Pediococcus pentosaceus and application thereof

Abstract

The invention discloses a Pediococcus pentosaceus strain and an application thereof. The invention provides a Pediococcus pentosaceus FeiHeP06 strain, the Pediococcus pentosaceus FeiHeP06 strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31506. The Pediococcus pentosaceus FeiHeP06 strain has the advantages that the Pediococcus pentosaceus FeiHeP06 strain can be used for preparing the Pediococcus pentosaceus FeiHeP06 strain, and the Pediococcus pentosaceus FeiHeP06 strain can be used for preparing the Pediococcus pentosaceus FeiHeP06 strain; the pediococcus pentosaceus provided by the invention has good gastrointestinal tract adaptive capacity, good stability, good bacteriostatic activity and good immune cell cytokine regulating capacity, can improve the intestinal health of a host and enhance the immunity of the organism, and has a huge application prospect.

Description

Technical Field

[0001] The invention relates to a Pediococcus pentosaceus strain and an application thereof, and in particular to a Pediococcus pentosaceus strain capable of promoting BMDC cells to secrete IFN-β and an application thereof in the production of food, functional food, feed additives and medicines, and belongs to the fields of microbial technology and pharmaceutical technology. Background Art

[0002] As a type of lactic acid bacteria, Pediococcus pentosaceus has been added to the list of probiotic strains that can be used in food. Studies have shown that Pediococcus pentosaceus isolated from fermented foods can significantly inhibit the growth of foodborne pathogens, showing good development prospects as a new antibacterial agent. In addition, there are also literature reports and confirmations that Pediococcus pentosaceus has the effects of lowering blood lipids, anti-obesity, anti-oxidation, anti-cancer, anti-inflammatory, improving alcoholic liver damage, relieving colitis and intestinal flora imbalance.

[0003] Studies have shown that in order for probiotics to play a beneficial role, they must first be more than 10 6 CFU / mL (colony-forming units per milliliter) of live bacteria reach the human intestine. The human gastrointestinal tract is a high-acid, high-bile salt environment. Whether probiotics can successfully pass through the gastrointestinal environment after entering the human body is a key factor in determining whether they can play a health-care function. In addition, probiotics can secrete some organic acids, hydrogen peroxide, bacteriocins and other substances during their growth and reproduction. 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.) are a class of cytokines that can trigger the protective defense of the immune system and activate immune cells, playing a key role in responding to most viral infections. Its expression is regulated at the transcriptional level and can be induced at high levels 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, cancer, and autoimmune diseases. In addition, unlike IFN-α, IFN-β is not associated with systemic diseases such as autoimmune diseases, but is dedicated to stabilizing protective immunity against infection and inflammation. Therefore, IFN-β is a potential way for the body to prevent bacterial and viral infections.

[0005] At present, there are literature reports that Lactobacillus rhamnosus GG can induce the production of type I interferon through Toll-like receptor 4 (TLR4) (reference 1), and Lactobacillus plantarum and Pseudomonas pentosaceus can induce human macrophage-like cells and human primary phagocytes isolated from PBMC to produce IFN-β (reference 2). However, there are few studies on whether Pediococcus pentosaceus can be used as a probiotic to stimulate immune cells to produce IFN-β, and there are no reports on the dendritic cell immune pathway.

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

[0007] Citations Cited literature 1. PLoS pathogens, 2019: Lung transcriptional unresponsiveness and loss of early influenza virus control in infected neonates is prevented by intranasal Lactobacillus rhamnosus GG. Cited literature 2. Gut microbes, 2020: Beneficial bacteria activate type-Iinterferon production via the intracellular cytosolic sensors STING and MAVS. Summary of the invention

[0008] Problems to be Solved by the Invention The technical problem to be solved by the present invention is to provide a strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, which can be used in products that improve the host's intestinal health, enhance the body's immunity or regulate the immune response.

[0009] Solutions to Solve the Problems [1]. A strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, wherein the Pediococcus pentosaceus ( Pediococcus pentosaceus) The FeiHeP06 strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No. 31506.

[0010] [2]. A culture, wherein the culture is obtained by culturing the strain described in [1].

[0011] [3]. A microbial preparation, wherein the microbial preparation comprises the strain described in [1] or the culture described in [2].

[0012] [4] The microbial preparation according to [3], wherein in the microbial preparation, the Pediococcus pentosaceus ( Pediococcus pentosaceus ) The viable count of 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 ( Pediococcus pentosaceus ) The FeiHeP06 strain is inactivated.

[0013] [5] The method for preparing a microbial preparation as described in [3] or [4], wherein the method for preparing a microbial preparation comprises the step of culturing the strain as described in [1].

[0014] [6]. A fermentation agent, wherein the fermentation agent comprises the strain described in [1], the culture described in [2], the microbial preparation described in [3] or [4], or the microbial preparation prepared by the preparation method described in [5].

[0015] [7]. A product, wherein the product comprises the strain described in [1], the culture described in [2], the microbial preparation described in [3] or [4], or the microbial preparation prepared by the preparation method described in [5], or the product is obtained by fermentation using the starter described in [6].

[0016] [8] The product according to [7], wherein the product comprises a probiotic product, a food, a food additive or a feed additive.

[0017] [9]. The product according to [8], wherein 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 excipients.

[0018]

[10] The product according to [8] or [9], wherein the product comprises a nutritional food, a functional food or a health food.

[0019]

[11] The strain described in [1], the culture described in [2], the microbial preparation described in [3] or [4], the microbial preparation prepared by the preparation method described in [5], or the product described in any one of [7] to

[10] , for any of the following (1) to (5): (1) Helps regulate intestinal flora; (2) Uses for improving intestinal health; (3) Uses that help enhance immunity; (4) Use in the inhibition of Escherichia coli, Staphylococcus aureus and / or Listeria monocytogenes for non-disease treatment purposes; (5) Use in regulating cytokine secretion of immune cells.

[0020]

[12] The use as described in

[11] , wherein the regulation of immune cell cytokine secretion includes promoting dendritic cells to secrete the cytokine IFN-β.

[0021]

[13] The use of the strain described in [1], the culture described in [2], the microbial preparation described in [3] or [4], or the microbial preparation prepared by the preparation method described in [5] in any of the following (1) to (5): (1) Use in the preparation of products for helping to regulate intestinal flora; (2) Use in the preparation of products for improving intestinal health; (3) Use in the preparation of products for helping to enhance immunity; (4) Use in the preparation of a product for inhibiting Escherichia coli, Staphylococcus aureus and / or Listeria monocytogenes for non-disease treatment purposes; (5) Use in the preparation of products for regulating cytokine secretion in immune cells.

[0022]

[14] The use as described in

[13] , wherein the regulation of immune cell cytokine secretion includes promoting dendritic cells to secrete the cytokine IFN-β.

[0023] Effects of the Invention The present invention provides a strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, this Pediococcus pentosaceus ( Pediococcus pentosaceus )The FeiHeP06 strain can play a probiotic role in the intestine and has the function of promoting bone marrow-derived dendritic cells (BMDC, also referred to as myeloid dendritic cells) to secrete the immunomodulatory cytokine IFN-β, which is specifically reflected in: (1) Pediococcus pentosaceus is a type of lactic acid bacteria. Studies have found that Pediococcus pentosaceus has strong immunoregulatory ability. Using mouse BMDC cells as a model and animal Bifidobacterium BB-12 as a control strain, the Pediococcus pentosaceus, animal Bifidobacterium BB-12 and other 10 strains of Bifidobacterium and Lactobacillus were co-cultured with BMDC, and the concentration of IFN-β was determined 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.

[0024] (2) Acid and bile resistance are necessary properties for probiotics to reach the intestine and maintain a certain number of viable bacteria. When cultured in the simulated gastric fluid and simulated intestinal fluid of the present invention for 3 to 4 hours, Pediococcus pentosaceus FeiHeP06 was able to maintain a viable bacterial count of 10 8 CFU / mL and above, reaching the standards of acid and bile salt resistance.

[0025] (3) During the growth and reproduction process, lactic acid bacteria can secrete some organic acids, hydrogen peroxide, bacteriocins and other substances. 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 BB-12 as the control strain, the antibacterial activity of Pediococcus pentosaceus FeiHeP06 was significantly higher than that of Bifidobacterium animalis BB-12, and it had good antibacterial ability.

[0026] The present invention provides Pediococcus pentosaceus ( Pediococcus pentosaceus ) The FeiHeP06 strain has good intestinal adaptability, good antibacterial activity, good production stability, and good immune cell cytokine regulation ability. It has great application prospects in products such as improving host intestinal health, enhancing the body's immunity or regulating immune responses (in food, functional food, feed additives and medicines). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The bar graph shows the results of ELISA assay of IFN-β produced by co-culture of 12 bacterial strains with BMDC. Different letters af in the figure indicate significant differences among the groups in Duncan's multiple comparison test (P < 0.05).

[0028] Figure 2 The bar graph shows the ELISA results of IFN-β produced by co-culture of Pediococcus pentosaceus FeiHeP06 with BMDC of 4-week-old, 6-week-old, and 10-week-old mice. In the figure, "****" indicates that P < 0.001 compared with the live bacteria group.

[0029] Figure 3 Pediococcus pentosaceus ( Pediococcus pentosaceus) Colony morphology of FeiHeP06 strain on MRS solid medium.

[0030] Figure 4 The growth curve of Pediococcus pentosaceus FeiHeP06 is shown. BB12 in the figure is Bifidobacterium animalis BB-12. DETAILED DESCRIPTION

[0031] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0032] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0033] 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.

[0034] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0035] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0036] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoint numerical values ​​A and B.

[0037] In this specification, "culture" refers to growing a microbial cell population under any suitable conditions (for example, using a liquid, gel or solid culture medium), 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 actual conditions.

[0038] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] Biomaterial Deposit A strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, taxonomically named Pediococcus pentosaceus Pediococcus pentosaceus The Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number CGMCC No. 31506, the deposit date is August 1, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0040] strain The present invention provides a strain of Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, the Pediococcus pentosaceus ( Pediococcus pentosaceus ) The FeiHeP06 strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 31506 and the deposit date being August 1, 2024.

[0041] The Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, which is derived from the fecal sample of a healthy pregnant woman in Wuxi City, Jiangsu Province, was sequenced and analyzed. The sequence obtained by sequencing was compared with the nucleic acid sequence in NCBI Standard Nucleotide BLAST. Pediococcus pentosaceus The homology of strainSMFM2016-WK1 was as high as 98.58%, and the strain was determined to be Pediococcus pentosaceus and was named Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06, whose original number in the laboratory is CCFM1443. In this specification, Pediococcus pentosaceus ( Pediococcus pentosaceus ) The FeiHeP06 strain is also referred to as Pediococcus pentosaceus FeiHeP06.

[0042] In some specific embodiments, mouse BMDC cells (mouse bone marrow derived dendritic cells) are used as a model, animal Bifidobacterium BB-12 is used as a control strain, the Pediococcus pentosaceus, animal Bifidobacterium BB-12 and other 10 strains of Bifidobacterium and Lactobacillus are co-cultured with BMDC, and the concentration of IFN-β is determined by ELISA. Compared with the other strains, Pediococcus pentosaceus FeiHeP06 has the best effect, and the heat sterilization effect is better than that of live bacteria (such as Figures 1 - 2 As shown in Figure 3 ), Pediococcus pentosaceus FeiHeP06 plays a key role in immune regulation.

[0043] The colonies of the Pediococcus pentosaceus FeiHeP06 on the MRS solid medium are milky white round protrusions with a smooth, translucent surface and a diameter of 1 to 2 mm (mm). Figure 3 as shown).

[0044] In some specific embodiments, Pediococcus pentosaceus FeiHeP06 can maintain a viable count of 10 8 CFU / mL and above, reaching the standards of acid and bile salt resistance.

[0045] In some specific embodiments, the Pediococcus pentosaceus FeiHeP06 has good antibacterial ability. The Pediococcus pentosaceus FeiHeP06 can antagonize the growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, regulate intestinal flora, and promote intestinal health.

[0046] In some specific embodiments, the 16S rDNA sequence of Pediococcus pentosaceus FeiHeP06 is as shown in SEQ ID NO.1.

[0047] SEQ ID NO.1: 16S rDNA Culture In some aspects, the present invention provides a culture, wherein the culture is obtained by culturing the above-mentioned Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain was obtained.

[0048] In the present invention, the term "culture" refers to a liquid or solid product (at least a part of the material in the culture container, such as the supernatant) containing a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying the microorganisms, which may be a biologically pure culture of the microorganism, or may contain a certain amount of culture medium, metabolites or other components produced during the culture process, or the supernatant obtained by centrifugation.

[0049] In some embodiments, the culture is a product obtained by growing and / or amplifying a microorganism or a supernatant (cell-free supernatant) obtained by centrifuging the product.

[0050] In some embodiments, the culturing is performed by the following steps: (1) Separately separate Pediococcus pentosaceus ( Pediococcus pentosaceus ) The FeiHeP06 strain was streaked on MRS solid medium and a single colony was obtained after culture; (2) picking a single colony and inoculating it into MRS liquid culture medium, culturing it to activate it, and continuously activating it for at least one generation, for example, two generations, to obtain an activated liquid; Optionally, (3) the activation solution is inoculated into MRS liquid culture medium at an inoculum amount of about 2% (v / v), and cultured to a stable phase to obtain a culture solution; (4) Centrifuging the activation solution obtained in step (2) or the culture solution obtained in step (3) to obtain a supernatant.

[0051] Optionally, (5) collecting the supernatant and filtering it to obtain a cell-free supernatant for use.

[0052] In some specific embodiments, the MRS solid culture medium includes 10 grams / 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 MgSO4·7 H2O, 0.25 g / L of MnSO4·H2O, 1 milliliter / liter (mL / L) of Tween 80, and 20 g / L of agar; the pH is 6.8.

[0053] In some specific embodiments, the MRS liquid culture medium includes 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 2 g / L anhydrous sodium acetate, 5 g / L yeast extract, 2 g / L diammonium hydrogen citrate, 2.6 g / L K2PO4·3H2O, 0.5 g / L MgSO4·7 H2O, 0.25 g / L MnSO4·H2O, and 1 mL / L Tween 80; the pH is 6.8.

[0054] Microbial agents In some aspects, the present invention also provides a method comprising the above-mentioned Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain or microbial preparations of the above cultures.

[0055] In some embodiments, the microbial preparation of the present invention is solid, and illustratively, can be lyophilized powder or the like.

[0056] Furthermore, the microbial preparation may also contain a protective agent (such as a freeze-dried protective agent), which includes but is not limited to skimmed milk powder, trehalose, sodium glutamate and / or glycerol, etc.; preferably, the protective agent is skimmed milk powder.

[0057] In other embodiments, the microbial preparation of the present invention is semi-solid, and can be, for example, bacterial mud, which refers to bacterial bodies containing a small amount of water.

[0058] In some other embodiments, the microbial preparation of the present invention is a liquid, for example, a suspension, a culture. Furthermore, the microbial preparation may also contain a solvent, including but not limited to water or a culture medium.

[0059] In some embodiments, in the microbial preparation, the Pediococcus pentosaceus ( Pediococcus pentosaceus ) The viable count of FeiHeP06 strain is not less than 1×10 6 CFU / mL or 1×10 6 Colony-Forming Units per Gram (CFU / g).

[0060] In some embodiments, in the microbial preparation, the Pediococcus pentosaceus ( Pediococcus pentosaceus ) The FeiHeP06 strain is inactivated, for example by heat sterilization.

[0061] Preparation method of microbial preparation In some embodiments, the method for preparing the microbial preparation comprises: Pediococcus pentosaceus ) The step of inoculating the FeiHeP06 strain into a suitable culture medium for cultivation, and optionally, the steps of fermentation, centrifugation, collecting bacterial sludge, adding a protective agent (such as a freeze-drying protective agent), etc. Centrifugation can increase the bacterial concentration and shorten the freeze-drying time. By adding a protective agent, the death or loss of activity of the strain during the freeze-drying process can be avoided as much as possible, and the fermentation effect of the microbial preparation can be improved.

[0062] In some embodiments, after the step of adding the protective agent, a freeze-drying step is further included. Freeze-drying facilitates the transportation and storage of the microbial preparation.

[0063] In some specific embodiments, the method for preparing the microbial preparation of the present invention comprises the following steps: Pediococcus pentosaceus ( Pediococcus pentosaceus ) The FeiHeP06 strain is inoculated into any solid culture medium suitable for lactic acid bacteria culture for anaerobic culture to obtain a single colony, which is then inoculated into a culture solution; The culture solution was centrifuged to obtain bacterial sludge; After washing the bacterial cells, resuspend them in a freeze-drying protective agent to obtain a bacterial suspension; The bacterial suspension is freeze-dried by vacuum freezing method to obtain a microbial preparation.

[0064] Optionally, single colony post cultures can be subjected to activation treatment.

[0065] In some embodiments, the method for preparing the culture medium is: using 85% to 90% water based on the total weight of the culture medium, dissolving 8% to 3% enzymatically hydrolyzed skim milk, 0.4% to 0.8% glucose, 1% to 2% tryptone and 0.2% to 0.5% yeast extract, and then adjusting its pH to obtain a culture medium.

[0066] In some preferred embodiments, the method for preparing the culture medium is: using 87.7% water based on the total weight of the culture medium, dissolving 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract, and then adjusting the pH thereof to obtain the culture medium.

[0067] In some embodiments of the present invention, the mass ratio of the lyoprotectant to the bacterial cells is (1-5):1, preferably (1-3):1, for example 1:1, 2:1, 3:1.

[0068] In some embodiments, the lyoprotectant comprises 100-150 g / L, such as 110 g / L, 130 g / L, or 150 g / L of skim milk powder in water.

[0069] In some embodiments, the pH of the culture medium is 6.5-7.5, preferably 6.8-7.2, for example 6.8, 7.0.

[0070] Fermentation Agents In some aspects, the present invention also provides a method comprising the above-mentioned Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, the above culture, the above microbial preparation, or the starter of the microbial preparation prepared by the above preparation method.

[0071] In some embodiments, the starter culture further comprises lactic acid bacteria.

[0072] In the present invention, the lactic acid bacteria used in the starter are not particularly limited as long as they do not hinder the effects of the present invention, and may be animal-derived lactic acid bacteria or plant-derived lactic acid bacteria.

[0073] Preferred lactic acid bacteria of the present invention include Lactobacillus ( Lactobacillus ) bacteria, Streptococcus ( Streptococcus ) bacteria, Lactococcus ( Lactococcus ) bacteria, Enterococcus ( Enterococcus )genus, Leuconostoc ( Leuconostoc ) genus bacteria, and their combination, preferably comprises the lactic acid bacteria of the genus Lactobacillus. As the lactic acid bacteria comprising the genus Lactobacillus, for example, can list the combination of the genus Lactobacillus, the genus Lactobacillus and the streptococcus, the combination of the genus Lactobacillus and the genus Lactococcus etc. These lactic acid bacteria for example can be obtained from storage institutions such as ATCC, etc., also can suitably use commercially available products.

[0074] Examples of Streptococcus bacteria include thermophilic Streptococcus ( Streptococcus thermophilus )wait.

[0075] Preferred examples of the combination of bacteria of the genus Lactobacillus and bacteria of the genus Streptococcus include Lactobacillus bulgaricus and Streptococcus thermophilus.

[0076] In some embodiments, in the fermentation agent, the Pediococcus pentosaceus ( Pediococcus pentosaceus ) The viable count of FeiHeP06 strain is not less than 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0077] product The present invention further provides a product, wherein the product comprises the above-mentioned Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, the above-mentioned culture, the above-mentioned microbial preparation, the microbial preparation prepared by the above-mentioned preparation method, or obtained by fermentation using the above-mentioned starter.

[0078] In some embodiments, the product is a probiotic product.

[0079] In some specific embodiments, the probiotic product is a probiotic product with good intestinal adaptability, and the product contains the above-mentioned Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, the above-mentioned microbial preparation or the microbial preparation prepared by the above-mentioned preparation method.

[0080] For different product categories, the present invention does not specifically limit the specific form of the product. Exemplarily, it can be in powder form or liquid form.

[0081] The present invention does not specifically limit the target group of the product. For example, the product can be used for infants, children, teenagers or adults.

[0082] In some embodiments, the product may further include 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.

[0083] In some embodiments, the plant product ingredients include plants or plant extract ingredients, for example: fruits or extracts thereof such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, rubber, grapes, pears, cherries, blueberries, blackberries, black currants, cranberries, raspberries, melons, emblica chinensis and bilberry; fruit and vegetable substances or extracts thereof such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano; grains or extracts thereof such as rice (indica rice, japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, yellow rice, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans; nuts or extracts thereof such as walnuts, pistachios, cashews, hazelnuts, almonds, almonds, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts; coffee or its extract.

[0084] In some embodiments, the animal-based ingredients, such as animal meat product ingredients, include meat product ingredients of cattle, sheep, fish or poultry.

[0085] In some embodiments, the product may further comprise a fat component, and the fat may include at least one of saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, OPO structured lipids, DHA, EPA, ARA, and phospholipids. More specifically, the fat includes safflower seed 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, butter, cream, lard, medium-chain triglycerides or lecithin, etc.

[0086] In some embodiments, the animal dairy product ingredients include milk-containing ingredients or protein ingredients. The milk-containing ingredients include dairy products such as fresh milk from raw cow (sheep) milk, milk powder, whey protein or cheese, etc.; the protein ingredients can come from plant proteins, such as soy protein, peanut protein, etc.

[0087] In some embodiments, the functional added 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, biotin), starch, modified starch, amino acids (L-lysine-L-glutamic acid, L-glutamic acid, L-arginine, L-tryptophan, L-glutamine, taurine, L-valine, L-isoleucine or L-leucine, etc.), traditional Chinese medicine or traditional Chinese medicine extract, dietary fiber (inulin, konjac flour, galacto-oligosaccharides, fructo-oligosaccharides, isomalto-oligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin or soybean fiber, etc.).

[0088] In some embodiments, the product may also contain trace element supplements, which may include metal ion salts of organic acids, such as one or more of calcium citrate, L-calcium 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.

[0089] In some embodiments, the arbitrary acceptable excipients include, but are not limited to, solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible colorings, etc.

[0090] The present invention does not specifically limit the specific types of the above products.

[0091] In some embodiments, the product is a food, which comprises the above-mentioned strain, culture, microbial preparation or the microbial preparation prepared by the above-mentioned preparation method, or the food is a food obtained by fermentation using the above-mentioned starter.

[0092] In some embodiments, the product is a food additive.

[0093] In some embodiments, the product is a nutraceutical, a functional food, or a health food.

[0094] In some preferred embodiments, the food is: dairy products, such as yogurt, cheese, butter, ice cream, etc.; soy products, such as tofu, soy milk, dried tofu, tofu skin, etc.; fruit and vegetable products, such as fresh fruits, vegetables, fruit juice, vegetable juice, jam, vegetable puree, etc.; beverages, such as tea, coffee, fruit juice, carbonated drinks, mineral water, etc.; snacks, such as nuts, dried fruits, potato chips, biscuits, candies, etc.

[0095] In some exemplary embodiments of the present invention, the food is a food containing Pediococcus pentosaceus ( Pediococcus pentosaceus ) Fermented dairy products produced from FeiHeP06 strain or the above-mentioned microbial preparations, or fermented dairy products obtained by fermentation with the above-mentioned starter.

[0096] In some exemplary embodiments of the present invention, the food is a food containing Pediococcus pentosaceus ( Pediococcus pentosaceus ) Fruit and vegetable beverages containing FeiHeP06 strain or the above-mentioned microbial preparations.

[0097] In some exemplary embodiments of the present invention, the food is a food containing Pediococcus pentosaceus ( Pediococcus pentosaceus ) Fermented pork (e.g., fermented sausage) produced by fermenting the FeiHeP06 strain or the above-mentioned microbial preparation, or fermented pork obtained by fermentation with the above-mentioned starter.

[0098] In some specific embodiments, the food of the present invention can be infant formula milk powder, baby complementary food, children's formula milk powder, children's snacks, milk powder for pregnant women, milk powder for the middle-aged and elderly, or nutritional or dietary supplements.

[0099] In other specific embodiments, the food described in the present invention can be powdered reconstituted food (solid beverages, instant coffee, cereal powder, nut powder or lotus root powder, etc.), baked food (bread, cake or biscuit baked food, etc.), beverages (carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages, milk beverages or alcoholic beverages, etc.), candy (jelly candy, hard candy, compressed candy, etc.), milk and dairy products (fresh milk from fresh cow (sheep) milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc.), pasta products (noodles, instant noodles, steamed buns, dumplings or wontons, etc.), etc.

[0100] In some embodiments, the product is a pharmaceutical.

[0101] In some embodiments of the present invention, the medicament contains the above-mentioned Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, drug carrier and / or pharmaceutical excipient.

[0102] In some embodiments of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0103] In some embodiments of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0104] In some embodiments of the present invention, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0105] In some embodiments of the present invention, the additive comprises a solubilizer, a co-solvent, a latent solvent and / or a preservative.

[0106] In some embodiments of the present invention, the drug is in the form of powder, granules, capsules, tablets, pills or oral liquid.

[0107] In some other specific embodiments, the product of the present invention is an oral preparation, which includes but is not limited to tablets, pills, granules, powders, teas, capsules or oral liquids.

[0108] In some embodiments, the product is a feed additive.

[0109] In some embodiments, the feed additive contains the above-mentioned Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain or the above-mentioned microbial preparations.

[0110] In some embodiments, the feed additive can stimulate the growth of beneficial bacteria in animals and resist the growth of harmful bacteria. After entering the body of livestock and poultry as feed, it can reproduce rapidly and form a normal microbial flora in the host, providing nutrition for the host and preventing the invasion of pathogenic bacteria.

[0111] In other embodiments, the product is a non-food product.

[0112] In other embodiments, the product is a non-nutraceutical food.

[0113] In other embodiments, the product is a non-drug.

[0114] use Use for Helping to Regulate Intestinal Flora The present invention provides Pediococcus pentosaceus ( Pediococcus pentosaceus) FeiHeP06 strain and its culture 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 regulate intestinal flora and promote intestinal health.

[0115] Therefore, the present invention provides Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, culture, microbial preparation, preparation method The microbial preparation prepared can be used to prepare products that help regulate intestinal flora. The product of the present invention also helps regulate intestinal flora.

[0116] The present invention does not specifically limit the specific product categories that help regulate intestinal flora. In some embodiments, the purpose of regulating intestinal flora is not to prevent and / or treat diseases. For example, when there is a disorder in intestinal flora, or when intestinal pathogenic microorganisms (Staphylococcus aureus, Escherichia coli and Listeria monocytogenes) are present but not to the extent of causing disease, or when there is a disorder in intestinal flora or an increase in intestinal pathogenic microorganisms that is not caused by disease, the Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, culture, microbial preparation, microbial preparation prepared by the preparation method, etc. In some embodiments, the product that helps regulate intestinal flora is food. In some embodiments, the product that helps regulate intestinal flora is health food. In some embodiments, the product that helps regulate intestinal flora is a probiotic product.

[0117] In other embodiments, the product is a drug. In some specific embodiments, the drug is used to inhibit intestinal pathogens.

[0118] Uses to enhance immunity, regulate immune cell cytokine secretion, and prevent infection The present invention provides Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain and its culture significantly promote the secretion of IFN-β by myeloid dendritic cells (BMDC cells), and can also increase the secretion of IFN-β. In addition, Pediococcus pentosaceus FeiHeP06 has a significant thermal sterilization effect and can also play an immunomodulatory function. Pediococcus pentosaceus FeiHeP06 has the ability to regulate the immune system for organisms of different ages at different times.

[0119] Therefore, the present invention provides Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, culture, microbial preparation, preparation method The microbial preparation prepared can be used to prepare products that help enhance immunity and regulate the secretion of cytokines in immune cells. The product of the present invention also helps enhance immunity and regulate the secretion of cytokines in immune cells.

[0120] The present invention does not particularly limit the specific categories of products that help enhance immunity and regulate the secretion of cytokines in immune cells.

[0121] Furthermore, in some embodiments, the purpose of helping to enhance immunity and regulating the secretion of cytokines in immune cells is not to prevent and / or treat diseases. For example, in the case of reduced immunity not caused by diseases, the use of the Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, culture, microbial preparation, microbial preparation prepared by the preparation method, etc. In some embodiments, the product that helps to enhance immunity and regulate the secretion of cytokines in immune cells is a food. In some embodiments, the product that helps to enhance immunity and regulate the secretion of cytokines in immune cells is a health food. In some embodiments, the product that helps to enhance immunity and regulate the secretion of cytokines in immune cells is a probiotic product.

[0122] In some specific embodiments, regulating immune cell cytokine secretion includes increasing immune cell IFN-β secretion.

[0123] In other embodiments, the product is a drug. In some specific embodiments, the drug is used for anti-infection, etc.

[0124] In some specific embodiments, the anti-infection comprises inhibiting the growth of enteric pathogenic microorganisms. In some specific embodiments, the anti-infection comprises antagonizing the growth of Staphylococcus aureus, Escherichia coli and / or Listeria monocytogenes.

[0125] Uses of feed additives The present invention provides Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain and its culture can inhibit the growth of intestinal pathogenic microorganisms, antagonize the growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, regulate intestinal flora, promote intestinal health, and increase the secretion of IFN-β to exert immune regulatory function.

[0126] Therefore, the present invention provides Pediococcus pentosaceus ( Pediococcus pentosaceus ) FeiHeP06 strain, culture, microbial preparation, preparation method The microbial preparation prepared can be used to prepare feed additive products. The product of the present invention also helps the growth of beneficial bacteria in livestock and poultry, provides nutrition and prevents the invasion of pathogenic bacteria.

[0127] The above products can be sprayed, mixed or coated on the feed or mixed into feed additives. The products can also be added during the production of the feed.

[0128] The present invention does not specifically limit the product category and usage of the specific feed additive.

[0129] Example

[0130] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0131] The culture medium involved in the following examples is as follows: mMRS liquid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7 H2O 0.5 g / L, MnSO4·H2O 0.25 g / L, Tween 80 1 mL / L, L-cysteine ​​0.5 g / L; pH 6.8. (MRS liquid medium does not contain L-cysteine, and the rest is the same).

[0132] mMRS solid medium: Add 20 g / L agar to the mMRS (MRS containing L-cysteine) liquid medium. (MRS solid medium does not contain L-cysteine, and the rest is the same).

[0133] LBS liquid culture medium: tryptone 10 g / L, anhydrous sodium acetate 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 was 5.4±0.2.

[0134] LBS solid medium: Add 20 g / L agar to LBS liquid medium.

[0135] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L.

[0136] LB solid medium: Add 20 g / L agar to LB liquid medium.

[0137] The preparation method of the Pediococcus pentosaceus cells involved in the following examples is as follows: Streak Pediococcus pentosaceus on MRS solid culture medium, culture under anaerobic conditions at 37 degrees Celsius (℃) for 48 hours (h) to obtain a single colony; pick a single colony and inoculate it in 5 milliliters (mL) of MRS liquid culture medium, culture it under anaerobic conditions at 37℃ for 18 hours for activation, and activate it for two generations in succession to obtain an activated solution; inoculate the activated solution into MRS liquid culture medium at an inoculation rate of 2% (v / v), culture it under anaerobic conditions at 37℃ for 18 hours to obtain a bacterial solution; centrifuge the bacterial solution at 5000 revolutions / minute (r / min) for 15 minutes (min), discard the upper culture medium, and obtain the Pediococcus pentosaceus bacteria.

[0138] Experimental Materials: Red blood cell lysis buffer (FMS-RBC100), Granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech, 315-03), ELISA kit (R&D, VAL612), Pepsin (McLean, P909753), Trypsin (McLean, T819003), Oxford cup (Shanghai Shendi, SDA-477131), Bifidobacterium animalis BB-12 (Chr. Hansen, Denmark, cultured in mMRS medium).

[0139] Example 1: Isolation, identification and cultivation of strains The specific steps are as follows: 1. Separation 0.5 mL of fecal sample was taken from healthy pregnant women and infants and added to 4.5 mL of sterile saline for gradient dilution. The sample was divided into two portions, and 100 μL of each portion was diluted to a gradient of 10. -4 , 10 -5 , 10 -6 The dilution was plated on mMRS solid medium with pH = 6.8 and added with mupirocin. Another 100 μL dilution was plated with a gradient of 10 -4 , 10 -5 , 10 -6 The dilution was plated on LBS solid medium with vancomycin added at pH = 5.6, cultured anaerobically at 37°C for 48 h, and the colony morphology was observed and recorded; The colonies with different morphologies on the solid culture medium were picked for streaking separation. After anaerobic culture at 37°C for 48 h, the single colonies with different morphologies on the solid culture medium were picked for streaking separation again until a pure single colony with consistent morphology was obtained. Pure colonies on the solid culture medium were picked and inoculated into 5 mL of liquid culture medium and cultured anaerobically at 37°C for 18 h. 1 mL of bacterial solution was taken into a sterile centrifuge tube and centrifuged at 5000 r / min for 15 min, then the upper culture medium was discarded and the bacterial sludge was resuspended in 30% glycerol solution and stored at -80°C to obtain 11 strains.

[0140] 2. Identification The isolated strains were subjected to PCR amplification of 16S rDNA, and the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The primer sequences were: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 2); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3).

[0141] The sequencing results were compared using 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 (whose 16S rDNA sequence is shown in SEQ ID NO.1), Pediococcus pentosaceus BB29L6, and Pediococcus pentosaceus BB76L11.

[0142] Example 2: Screening of Pediococcus pentosaceus FeiHeP06 strain In order to screen probiotics with antiviral and immune-regulating abilities, a BMDC cell model was used to co-culture 11 bacterial strains and Bifidobacterium animalis BB-12 with BMDCs. Pediococcus pentosaceus FeiHeP06 produced the highest level of IFN-β. The specific steps are as follows: 1. Obtaining BMDC cells (1) Bone marrow cells were collected from the tibia and femur of 4-week-old mice, passed through a 200-mesh cell sieve, and centrifuged at 1000 revolutions per minute (rpm) for 5 min, and the supernatant was discarded; 3 mL of red blood cell lysis buffer was added to the centrifuged cell pellet, and the cell pellet was quickly blown off. After standing for 10 min, 10 mL of complete culture medium was added to neutralize it, and the supernatant was discarded after centrifugation at 1000 rpm for 5 min. Complete culture medium containing 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF) was added to resuspend the cells (2×10 6 The cells were inoculated into 10 cm cell culture dishes and cultured in a 37°C 5% CO2 incubator.

[0143] (2) On the third day, half of the cell culture medium was replaced (centrifugation at 1000 rpm for 5 min, half of the culture medium was discarded, and half of the new culture medium was added). The culture medium was replaced every other day until the ninth day, when suspended and loosely adhered cells were obtained, namely BMDC cells.

[0144] 2. Co-culture of strains and BMDC to measure IFN-β concentration (1) Strain culture: 1 mL of bacterial solution was inoculated into 5 mL of MRS liquid medium and cultured in a 37℃ 5% CO2 incubator for 1-2 days. 200 µL of bacterial solution from each of the 11 strains was inoculated into 5 mL of MRS liquid medium and cultured in a 37℃ 5% CO2 incubator for 24 h.

[0145] (2) Bacterial pretreatment: Resuspend the bacterial solution and take 200 μL into a 96-well plate to measure OD 600 1 mL of bacterial solution was centrifuged and the concentration was adjusted to 10 with 1640 basal medium. 8 , and then dilute 1 mL of bacterial solution 10 times. After dilution, wash the bacterial solution 2-3 times with 1640 basal medium and centrifuge (8000 r / min, 4°C, 3 min). Take 500 µL for co-culture of live bacteria with cells, and another 500 µL is treated in a metal bath at 98°C for 20 min (i.e., heat sterilization) and co-cultured with cells.

[0146] (3) Co-culture: After centrifugation, discard the supernatant of BMDC cells, resuspend them in 1640 complete medium, blow evenly, and add 200 µL per well to a 96-well cell culture plate. Add 20 µL of bacterial solution to each well, with 4 parallels per group. After addition, place the plates in a 37°C 5% CO2 incubator for 24 h.

[0147] (4) After 24 h of co-culture, the 96-well plate was centrifuged (2000 r / min, 20 min), the supernatant was collected, and the IFN-β level was measured by ELISA.

[0148] The results of the ELISA assay of IFN-β produced by co-culture of 11 strains and animal Bifidobacterium BB-12 (denoted as BB12 in the attached figure) with BMDC from 4-week-old mice are shown in Table 1 , and the bar graph is referenced to Figure 1 .

[0149] Table 1: ELISA results of IFN-β produced by co-culture of 12 bacterial strains with BMDC (mean ± standard error)

[0150] Note: “—” indicates that the ELISA detection limit was not reached; a-f Different letters indicate significant differences among the groups in Duncan's multiple comparison test (P < 0.05).

[0151] The results of ELISA experiment ( Figure 1 , Table 1) shows that different strains have significant differences in promoting BMDC to secrete IFN-β. Among them, the live bacteria effect and heat sterilization effect 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 sterilization is more than 35 times that of BB-12 heat sterilization; compared with other Pediococcus pentosaceus, FeiHeP06 stimulates BMDC to produce the highest level of IFN-β, which is significantly different from the other two strains of Pediococcus pentosaceus, and Pediococcus pentosaceus FeiHeP06 produces the highest level of IFN-β. Therefore, the Pediococcus pentosaceus FeiHeP06 of the present invention can significantly promote the secretion of immune regulatory factor IFN-β by immune cells-dendritic cells, and increase the secretion of IFN-β, indicating that Pediococcus pentosaceus FeiHeP06 has potential immunoregulatory ability, which can enhance the body's resistance to infection by pathogens such as viruses. In addition, the thermal sterilization effect of Pediococcus pentosaceus FeiHeP06 is significant, which further broadens the application potential of the strain. It can be added to other foods sold at room temperature, reducing storage and transportation costs. The live bacteria can not only exert immune regulation functions, but also play an antibacterial role and regulate intestinal flora, and has good development prospects.

[0152] 3. Pediococcus pentosaceus FeiHeP06 was co-cultured with BMDC cells from mice of different ages according to the above steps, and the IFN-β level was determined by ELISA. The experimental results are shown in Table 2, and the bar graph is referenced to Figure 2 .

[0153] Table 2: IFN-β production by co-culture of Pediococcus pentosaceus FeiHeP06 with BMDC strains of different ages

[0154] Note: **** indicates P < 0.001 compared with the live bacteria group.

[0155] The experimental results showed that the level of IFN-β produced by BMDC cells from mice of different ages stimulated by the Pediococcus pentosaceus FeiHeP06 strain was significantly higher than that of live bacteria after heat sterilization. Therefore, the Pediococcus pentosaceus FeiHeP06 strain can regulate the immune function of mice of different ages, suggesting that it may have immune regulation capabilities for organisms of different ages, including infants, adolescents, and adults, and has a wider range of application scenarios.

[0156] Example 3: Study on the basic probiotic properties of Pediococcus pentosaceus FeiHeP06 The specific steps are as follows: 1. Cultivation of Pediococcus pentosaceus FeiHeP06 Pediococcus pentosaceus was inoculated onto MRS solid medium and cultured anaerobically at 37℃ for 48 h, and then its colonies were observed.

[0157] Experimental results: The colonies were found to be milky white round protrusions with a smooth, opaque surface and a diameter of 1 to 2 mm. Figure 3 The pH of the blank culture medium and the culture medium with bacteria was measured by a pH meter before and after the culture, and it was found that Pediococcus pentosaceus produced acid during the culture process. The Pediococcus pentosaceus strain was inoculated into MRS liquid culture medium, placed in an ELISA instrument, and cultured anaerobically at 37°C for 24 h. During the culture process, the OD of the culture medium was measured at intervals of 15 min. 600 It was found that Pediococcus pentosaceus reached a stable growth period after 18-19 hours of cultivation, and the growth curve was as follows Figure 4 .

[0158] 2. Acid and bile salt resistance of Pediococcus pentosaceus FeiHeP06 1. Strain activation 200 μL of Pediococcus pentosaceus FeiHeP06 bacterial solution was aspirated from the bacterial preservation tube and inoculated into 5 mL of MRS liquid culture medium, and cultured in a constant temperature incubator at 37°C until the early stage of the plateau to obtain the first-generation activated bacterial solution; in the second step, 100 μL of the first-generation activated bacterial solution was transferred into 5 mL of MRS liquid culture medium, and cultured in a constant temperature incubator at 37°C until the early stage of the plateau to obtain the second-generation activated bacterial solution.

[0159] 2. Preparation of simulated gastric juice and simulated intestinal juice (1) Dissolve pepsin in sterile saline (0.9% w / v, pH adjusted to 3.0 with hydrochloric acid) to a final concentration of 3 g / L. Filter through a 0.22 μm sterile filter membrane and prepare for use immediately.

[0160] (2) Dissolve trypsin in sterile saline (0.9% w / v, adjust pH to 8.0 with NaOH) to a final concentration of 1 g / L, and add bile salt to a final concentration of 0.3%. Filter with a 0.22 μm sterile filter membrane and use immediately after preparation.

[0161] 3. Acid and bile salt resistance test (1) Take out 200 μL of bacterial solution at the stationary stage and measure the OD 600 OD of bacterial solution 600 The CFU of Pediococcus pentosaceus was considered to be 10 9 , calculate the volume of physiological saline / simulated gastric fluid / simulated intestinal fluid added according to the OD value.

[0162] (2) Acid resistance: Take 1 mL of the plateau bacterial solution and centrifuge at 5000 r / min for 5 min. Collect the cells in two 1.5 mL centrifuge tubes, discard the supernatant, wash twice with saline, centrifuge, discard the supernatant, and calculate the OD value. 600 The value of was used to calculate the amount of saline / simulated gastric fluid to be added, and the bacteria in the two centrifuge tubes were resuspended. After culturing at 37°C for 3 h, the live bacteria were counted on the plate. 10 samples were taken from the control group (resuspended in saline) and the experimental group (resuspended in simulated gastric fluid). -6 , 10 -7 , 10 -8 Three dilution concentrations of bacterial solution were used for counting, and 2 parallels were made for each dilution.

[0163] (3) Bile salt resistance: Take 1 mL of the plateau bacterial solution and centrifuge at 5000 r / min for 5 min. Collect the cells in two 1.5 mL centrifuge tubes, discard the supernatant, wash twice with physiological saline, centrifuge, discard the supernatant, and calculate the OD value. 600 The value of was used to calculate the amount of physiological saline / simulated intestinal fluid to be added, and the bacteria in the two centrifuge tubes were resuspended. After culturing at 37℃ for 4 h, the live bacteria were counted on the plate. 10 samples were taken from the control group (resuspended with physiological saline) and the experimental group (resuspended with simulated intestinal fluid). -6 , 10 -7 , 10 -8 Three dilution concentrations of bacterial solution were used for counting, and 2 parallels were made for each dilution.

[0164] Survival rate (%) = viable bacterial concentration after culture (CFU / mL) / viable bacterial concentration at 0 h (CFU / mL) × 100; the colony count after gastric juice / intestinal juice treatment decreased by less than 3 logarithmically compared with that of untreated, and the viable bacterial count was still above 106 CFU / mL or above indicates 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 Chuan, Zhang Chaowu, Sun Xiaofan, et al. Preliminary study on the acid and bile resistance of lactobacilli from human stomach and intestines [J]. Modern Preventive Medicine, 2006, (10):1792-1794.).

[0165] The experimental results showed that the live bacterial concentration of Pediococcus pentosaceus at 0 h was 8.65×10 8 CFU / mL, the logarithmic decrease after 3 h of digestion in simulated gastric fluid at pH = 3 was only 0.44, and the concentration of viable bacteria after 3 h was 3.15×10 8 CFU / mL; after 4 h of digestion in simulated intestinal fluid at pH = 8, the logarithmic decrease was only 0.52, and the viable bacterial concentration after 4 h was 2.60×10 8 CFU / mL. Therefore, it can be seen that the Pediococcus pentosaceus has good tolerance to gastric acid and bile salts, and can effectively reach the downstream digestive tract to exert its effect. Therefore, Pediococcus pentosaceus FeiHeP06 meets the basic requirements as a probiotic.

[0166] 3. Antibacterial activity of Pediococcus pentosaceus FeiHeP06 against four pathogenic bacteria During the growth and reproduction process, lactic acid bacteria can secrete some organic acids, hydrogen peroxide, bacteriocins and other substances. 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. Animal Bifidobacterium BB-12 was used as the control strain. The antibacterial activity of Pediococcus pentosaceus FeiHeP06 was significantly higher than that of Animal Bifidobacterium BB-12, and it had good antibacterial ability. The specific experimental steps are as follows: 1. Strain culture Take Pediococcus pentosaceus FeiHeP06 and Bifidobacterium animalis BB-12 from the bacterial preservation tube and streak them on MRS solid medium and mMRS solid medium respectively, culture them at 37℃ for 48 h, pick a single colony and transfer it to 5 mL MRS liquid medium and mMRS liquid medium, culture them at 37℃ for 24 h; transfer 2% of the colony to 5 mL MRS liquid medium and mMRS liquid medium, and culture them at 37℃ for 24 h.

[0167] Take pathogenic bacteria (taking Staphylococcus aureus as an example) from the bacterial preservation tube and streak it onto LB solid culture medium, and culture it at 37℃ for 48h; pick a single colony and transfer it to 5 mL LB liquid culture medium, and culture it at 37℃ for 24h; transfer it to 5 mL LB liquid culture medium at a 2% inoculation volume, and culture it at 37℃ for 24h.

[0168] 2. Determination of in vitro antibacterial ability: Oxford cup method (1) Preparation of fermentation supernatant of strains: Pediococcus pentosaceus FeiHeP06 and Bifidobacterium animalis BB-12 activated for three generations were inoculated into MRS liquid medium and mMRS liquid medium, respectively, and cultured at 37°C for 24 h. 4 mL of bacterial culture was placed in a 5 mL centrifuge tube and centrifuged at 8000 rpm for 20 min. The supernatant was collected and filtered through a 0.22 μm microporous filter to obtain the cell-free fermentation supernatant of Pediococcus pentosaceus FeiHeP06 and the cell-free fermentation supernatant of Bifidobacterium animalis BB-12.

[0169] (2) Pour 10 mL of sterilized plain agar solid medium into a sterile plate and use it as the bottom medium after it is completely solidified. Place three sterile Oxford cups at appropriate distances on the plate. 8 Add the bacterial solution of Staphylococcus aureus, Salmonella, Escherichia coli or Listeria monocytogenes at CFU / mL to the LB solid culture medium that has been melted and cooled to 40-50℃. Mix carefully and pour it on the bottom culture medium. Be careful not to add it into the Oxford cup. Allow it to solidify completely as the bacterial layer culture medium.

[0170] (3) Carefully remove the Oxford cup, taking care not to damage the formed hole. Add 0.2 mL of the cell-free fermentation supernatant of Pediococcus pentosaceus FeiHeP06 and Bifidobacterium animalis BB-12 and the negative control (0.2 mL of MRS medium) into the hole with a diameter of about 8 mm, and culture at 37°C for 24 h. Measure the size of the inhibition zone with a ruler. The experimental results are shown in Table 3: Table 3: Inhibitory effect of Pediococcus pentosaceus FeiHeP06 on four pathogenic bacteria

[0171] Note: “ / ” indicates no inhibitory effect.

[0172] The results showed that Bifidobacterium animalis 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 BB-12, and it had good antibacterial ability. Therefore, Pediococcus pentosaceus FeiHeP06 has certain antibacterial activity and has advantages in production applications such as fermentation.

[0173] Example 4: Pediococcus pentosaceus ( Pediococcus pentosaceus ) Preparation of bacterial powder of FeiHeP06 strain Pediococcus pentosaceus FeiHeP06 can be used to prepare bacterial powder. The specific preparation process of bacterial powder is as follows: Pediococcus pentosaceus FeiHeP06 was streaked on MRS solid medium and cultured anaerobically at 37°C for 48 h to obtain a single colony; a single colony was picked and inoculated into MRS liquid medium, cultured anaerobically at 37°C for 18 h, and then activated, and activated for two generations to obtain an activated solution; the activated solution was inoculated into the medium at an inoculum of 2% (v / v), and cultured anaerobically at 37°C for 18 h to obtain a bacterial solution; the bacterial solution was centrifuged at 5000 r / min for 15 min to obtain bacterial mud; the solution was washed three times with physiological saline and resuspended with a protective agent (wet bacterial mud mass g: protective agent volume mL = 1:1) to a concentration of 1×10 10 CFU / mL, and obtain a bacterial suspension; the bacterial suspension was incubated at 37°C for 60 min and then freeze-dried to obtain Pediococcus pentosaceus FeiHeP06 bacterial powder; The preparation method of the culture medium is as follows: 10% enzyme-hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract are dissolved in 87.7% water based on the total weight of the culture medium, and then the pH thereof is adjusted to 6.8 to obtain the culture medium; The protective agent comprises: 130 g / L skimmed milk powder aqueous solution.

[0174] Example 5: Pediococcus pentosaceus ( Pediococcus pentosaceus ) Preparation of fermented milk with FeiHeP06 strain The powder of Pediococcus pentosaceus FeiHeP06 was mixed with commercial dry powder starter Lactobacillus bulgaricus and commercial dry powder starter Streptococcus thermophilus in a mass ratio of 1:1:1 to obtain a starter; sugar was added to fresh milk to a concentration of 50 g / L to obtain a mixed solution; the mixed solution was homogenized under the conditions of 65°C and 20 MPa, and then sterilized at 95°C for 5 min to obtain a fermentation raw material; the fermentation raw material was cooled to 35°C, and the starter was inoculated into the fermentation raw material at an inoculation rate of 0.03% (v / v), and the fermentation was carried out at 35°C for 16 h to obtain fermented milk; the fermented milk was placed at 42°C for 4 h for coagulation, and then refrigerated at 4°C for 24 h for post-ripening to obtain a fermented milk product.

[0175] Example 6: Pediococcus pentosaceus ( Pediococcus pentosaceus ) Preparation of fruit and vegetable beverages with FeiHeP06 strain 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: Wash fresh fruits and vegetables and squeeze out the juice, add food-grade peptone at a ratio of 5 g / L, and sterilize at 140°C for 2 seconds; cool the sterilized fruit and vegetable juice to about 37°C and then add the FeiHeP06 bacterial powder of the present invention to a concentration of not less than 1×10 6 CFU / mL, a fruit and vegetable beverage is obtained, and the beverage is refrigerated and stored at 4° C. A fruit and vegetable beverage containing the live bacteria of Pediococcus pentosaceus FeiHeP06 of the present invention can be obtained.

[0176] Example 7: Pediococcus pentosaceus ( Pediococcus pentosaceus )Application of FeiHeP06 strain in fermented sausage Formula of fermented sausage: fresh pork fat-to-lean ratio 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%. Preparation method is as follows: The raw meat was sorted and cut into pieces, marinated for 12 hours, minced, chopped, and mixed, and spices, seasonings, and fermentation agents were added and stirred, wherein 10 7 The bacterial powder of Pediococcus pentosaceus FeiHeP06 with a CFU / g is stirred, then enema is performed, fermented, smoked, and finally vacuum-packed to obtain a finished product of fermented sausage.

[0177] In summary, the present invention discloses a strain of Pediococcus pentosaceus FeiHeP06 that can promote myeloid dendritic cells (BMDC cells) to secrete IFN-β and its application. Pediococcus pentosaceus ) strain can promote BMDC cells to secrete IFN-β, which helps to prevent and resist infection by 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 food, medicine or health products, etc.) that improve host immunity and prevent infection.

[0178] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

[0179] It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0180] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

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

1.

2. A culture, characterized in that The culture is obtained by culturing the strain according to claim 1.

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

4. The microbial preparation according to claim 3, characterized in that In the microbial preparation, the Pediococcus pentosaceus ( Pediococcus pentosaceus ) The viable count of 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 ( Pediococcus pentosaceus ) The FeiHeP06 strain is inactivated.

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

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

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

8. The product according to claim 7, characterized in that The product comprises a probiotic product, a food, a food additive or a feed additive.

9. The product according to claim 8, 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.

10. The product according to claim 8 or 9, characterized in that The products include nutritional foods, functional foods or health foods.

11. The strain according to claim 1, the culture according to claim 2, the microbial preparation according to claim 3 or 4, the microbial preparation prepared by the preparation method according to claim 5, or the product according to any one of claims 7 to 10, for any use in (1) to (5): (1) Helps regulate intestinal flora; (2) Uses for improving intestinal health; (3) Uses that help enhance immunity; (4) Use in the inhibition of Escherichia coli, Staphylococcus aureus and / or Listeria monocytogenes for non-disease treatment purposes; (5) Use in regulating cytokine secretion of immune cells.

12. The use according to claim 11, characterized in that The regulating immune cell cytokine secretion includes promoting dendritic cells to secrete the cytokine IFN-β.

13. Use of the strain according to claim 1, the culture according to claim 2, the microbial preparation according to claim 3 or 4, or the microbial preparation prepared by the preparation method according to claim 5 in any of the following (1) to (5): (1) Use in the preparation of products for helping to regulate intestinal flora; (2) Use in the preparation of products for improving intestinal health; (3) Use in the preparation of products for helping to enhance immunity; (4) Use in the preparation of a product for inhibiting Escherichia coli, Staphylococcus aureus and / or Listeria monocytogenes for non-disease treatment purposes; (5) Use in the preparation of products for regulating cytokine secretion in immune cells.

14. The use according to claim 13, characterized in that The regulating immune cell cytokine secretion includes promoting dendritic cells to secrete the cytokine IFN-β.

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