhea pathogens are proposed, and the preparation method includes the following steps: S1, Lactobacillus paracasei Postbio-P8 and fermented mucus lactobacillus Postbio-Q8 strains are activated and cultured respectively, and then the activated Lactobacillus paracasei Postbio-P8 and fermented mucus lactobacillus Postbio-Q8 are fermented sequentially in stages, and after fermentation, the mixed fermentation liquid is sterilized to obtain a postbiotic fermentation liquid; S2, the postbiotic fermentation liquid is clarified and impurities removed, concentrated and desalted, and freeze-dried to obtain a postbiotic freeze-dried powder. The present invention adopts staged fermentation, which not only improves nutrient utilization efficiency, reduces the accumulation of metabolic inhibitors, but also alleviates space and competition with each other, thereby significantly improving the yield of metabolites such as total bacterial volume and organic acids.

Description

Technical Field

[0001] The present invention relates to the technical field of postbiotic fermentation, and in particular to a preparation method and application of a postbiotic capable of inhibiting bacterial diarrhea pathogens. Background Art

[0002] Diarrhea is a common health problem in the world. Every year, about 1.3 million people die from diarrhea of ​​various causes, especially infants and young children. Diarrhea caused by bacterial pathogens is a global health problem. Enteric bacterial pathogens such as Escherichia coli, Shigella, Salmonella, Campylobacter, Clostridium difficile and Aeromonas are the main causes of bacterial diarrhea. The first choice for clinical treatment of bacterial diarrhea is antibacterial drugs, but the types of bacterial diarrhea pathogens are complex, and the drug resistance of most bacteria has generally increased. In addition, new reports of antibiotic-associated diarrhea have also appeared. Traditional treatment methods include the use of antibiotics to kill pathogenic bacteria, but the combined use of antibiotics will lead to the development of bacterial resistance and destroy the balance of intestinal microbial communities, causing repeated diarrhea and making clinical treatment more difficult.

[0003] Studies have shown that probiotics and their metabolites can treat diarrhea by inhibiting pathogenic bacteria. By increasing the intake of probiotics, the number and activity of probiotics in the intestine can be enhanced, thereby inhibiting the growth and activity of pathogenic bacteria. Postbiotics refer to preparations of inanimate microorganisms and / or their components that are beneficial to the health of the host. Compared with probiotics, postbiotics have a longer shelf life and higher safety, and are also suitable for special populations such as newborns and sensitive populations. They are not interfered with by antibiotics, providing new ideas for the clinical treatment of bacterial diarrhea. However, the existing methods for preparing postbiotics have the defects of introducing a small amount of bacteria and a small number of metabolites, which affects their efficacy. Summary of the invention

[0004] In view of this, the present invention proposes a preparation method and application of a postbiotic that has a large amount of introduced bacteria and a large number of metabolites and has the function of inhibiting bacterial diarrhea pathogens.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides a method for preparing a postbiotic having the function of inhibiting bacterial diarrhea pathogens, comprising the following steps:

[0006] S1, Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Postbio-P8 and fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) The Postbio-Q8 strains are inoculated into MRS liquid culture medium for activation and culture, and then the activated Lactobacillus paracasei Postbio-P8 is inoculated into fermentation medium for fermentation; after the fermentation is completed, the activated Lactobacillus mucilaginosus Postbio-Q8 is continuously inoculated for fermentation, and after the fermentation is completed, the mixed fermentation liquid is sterilized to obtain a postbiotic fermentation liquid;

[0007] The deposit number of the Lactobacillus paracasei Postbio-P8 is CGMCC No.31112, and the deposit number of the fermented Lactobacillus mucilaginosus Postbio-Q8 is CGMCC No.31113;

[0008] S2, the postbiotic fermentation liquid of step S1 is clarified, decontaminated, concentrated, desalted and freeze-dried to obtain postbiotic freeze-dried powder.

[0009] The present invention adopts the method that Lactobacillus paracasei Postbio-P8 is first fermented and Lactobacillus mucilaginosus Postbio-Q8 is then fermented to prepare postbiotics, because there is a mutual inhibition effect of microorganisms when the two are fermented simultaneously, resulting in less bacterial volume and secondary metabolites of the fermentation end product. During the staged fermentation, the fermentation product of Lactobacillus paracasei Postbio-P8 can be used for the fermentation of Lactobacillus mucilaginosus Postbio-Q8, and the bacterial volume is increased, specifically:

[0010] In the staged fermentation process, Lactobacillus paracasei first consumes some simple, easily available nutrients and produces metabolites, which include growth factors or secondary nutrients required by Lactobacillus mucilaginosus. For example, simple polysaccharides and proteins are broken down by Lactobacillus paracasei into more easily absorbed small molecules such as monosaccharides, peptones and amino acids, which are more easily utilized by Lactobacillus mucilaginosus. At the same time, it avoids the two strains from competing for the same nutrients at the same time. In addition, the fermentation metabolites of Lactobacillus paracasei, such as lactic acid, short-chain fatty acids and certain functional peptides, not only improve the fermentation environment, but also have an incentive effect on Lactobacillus mucilaginosus, providing additional energy and metabolic signals for its fermentation, and promoting Lactobacillus mucilaginosus to proliferate more efficiently.

[0011] During the fermentation process, Lactobacillus paracasei Postbio-P8 synthesizes and releases some biologically active metabolites, such as lactic acid, short-chain fatty acids, functional peptides, etc. These metabolites not only have prebiotic effects, but also can provide additional nutrients and growth factors for the subsequent fermentation of Lactobacillus mucilaginosus Postbio-Q8, promoting its growth and metabolic activity. Secondly, through the early fermentation, Lactobacillus paracasei Postbio-P8 can change the environmental conditions of the culture medium, such as lowering the pH value and accumulating an appropriate amount of acidity. This slightly acidic environment is more suitable for the activity and subsequent fermentation of Lactobacillus mucilaginosus Postbio-Q8, thereby achieving the purpose of optimizing the fermentation environment and further promoting the generation of postbiotics.

[0012] On the basis of the above technical scheme, preferably, the formula of the fermentation medium includes: 15-25 g / L glucose, 10-20 g / L whey protein, 15-25 g / L soy flour, 5-15 g / L skim milk powder and 5-15 g / L yeast powder, and the initial pH=7.0-7.5.

[0013] Glucose, soy flour, whey protein, skim milk powder and yeast powder provide sugars, proteins and other trace nutrients, which are rich in amino acids, vitamins and minerals, which are beneficial to the growth and metabolic activity of lactic acid bacteria. In addition, by adjusting the initial pH to 7.0-7.5, the stability of the fermentation start stage and the efficient growth of the strains are ensured.

[0014] On the basis of the above technical solution, preferably, the fermentation medium also includes a growth factor formula: Tween 800.2~0.8g / L, disodium hydrogen phosphate 0.5~1.3g / L, manganese sulfate 0.01~0.03g / L and magnesium sulfate 0.1~0.2g / L.

[0015] Tween 80 acts as a surfactant, which can help lactic acid bacteria better utilize lipids. Disodium hydrogen phosphate, manganese sulfate and magnesium sulfate are essential elements for bacterial growth and can increase bacterial reproduction speed and metabolic activity.

[0016] On the basis of the above technical solution, preferably, the fermentation medium also includes a growth interaction factor formula: 0.01-0.03 g / L leucine and 0.01-0.03 g / L isoleucine.

[0017] Leucine and isoleucine are two branched-chain amino acids. Leucine and isoleucine can work together in cells through different metabolic pathways to achieve complementary and mutual assistance effects, fully meeting the various physiological needs of bacteria. In addition, their metabolic pathways in cells are interrelated, which can effectively regulate and optimize the growth environment of bacteria, ensure sufficient nutrition supply at different stages, and avoid metabolic imbalance problems that may be caused by excessive or insufficient single amino acids. After the bacteria accumulate to a certain amount, these amino acids can also promote the production of secondary metabolites, such as organic acids, cell wall components, extracellular metabolites, etc., to improve the quality and functionality of the final postbiotic products.

[0018] Lactobacillus paracasei requires a large amount of branched-chain amino acids during metabolic activity and cell growth, especially leucine, which can enhance the stability and function of bacterial membrane proteins. By adding leucine, its growth rate is accelerated and the cell mass is increased. Fermented mucus lactobacillus has a strong secondary metabolic capacity. Adding isoleucine can significantly promote the activity of its metabolic pathway, generate more lactic acid and other secondary metabolites, and increase the organic acid content of the final fermentation broth.

[0019] Based on the above technical solution, preferably, the number of viable bacteria after activation of each strain is 5×10 6 ~10×10 6 CFU / mL.

[0020] On the basis of the above technical solution, preferably, the inoculation amount of the Lactobacillus paracasei Postbio-P8 is 1wt%~3wt%.

[0021] On the basis of the above technical solution, preferably, the pH value of the culture medium during the fermentation of Lactobacillus paracasei Postbio-P8 is 4-4.5.

[0022] On the basis of the above technical solution, preferably, the inoculation amount of the fermented mucus lactobacillus Postbio-Q8 is 4wt%~6wt%.

[0023] On the basis of the above technical solution, preferably, the pH value of the culture medium during the fermentation of the fermented Lactobacillus mucilaginosus Postbio-Q8 is 3.2-3.8.

[0024] On the other hand, the present invention also provides a use of a postbiotic in the preparation of a medicament for preventing and treating bacterial diarrhea, wherein the postbiotic is prepared by the above-mentioned preparation method.

[0025] The preparation method and application of a postbiotic capable of inhibiting bacterial diarrhea pathogens of the present invention have the following beneficial effects compared with the prior art:

[0026] The invention adopts Lactobacillus paracasei Postbio-P8 and Lactobacillus mucilaginosus Postbio-Q8 for staged fermentation, which not only improves nutrient utilization efficiency, optimizes the fermentation environment, reduces the accumulation of metabolic inhibitors, but also alleviates space and mutual competition, thereby significantly increasing the total bacterial volume and the yield of metabolites such as organic acids.

[0027] Leucine and isoleucine work together in the cell through different metabolic pathways to achieve a complementary and mutually supportive effect, fully meeting the various physiological needs of the bacteria, accelerating the growth rate of the bacteria and increasing the cell mass; at the same time, more lactic acid and other secondary metabolites are generated, increasing the organic acid content of the final fermentation liquid.

[0028] The postbiotics prepared by the present invention can damage the morphology of Escherichia coli, Salmonella and Campylobacter jejuni, destroy the integrity of their cell membranes, cause the leakage of cell contents, and can be used to prevent and treat bacterial diarrhea; compared with antibiotics, the postbiotics of the present application will not cause the generation of bacterial resistance, will not destroy the balance of intestinal microbial communities, and have a good application effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 This is a diagram showing the antibacterial effects of the postbiotics of the present invention on Escherichia coli, Salmonella and Campylobacter jejuni, wherein a represents Escherichia coli, b represents Salmonella, and c represents Campylobacter jejuni;

[0031] Figure 2 This is a diagram showing the effect of the postbiotics of the present invention on inhibiting the biofilm formation of Escherichia coli, Salmonella and Campylobacter jejuni, wherein a is Escherichia coli, b is Salmonella, and c is Campylobacter jejuni;

[0032] Figure 3 This is a diagram showing the effect of the postbiotics of the present invention on the morphology of Escherichia coli, wherein a is the control group and b is the postbiotic treatment group;

[0033] Figure 4 This is a diagram showing the effect of the postbiotics of the present invention on the morphology of Salmonella, wherein a is the control group and b is the postbiotic treatment group;

[0034] Figure 5 This is a diagram showing the effect of the postbiotics of the present invention on the morphology of Campylobacter jejuni, wherein a is the control group and b is the postbiotic treatment group;

[0035] Figure 6 This is a graph showing the intracellular DNA leakage of the postbiotics of the present invention on Escherichia coli, Salmonella and Campylobacter jejuni, wherein a is Escherichia coli, b is Salmonella, and c is Campylobacter jejuni;

[0036] Figure 7 This is a diagram showing the effect of the postbiotics of the present invention on the transcriptional expression of Escherichia coli genes;

[0037] Figure 8 for Figure 7 Pathways enriched for up-regulated genes;

[0038] Fig. 9 for Figure 7 pathways enriched for moderately downregulated genes;

[0039] Fig.10 This is a diagram showing the effect of the postbiotics of the present invention on the transcriptional expression of Salmonella genes;

[0040] Fig.11 for Fig.10 Pathways enriched for up-regulated genes;

[0041] Fig.12 for Fig.10 pathways enriched for moderately downregulated genes;

[0042] Fig.13 This is a diagram showing the effect of the postbiotics of the present invention on the transcriptional expression of Campylobacter jejuni genes;

[0043] Fig.14 for Fig.13 Pathways enriched for up-regulated genes;

[0044] Fig.15 for Fig.13 Pathways enriched for moderately downregulated genes. DETAILED DESCRIPTION

[0045] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The present invention uses Lactobacillus paracasei ( Lacticaseibacillus paracasei )Postbio-P8 was deposited on June 27, 2024 at the General Microbiology Center of China Microorganism Culture Collection (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101. The deposit number is CGMCC No. 31112. The collection center tested the strain on June 27, 2024, and the result was in a viable state.

[0047] The fermented mucus lactobacillus used in the present invention ( Limosilactobacillus fermentum )Postbio-Q8 was deposited on June 27, 2024 at the General Microbiology Center of China Microorganism Culture Collection (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101. The deposit number is CGMCC No. 31113. The collection center tested the strain on June 27, 2024, and the result was in a viable state.

[0048] The present invention verifies the ability of the postbiotics prepared by the present invention to inhibit bacterial diarrhea pathogens from two aspects: the preparation method of the postbiotics and the antibacterial property of the postbiotics.

[0049] 1. Preparation of postbiotics

[0050] Example 1

[0051] The preparation method of the postbiotics of Example 1 comprises the following steps:

[0052] S1, Lactobacillus paracasei Postbio-P8 and Lactobacillus fermentans Postbio-Q8 were inoculated into MRS liquid culture medium, cultured at 37°C for 18 h, and subcultured twice to make the number of viable bacteria of each strain after activation reach 5×10 6 ~10×10 6 CFU / mL.

[0053] S2, firstly, the activated Lactobacillus paracasei Postbio-P8 was inoculated into the fermentation medium at an inoculation rate of 2wt%, and fermented at 37°C for 20 h, with a rotation speed of 150 rpm / min and a fermentation pH of 4.0; then, the activated Lactobacillus mucilaginosus Postbio-Q8 was inoculated into the fermentation medium at an inoculation rate of 5wt% and continued to ferment for 20 h, with a fermentation pH of 3.2.

[0054] The formula of the fermentation medium is: glucose 20g / L, whey protein 15g / L, soy powder 20g / L, skim milk powder 10g / L, yeast powder 10g / L, solvent is water, initial pH = 7.0. Before inoculation and fermentation, the fermentation medium needs to be sterilized at 105℃ for 30min.

[0055] S3, after the fermentation is completed, the mixed probiotic fermentation liquid is sterilized and pasteurized at 95°C for 40 minutes to obtain a postbiotic fermentation liquid. The postbiotic fermentation liquid is then clarified and impurity-removed, nanofiltration membrane concentrated and desalted, and vacuum-freeze-dried to obtain a postbiotic freeze-dried powder.

[0056] Example 2

[0057] The preparation method of the postbiotics of Example 2 comprises the following steps:

[0058] S1, Lactobacillus paracasei Postbio-P8 and Lactobacillus fermentans Postbio-Q8 were inoculated into MRS liquid culture medium, cultured at 37°C for 18 h, and subcultured twice to make the number of viable bacteria of each strain after activation reach 5×10 6 ~10×10 6 CFU / mL.

[0059] S2, firstly, the activated Lactobacillus paracasei Postbio-P8 was inoculated into the fermentation medium at an inoculation rate of 2wt%, and fermented at 37°C for 20 h, with a rotation speed of 150 rpm / min and a fermentation pH of 4.0; then, the activated Lactobacillus mucilaginosus Postbio-Q8 was inoculated into the fermentation medium at an inoculation rate of 5wt% and continued to ferment for 20 h, with a fermentation pH of 3.2.

[0060] The formula of the fermentation medium is: glucose 20g / L, whey protein 15g / L, soy flour 20g / L, skim milk powder 10g / L, yeast powder 10g / L, Tween 80 0.5g / L, disodium hydrogen phosphate 1g / L, manganese sulfate 0.01g / L, magnesium sulfate 0.1g / L, solvent is water, initial pH = 7.0. Before inoculation and fermentation, the fermentation medium needs to be sterilized at 105°C for 30 minutes.

[0061] S3, after the fermentation is completed, the mixed probiotic fermentation liquid is sterilized and pasteurized at 95°C for 40 minutes to obtain a postbiotic fermentation liquid. The postbiotic fermentation liquid is then clarified and impurity-removed, nanofiltration membrane concentrated and desalted, and vacuum-freeze-dried to obtain a postbiotic freeze-dried powder.

[0062] Example 3

[0063] The preparation method of the postbiotics of Example 3 comprises the following steps:

[0064] S1, Lactobacillus paracasei Postbio-P8 and Lactobacillus fermentans Postbio-Q8 were inoculated into MRS liquid culture medium, cultured at 37°C for 18 h, and subcultured twice to make the number of viable bacteria of each strain after activation reach 5×10 6 ~10×10 6 CFU / mL.

[0065] S2, firstly, the activated Lactobacillus paracasei Postbio-P8 was inoculated into the fermentation medium at an inoculation rate of 2wt%, and fermented at 37°C for 20 h, with a rotation speed of 150 rpm / min and a fermentation pH of 4.0; then, the activated Lactobacillus mucilaginosus Postbio-Q8 was inoculated into the fermentation medium at an inoculation rate of 5wt% and continued to ferment for 20 h, with a fermentation pH of 3.2.

[0066] The formula of the fermentation medium is: glucose 20g / L, whey protein 15g / L, soy flour 20g / L, skim milk powder 10g / L, yeast powder 10g / L, Tween 80 0.5g / L, disodium hydrogen phosphate 1g / L, manganese sulfate 0.01g / L, magnesium sulfate 0.1g / L, leucine 0.02g / L, isoleucine 0.01g / L, solvent is water, initial pH = 7.0. Before inoculation and fermentation, the fermentation medium needs to be sterilized at 105°C for 30 minutes.

[0067] S3, after the fermentation is completed, the mixed probiotic fermentation liquid is sterilized and pasteurized at 95°C for 40 minutes to obtain a postbiotic fermentation liquid. The postbiotic fermentation liquid is then clarified and impurity-removed, nanofiltration membrane concentrated and desalted, and vacuum-freeze-dried to obtain a postbiotic freeze-dried powder.

[0068] Example 4

[0069] The preparation method of the postbiotics of Example 4 comprises the following steps:

[0070] S1, Lactobacillus paracasei Postbio-P8 and Lactobacillus fermentans Postbio-Q8 were inoculated into MRS liquid culture medium, cultured at 37°C for 18 h, and subcultured twice to make the number of viable bacteria of each strain after activation reach 5×10 6 ~10×10 6 CFU / mL.

[0071] S2, firstly, the activated Lactobacillus paracasei Postbio-P8 is inoculated into the fermentation medium at an inoculation amount of 1wt%, and fermented at 37°C for 25 h, with a rotation speed of 150 rpm / min and a fermentation pH of 4.3; then, the activated Lactobacillus mucilaginosus Postbio-Q8 is inoculated into the fermentation medium at an inoculation amount of 4wt% and continued to ferment, and the fermentation pH is 3.5.

[0072] The formula of the fermentation medium includes: glucose 15g / L, whey protein 20g / L, soy flour 15g / L, skim milk powder 15g / L, yeast powder 5g / L, Tween 80 0.8g / L, disodium hydrogen phosphate 0.5g / L, manganese sulfate 0.03g / L, magnesium sulfate 0.1g / L, leucine 0.03g / L, isoleucine 0.01g / L, solvent is water, initial pH = 7.3. Before inoculation and fermentation, the fermentation medium needs to be sterilized at 105°C for 30 minutes.

[0073] S3, after the fermentation is completed, the mixed probiotic fermentation liquid is sterilized and pasteurized at 95°C for 60 minutes to obtain a postbiotic fermentation liquid. The postbiotic fermentation liquid is then clarified and impurity-removed and concentrated and desalted by a nanofiltration membrane, and then vacuum-freeze-dried to obtain a postbiotic freeze-dried powder.

[0074] Example 5

[0075] The preparation method of the postbiotics of Example 5 comprises the following steps:

[0076] S1, Lactobacillus paracasei Postbio-P8 and Lactobacillus fermentans Postbio-Q8 were inoculated into MRS liquid culture medium, cultured at 37°C for 18 h, and subcultured twice to make the number of viable bacteria of each strain after activation reach 5×10 6 ~10×10 6 CFU / mL.

[0077] S2, firstly, the activated Lactobacillus paracasei Postbio-P8 was inoculated into the fermentation medium at an inoculation rate of 3wt%, and fermented at 37°C for 30 h, with a rotation speed of 150 rpm / min and a fermentation pH of 4.5; then, the activated Lactobacillus mucilaginosus Postbio-Q8 was inoculated into the fermentation medium at an inoculation rate of 6wt% and fermented for 30 h, with a fermentation pH of 3.8.

[0078] The formula of the fermentation medium includes: glucose 25g / L, whey protein 10g / L, soy flour 25g / L, skim milk powder 5g / L, yeast powder 15g / L, Tween 80 0.2g / L, disodium hydrogen phosphate 1.3g / L, manganese sulfate 0.01g / L, magnesium sulfate 0.2g / L, leucine 0.01g / L, isoleucine 0.03g / L, solvent is water, initial pH = 7.5. Before inoculation and fermentation, the fermentation medium needs to be sterilized at 105°C for 30 minutes.

[0079] S3, after the fermentation is completed, the mixed probiotic fermentation liquid is sterilized and pasteurized at 95°C for 100 minutes to obtain a postbiotic fermentation liquid. The postbiotic fermentation liquid is then clarified and impurity-removed, concentrated and desalted by a nanofiltration membrane, and vacuum-freeze-dried to obtain a postbiotic freeze-dried powder.

[0080] Example 6

[0081] The preparation method of the postbiotics of Example 6 comprises the following steps:

[0082] S1, Lactobacillus paracasei Postbio-P8 and Lactobacillus fermentans Postbio-Q8 were inoculated into MRS liquid culture medium, cultured at 37°C for 18 h, and subcultured twice to make the number of viable bacteria of each strain after activation reach 5×10 6 ~10×10 6 CFU / mL.

[0083] S2, firstly, the activated Lactobacillus paracasei Postbio-P8 was inoculated into the fermentation medium at an inoculation amount of 1.5wt%, and fermented at 37°C for 30 h, with a rotation speed of 150 rpm / min and a fermentation pH of 4.5; then, the activated Lactobacillus mucilaginosus Postbio-Q8 was inoculated into the fermentation medium at an inoculation amount of 4.5wt% and continued to ferment for 30 h, with a fermentation pH of 3.5.

[0084] The formula of the fermentation medium includes: glucose 18g / L, whey protein 14g / L, soy flour 23g / L, skim milk powder 13g / L, yeast powder 14g / L, Tween 80 0.6g / L, disodium hydrogen phosphate 1.2g / L, manganese sulfate 0.025g / L, magnesium sulfate 0.15g / L, leucine 0.015g / L, isoleucine 0.025g / L, solvent is water, initial pH = 7.5. Before inoculation and fermentation, the fermentation medium needs to be sterilized at 105°C for 30 minutes.

[0085] S3, after the fermentation is completed, the mixed probiotic fermentation liquid is sterilized and pasteurized at 95°C for 100 minutes to obtain a postbiotic fermentation liquid. The postbiotic fermentation liquid is then clarified and impurity-removed, concentrated and desalted by a nanofiltration membrane, and vacuum-freeze-dried to obtain a postbiotic freeze-dried powder.

[0086] Comparative Example 1

[0087] The difference between Comparative Example 1 and Example 1 is that Lactobacillus paracasei Postbio-P8 and Lactobacillus mucilaginosus fermentation Postbio-Q8 are inoculated and fermented simultaneously, the fermentation time is 20 h, the fermentation pH value is 3.2, and the rest of the contents are the same.

[0088] Comparative Example 2

[0089] The difference between Comparative Example 1 and Example 1 is that the strains selected are Lactobacillus paracasei Postbio-P6 and Lactobacillus mucilaginosus fermentans Postbio-Q7, and the rest of the contents are the same.

[0090] Comparative Example 3

[0091] The difference between Comparative Example 3 and Example 3 is that Lactobacillus paracasei Postbio-P8 and Lactobacillus mucilaginosus fermentation Postbio-Q8 are inoculated and fermented simultaneously, the fermentation time is 20 h, the fermentation pH value is 3.2, and the rest of the contents are the same.

[0092] The postbiotic freeze-dried powder prepared in the examples and comparative examples was dissolved in water, and the bacterial cell amount and metabolite content thereof were detected. The results are shown in Tables 1-2.

[0093] Table 1 Bacterial mass of postbiotic freeze-dried powder

[0094]

[0095] As shown in Table 1, it can be seen from Example 1 and Comparative Example 1 that the amount of postbiotic bacteria prepared in stages by Lactobacillus paracasei Postbio-P8 and Lactobacillus fermentans Postbio-Q8 was significantly increased. It can be seen from Examples 1 to 3 that after the growth factor formula and the growth interaction factor formula were added to the culture medium, the amount of bacteria increased to 4.3× and 3.8× , which proves that the growth factor formula and the growth interaction factor formula have the technical effect of increasing the amount of bacteria.

[0096] Table 2 Metabolites of postbiotic freeze-dried powder

[0097]

[0098] As shown in Table 2, the use of staged fermentation, the addition of growth factor components and growth interaction factor components all played a role in increasing the content of metabolites, among which Example 6 had the best effect; and compared with the existing strains: Lactobacillus paracasei Postbio-P6 and fermented Lactobacillus mucosa Postbio-Q7, the Lactobacillus paracasei Postbio-P8 and fermented Lactobacillus mucosa Postbio-Q8 used in the present invention had better effects.

[0099] 2. Antibacterial Test of Postbiotics

[0100] 2.1 Determination of inhibition zone

[0101] The postbiotics prepared in Example 1 were centrifuged at 4000 rpm / min for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The antibacterial activity of the postbiotics against Escherichia coli, Salmonella and Campylobacter jejuni was verified by the Oxford cup method. Escherichia coli, Salmonella and Campylobacter jejuni were diluted to 1×10 6 After reaching CFU / mL, add the bacterial solution to the unsolidified culture medium (below 45°C) at a ratio of 1%, mix thoroughly and pour into the culture dish with the Oxford cup. After the culture medium solidifies, take out the Oxford cup. Pipette 150μL of the above-mentioned sample into the agar well, repeat 3 times, and add an equal amount of PBS to the control well plate. Place the culture dish at 4°C for 4h and then place it in a 37°C constant temperature incubator to culture and measure the size of the inhibition zone. The experimental results are as follows Figure 1 shown.

[0102] Figure 1 The experimental results showed that Escherichia coli ( Figure 1 The diameter of the inhibition zone of (a) is 25 ± 0.5 mm, and the diameter of the inhibition zone of Salmonella ( Figure 1 The diameter of the inhibition zone in b) was 27 ± 0.5 mm, and Campylobacter jejuni ( Figure 1 The diameter of the inhibition zone in (c) is 30±0.5 mm, indicating that the postbiotics of the present invention have significant antibacterial effects on Escherichia coli, Salmonella and Campylobacter jejuni.

[0103] 2.2 Effects of postbiotics on biofilm formation of Escherichia coli, Salmonella, and Campylobacter jejuni

[0104] The inhibitory effect of postbiotics on biofilm formation of Escherichia coli, Salmonella and Campylobacter jejuni was determined by crystal violet staining. 100 μL of 10 6 CFU / mL bacterial suspension and 100μL postbiotics, an equal amount of water was added to the control group, and after culturing in a constant temperature incubator at 37°C, the upper culture medium and free bacteria were removed, and washed three times with 200μL sterilized PBS, 100μL 1% crystal violet was added for staining for 15 minutes, and then the crystal violet was absorbed; washed with sterile water until colorless, and after drying, decolorized with 33% acetic acid solution, and the absorbance of OD600 was measured with an enzyme reader. The inhibition rate was determined using the formula: Inhibition rate (%) = (ODc-ODt) / ODc×100%, where ODc is the OD value of the control group; ODt is the OD value of the postbiotic treatment group. The experimental results are as follows Figure 2 shown.

[0105] The experimental results showed that compared with the control group, postbiotics had an Figure 2 a) The inhibition rate of biofilm reached 70.25%, and the inhibition rate of Salmonella ( Figure 2 b) The inhibition rate of biofilm reached 71.42%, which was effective against Campylobacter jejuni ( Figure 2 c) The inhibition rate of biofilm reached 52.37%, indicating that the postbiotics have a significant inhibitory effect on the formation of biofilms of Escherichia coli, Salmonella and Campylobacter jejuni (p<0.01). It is reported that bacterial biofilms are closely related to the pathogenicity and virulence of bacteria, especially their antibiotic resistance. It can be seen that the postbiotics of the present invention have a good effect on anti-biofilm of Escherichia coli, Salmonella and Campylobacter jejuni.

[0106] 2.3 Effects of postbiotics on the morphology of Escherichia coli, Salmonella and Campylobacter jejuni

[0107] Escherichia coli, Salmonella and Campylobacter jejuni strains were activated and diluted to 10 6 ~10 7CFU / mL, centrifuge at 8000r / min for 10min, wash three times with PBS, remove the supernatant, add 100μL postbiotics, and add an equal amount of water to the control group. After placing in a 37℃ water bath for 30min, centrifuge at 8000r / min for 10min, collect the precipitate, wash three times with PBS, remove the supernatant, and fix with 2.5% glutaraldehyde solution at 4℃ for 24h; then centrifuge at 8000r / min for 10min, wash three times with PBS, remove the supernatant; use increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90% and 100%) for gradient dehydration in sequence, and after dehydration, vacuum freeze-dry the bacterial cells, spray gold after drying, and observe the changes in bacterial cell morphology by scanning electron microscopy. The experimental results are as follows Figures 3-5 shown.

[0108] Figure 3 In the control group ( Figure 3 a) The bacterial cells without postbiotic treatment have a typical cell structure, with intact cells, a round and smooth surface, no damage on the cell surface, and a complete structure. The cells grow well without aggregation and shrinkage. Figure 3 In b), the cell wall and cell membrane of the bacteria are destroyed, the bacteria are deformed and dissolved, the cell integrity is lost, and the overall cell structure collapses. This shows that postbiotics can effectively destroy the bacterial structure, have a damaging effect on E. coli bacteria, and can cause leakage of cell contents.

[0109] Figure 4 Without postbiotic treatment ( Figure 4 The bacterial cells in a) are typical cell structures, the bacteria are intact, the cell surface is completely intact, the structure is intact, and the bacteria grow well; while the postbiotic treatment ( Figure 4 In the middle (b), the bacteria are deformed and dissolved, the cell membrane permeability increases, the cell contents are clearly seen flowing out, and the overall cell structure collapses. This shows that postbiotics can effectively destroy the integrity of the Salmonella cell membrane and have a significant damaging effect on Salmonella bacteria.

[0110] Figure 5 In the control group ( Figure 5 The bacterial cells in the group (a) without postbiotic treatment showed a typical cell structure, with intact cells, a round and smooth surface, no damage to the cell surface, and a complete structure. The cells grew well without aggregation and shrinkage. Figure 5 The cells of b) are deformed, wrinkled and dissolved, and the length of the cells is shortened. This shows that postbiotics can destroy the cell structure of Campylobacter jejuni and have a significant damaging effect on Campylobacter jejuni.

[0111] 2.4 Intracellular DNA leakage test of Escherichia coli, Salmonella and Campylobacter jejuni

[0112] Escherichia coli, Salmonella and Campylobacter jejuni were diluted to 10 6 ~10 7 CFU / mL, centrifuge at 8000r / min for 10min, wash with PBS 3 times, remove the supernatant, add 100μL postbiotics, and add an equal amount of water to the control group. After placing in a 37℃ water bath for 30min, use an ELISA reader to compare the changes in absorbance values ​​of the negative control and the postbiotic-treated bacteria at a wavelength of 260nm to detect DNA leakage after postbiotic treatment. The test results are as follows Figure 6 shown.

[0113] Figure 6 As shown in Figure a, after treatment with postbiotics, the amount of extracellular DNA leakage of E. coli in the postbiotic group increased significantly compared with the control group, reaching 26.85 times that of the control group (P<0.01), indicating that postbiotics destroyed the integrity of the E. coli cell membrane, leading to the leakage of cell contents.

[0114] Figure 6 As shown in middle b, after postbiotic treatment, the amount of extracellular DNA leakage of Salmonella in the postbiotic group increased significantly compared with that in the control group, reaching 6 times that of the control group, indicating that postbiotics destroyed the integrity of the Salmonella cell membrane and caused the leakage of cell contents.

[0115] Figure 6 As shown in Figure c, after treatment with postbiotics, the amount of extracellular DNA leakage of Campylobacter jejuni in the YDFF group increased significantly, reaching 6 times that of the control group (P<0.01), indicating that postbiotics affected the integrity of the cell membrane of Campylobacter jejuni, increased the permeability of its cell membrane, and caused the leakage of cell contents.

[0116] 2.5 Effects of postbiotics on gene transcription expression in Escherichia coli, Salmonella, and Campylobacter jejuni

[0117] The postbiotics were centrifuged at 8000 r / min for 5 min, and the supernatant was taken. It was filtered with a 0.22 μL filter membrane and set aside. The bacterial liquid of Escherichia coli, Salmonella and Campylobacter jejuni was centrifuged at 8000 r / min for 10 min, and the supernatant was removed to collect the bacteria. The cells were redissolved in PBS, and the bacterial liquid and postbiotics were added in a 2 mL centrifuge tube at a ratio of 1:1. PBS was added to the control group. After reacting at 37 °C for 30 min, the supernatant was centrifuged to collect the bacteria, and they were immediately placed in liquid nitrogen and transferred to -80 °C for storage. The above-mentioned treated samples were placed in dry ice and sent to Tianjin Novo Biotechnology Co., Ltd. to complete transcriptome sequencing. The LLL Lumina HiSeq platform was used for sequencing, Bowtie2 software was used for reference sequence comparison analysis, and HTSeq and DESeq 2 software were used to calculate gene expression and differential analysis expression. The experimental results are as follows Figures 7 to 15 shown.

[0118] Figures 7-9 As shown in the figure, a total of 311 significantly differentially expressed genes were screened, of which 112 genes were upregulated and 199 genes were downregulated. KEGG enrichment analysis was performed on the upregulated and downregulated differentially expressed genes, respectively. The upregulated differentially expressed genes were enriched in 19 metabolic pathways, and the downregulated genes were enriched in 61 metabolic pathways. The enrichment of upregulated genes was mainly concentrated in pathways such as ribosomes, bacterial secretion systems, and protein export. The downregulated genes were significantly enriched in pathways such as phenylalanine, tyrosine and tryptophan biosynthesis, and propionate metabolism, which are essential for lipid metabolism, energy metabolism, translation, and replication and repair.

[0119] Figures 10-12 As shown in the figure, a total of 1527 differentially expressed genes were screened, of which 701 genes were up-regulated and 826 genes were down-regulated. KEGG enrichment analysis was performed on the up-regulated and down-regulated differentially expressed genes. The up-regulated gene enrichment was mainly concentrated in pathways such as ribosomes, bacterial secretion systems, protein export, and lipopolysaccharide biosynthesis. These pathways are closely related to the synthesis of glycosomes and enzyme systems such as glycosyltransferases and sugar nucleic acid invertases in cells, bacterial pathogenicity, biofilm formation, and interactions. Down-regulated genes were significantly enriched in pathways such as fatty acid degradation, lysine degradation, and phosphotransferase systems. The normal operation of these pathways is very important for maintaining the energy balance and metabolic balance of organisms, and is closely related to the growth and development, energy supply, and material synthesis of organisms.

[0120] Figures 13-15As shown in the figure, a total of 117 differentially expressed genes were screened, of which 49 genes were up-regulated and 68 genes were down-regulated. KEGG enrichment analysis was performed on the up-regulated and down-regulated differentially expressed genes. The up-regulated genes were mainly enriched in pathways such as ribosome, butyrate metabolism, citric acid cycle (TCA cycle), glycine, serine and threonine metabolism, which are related to important biological processes such as protein synthesis, energy metabolism and signal transduction. The down-regulated genes were mainly enriched in pathways such as ABC transport, biosynthesis of valine, leucine and isoleucine, oxidative phosphorylation, and 2-oxocarboxylic acid metabolism, which play an important role in the regulation of cellular energy metabolism, organic matter synthesis and metabolic pathways.

[0121] Figures 1 to 15 As shown, the postbiotics prepared by the present invention can damage the morphology of Escherichia coli, Salmonella and Campylobacter jejuni, destroy the integrity of the cell membranes of Escherichia coli, Salmonella and Campylobacter jejuni, cause the leakage of cell contents, and prevent and treat bacterial diarrhea; compared with antibiotics, the postbiotics of the present application will not cause the development of bacterial resistance, will not destroy the balance of intestinal microbial communities, and have a good application effect.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a postbiotic having the ability to inhibit bacterial diarrhea pathogens, characterized in that: The steps include: S1, inoculating Lactobacillus paracasei Postbio-P8 and Lactobacillus fermentans Postbio-Q8 into MRS liquid culture medium for activation and culture respectively, and then inoculating the activated Lactobacillus paracasei Postbio-P8 into fermentation medium for fermentation; after the fermentation is completed, continue to inoculate the activated Lactobacillus fermentans Postbio-Q8 for fermentation, and after the fermentation is completed, sterilize the mixed fermentation liquid to obtain a postbiotic fermentation liquid; The deposit number of the Lactobacillus paracasei Postbio-P8 is CGMCC No.31112, and the deposit number of the fermented Lactobacillus mucilaginosus Postbio-Q8 is CGMCC No.31113; S2, subjecting the postbiotic fermentation broth of step S1 to clarification, impurity removal, concentration, desalination and freeze-drying to obtain a postbiotic freeze-dried powder; The formula of the fermentation medium includes: 15-25 g / L glucose, 10-20 g / L whey protein, 15-25 g / L soybean powder, 5-15 g / L skim milk powder and 5-15 g / L yeast powder, and the initial pH is 7.0-7.5; The fermentation medium also includes a growth factor formula: Tween 80 0.2-0.8 g / L, disodium hydrogen phosphate 0.5-1.3 g / L, manganese sulfate 0.01-0.03 g / L and magnesium sulfate 0.1-0.2 g / L; The fermentation medium also includes a growth interaction factor formula: 0.01-0.03 g / L leucine and 0.01-0.03 g / L isoleucine.

2. The method for preparing a postbiotic having the function of inhibiting bacterial diarrhea pathogens according to claim 1, characterized in that: The number of viable bacteria of each strain after activation was 5×10 6 ~10×10 6 CFU / mL.

3. The method for preparing a postbiotic having the function of inhibiting bacterial diarrhea pathogens according to claim 1, characterized in that: The inoculation amount of the Lactobacillus paracasei Postbio-P8 is 1wt% to 3wt%.

4. The method for preparing a postbiotic having the function of inhibiting bacterial diarrhea pathogens according to claim 1, characterized in that: The pH value of the culture medium during the fermentation of the Lactobacillus paracasei Postbio-P8 is 4-4.

5.

5. The method for preparing a postbiotic having the function of inhibiting bacterial diarrhea pathogens according to claim 1, characterized in that: The inoculation amount of the fermented mucus lactobacillus Postbio-Q8 is 4wt%-6wt%.

6. The method for preparing a postbiotic having the function of inhibiting bacterial diarrhea pathogens according to claim 1, characterized in that: The pH value of the culture medium during fermentation of the fermented mucus lactobacillus Postbio-Q8 is 3.2-3.

8.

7. Use of a postbiotic in the preparation of a drug for preventing and treating bacterial diarrhea, characterized in that: The postbiotics are prepared by the preparation method according to any one of claims 1 to 6.

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