A strain of Pediococcus pentosaceus and its applications

Abstract

The present invention discloses a strain of Pediococcus pentosaceus and its application. The present invention provides a strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ) strain FeiHeP06. The Pediococcus pentosaceus ( Pediococcus pentosaceus ) strain FeiHeP06 is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 31506. The Pediococcus pentosaceus provided by the present invention has good gastrointestinal adaptability, good stability, good antibacterial activity, and good ability to regulate immune cell cytokines, can improve the intestinal health of the host and enhance the body's immunity, and has great application prospects.

Description

Technical Field

[0001] The present invention relates to a strain of Pediococcus pentosaceus and its applications. Specifically, it relates to a strain of Pediococcus pentosaceus that can promote the secretion of IFN-β by BMDC cells and its applications in the production of health foods, feed additives, and pharmaceuticals, belonging to the fields of microbial technology and pharmaceutical technology. Background Art

[0002] As a kind of lactic acid bacteria, Pediococcus pentosaceus has been added to the list of probiotic strains that can be used in foods. Research shows that Pediococcus pentosaceus isolated from fermented foods can significantly inhibit the growth of foodborne pathogenic bacteria, showing good prospects for development as a new antibacterial agent. In addition, there are also literature reports and confirmations that Pediococcus pentosaceus has the effects of reducing blood lipids, anti-obesity, antioxidant, anti-cancer, anti-inflammatory, improving alcoholic liver injury, alleviating colitis and intestinal flora imbalance, etc.

[0003] Research shows that for probiotics to exert their probiotic functions, it is first required that more than 10 6 CFU / mL (Colony-Forming Units per Milliliter) of viable bacteria reach the human intestine. The human gastrointestinal tract is a high-acid and high-bile salt environment. Whether probiotics can successfully pass through the gastrointestinal environment after entering the human body is the key factor determining whether they can exert their health care functions. In addition, probiotics can secrete some substances such as organic acids, hydrogen peroxide, bacteriocins, etc. during their growth and reproduction, and these antibacterial substances can inhibit the growth of certain pathogenic bacteria, thereby preventing some diseases.

[0004] Type I interferons (Interferon alpha (IFN-α), Interferon beta (IFN-β), etc.), as a class of cytokines, can trigger the protective defenses of the immune system and can also activate immune cells, playing a key role in responding to most viral infections. Its expression is regulated at the transcriptional level and can be highly induced in all nucleated cells in response to the perception of pathogens by Pattern Recognition Receptors (PRRs). At the same time, type I interferons have also been reported to play a protective role against bacterial infections, cancers, and autoimmune diseases. In addition, different from IFN-α, IFN-β is not related to systemic diseases such as autoimmune diseases, but is committed to stabilizing the protective immunity against infections and inflammations. Therefore, IFN-β is a potential way for the body to prevent bacterial and viral infections.

[0005] It has been reported in the literature that Lactobacillus rhamnosus GG can induce the production of type I interferon through Toll-like receptor 4 (TLR4) (citing reference 1), and Lactobacillus plantarum and Pseudomonas pentosaceus can induce IFN-β production in human macrophage-like cells and human primary phagocytes isolated from PBMCs (citing reference 2). However, there is little research on whether Pediococcus pentosaceus can be used as a probiotic to stimulate immune cells to produce IFN-β, and there is no report on this immune pathway in dendritic cells.

[0006] Therefore, studying the ability to stimulate the body's immune cells to produce IFN-β has greater potential and value for the development and utilization of Pediococcus pentosaceus. At the same time, there is also a need to develop products that can improve host intestinal health and regulate immune responses with good intestinal adaptability and production stability.

[0007] Citing references

[0008] Citing reference 1. PLoS pathogens, 2019: Lung transcriptional unresponsivenessand loss of early influenza virus control in infected neonates is preventedby intranasal Lactobacillus rhamnosus GG.

[0009] Citing reference 2. Gut microbes, 2020: Beneficial bacteria activate type-Iinterferon production via the intracellular cytosolic sensors STING and MAVS. Summary of the invention

[0010] Problems to be Solved by the Invention

[0011] The technical problem to be solved by the present invention is to provide a strain of Pediococcus pentosaceus FeiHeP06 with good intestinal adaptability, good production stability, good antibacterial activity, and good immune cell cytokine regulation ability, which can be used in products for improving host intestinal health, enhancing the body's immunity, or regulating immune responses.

[0012] Solutions for Solving the Problems

[0013] [1]. A strain of Pediococcus pentosaceus FeiHeP06, which is deposited in the General Microbiology Center of the China Microbial Culture Collection Center with the deposit number CGMCC No. 31506;

[0014] The 16S rDNA sequence of the Pediococcus pentosaceus FeiHeP06 strain is as shown in SEQ ID NO.1.

[0015] [2]. A microbial preparation, which contains the strain described in [1].

[0016] [3]. According to the microbial preparation described in [2], in the microbial preparation, the viable count of the Pediococcus pentosaceus FeiHeP06 strain is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g, or, in the microbial preparation, the Pediococcus pentosaceus FeiHeP06 strain is inactivated.

[0017] [4]. The preparation method of the microbial preparation as described in [2] or [3], which includes the step of culturing the strain described in [1].

[0018] [5]. A starter, which contains the strain described in [1], the microbial preparation described in [2] or [3], or the microbial preparation prepared by the preparation method described in [4].

[0019] [6]. A product, which contains the strain described in [1], the microbial preparation described in [2] or [3], or the microbial preparation prepared by the preparation method described in [4], or the product is obtained by fermentation using the starter described in [5];

[0020] The product is a food, a food additive or a feed additive.

[0021] [7]. According to the product described in [6], the product further contains any one or more of the following components: plant product components, animal meat product components, animal dairy product components, functional additive components and any acceptable excipients.

[0022] [8]. The use of the strain as described in [1], the microbial preparation as described in [2] or [3], or the microbial preparation prepared by the preparation method as described in [4] in any of the following (1) to (2):

[0023] (1) Use in the preparation of health foods that help regulate the intestinal flora;

[0024] (2) Use in the preparation of health foods that help enhance immunity.

[0025] Effects of the Invention

[0026] The present invention provides a strain of Pediococcus pentosaceus FeiHeP06. This Pediococcus pentosaceus FeiHeP06 strain can play a probiotic role in the intestine and has the function of promoting bone marrow-derived dendritic cells (BMDCs, also simply referred to as myeloid dendritic cells) to secrete the immunomodulatory cytokine IFN-β, specifically manifested in:

[0027] (1) Pediococcus pentosaceus is a kind of lactic acid bacteria. It has been found that Pediococcus pentosaceus has strong immunomodulatory ability. Using mouse BMDC cells as a model and Bifidobacterium animalis BB-12 as a control strain, the Pediococcus pentosaceus, Bifidobacterium animalis BB-12, and 10 other strains of Bifidobacterium and Lactobacillus were co-cultured with BMDCs respectively, and the concentration of IFN-β was measured by ELISA. Compared with the other strains, Pediococcus pentosaceus FeiHeP06 had the best effect, and the heat sterilization effect was better than that of live bacteria.

[0028] (2) Acid and bile salt tolerance are necessary characteristics for probiotics to reach the intestine and maintain a certain number of live bacteria. In the case of culturing in simulated gastric juice and simulated intestinal juice of the present invention for 3 to 4 h, Pediococcus pentosaceus FeiHeP06 can maintain the number of live bacteria still above 10 8 CFU / mL, meeting the standard of acid and bile salt tolerance.

[0029] (3) During the growth and reproduction process, lactic acid bacteria can secrete some substances such as organic acids, hydrogen peroxide, and bacteriocins. These antibacterial substances can inhibit the growth of certain pathogenic bacteria, thereby preventing some diseases. In the present invention, four pathogenic bacteria were used to measure the antibacterial activity of Pediococcus pentosaceus. Taking Bifidobacterium animalis BB-12 as a control strain, the antibacterial activity of Pediococcus pentosaceus FeiHeP06 was significantly higher than that of Bifidobacterium animalis BB-12, showing good antibacterial ability.

[0030] The Pediococcus pentosaceus strain FeiHeP06 provided by the present invention has good intestinal adaptability, good antibacterial activity, good production stability, and good ability to regulate immune cell cytokines, and has great application prospects in products for improving host intestinal health, enhancing body immunity or regulating immune response (such as health foods, feed additives and pharmaceuticals). Brief Description of the Drawings

[0031] Figure 1 The bar graph shows the ELISA assay results of IFN-β produced by co-culturing 12 strains with BMDC. Different letters a-f in the figure indicate significant differences between groups in the Duncan multiple comparison test (P < 0.05).

[0032] Figure 2 The bar graph shows the ELISA assay results of IFN-β produced by co-culturing Pediococcus pentosaceus FeiHeP06 with BMDC from 4-week-old, 6-week-old, and 10-week-old mice, respectively. "****" in the figure indicates P < 0.001 compared with the live bacteria group.

[0033] Figure 3 The figure shows the colony morphological characteristics of Pediococcus pentosaceus strain FeiHeP06 on MRS solid medium.

[0034] Figure 4 The figure shows the growth curve of Pediococcus pentosaceus FeiHeP06. BB12 in the figure is Bifidobacterium animalis subsp. lactis BB-12. Detailed Embodiments

[0035] The following will detail various exemplary embodiments, features and aspects of the present invention. The special term "exemplary" here means "serving as an example, embodiment or illustrative". Any embodiment described as "exemplary" here does not have to be construed as superior or better than other embodiments.

[0036] In addition, for better illustration of the present invention, numerous specific details are given in the following detailed embodiments. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In other instances, methods, means, equipment and steps well-known to those skilled in the art are not described in detail so as to highlight the gist of the present invention.

[0037] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0038] In this specification, the meaning expressed by "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0039] In this specification, the "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc. mentioned refer to the specific elements related to the embodiment (for example, features, structures, properties, and / or characteristics) are included in at least one of the embodiments described herein, and may or may not exist in other embodiments. Additionally, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0040] In this specification, the numerical range expressed by "numerical value A to numerical value B" refers to the range including the endpoint numerical values A and B.

[0041] In this specification, "culturing" refers to growing a population of microbial cells under any suitable conditions (such as using liquid, gel, or solid culture media), including but not limited to well plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, etc., and various culture conditions such as temperature, time, and pH value of the culture medium can be appropriately adjusted according to the actual situation.

[0042] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0043] Biological material preservation

[0044] A strain of Pediococcus pentosaceus FeiHeP06, taxonomically named Pediococcus pentosaceus. The Pediococcus pentosaceus FeiHeP06 strain was deposited with the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No. 31506, the deposit date being August 1, 2024, and the deposit address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0045] Strain

[0046] The present invention provides a strain of Pediococcus pentosaceus FeiHeP06. The Pediococcus pentosaceus FeiHeP06 strain is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 31506 and the deposit date of August 1, 2024.

[0047] The Pediococcus pentosaceus FeiHeP06 strain is derived from a fecal sample of a healthy pregnant woman in Wuxi, Jiangsu Province. After sequencing analysis of the strain, the obtained sequence is subjected to nucleic acid sequence alignment in NCBI Standard Nucleotide BLAST. The strain has a homology of up to 98.58% with Pediococcus pentosaceus strain SMFM2016-WK1. It is determined that the strain is Pediococcus pentosaceus and named Pediococcus pentosaceus FeiHeP06, and its original number in the laboratory is CCFM1443. In this specification, the Pediococcus pentosaceus FeiHeP06 strain is also simply referred to as Pediococcus FeiHeP06.

[0048] In some specific embodiments, using mouse BMDC cells (mouse bone marrow-derived dendritic cells) as a model and Bifidobacterium animalis subsp. lactis BB-12 as a control strain, the Pediococcus pentosaceus, Bifidobacterium animalis subsp. lactis BB-12, and 10 other strains of Bifidobacterium and Lactobacillus are co-cultured with BMDC respectively, and the concentration of IFN-β is measured by ELISA. Compared with the other strains, Pediococcus FeiHeP06 has the best effect, and its heat sterilization effect is better than that of live bacteria (as Figures 1 - 2 shown), and Pediococcus FeiHeP06 plays a key role in immunomodulation.

[0049] The colonies of the Pediococcus FeiHeP06 on MRS solid medium are milky white, round and raised, with a smooth and semi-transparent surface, and a diameter of 1-2 millimeters (mm) (as Figure 3 shown).

[0050] In some specific embodiments, when Pediococcus FeiHeP06 is cultured in simulated gastric juice and simulated intestinal juice for 3-4 hours, the viable cell count can still be maintained above 10 8 CFU / mL, meeting the standard of acid and bile salt tolerance.

[0051] In some specific embodiments, the Pediococcus pentosaceus FeiHeP06 has good antibacterial ability. Pediococcus pentosaceus FeiHeP06 can antagonize the growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, regulate the intestinal flora and promote intestinal health.

[0052] In some specific embodiments, the 16S rDNA sequence of the Pediococcus pentosaceus FeiHeP06 is as shown in SEQ ID NO.1.

[0053] SEQ ID NO.1: 16S rDNA

[0054]

[0055] Culture

[0056] In some aspects, the present invention provides a culture, wherein the culture is obtained by culturing the above-mentioned Pediococcus pentosaceus strain FeiHeP06.

[0057] In the present invention, the term "culture" refers to the general term for liquid or solid products (at least a part of the substances in the culture container, such as the supernatant) with a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or can contain a certain amount of culture medium, metabolites or other components generated during the culture process, or the supernatant obtained by centrifugation.

[0058] In some embodiments, the culture is a product obtained by growing and / or amplifying microorganisms or a supernatant (cell-free supernatant) obtained by centrifuging the product.

[0059] In some embodiments, the culture is carried out by the following steps:

[0060] (1) Streak the Pediococcus pentosaceus strain FeiHeP06 on MRS solid medium respectively, and obtain single colonies after culture;

[0061] (2) Pick single colonies and inoculate them into MRS liquid medium, culture for activation, and continuously activate for at least one generation or more, such as two generations, to obtain an activation solution;

[0062] Optionally, (3) inoculate the activation solution into MRS liquid medium at an inoculation amount of about 2% (v / v), and culture until the stationary phase to obtain a culture solution;

[0063] (4) Centrifuge the activation solution obtained in step (2) or the culture solution obtained in step (3) to obtain a supernatant.

[0064] Optionally, (5) collect the supernatant, filter it to obtain a cell-free supernatant for standby.

[0065] In some specific embodiments, the MRS solid medium comprises 10 grams per liter (g / L) of peptone, 10 g / L of beef extract, 20 g / L of glucose, 2 g / L of anhydrous sodium acetate, 5 g / L of yeast extract, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of K2PO4·3H2O, 0.5 g / L of MgSO4·7H2O, 0.25 g / L of MnSO4·H2O, 1 milliliter per liter (mL / L) of Tween 80, and 20 g / L of agar; the pH is 6.8.

[0066] In some specific embodiments, the MRS liquid medium comprises 10 g / L of peptone, 10 g / L of beef extract, 20 g / L of glucose, 2 g / L of anhydrous sodium acetate, 5 g / L of yeast extract, 2 g / L of diammonium hydrogen citrate, 2.6 g / L of K2PO4·3H2O, 0.5 g / L of MgSO4·7H2O, 0.25 g / L of MnSO4·H2O, 1 mL / L of Tween 80; the pH is 6.8.

[0067] Microbial preparation

[0068] In some aspects, the present invention further provides a microbial preparation comprising the above-mentioned Pediococcus pentosaceus FeiHeP06 strain or the above-mentioned culture.

[0069] In some embodiments, the microbial preparation of the present invention is solid, and exemplarily, it can be freeze-dried powder, etc.

[0070] Furthermore, the microbial preparation may further comprise a protective agent (such as a freeze-drying protective agent), and the protective agent includes but is not limited to skim milk powder, trehalose, sodium glutamate, and / or glycerol, etc.; preferably, the protective agent is skim milk powder.

[0071] In some other embodiments, the microbial preparation of the present invention is semi-solid, and exemplarily, it can be bacterial sludge. The bacterial sludge refers to bacteria containing a small amount of water.

[0072] In some other embodiments, the microbial preparation of the present invention is liquid, and exemplarily, it can be a suspension, a culture. Furthermore, the microbial preparation may further comprise a solvent, and the solvent includes but is not limited to water or a culture medium, etc.

[0073] In some embodiments, in the microbial preparation, the viable count of the Pediococcus pentosaceus FeiHeP06 strain is not less than 1×10 6 CFU / mL or 1×10 6Colony-Forming Units per Gram (CFU / g).

[0074] In some embodiments, in the microbial preparation, the Pediococcus pentosaceus strain FeiHeP06 is inactivated, for example, by heat sterilization.

[0075] Preparation method of microbial preparation

[0076] In some embodiments, the preparation method of the microbial preparation includes the steps of inoculating the above-mentioned Pediococcus pentosaceus strain FeiHeP06 into a suitable culture medium for cultivation, and optionally, steps such as fermentation, centrifugation, collecting the bacterial sludge, and adding a protective agent (such as a lyophilization protective agent). Centrifugation can increase the cell concentration and shorten the lyophilization time. By adding a protective agent, the death or loss of activity of the bacterial strain during freeze-drying can be minimized, and the fermentation effect of the microbial preparation can be improved.

[0077] In some embodiments, after the step of adding the protective agent, a lyophilization step is further included. Lyophilization facilitates the transportation and storage of the microbial preparation.

[0078] In some specific embodiments, the preparation method of the microbial preparation described in the present invention includes the following steps:

[0079] Inoculate the Pediococcus pentosaceus strain FeiHeP06 into any solid medium suitable for lactic acid bacteria culture for anaerobic cultivation to obtain single colonies, and then inoculate the single colonies into the culture solution;

[0080] Centrifuge the culture solution to obtain bacterial sludge;

[0081] Wash the cells and resuspend them with a lyophilization protective agent to obtain a cell suspension;

[0082] Lyophilize the cell suspension by vacuum freezing to obtain the microbial preparation.

[0083] Optionally, the post-culture of single colonies can be subjected to activation treatment.

[0084] In some embodiments, the preparation method of the culture medium is as follows: Dissolve 8% - 3% enzymatically hydrolyzed skim milk, 0.4% - 0.8% glucose, 1% - 2% tryptone, and 0.2% - 0.5% yeast extract with 85 - 90% of water by the total weight of the culture medium, and then adjust its pH to obtain the culture medium.

[0085] In some preferred embodiments, the method for preparing the culture medium is as follows: Dissolve 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone, and 0.3% yeast extract in 87.7% water based on the total weight of the culture medium, and then adjust its pH to obtain the culture medium.

[0086] In some embodiments of the present invention, the mass ratio of the cryoprotectant to the bacterial cells is (1 - 5):1, preferably (1 - 3):1, such as 1:1, 2:1, 3:1.

[0087] In some embodiments, the cryoprotectant contains an aqueous solution of skim milk powder at 100 - 150 g / L, such as 110 g / L, 130 g / L, 150 g / L.

[0088] In some embodiments, the pH of the culture medium is 6.5 - 7.5, preferably 6.8 - 7.2, such as 6.8, 7.0.

[0089] starter

[0090] In some aspects, the present invention also provides a starter containing the above-mentioned Pediococcus pentosaceus FeiHeP06 strain, the above-mentioned culture, the above-mentioned microbial preparation, or the microbial preparation prepared by the above-mentioned preparation method.

[0091] In some embodiments, the starter further contains lactic acid bacteria.

[0092] In the present invention, the lactic acid bacteria used in the starter are not particularly limited as long as they do not interfere with the effects of the present invention, and they can be lactic acid bacteria derived from animals or lactic acid bacteria derived from plants.

[0093] As preferred lactic acid bacteria of the present invention, bacteria belonging to the genus Lactobacillus, Streptococcus, Lactococcus, Enterococcus, Leuconostoc, and combinations thereof can be listed, and lactic acid bacteria containing bacteria belonging to the genus Lactobacillus are preferred. As lactic acid bacteria containing bacteria belonging to the genus Lactobacillus, for example, bacteria belonging to the genus Lactobacillus, combinations of bacteria belonging to the genus Lactobacillus and Streptococcus, combinations of bacteria belonging to the genus Lactobacillus and Lactococcus, etc. can be listed. These lactic acid bacteria can be obtained from storage institutions such as ATCC, etc., or commercially available products can be appropriately used.

[0094] As bacteria belonging to the genus Streptococcus, for example, Streptococcus thermophilus, etc. can be listed.

[0095] As a combination of Lactobacillus spp. and Streptococcus spp., preferably, Lactobacillus bulgaricus and Streptococcus thermophilus can be cited.

[0096] In some embodiments, in the starter culture, the viable count of Pediococcus pentosaceus FeiHeP06 strain is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0097] Product

[0098] The present invention further provides a product, which comprises the above-mentioned Pediococcus pentosaceus FeiHeP06 strain, the above-mentioned culture, the above-mentioned microbial preparation, the microbial preparation prepared by the above-mentioned preparation method, or is obtained by fermenting with the above-mentioned starter culture.

[0099] In some embodiments, the product is a probiotic product.

[0100] In some specific embodiments, the probiotic product is a probiotic product with good intestinal adaptability, and the product contains the above-mentioned Pediococcus pentosaceus FeiHeP06 strain, the above-mentioned microbial preparation or the microbial preparation prepared by the above-mentioned preparation method.

[0101] For different product categories, the specific form of the product is not particularly limited in the present invention. Exemplarily, it can be in powder form or liquid form.

[0102] The population targeted by the product is not particularly limited in the present invention. Exemplarily, the product can be used for infants, children, adolescents or adults.

[0103] In some embodiments, the product may further contain any one or more of the following components: plant product components, animal meat product components, animal dairy product components, functional additive components and any acceptable excipients.

[0104] In some embodiments, the plant product component includes a plant or a plant extract component, such as fruits or their extracts like fig, pomegranate, kiwifruit, tangerine, orange, pineapple, strawberry, apple, rubber, grape, pear, cherry, blueberry, blackberry, blackcurrant, cranberry, raspberry, melon, Phyllanthus emblica, and cornel; fruit and vegetable substances or their extracts like onion, cucumber, tomato, cauliflower, carrot, spinach, Chinese kale, Brussels sprout, garlic, basil, and oregano; grains or their extracts like rice (indica rice, japonica rice, glutinous rice), wheat (common wheat, barley, oats, rye), corn, sorghum, millet, broomcorn millet, buckwheat, soybean, broad bean, pea, mung bean, adzuki bean, kidney bean; nut substances or their extracts like walnut, pistachio, cashew nut, hazelnut, almond, pine nut, peanut, melon seed, chestnut, macadamia nut, and ginkgo nut; coffee or its extract.

[0105] In some embodiments, the animal component, such as the animal meat product component, includes the meat product components of cattle, sheep, fish, or poultry.

[0106] In some embodiments, the product may further contain a fat component, and the fat may include at least one of saturated fatty acid, polyunsaturated fatty acid, monounsaturated fatty acid, OPO structured lipid, DHA, EPA, ARA, and phospholipid. More specifically, the fat includes safflower oil, walnut oil, peanut oil, corn oil, soybean oil, argan oil, olive oil, tea oil, Sacha inchi oil, olive oil, coconut oil, perilla oil, deep-sea fish oil, cocoa butter, palm oil, beef tallow, cream, lard, medium-chain triglyceride, or lecithin, etc.

[0107] In some embodiments, the animal dairy product component contains a milk-containing component or a protein component. The milk-containing component includes dairy products such as fresh milk, milk powder, whey protein, or cheese derived from fresh raw cow (sheep) milk; for the protein component, it can come from plant proteins such as soybean protein and peanut protein.

[0108] In some embodiments, the functional additive components include vitamins (one or more of vitamin A, β-carotene, vitamin D3, vitamin E, vitamin K1, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, pantothenic acid, folic acid, niacin, choline, inositol, and biotin), starch, modified starch, amino acids (such as L-lysine-L-glutamate, L-glutamate, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine, or L-leucine), traditional Chinese medicine or traditional Chinese medicine extracts, dietary fiber (such as inulin, konjac powder, galactooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, soybean polysaccharide, cyclodextrin, resistant dextrin, or soybean fiber).

[0109] In some embodiments, the product may further comprise a trace element supplement, which may include metal ion salts of organic acids, such as one or more of calcium citrate, calcium L-lactate, calcium hydrogen phosphate, potassium gluconate, sodium citrate, ferrous gluconate, potassium iodide, zinc gluconate, sodium selenite, copper gluconate, chromium sulfate, manganese gluconate, and magnesium gluconate.

[0110] In some embodiments, any acceptable excipients include, but are not limited to, solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible pigments, etc.

[0111] The present invention does not make any particular limitation on the specific type of the above product.

[0112] In some embodiments, the product is a food, which contains the above-mentioned strains, cultures, microbial preparations or microbial preparations prepared by the above preparation method, or the food is a food obtained by fermenting with the above-mentioned starter.

[0113] In some embodiments, the product is a food additive.

[0114] In some embodiments, the product is a nutritional food, a functional food or a health food.

[0115] In some preferred embodiments, the food is: dairy products, such as: yogurt, cheese, butter, ice cream, etc.; soy products, such as: including tofu, soy milk, dried tofu, tofu skin, etc.; fruit and vegetable products, such as: including fresh fruits, vegetables, fruit juices, vegetable juices, jams, vegetable purees, etc.; beverages, such as: including tea, coffee, fruit juices, carbonated beverages, mineral water, etc.; snacks, such as: including nuts, dried fruits, potato chips, biscuits, candies, etc.

[0116] In some exemplary embodiments of the present invention, the food is a fermented dairy product containing Pediococcus pentosaceus strain FeiHeP06 or the above-mentioned microbial preparation, or a fermented dairy product obtained by fermenting with the above-mentioned starter.

[0117] In some exemplary embodiments of the present invention, the food is a fruit and vegetable beverage containing Pediococcus pentosaceus strain FeiHeP06 or the above-mentioned microbial preparation.

[0118] In some exemplary embodiments of the present invention, the food is fermented pork (such as fermented sausage) containing Pediococcus pentosaceus strain FeiHeP06 or the above-mentioned microbial preparation, or fermented pork obtained by fermenting with the above-mentioned fermenting agent.

[0119] In some specific embodiments, the food of the present invention may be infant formula milk powder, baby complementary food, children's formula milk powder, children's snacks, pregnant women's formulated milk powder, middle-aged and elderly milk powder, or nutritional or dietary supplements.

[0120] In some other specific embodiments, the food of the present invention may be powdered reconstitutable foods (such as solid beverages, instant coffee, cereal powder, nut powder or lotus root powder, etc.), baked foods (such as bread, cake or biscuit baked foods, etc.), beverages (such as carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages, milk beverages or alcoholic beverages, etc.), candies (such as gel candies, hard candies, tablet candies, etc.), milk and dairy products (such as fresh milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc. derived from fresh cow (sheep) milk), pasta products (such as noodles, instant noodles, steamed buns, buns, dumplings or wontons, etc.), etc.

[0121] In some embodiments, the product is a drug.

[0122] In some embodiments of the present invention, the drug contains the above-mentioned Pediococcus pentosaceus strain FeiHeP06, a drug carrier and / or pharmaceutical excipients.

[0123] In some embodiments of the present invention, the drug carrier includes microcapsules, microspheres, nanoparticles and / or liposomes.

[0124] In some embodiments of the present invention, the pharmaceutical excipients include excipients and / or additives.

[0125] In some embodiments of the present invention, the excipients include binders, fillers, disintegrants and / or lubricants.

[0126] In some embodiments of the present invention, the additives include solubilizers, cosolvents, latent solvents and / or preservatives.

[0127] In some embodiments of the present invention, the dosage form of the drug is powder, granule, capsule, tablet, pill or oral liquid.

[0128] In some other specific embodiments, the product of the present invention is an oral preparation, and the oral preparation includes but is not limited to tablets, pills, granules, powders, tea preparations, capsules or oral liquids, etc.

[0129] In some embodiments, the product is a feed additive.

[0130] In some embodiments, the feed additive contains the above-mentioned Pediococcus pentosaceus strain FeiHeP06 or the above-mentioned microbial preparation.

[0131] In some embodiments, the feed additive is a microbial product that can stimulate the reproduction and growth of beneficial bacteria in animals while inhibiting the growth of harmful bacterial strains. After entering the livestock and poultry body as feed, it can quickly reproduce, form a normal microbial flora in the host body, provide nutrition for the host, and prevent the invasion of pathogenic bacteria.

[0132] In other embodiments, the product is non-food.

[0133] In other embodiments, the product is a non-health food.

[0134] In other embodiments, the product is non-medicinal.

[0135] Use

[0136] Use for Facilitating the Regulation of Intestinal Flora

[0137] The Pediococcus pentosaceus strain FeiHeP06 and its culture provided by the present invention can inhibit the growth of intestinal pathogenic microorganisms, and can antagonize the growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes. It has good tolerance to gastric acid and bile salts and can also regulate the intestinal flora to promote intestinal health.

[0138] Therefore, the Pediococcus pentosaceus strain FeiHeP06, culture, microbial preparation, and microbial preparation prepared by the preparation method provided by the present invention can be used to prepare products that help regulate the intestinal flora. The products described in the present invention also help regulate the intestinal flora.

[0139] The present invention does not particularly limit the specific product categories that are helpful for regulating the intestinal flora. In some embodiments, the regulation of the intestinal flora is not for the purpose of preventing and / or treating diseases. For example, in the case of intestinal flora disorder, or the presence of intestinal pathogenic microorganisms (Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes) but not to the extent of causing diseases, or in the case of intestinal flora disorder and increase in intestinal pathogenic microorganisms not caused by diseases, the microbial preparations prepared by using the Pediococcus pentosaceus FeiHeP06 strain, culture, microbial preparation, and preparation method provided by the present invention are used. In some embodiments, the products that are helpful for regulating the intestinal flora are foods. In some embodiments, the products that are helpful for regulating the intestinal flora are health foods. In some embodiments, the products that are helpful for regulating the intestinal flora are probiotic products.

[0140] In other embodiments, the product is a drug. In some specific embodiments, the drug is used to inhibit intestinal pathogenic bacteria.

[0141] Use for Facilitating Immunity Enhancement, Facilitating the Regulation of Immune Cell Cytokine Secretion, and Preventing Infections

[0142] The Pediococcus pentosaceus FeiHeP06 strain and its culture provided by the present invention significantly promote the secretion of IFN-β by bone marrow-derived dendritic cells (BMDC cells) and can also increase the secretion amount of IFN-β. In addition, the heat sterilization effect of Pediococcus pentosaceus FeiHeP06 is significant, and it can also play an immunomodulatory function. Pediococcus pentosaceus FeiHeP06 has the ability to regulate immunity in organisms of different ages at different times.

[0143] Therefore, the microbial preparations prepared by using the Pediococcus pentosaceus FeiHeP06 strain, culture, microbial preparation, and preparation method provided by the present invention can be used to prepare products that are helpful for enhancing immunity and regulating the secretion of cytokines by immune cells. The products described in the present invention are also helpful for enhancing immunity and regulating the secretion of cytokines by immune cells.

[0144] The present invention does not particularly limit the specific product categories that are helpful for enhancing immunity and regulating the secretion of cytokines by immune cells.

[0145] Moreover, in some embodiments, the enhancement of immunity and the regulation of cytokine secretion by immune cells are not for the purpose of preventing and / or treating diseases. For example, in cases of reduced immunity not caused by diseases, etc., the microbial preparations prepared using the Pediococcus pentosaceus FeiHeP06 strain, cultures, microbial preparations, and preparation methods provided by the present invention are used. In some embodiments, the products that help enhance immunity and regulate cytokine secretion by immune cells are foods. In some embodiments, the products that help enhance immunity and regulate cytokine secretion by immune cells are health foods. In some embodiments, the products that help enhance immunity and regulate cytokine secretion by immune cells are probiotic products.

[0146] In some specific embodiments, the regulation of cytokine secretion by immune cells includes increasing the secretion of IFN-β by immune cells.

[0147] In some other embodiments, the product is a drug. In some specific embodiments, the drug is used for anti-infection and the like.

[0148] In some specific embodiments, the anti-infection includes inhibiting the growth of intestinal pathogenic microorganisms. In some specific embodiments, the anti-infection includes antagonizing the growth of Staphylococcus aureus, Escherichia coli, and / or Listeria monocytogenes.

[0149] Use as a Feed Additive

[0150] The Pediococcus pentosaceus FeiHeP06 strain and its culture provided by the present invention can inhibit the growth of intestinal pathogenic microorganisms, can antagonize the growth of Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes, regulate the intestinal flora, promote intestinal health, and can also increase the secretion amount of IFN-β to exert an immune regulatory function.

[0151] Therefore, the Pediococcus pentosaceus FeiHeP06 strain, cultures, microbial preparations prepared by the preparation method, and microbial preparations provided by the present invention can be used to prepare products for feed additives. The products of the present invention also help the reproduction and growth of beneficial livestock and poultry strains, provide nutrition, and prevent the invasion of pathogenic bacteria.

[0152] The above products can be sprayed, mixed, or coated onto feeds or the above products can be mixed into feed additives. The products can also be added during the production of feeds.

[0153] The present invention does not make specific limitations on the specific product categories for feed additives and their usage methods.

[0154] Example

[0155] The following will describe the implementation scheme of the present invention in detail in combination with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0156] The culture media involved in the following examples are as follows:

[0157] mMRS liquid medium: Peptone 10 g / L, Beef extract 10 g / L, Glucose 20 g / L, Sodium acetate anhydrous 2 g / L, Yeast extract 5 g / L, Diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.25 g / L, Tween 80 1 mL / L, L-cysteine 0.5 g / L; pH is 6.8. (For MRS liquid medium, L-cysteine is not added, and the rest is the same).

[0158] mMRS solid medium: On the basis of mMRS (MRS containing L-cysteine) liquid medium, add agar 20 g / L. (For MRS solid medium, L-cysteine is not added, and the rest is the same).

[0159] LBS liquid medium: Tryptone 10 g / L, Sodium acetate anhydrous 17 g / L, Yeast extract 5 g / L, Magnesium sulfate 0.575 g / L, Glucose 20 g / L, Manganese sulfate 0.12 g / L, Tween 80 1mL / L, Ferrous sulfate 0.034 g / L, Potassium dihydrogen phosphate 6 g / L, Ammonium citrate 2 g / L; pH is 5.4±0.2.

[0160] LBS solid medium: On the basis of LBS liquid medium, add agar 20 g / L.

[0161] LB liquid medium: Tryptone 10g / L, Yeast extract 5g / L, Sodium chloride (NaCl) 10g / L.

[0162] LB solid medium: On the basis of LB liquid medium, add agar 20 g / L.

[0163] The preparation method of Pediococcus pentosaceus cells involved in the following examples is as follows:

[0164] Streptococcus pentosaceus was streaked on MRS solid medium and cultured under anaerobic conditions at 37 °C for 48 h to obtain single colonies; single colonies were picked and inoculated into 5 mL of MRS liquid medium, and cultured under anaerobic conditions at 37 °C for 18 h for activation. After two consecutive generations of activation, an activated solution was obtained; the activated solution was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v) and cultured under anaerobic conditions at 37 °C for 18 h to obtain a bacterial solution; the bacterial solution was centrifuged at 5000 r / min for 15 min, and the upper layer of the culture medium was discarded to obtain Streptococcus pentosaceus cells.

[0165] Experimental materials:

[0166] Red blood cell lysate (Formax, FMS-RBC100),

[0167] Granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech, 315-03),

[0168] ELISA kit (R&D, VAL612),

[0169] Pepsin (Maclean, P909753),

[0170] Trypsin (Maclean, T819003),

[0171] Oxford cup (Shanghai Shendi, SDA-477131), Bifidobacterium animalis subsp. lactis BB-12 (Chr. Hansen, cultured using mMRS medium).

[0172] Example 1: Isolation, identification and culture of strains

[0173] The specific steps are as follows:

[0174] 1. Isolation

[0175] 0.5 mL of samples were taken from the fecal samples of healthy pregnant women and infants and added to 4.5 mL of sterilized normal saline for gradient dilution. The diluted samples were divided into two parts. One part took 100 μL of the diluted solutions with dilution gradients of 10 -4 、10 -5 、10 -6 and spread on mMRS solid medium with pH = 6.8 supplemented with mupirocin. The other part took 100 μL of the diluted solutions with dilution gradients of 10 -4 、10 -5 、10 -6 and spread on LBS solid medium with pH = 5.6 supplemented with vancomycin. The plates were cultured anaerobically at 37 °C for 48 h, and the colony morphology was observed and recorded;

[0176] Colonies with different morphologies on the solid medium were picked for streak isolation. After anaerobic culture at 37 °C for 48 h, single colonies with different morphologies on the solid medium were picked again for streak isolation until pure single colonies with consistent morphology were obtained;

[0177] The pure colonies on the solid medium were picked and inoculated into 5 mL of liquid medium, and anaerobically cultured at 37 °C for 18 h; 1 mL of the bacterial solution was taken into a sterile centrifuge tube, centrifuged at 5000 r / min for 15 min, and then the upper-layer medium was discarded. The bacterial pellet was resuspended in 30% glycerol solution and stored at -80 °C, obtaining 11 strains.

[0178] 2. Identification

[0179] The 16S rDNA of the separated bacterial strains was amplified by PCR respectively, and the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The primer sequences were:

[0180] 27F: 5’-AGAGTTTGATCMTGGCTCAG-3’ (SEQ ID NO.2);

[0181] 1492R: 5’-GGTTACCTTGTTACGACTT-3’ (SEQ ID NO.3).

[0182] The obtained sequencing results were subjected to nucleic acid sequence alignment in NCBI standard Nucleotide BLAST. The 11 strains were Bifidobacterium breve BB27M16, Bifidobacterium breve BB30M4, Bifidobacterium bifidum YF5M8, Bifidobacterium bifidum YF9M10, Bifidobacterium longum subsp. longum BB16M6, Bifidobacterium longum subsp. longum BB21M8, Lactobacillus rhamnosus BB6L9, Lactobacillus rhamnosus BB15L10, Pediococcus pentosaceus FeiHeP06 (its 16S rDNA sequence is shown in SEQ ID NO.1), Pediococcus pentosaceus BB29L6, and Pediococcus pentosaceus BB76L11.

[0183] Example 2: Screening of Pediococcus pentosaceus FeiHeP06 strain

[0184] To screen probiotics with antiviral and immune-regulating abilities, using the BMDC cell model, 11 strains and Bifidobacterium animalis subsp. lactis BB-12 were co-cultured with BMDC, and Pediococcus pentosaceus FeiHeP06 produced the highest level of IFN-β. The specific steps are as follows:

[0185] 1. Obtaining of BMDC cells

[0186] (1)Collect bone marrow cells from the tibias and femurs of 4-week-old mice. Pass the cells through a 200-mesh cell sieve, centrifuge at 1000 revolutions per minute (rpm) for 5 minutes, and discard the supernatant. Add 3 mL of red blood cell lysis buffer to the centrifuged cell pellet, quickly disperse the cell pellet, let it stand for 10 minutes, then add 10 mL of complete medium to neutralize. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Add complete medium containing 20 nanograms per milliliter (ng / mL) of granulocyte-macrophage colony-stimulating factor (GM-CSF) to resuspend the cells (2×10 6 cells / dish) and inoculate them into 10-centimeter (cm) cell culture dishes. Incubate the dishes in a 37°C, 5% CO2 incubator.

[0187] (2)On the 3rd day, perform a half-medium change for the cells (centrifuge at 1000 rpm for 5 minutes, discard half of the medium, and supplement with half the amount of fresh medium). Thereafter, change the medium every other day until the 9th day to obtain suspended and loosely adherent cells, which are BMDC cells.

[0188] 2. Co-culture of the strain and BMDC to measure the concentration of IFN-β

[0189] (1)Strain culture: Pipette 1 mL of the bacterial solution into 5 mL of MRS liquid medium and incubate it in a 37°C, 5% CO2 incubator for 1 - 2 days. Take 200 µL of the bacterial solution from each of the 11 strains and inoculate it into 5 mL of MRS liquid medium, then incubate it in a 37°C, 5% CO2 incubator for 24 hours.

[0190] (2)Bacterial pretreatment: Resuspend the bacterial solution. Take 200 μL of the solution and transfer it to a 96-well plate to measure the OD 600 . Centrifuge 1 mL of the bacterial solution, adjust the concentration to 10 8 using 1640 basal medium, and then dilute 1 mL of the bacterial solution 10-fold. Wash the diluted bacterial solution 2 - 3 times with 1640 basal medium and centrifuge (8000 r / min, 4°C, 3 minutes). Take 500 µL for co-culture with live bacteria and cells, and take another 500 µL for co-culture with cells after treating it in a metal bath at 98°C for 20 minutes (i.e., heat sterilization).

[0191] (3)Co-culture: After centrifuging the BMDC cells, discard the supernatant, resuspend them with 1640 complete medium, and pipette 200 µL per well into a 96-well cell culture plate after homogenizing. Add the bacterial solution to each well at a volume of 20 µL per well, with 4 parallels in each group. After adding, place the plate in a 37°C, 5% CO2 incubator and incubate for 24 hours.

[0192] After co - culturing for 24 h, centrifuge the 96 - well plate (2000 r / min, 20 min), take the supernatant, and measure the IFN - β level by ELISA.

[0193] The experimental results of ELISA for IFN - β produced by co - culturing 11 strains and Bifidobacterium animalis subsp. lactis BB - 12 (denoted as BB12 in the figure) with BMDCs derived from 4 - week - old mice are shown in Table 1, and the bar chart is for reference Figure 1 .

[0194] Table 1: ELISA determination results of IFN - β produced by co - culturing 12 strains with BMDCs (mean ± standard error)

[0195]

[0196] Note: “—” indicates not reaching the ELISA detection limit; a-f Different letters indicate significant differences between groups in the Duncan multiple comparison test (P < 0.05).

[0197] From the ELISA experimental results ( Figure 1 and Table 1), it can be seen that the effects of different strains on promoting BMDCs to secrete IFN - β are significantly different. Among them, the live bacteria and heat - killed bacteria effects of Pediococcus pentosaceus FeiHeP06 are significantly stronger than those of other strains. Compared with the control strain BB - 12, the effect intensity of FeiHeP06 live bacteria is more than 20 times that of BB - 12 live bacteria, and the effect intensity of FeiHeP06 heat - killed bacteria is more than 35 times that of BB - 12 heat - killed bacteria; compared with other Pediococcus pentosaceus, the level of IFN - β produced by FeiHeP06 stimulating BMDCs is the highest, showing significant differences compared with the other two Pediococcus pentosaceus strains, and the IFN - β level produced by Pediococcus pentosaceus FeiHeP06 is the highest. Therefore, Pediococcus pentosaceus FeiHeP06 of the present invention can significantly promote immune cells - dendritic cells to secrete the immunomodulatory factor IFN - β, increase the secretion amount of IFN - β, suggesting the potential immunomodulatory ability of Pediococcus pentosaceus FeiHeP06, and enabling the body to enhance the resistance to virus and other pathogen infections. In addition, the heat - killing effect of Pediococcus pentosaceus FeiHeP06 is significant, further broadening the application potential of the strain, which can be added to other foods sold at room temperature, reducing costs such as storage and transportation; live bacteria can not only play an immunomodulatory function, but also play roles such as antibacterial and regulating the intestinal flora, and have good development prospects.

[0198] 3. Co - culture Pediococcus pentosaceus FeiHeP06 with BMDC cells from mice of different weeks of age according to the above steps respectively, and measure the IFN - β level by ELISA. The experimental results are shown in Table 2, and the bar chart is for reference Figure 2 .

[0199] Table 2: ELISA assay results of IFN-β production by co-culturing Pediococcus pentosaceus FeiHeP06 with BMDC strains of different weeks of age (mean ± standard error)

[0200]

[0201] Note: **** indicates P < 0.001 compared with the live bacteria group.

[0202] The experimental results showed that the level of IFN-β produced by stimulating BMDC cells derived from mice of different weeks of age with Pediococcus pentosaceus FeiHeP06 strain was significantly higher in the heat-killed bacteria group than in the live bacteria group. Therefore, Pediococcus pentosaceus FeiHeP06 strain can regulate the immune function of mice of different weeks of age, suggesting that it may have immune regulatory ability for the body at different times of the population, including infants, adolescents, and adults, etc., and has a wider application scenario.

[0203] Example 3: Basic probiotic characteristics study of Pediococcus pentosaceus FeiHeP06

[0204] The specific steps are as follows:

[0205] I. Culture of Pediococcus pentosaceus FeiHeP06

[0206] Inoculate Pediococcus pentosaceus into MRS solid medium and anaerobically culture it at 37 °C for 48 h, then observe its colonies.

[0207] Experimental results: It was found that its colonies were milky white, round, convex, with a smooth and opaque surface, and the diameter was 1 - 2 mm. Refer to Figure 3 . Before and after culture, measure the pH of the blank culture solution and the culture solution with added bacteria using a pH meter, and it was found that Pediococcus pentosaceus produced acid during the culture process. Inoculate the Pediococcus pentosaceus strain into MRS liquid medium, place it in an enzyme-labeled instrument, and anaerobically culture it at 37 °C for 24 h. During the culture process, measure the OD of the culture solution every 15 min 600 , and it was found that Pediococcus pentosaceus reached the growth stationary phase after 18 - 19 h of culture, and the growth curve was as shown in Figure 4 .

[0208] II. Acid and bile salt tolerance of Pediococcus pentosaceus FeiHeP06

[0209] 1. Strain activation

[0210] Absorb 200 μL of Pediococcus pentosaceus FeiHeP06 bacterial solution from the bacteria preservation tube, inoculate it into 5 mL of MRS liquid medium, and culture it in a constant temperature incubator at 37 °C until the initial stage of the plateau to obtain the first-generation activated bacterial solution; in the second step, transfer 100 μL of the first-generation activated bacterial solution into 5 mL of MRS liquid medium, and culture it in a constant temperature incubator at 37 °C until the initial stage of the plateau to obtain the second-generation activated bacterial solution.

[0211] 2. Prepare simulated gastric juice and simulated intestinal juice

[0212] (1) Dissolve pepsin in sterilized normal saline (0.9% w / v, adjust the pH to 3.0 with hydrochloric acid), and the final concentration is 3 g / L. Filter with a 0.22 μm sterile filter membrane and use it immediately after preparation.

[0213] (2) Dissolve trypsin in sterilized normal saline (0.9% w / v, adjust the pH to 8.0 with NaOH), make the final concentration 1 g / L, and add bile salts to make the final concentration 0.3%. Filter with a 0.22 μm sterile filter membrane and use it immediately after preparation.

[0214] 3. Acid and bile salt tolerance experiment

[0215] (1) Take out 200 μL of the bacterial solution at the plateau stage to measure OD 600 , when the OD 600 value of the bacterial solution is within the range of 1.0 ± 0.2, it is considered that the CFU of Pediococcus pentosaceus is 10 9 , and calculate the volume of normal saline / simulated gastric juice / simulated intestinal juice to be added according to the OD value.

[0216] (2) Acid tolerance: Take 1 mL of the bacterial solution at the plateau stage and centrifuge it at 5000 r / min for 5 min respectively. Collect the bacterial cells in 2 1.5 mL centrifuge tubes, discard the supernatant, wash twice with normal saline, centrifuge, and pour out the supernatant. Calculate the amount of normal saline / simulated gastric juice to be added according to the OD 600 value, and resuspend the bacterial cells in the two centrifuge tubes respectively. After culturing at 37 °C for 3 h, perform plate viable cell counting. Take 10 -6 , 10 -7 , 10 -8 three dilution concentrations of the bacterial solution for counting from the control group (resuspended with normal saline) and the experimental group (resuspended with simulated gastric juice), and make 2 parallels for each dilution.

[0217] (3) Bile salt tolerance: Take 1 mL of the bacterial solution at the plateau stage and centrifuge it at 5000 r / min for 5 min respectively. Collect the bacterial cells in 2 1.5 mL centrifuge tubes, discard the supernatant, wash twice with normal saline, centrifuge, and pour out the supernatant. Calculate according to the OD 600The value was used to calculate the amount of normal saline / simulated intestinal fluid to be added, and the bacterial cells in the two centrifuge tubes were resuspended respectively. After culturing at 37 °C for 4 h, plate viable cell counting was performed. For the control group (resuspended with normal saline) and the experimental group (resuspended with simulated intestinal fluid), 10 -6 、10 -7 、10 -8 bacterial suspensions at three dilution concentrations were used for counting, and 2 replicates were made for each dilution.

[0218] Survival rate (%) = viable cell concentration after culture (CFU / mL) / viable cell concentration at 0 h (CFU / mL) × 100; the logarithmic decrease in the number of colonies after gastric juice / intestinal fluid treatment compared with that without treatment was less than 3, and the viable cell number was still above 10 6 CFU / mL, indicating good acid resistance / bile salt resistance (References: Jensen H, Grimmer S, Naterstad K, Axelsson L. In vitro testing of commercial and potential probiotic lactic acid bacteria. Int J Food Microbiol. 2012 Feb 1;153(1-2):216-22.; Wang C, Zhang C W, Sun X F, et al. Preliminary study on acid and bile salt tolerance of Lactobacillus from human stomach and intestine [J]. Modern Preventive Medicine, 2006, (10):1792-1794.).

[0219] The experimental results showed that the viable cell concentration of Pediococcus pentosaceus at 0 h was 8.65×10 8 CFU / mL, and the logarithmic decrease after digestion in simulated gastric juice at pH = 3 for 3 h was only 0.44. The viable cell concentration after 3 h was 3.15×10 8 CFU / mL; the logarithmic decrease after digestion in simulated intestinal fluid at pH = 8 for 4 h was only 0.52. The viable cell concentration after 4 h was 2.60×10 8 CFU / mL. Therefore, it can be known that Pediococcus pentosaceus has good tolerance to gastric acid and bile salts and can effectively reach the downstream digestive tract to play its role. Therefore, Pediococcus pentosaceus FeiHeP06 meets the basic requirements of a probiotic.

[0220] III. Bacteriostatic Activity of Pediococcus pentosaceus FeiHeP06 against Four Pathogenic Bacteria

[0221] During the growth and reproduction process of lactic acid bacteria, they can secrete some substances such as organic acids, hydrogen peroxide, bacteriocins, etc. These antibacterial substances can inhibit the growth of certain pathogenic bacteria, thereby preventing some diseases. In the present invention, four pathogenic bacteria were used to determine the antibacterial activity of Pediococcus pentosaceus, with Bifidobacterium animalis subsp. lactis BB-12 as the control strain. The antibacterial activity of Pediococcus pentosaceus FeiHeP06 was significantly higher than that of Bifidobacterium animalis subsp. lactis BB-12, showing good antibacterial ability. The specific experimental steps are as follows:

[0222] 1. Strain culture

[0223] Take Pediococcus pentosaceus FeiHeP06 and Bifidobacterium animalis subsp. lactis BB-12 from the preservation tubes and streak them on MRS solid medium and mMRS solid medium respectively, and culture at 37 °C for 48 h. Pick a single colony into 5 mL of MRS liquid medium and mMRS liquid medium, and culture at 37 °C for 24 h; transfer according to 2% volume to 5 mL of MRS liquid medium and mMRS liquid medium, and culture at 37 °C for 24 h.

[0224] Take the pathogenic bacteria (taking Staphylococcus aureus as an example) from the preservation tube and streak them on LB solid medium, and culture at 37 °C for 48 h; pick a single colony into 5 mL of LB liquid medium, and culture at 37 °C for 24 h; transfer according to 2% inoculation volume to 5 mL of LB liquid medium, and culture at 37 °C for 24 h.

[0225] 2. Determination of in vitro antibacterial ability: Oxford cup method

[0226] (1) Preparation of strain fermentation supernatant: Inoculate the activated Pediococcus pentosaceus FeiHeP06 and Bifidobacterium animalis subsp. lactis BB-12 for 3 generations into MRS liquid medium and mMRS liquid medium respectively, and culture at 37 °C for 24 h. Take 4 mL of the bacterial liquid into a 5 mL centrifuge tube, centrifuge at 8000 rpm for 20 min, take the supernatant, and collect the filtrate after passing through a 0.22 μm microporous filter membrane to obtain the cell-free fermentation supernatant of Pediococcus pentosaceus FeiHeP06 and the cell-free fermentation supernatant of Bifidobacterium animalis subsp. lactis BB-12.

[0227] (2) Pour 10 mL of sterilized plain agar solid medium into a sterile petri dish. After it completely solidifies, it serves as the bottom medium, and place 3 sterile Oxford cups at appropriate positions on the petri dish. Add 0.1 mL of Staphylococcus aureus, Salmonella, Escherichia coli or Listeria monocytogenes bacterial liquid containing 10 8 CFU / mL to the melted and cooled LB solid medium at 40 - 50 °C, carefully mix it and pour it on the bottom medium, taking care not to add it into the Oxford cups, and let it completely solidify to serve as the bacterial layer medium.

[0228] (3)Carefully remove the Oxford cup, taking care not to damage the formed holes. Add 0.2 mL of the cell-free fermentation supernatant of Pediococcus pentosaceus FeiHeP06 and Bifidobacterium animalis subsp. lactis BB-12, and the negative control (0.2 mL of MRS medium) into the holes with a diameter of approximately 8 mm, and incubate at 37 °C for 24 h. Measure the size of the inhibition zone with a ruler. The experimental results are shown in Table 3:

[0229] Table 3: Inhibitory effect of Pediococcus pentosaceus FeiHeP06 on four pathogenic bacteria

[0230]

[0231] Note: " / " indicates no inhibitory effect.

[0232] The results showed that Bifidobacterium animalis subsp. lactis BB-12 had no inhibitory effect on the four pathogenic bacteria, Pediococcus pentosaceus FeiHeP06 had no inhibitory effect on Salmonella, but had inhibitory effects on Staphylococcus aureus, Escherichia coli and Listeria monocytogenes. The antibacterial activity of Pediococcus pentosaceus FeiHeP06 was significantly higher than that of Bifidobacterium animalis subsp. lactis BB-12, indicating good antibacterial ability. Therefore, Pediococcus pentosaceus FeiHeP06 has certain antibacterial activity and has advantages in production applications such as fermentation.

[0233] Example 4: Preparation of the bacterial powder of Pediococcus pentosaceus FeiHeP06 strain

[0234] Pediococcus pentosaceus FeiHeP06 can be used to prepare bacterial powder, and the specific preparation process is as follows:

[0235] Streak Pediococcus pentosaceus FeiHeP06 on MRS solid medium and incubate anaerobically at 37 °C for 48 h to obtain single colonies; pick single colonies and inoculate them into MRS liquid medium, activate them after anaerobic incubation at 37 °C for 18 h, and activate them continuously for two generations to obtain an activated solution; inoculate the activated solution into the medium at an inoculation amount of 2% (v / v), and incubate anaerobically at 37 °C for 18 h to obtain a bacterial solution; centrifuge the bacterial solution at 5000 r / min for 15 min to obtain bacterial sludge; wash it 3 times with physiological saline and resuspend it with a protective agent (wet bacterial sludge mass g: protective agent volume mL = 1:1) to a concentration of 1×10 10 CFU / mL to obtain a bacterial suspension; incubate the bacterial suspension at 37 °C for 60 min and then freeze-dry it to obtain the bacterial powder of Pediococcus pentosaceus FeiHeP06;

[0236] Among them, the preparation method of the medium is: dissolve 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% of water by the total weight of the medium, and then adjust its pH to 6.8 to obtain the medium;

[0237] The components of the protective agent include: 130 g / L skim milk powder aqueous solution.

[0238] Example 5: Preparation of fermented milk of Pediococcus pentosaceus strain FeiHeP06

[0239] Mix the Pediococcus pentosaceus FeiHeP06 bacterial powder with the commercial dry fermenting agent Lactobacillus bulgaricus and the commercial dry fermenting agent Streptococcus thermophilus in a mass ratio of 1:1:1 to obtain a fermenting agent; add sugar to fresh milk to a concentration of 50 g / L to obtain a mixed solution; homogenize the mixed solution at 65 °C and 20 MPa and then keep it warm and sterilize at 95 °C for 5 min to obtain a fermentation raw material; cool the fermentation raw material to 35 °C and then inoculate the fermenting agent into the fermentation raw material at an inoculation amount of 0.03% (v / v), and keep it warm and ferment at 35 °C for 16 h to obtain fermented milk; place the fermented milk at 42 °C for 4 h for curdling and then refrigerate it at 4 °C for 24 h for after-ripening to obtain the finished fermented milk.

[0240] Example 6: Preparation of fruit and vegetable beverage of Pediococcus pentosaceus strain FeiHeP06

[0241] Pediococcus pentosaceus FeiHeP06 can be used to prepare fruit and vegetable beverages. The specific preparation process of the fruit and vegetable beverage is as follows:

[0242] Wash and juice fresh fruits and vegetables, add food-grade peptone at a ratio of 5 g / L, and perform high-temperature thermal sterilization at 140 °C for 2 seconds; cool the sterilized fruit and vegetable juice to about 37 °C and then add the Pediococcus pentosaceus FeiHeP06 bacterial powder of the present invention until the concentration is not less than 1×10 6 CFU / mL to obtain a fruit and vegetable beverage, and store it refrigerated at 4 °C. The fruit and vegetable beverage containing viable Pediococcus pentosaceus FeiHeP06 of the present invention can be obtained.

[0243] Example 7: Application of Pediococcus pentosaceus strain FeiHeP06 in fermented sausage

[0244] Formula of fermented sausage: fat-to-lean ratio of fresh pork 3:7, salt 2.8%, glucose 0.8%, sucrose 1.2%, sodium nitrate 0.12%, sodium nitrite 0.015%, phosphate 0.3% (sodium tripolyphosphate: sodium pyrophosphate: sodium hexametaphosphate = 2:2:1), monosodium glutamate 0.1%, starch 11.25%, soy protein 11.25%, carrageenan 0.3%, white pepper powder 0.25%. The preparation method is as follows:

[0245] Cut the raw meat into pieces, marinate for 12 h, then grind and chop it. Add spices, seasonings and starter culture and stir. Among them, 10 7 CFU / g of Pediococcus pentosaceus FeiHeP06 bacterial powder is added to the starter culture. After stirring, stuff into casings, ferment, smoke, and finally perform vacuum packaging to obtain the finished product of fermented sausage.

[0246] In summary, the present invention discloses a Pediococcus pentosaceus FeiHeP06 that can promote the secretion of IFN-β by bone marrow-derived dendritic cells (BMDC cells) and its application. The Pediococcus pentosaceus strain screened in the present invention can promote the secretion of IFN-β by BMDC cells, which helps to prevent and resist the infection of pathogens such as viruses, improve the body's immunity, and improve intestinal health. At the same time, it has good gastrointestinal fluid tolerance and good antibacterial activity. Therefore, this Pediococcus pentosaceus FeiHeP06 has great application prospects in the preparation of products (such as foods, drugs or health products, etc.) that improve host immunity and prevent infection.

[0247] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

[0248] It should be noted that although the technical solutions of the present invention are introduced with specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

[0249] The above have described the embodiments of the present invention. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art in the technical field without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, the practical application or the improvement of the technology in the market, or to enable other ordinary skilled persons in the technical field to understand the disclosed embodiments.

Claims

1. A strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ), strain FeiHeP06, characterized in that The Pediococcus pentosaceus Pediococcus pentosaceus strain FeiHeP06 is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 31506; The 16S rDNA sequence of the Pediococcus pentosaceus Pediococcus pentosaceus strain FeiHeP06 is shown in SEQ ID NO.

1.

2. A microbial preparation, characterized in that, The microbial preparation comprises the strain according to claim 1.

3. The microbial preparation according to claim 2, wherein In the microbial preparation, the viable count of the Pediococcus pentosaceus Pediococcus pentosaceus strain FeiHeP06 is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g. Alternatively, in the microbial preparation, the Pediococcus pentosaceus Pediococcus pentosaceus strain FeiHeP06 is inactivated.

4. The preparation method of the microbial preparation according to claim 2 or 3, characterized in that, The method for preparing the microbial preparation comprises the step of culturing the strain as claimed in claim 1.

5. A starter culture, characterized in that, The fermentation agent comprises the strain according to claim 1, the microbial preparation according to claim 2 or 3, or the microbial preparation prepared by the preparation method according to claim 4.

6. A product, characterized in that, The product comprises the strain according to claim 1, the microbial preparation according to claim 2 or 3, or the microbial preparation prepared by the preparation method according to claim 4, or the product is obtained by fermentation using the starter according to claim 5; The product is food, food additive or feed additive.

7. The product according to claim 6, characterized in that, The product further comprises any one or more of the following ingredients: plant product ingredients, animal meat product ingredients, animal dairy product ingredients, functional added ingredients and any acceptable auxiliary materials.

8. The use of the strain according to claim 1, the microbial preparation according to claim 2 or 3, or the microbial preparation prepared by the preparation method according to claim 4 in any of the following (1) to (2): (1) Use in the preparation of health foods that help regulate intestinal flora; (2) Use in the preparation of health foods that help enhance immunity.

Images