Breast milk-derived staphylococcus epidermidis and application thereof

Abstract

The invention discloses a breast milk-derived staphylococcus epidermidis strain and application thereof. The invention provides a staphylococcus epidermidis strain, and the preservation number of the staphylococcus epidermidis strain is CGMCC (China General Microbiological Culture Collection Center) No.31507. The staphylococcus epidermidis strain has the advantages that the staphylococcus epidermidis strain can be used for preparing the staphylococcus epidermidis strain; the strain of the staphylococcus epidermidis, provided by the invention, has good intestinal adaptive capacity, safety and stability, and can be used for inhibiting enteropathogenic bacteria and improving the immune response reaction of host immune cells.

Description

Technical Field

[0001] The invention relates to a breast milk-derived Staphylococcus epidermidis strain and application thereof, and belongs to the field of microorganisms. Background Art

[0002] Staphylococcus epidermidis ( Staphylococcus epidermidis ) is a Gram-positive bacterium that is commonly found in the human skin microbiome and is considered a commensal. Staphylococcus epidermidis is also found in breast milk, as reported in the literature (BMC Microbiology 2008: Staphylococcus epidermidis : A differential trait of the fecal microbiota of breast-fed infants) records that Staphylococcus epidermidis is the main species in the intestines of breastfed infants, and the presence of this species is a characteristic difference in the intestinal flora of breastfed infants and formula-fed infants. Past research on Staphylococcus epidermidis has mainly focused on its pathogenic mechanism and corresponding prevention / treatment strategies under specific conditions (impaired immune system, damaged skin barrier, artificial joint or catheter placement, etc.). With the development of multidisciplinary cross-border and multiple technologies, researchers have gained new insights into the nature of Staphylococcus epidermidis, but research on its potential host health benefits is still in its infancy.

[0003] Some literature and patents indicate that Staphylococcus epidermidis has the effect of promoting wound healing and improving skin barrier. For example, the literature (Journal of Cellular and Molecular Medicine 2019: Staphylococcus epidermidisrole in the skin microenvironment) discloses that Staphylococcus epidermidis can induce epithelial regeneration of damaged skin and accelerate wound closure. Similarly, the Chinese invention patent application with publication number CN113730648A (application number 202111038989.1) discloses that a hydrogel combined with Staphylococcus epidermidis can be used for rapid repair of superficial skin injuries and reduce scarring on wounds. Some other Chinese invention patents show the good application prospects of Staphylococcus epidermidis in skin health products. For example, the patent application with publication number CN115637235A (application number 202210704624.6) discloses that the fermentation supernatant of Staphylococcus epidermidis has good free radical scavenging activity and can reduce the ROS content of human keratinocytes caused by vitamin K3; the patent application with publication number CN115806899A (application number 202210967956.3) discloses that the fermentation supernatant of Staphylococcus epidermidis has good whitening effect, It can significantly reduce the activity of tyrosinase and has good anti-inflammatory effects, significantly reducing the expression level of inflammatory factor IL-6 produced by macrophages induced by lipopolysaccharide (LPS); the patent application text with publication number CN111763630A (application number 201910238361.2) discloses that Staphylococcus epidermidis can decompose triglycerides on the surface of the skin; the patent application text with publication number CN116496937A (application number 202310191536.5) discloses that Staphylococcus epidermidis can inhibit the activity of Staphylococcus aureus and Propionibacterium acnes.

[0004] In addition, the Chinese invention patent application with publication number CN104745501A (application number 201310752880.3) discloses a Staphylococcus epidermidis and its application in the production of fermented meat products. The meat products fermented by Staphylococcus epidermidis in this patent have excellent flavor, stable product quality, no odor, and good color development effect. Similarly, the Chinese invention patent application with publication number CN101717741A (application number 200910264536.3) discloses meat products in which raw meat is inoculated with Staphylococcus epidermidis and then fermented to maturity. Compared with traditional fermented meat products such as dry-cured ham, the processing cycle of fermented pork products by this method is shortened by nearly 5 months, and the sensory quality of the product is significantly improved, with a rich ham flavor and aroma. The Chinese invention patent application with publication number CN108823124A (application number 201810603967.7) discloses a strain of Staphylococcus epidermidis isolated from the nasal cavity of a healthy person, which has broad-spectrum antibacterial activity against common Gram-positive resistant bacteria such as Staphylococcus aureus, Streptococcus pneumoniae and Micrococcus luteus. It can be seen that Staphylococcus epidermidis has potential probiotic properties and application prospects as an edible beneficial bacterium.

[0005] For edible probiotics, survival in the host's digestive tract, reaching the colon and successfully colonizing in intestinal epithelial cells are important prerequisites for exerting probiotic effects. Shorter reproduction cycles (generation time), specific carbon source utilization capabilities, and stronger intestinal epithelial cell adhesion capabilities help improve the colonization ability of probiotics. In addition, as probiotics, maintaining strong antibiotic sensitivity and low pathogenic characteristics (virulence genes, pathogen-host interactions, secretion systems, etc.) are even more necessary conditions.

[0006] In recent years, people's health awareness has been continuously enhanced, and the coronavirus crisis has pushed people's interest in immune-related products to an unprecedented level. Therefore, the role of probiotics in inhibiting intestinal pathogens, maintaining the stability of the intestinal microecological system, and improving host immunity has become a hot topic in the health market and research.

[0007] Therefore, screening and isolating a strain of Staphylococcus epidermidis from breast milk and studying its intestinal adaptability, safety and probiotic properties have scientific value and practical significance for the development and application of Staphylococcus epidermidis. 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 new strain of Staphylococcus epidermidis isolated from breast milk ( Staphylococcus epidermidis ) and its applications.

[0009] Solutions to Solve the Problems To solve the above problems, in a first aspect of the present invention, a Staphylococcus epidermidis ( Staphylococcus epidermidis ) strain, wherein the strain is Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, the deposit number of the strain is CGMCC No. 31507. Specifically, the present invention provides a strain of Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, the Staphylococcus epidermidis FeiHeS06 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.31507 and the deposit date of August 1, 2024.

[0010] In the second aspect of the present invention, a culture is provided, wherein the culture is obtained by culturing the strain as described in the first aspect of the present invention.

[0011] In the third aspect of the present invention, a microbial preparation is provided, wherein the microbial preparation comprises the strain as described in the first aspect of the present invention or the culture as described in the second aspect of the present invention.

[0012] In a fourth aspect of the present invention, a fermentation agent is provided, wherein the fermentation agent comprises the strain as described in the first aspect of the present invention, the culture as described in the second aspect of the present invention, or the microbial preparation as described in the third aspect of the present invention.

[0013] In the fifth aspect of the present invention, a product is provided, wherein the product comprises the strain as described in the first aspect of the present invention, the culture as described in the second aspect of the present invention, or the microbial preparation as described in the third aspect of the present invention, or the product is obtained by fermentation using the starter as described in the fourth aspect of the present invention.

[0014] In some specific embodiments, the product is a probiotic product.

[0015] In some specific embodiments, the product is a food or a food additive.

[0016] In some optional embodiments, the food comprises a nutritional food, a functional food or a health food.

[0017] In the sixth aspect of the present invention, there is provided the use of the strain described in the first aspect of the present invention, the culture described in the second aspect of the present invention, the microbial preparation described in the third aspect of the present invention or the product described in the fifth aspect of the present invention to help regulate intestinal flora or help enhance immunity.

[0018] In some embodiments, the helping to regulate intestinal flora comprises inhibiting at least one of Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes.

[0019] In some embodiments, the helping to enhance immunity comprises at least one of promoting macrophages to produce the anti-inflammatory factor IL-10, reducing the secretion of macrophage inflammatory factor TNF-α, and reducing the secretion of inflammatory mediator NO.

[0020] In the seventh aspect of the present invention, there is provided the use of the strain described in the first aspect of the present invention, the culture described in the second aspect of the present invention, and the microbial preparation described in the third aspect of the present invention in the preparation of a product that helps regulate intestinal flora or helps enhance immunity.

[0021] In some embodiments, the helping to regulate intestinal flora comprises inhibiting at least one of Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes.

[0022] In some embodiments, the helping to enhance immunity comprises at least one of promoting macrophages to produce the anti-inflammatory factor IL-10, reducing the secretion of macrophage inflammatory factor TNF-α, and reducing the secretion of inflammatory mediator NO.

[0023] Effects of the Invention The present invention provides a strain of Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, this Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 has good intestinal adaptability, safety and stability, and can inhibit intestinal pathogens and improve the immune response of host immune cells, which is specifically reflected in: (1) The survival of probiotics after experiencing the gastrointestinal environment is a prerequisite for their colonization in the intestine. After the Staphylococcus epidermidis FeiHeS06 of the present invention and the control probiotic strain were cultured in simulated gastric fluid (pH = 3.0, pepsin addition amount 3 g / L) for 3 h, the colony count of Staphylococcus epidermidis FeiHeS06 treated with gastric fluid decreased by less than 2 logarithmically compared with that of the untreated strain, and the number of viable bacteria was still above 10 6 CFU / mL or more, indicating good tolerance to the gastric environment; after being cultured in simulated intestinal fluid (pH = 8.0, trypsin addition amount 1 g / L, bile salt addition amount 0.3% w / v) for 4 h, among the 5 strains, the colony count of the Staphylococcus epidermidis FeiHeS06 treated with intestinal fluid of the present invention decreased the least compared with that of the untreated one, and the number of viable bacteria was still above 10 6 CFU / mL or above indicates good tolerance to the intestinal environment and strong ability to survive the gastrointestinal tract.

[0024] (2) The colonization of probiotics in the intestine is the basis for their probiotic function. A shorter generation time, a higher adhesion rate to intestinal epithelial cells and a stronger ability to utilize host dietary oligosaccharides can improve the colonization ability of probiotics in the intestine. The growth characteristics of the Staphylococcus epidermidis FeiHeS06 of the present invention and the control probiotic strain in MRS liquid culture medium are different. The generation time of the Staphylococcus epidermidis FeiHeS06 of the present invention is shorter. When the strain is co-cultured with HT-29 cells in DMEM culture medium for 4 hours, the cell adhesion rate of the Staphylococcus epidermidis FeiHeS06 of the present invention is the highest. In the MRS liquid culture medium with oligofructose or oligogalactose as the carbon source, the Staphylococcus epidermidis FeiHeS06 of the present invention can utilize these two common oligosaccharides and has a strong ability to utilize oligofructose.

[0025] (3) Determination of antibiotic resistance of probiotics is an important part of safety assessment. When the minimum inhibitory concentration (MIC) of 14 common antibiotics was measured for the Staphylococcus epidermidis FeiHeS06 of the present invention and the control probiotic strain, the antibiotic sensitivity of the Staphylococcus epidermidis FeiHeS06 of the present invention was equivalent to that of the control probiotic strain, and the sensitivity to rifampicin and clindamycin was the strongest among the five strains; the whole genome data (virulence genes, secretion system, prophage, and interaction with the host) of the Staphylococcus epidermidis FeiHeS06 of the present invention were analyzed. The virulence-related genes carried by the Staphylococcus epidermidis FeiHeS06 of the present invention are mainly involved in metabolism, synthesis of extracellular components, and providing adhesion. There are no significant toxin-producing genes. The genes related to host interaction are mainly related to the phenotype of weakened virulence or no effect on pathogenicity. It only has a common non-toxic Sec-SRP type secretion system and no prophage. The possibility of horizontal transfer of resistance genes to other intestinal bacteria in the intestine is low, and its pathogenicity is low, and its safety is good.

[0026] (4) Stability is related to the product quality, cost and energy consumption of industrial production of probiotics. The Staphylococcus epidermidis FeiHeS06 of the present invention and the control probiotic strain were inoculated into MRS liquid culture medium, and the OD was measured every 1 h. 600 The value of is continuously monitored for its growth state. The growth rate of the Staphylococcus epidermidis FeiHeS06 of the present invention is relatively fast, and the biomass (the mass of the bacterial mud produced in the stable period) when it reaches the stable period is acceptable. The Staphylococcus epidermidis FeiHeS06 of the present invention and the control probiotic strain are inoculated in a series of MRS culture media with different osmotic pressures prepared by adding different contents of NaCl. The osmotic pressure when the growth rate of the Staphylococcus epidermidis FeiHeS06 of the present invention begins to be inhibited and the osmotic pressure when the growth is completely inhibited are relatively large, and the osmotic pressure tolerance is relatively strong. Osmotic pressure affects the water activity and cell membrane permeability in cells, and affects the structure of macromolecular substances such as proteins, thereby affecting the structure, physiological function and growth and reproduction of microbial cells.

[0027] (5) The organic acid produced by probiotics can reduce the intestinal pH and inhibit the growth of intestinal pathogenic microorganisms. The Staphylococcus epidermidis FeiHeS06 of the present invention produces acid during growth, and the pH of the culture medium is significantly reduced during the culture process. In the process of co-cultivation with pathogenic bacteria, the Staphylococcus epidermidis FeiHeS06 of the present invention can antagonize the growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, produce obvious inhibition zones, regulate intestinal flora, and promote intestinal health.

[0028] (6) Macrophages are important immune effector cells differentiated from monocytes, and they play a key role in innate and acquired immune responses. Activated macrophages RAW264.7, added LPS to induce macrophages to produce inflammatory responses, and the supernatant of Staphylococcus epidermidis FeiHeS06 of the present invention promoted macrophages RAW264.7 to produce anti-inflammatory factors (IL-10), and reduced the secretion of inflammatory factors (TNF-α) and inflammatory mediators (NO) by macrophages RAW264.7.

[0029] Therefore, the Staphylococcus epidermidis FeiHeS06 of the present invention has great application prospects in the preparation of products (such as food, medicine, functional food or health care products, etc.) that improve host intestinal health and regulate immune response with good intestinal adaptability, host safety and production stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 :Staphylococcus epidermidis( Staphylococcus epidermidis ) Characteristic colony morphology of FeiHeS06 on MRS solid medium.

[0031] Figures 2A - 2B :The changes of viable bacterial counts of Staphylococcus epidermidis FeiHeS06 and control probiotic strains before and after culture in simulated gastric and intestinal fluids. Figure 2A To simulate gastric fluid treatment, Figure 2B To simulate intestinal fluid treatment.

[0032] Figure 3 : Generation time of Staphylococcus epidermidis FeiHeS06 and control probiotic strains grown in MRS liquid medium. Among them, different letters "a, b, c" indicate that the generation time of the strains is significantly different ( P <0.05).

[0033] Figure 4 :Adhesion ability of Staphylococcus epidermidis FeiHeS06 and control probiotic strains to HT-29 cells. Among them, different letters "a, b, c, d" indicate that the strains have significant differences in adhesion ability to HT-29 cells ( P <0.05).

[0034] Figure 5 :Distribution of minimum inhibitory concentration (MIC) of Staphylococcus epidermidis FeiHeS06 and control probiotic strains against 14 common antibiotics. The darker the color of the square, the greater the MIC value of the strain to the antibiotic. The color shows the result after homogenization of each column; the value on the square is the actual MIC value of the strain to the antibiotic.

[0035] Figure 6: Growth patterns of Staphylococcus epidermidis FeiHeS06 and control probiotic strains in MRS liquid culture medium.

[0036] Figure 7 : The mass of bacterial sludge (maximum biomass) that can be obtained when Staphylococcus epidermidis FeiHeS06 and the control probiotic strain grow to the stable phase. Different letters "a, b" indicate that the maximum biomass of different strains has significant differences ( P <0.05).

[0037] Figures 8A - 8E :Osmotic pressure tolerance of Staphylococcus epidermidis FeiHeS06 and control probiotic strains. Among them, Figure 8A :Osmotic pressure resistance of Staphylococcus epidermidis FeiHeS06, Figure 8B :Osmotic pressure resistance of Staphylococcus epidermidis MRJSWX39M8, Figure 8C :Lactobacillus gasseri MRJSWX2L1 osmotic pressure resistance, Figure 8D :Osmotic pressure resistance of Bifidobacterium longum subspecies infantis MRJSWX29M3, Figure 8E : Osmotic pressure resistance of Bifidobacterium animalis subsp. lactis BB12.

[0038] Figure 9 : Changes in culture medium pH before and after culture of Staphylococcus epidermidis FeiHeS06 and control probiotics.

[0039] Figures 10A - 10C :The regulatory ability of Staphylococcus epidermidis FeiHeS06 and control probiotic strains on LPS-induced inflammatory response of macrophages RAW264.7. Figure 10A The regulatory ability of different strains on the anti-inflammatory factor IL-10 of macrophage RAW264.7. Figure 10B The regulatory ability of different strains on the inflammatory factor TNF-α of macrophage RAW264.7. Figure 10C The different strains regulate the inflammatory mediator NO in macrophage RAW264.7. Different letters "a, b, c, d" indicate that the strains have significant differences in their ability to regulate the production of anti-inflammatory factor IL-10, inflammatory factor TNF-α and inflammatory mediator NO in macrophage RAW264.7 ( P <0.05).

[0040] Figure 11 : The colors of bromocresol purple indicator in Table 1 are as follows, from left to right: ++++, +++, ++, +, control; the more "+" there are, the stronger the strain's ability to utilize this sugar is. DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] 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.

[0044] In this specification, the word "may" includes both performing a certain process and not performing a certain process.

[0045] 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.

[0046] 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.

[0047] Biomaterial Deposit A strain of Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, taxonomic name Staphylococcus epidermidis Staphylococcus epidermidis , was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on August 1, 2024, with the deposit number CGMCC No. 31507. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0048] strains In some aspects, the present invention provides a strain of Staphylococcus epidermidis ( Staphylococcus epidermidis) FeiHeS06, the Staphylococcus epidermidis FeiHeS06 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 31507 and the deposit date of August 1, 2024.

[0049] The Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 was derived from breast milk of healthy lactating women in Wuxi City, Jiangsu Province. The strain was sequenced and analyzed. The sequence obtained was compared with the nucleic acid sequence of Staphylococcus epidermidis in NCBI Standard Nucleotide BLAST. The results showed that the nucleic acid sequence similarity between the strain and Staphylococcus epidermidis was 100%. The results showed that the strain was Staphylococcus epidermidis, and it was named Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, whose original number in the laboratory is CCFM1371.

[0050] The Staphylococcus epidermidis ( Staphylococcus epidermidis The colonies of FeiHeS06 on MRS solid medium are white round protrusions with a smooth, opaque surface and a diameter of 1 to 2 mm (e.g. Figure 1 as shown).

[0051] In some embodiments of the present invention, the Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 has good tolerance in simulated gastric fluid and simulated intestinal fluid environments, and has a strong ability to survive in the gastrointestinal tract.

[0052] In some embodiments of the present invention, the Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 has a shorter generation time in MRS liquid culture medium, a stronger ability to adhere to HT-29 cells (a human colorectal cancer cell line), a stronger ability to utilize oligofructose, and a stronger intestinal adaptability.

[0053] In some embodiments of the present invention, the Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 showed strong sensitivity in the determination of minimum inhibitory concentration (MIC) of 14 common antibiotics. Whole genome data showed that the virulence-related genes it carried were mainly involved in metabolism, synthesis of extracellular components and providing adhesion, without significant toxin-producing genes. Genes related to host interaction were mainly related to phenotypes with weakened virulence or no effect on pathogenicity. It only has the common non-toxic Sec-SRP type secretion system, and there is no prophage. The risk of resistance gene transfer is small, its potential pathogenicity and toxicity are low, and its safety is good.

[0054] In some embodiments of the present invention, the Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 has a fast growth rate, a decent biomass in the stable period, a strong osmotic pressure tolerance, and good stability.

[0055] In some embodiments of the present invention, the Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 produces acid, and the organic acid produced can reduce the intestinal pH and inhibit the growth of intestinal pathogenic microorganisms. The Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 can antagonize the growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, regulate intestinal flora and promote intestinal health.

[0056] In some embodiments of the present invention, the Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 significantly reduced the levels of inflammatory factors Tumor Necrosis Factor-alpha (TNF-α) and inflammatory mediator nitric oxide (NO) produced by lipopolysaccharide (LPS)-induced macrophages RAW264.7, and promoted the production of anti-inflammatory cytokines (Interleukin-10, IL-10) by macrophages RAW264.7. Macrophages are important immune effector cells differentiated from monocytes, and play a key role in innate and acquired immune responses.

[0057] Culture In some aspects, the present invention provides a culture, wherein the culture is obtained by culturing the above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis )FeiHeS06 obtained.

[0058] 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 microorganisms, or may contain a certain amount of culture medium, metabolites or other components produced during the culture process, or the supernatant after centrifugation to remove cells.

[0059] In some embodiments, the culture is a supernatant (cell-free supernatant) obtained by centrifuging a product obtained by growing and / or amplifying a microorganism.

[0060] In some embodiments, the culturing is performed by the following steps: (1) Staphylococcus epidermidis ( Staphylococcus epidermidis) FeiHeS06 was streaked on MRS solid medium and a single colony was obtained; (2) picking a single bacterial colony and inoculating it into an MRS liquid culture medium, culturing it for activation, and continuously activating it for at least one generation, for example, two generations, to obtain an activation solution; 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.

[0061] Optionally, (5) collecting the supernatant and filtering it to obtain a cell-free supernatant for use.

[0062] In some specific embodiments, the MRS solid 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, 1 ml / L Tween 80, and 20 g / L agar.

[0063] 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.

[0064] Microbial agents In some aspects, the present invention also provides a method comprising the above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 or the microbial preparation of the above-mentioned culture.

[0065] In some embodiments, the microbial preparation of the present invention is solid, and can be, for example, a freeze-dried powder, etc. In these embodiments, for ease of use, the microbial preparation can be contained in a capsule.

[0066] Furthermore, the microbial preparation may also contain a protective agent (such as a freeze-dried protective agent), which includes but is not limited to skim milk powder, trehalose, sodium glutamate and / or glycerol, etc.; preferably, the protective agent is skim milk powder.

[0067] 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.

[0068] In some other embodiments, the microbial preparation of the present invention is a liquid, for example, a suspension, a culture (supernatant). Furthermore, the microbial preparation may also contain a solvent, including but not limited to water or a culture medium.

[0069] Preparation method of microbial preparation In some embodiments of the present invention, the method for preparing the microbial preparation comprises: Staphylococcus epidermidis ) The steps of inoculating FeiHeS06 into a suitable culture medium for cultivation.

[0070] In some embodiments, the method further comprises the step of centrifuging the culture solution obtained by the culture to collect bacterial sludge, and the step of mixing the bacterial sludge with a protective agent (e.g., a freeze-drying protective agent). Centrifugation can increase the bacterial concentration and shorten the freeze-drying time. In some embodiments, after the mixing step, a freeze-drying step is also included. Freeze-drying facilitates the transportation and storage of microbial preparations. By adding a protective agent, the death or loss of activity of the strains during the freeze-drying process can be avoided as much as possible, thereby improving the fermentation effect of the microbial preparation.

[0071] In some specific embodiments, the method for preparing the microbial preparation of the present invention comprises the following steps: The above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06 is inoculated into a suitable culture medium to obtain a culture solution; The culture solution was centrifuged to obtain bacterial cells; After washing the bacterial cells, resuspend them in a freeze-drying protective agent to obtain a resuspended liquid; The resuspension is freeze-dried by a vacuum freezing method to obtain a microbial preparation.

[0072] 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.

[0073] In some embodiments of the present invention, the lyoprotectant comprises 100-150 g / L, such as 110 g / L, 130 g / L, or 150 g / L of skim milk powder aqueous solution.

[0074] In some embodiments of the present invention, the pH of the culture medium is 6.5-7.5, preferably 6.8-7.2, for example 6.8, 7.0.

[0075] Fermentation Agents In some aspects, the present invention also provides a method comprising the above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, the above-mentioned culture, the above-mentioned microbial preparation or the starter of the microbial preparation prepared by the above-mentioned preparation method.

[0076] In some embodiments, the starter culture further comprises lactic acid bacteria.

[0077] 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.

[0078] 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.

[0079] Examples of Streptococcus bacteria include thermophilic Streptococcus ( Streptococcus thermophilus )wait.

[0080] Preferred examples of the combination of bacteria of the genus Lactobacillus and bacteria of the genus Streptococcus include Lactobacillus bulgaricus and Streptococcus thermophilus.

[0081] product The present invention further provides a product, wherein the product comprises the above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, the above-mentioned culture, the above-mentioned microbial preparation, the microbial preparation prepared by the above-mentioned preparation method, or the product is obtained by fermentation using the above-mentioned starter.

[0082] In addition to the above essential components, the product of the present invention may also include other optional ingredients according to the needs of the final product, which may include: Containing milk ingredients or protein ingredients, the milk ingredients include dairy products including fresh milk from fresh 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.

[0083] Plants or plant extracts, including fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, rubber, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblica and bilberry or their extracts; fruits and vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano or their extracts; grains 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 or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricots, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts or their extracts; coffee or its extracts.

[0084] Animal ingredients include meat products from beef, lamb, fish or poultry.

[0085] The fat component 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] 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 extracts, dietary fiber (inulin, konjac flour, galacto-oligosaccharides, fructo-oligosaccharides, isomaltooligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin or soybean fiber, etc.).

[0087] Trace element supplements 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.

[0088] Any 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.

[0089] The present invention does not specifically limit the specific types of the above products.

[0090] In some embodiments, the product is a food product.

[0091] In some embodiments, the product is a nutraceutical, a functional food, or a health food.

[0092] In some exemplary embodiments of the present invention, the food is a food containing the above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, the above-mentioned culture, the above-mentioned microbial preparation or the fermented dairy product of the microbial preparation prepared by the above-mentioned preparation method, or the fermented dairy product obtained by fermentation of the above-mentioned starter.

[0093] In some exemplary embodiments of the present invention, the food is a food containing the above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, fermented pork made from the above-mentioned culture, the above-mentioned microbial preparation or the microbial preparation prepared by the above-mentioned preparation method, or fermented pork obtained by fermentation with the above-mentioned starter.

[0094] In some exemplary embodiments of the present invention, the food is a food containing the above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, the above-mentioned culture, the above-mentioned microbial preparation or the fermented fish paste of the microbial preparation prepared by the above-mentioned preparation method, or the fermented fish paste obtained by fermentation of the above-mentioned starter.

[0095] In some embodiments, the product is a food additive.

[0096] In other embodiments, the product is a pharmaceutical.

[0097] In some embodiments of the present invention, the drug contains the above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, the above-mentioned culture, the above-mentioned microbial preparation or the microbial preparation prepared by the above-mentioned preparation method, and optional pharmaceutical carriers and / or pharmaceutical excipients.

[0098] In some embodiments of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0099] In some embodiments of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0100] In some embodiments of the present invention, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0101] In some embodiments of the present invention, the additive comprises a solubilizer, a co-solvent, a latent solvent and / or a preservative.

[0102] In some embodiments of the present invention, the drug is in the form of powder, granules, capsules, tablets, pills or oral liquid.

[0103] In other embodiments, the product is a non-food product.

[0104] In other embodiments, the product is a non-nutraceutical food.

[0105] In other embodiments, the product is a non-drug.

[0106] In some embodiments, the product is a probiotic product.

[0107] In some specific embodiments, the probiotic product is a probiotic product with good intestinal adaptability, and the product contains the above-mentioned Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, the above-mentioned culture, the above-mentioned microbial preparation or the microbial preparation prepared by the above-mentioned preparation method.

[0108] 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.

[0109] 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.

[0110] In some specific embodiments, the product of the present invention can be infant formula milk powder, baby food, children's formula milk powder, children's snacks, pregnant women's milk powder, middle-aged and elderly milk powder or nutritional or dietary supplements.

[0111] In other specific embodiments, the product 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.

[0112] 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.

[0113] Helps regulate intestinal flora The Staphylococcus epidermidis provided by the present invention ( Staphylococcus epidermidis ) FeiHeS06 produces acid, and the organic acid produced can lower the intestinal pH, inhibit the growth of intestinal pathogenic microorganisms, and antagonize the growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, regulate intestinal flora and promote intestinal health.

[0114] Therefore, the Staphylococcus epidermidis provided by the present invention ( Staphylococcus epidermidis ) FeiHeS06, 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.

[0115] 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, in the case of disorder of intestinal flora, or the presence of intestinal pathogenic microorganisms (Staphylococcus aureus, Escherichia coli and Listeria monocytogenes) but not to the extent of causing disease, or in the case of disorder of intestinal flora and increase of intestinal pathogenic microorganisms not caused by disease, the use of Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, culture, microbial preparation, microbial agent 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.

[0116] In other embodiments, the product is a drug. In some specific embodiments, the drug is used to inhibit intestinal pathogens.

[0117] Uses that help enhance immunity and regulate immune response The Staphylococcus epidermidis provided by the present invention ( Staphylococcus epidermidis ) FeiHeS06 and its culture significantly reduced the levels of inflammatory factors TNF-α and inflammatory mediator NO produced by lipopolysaccharide (LPS)-induced macrophages RAW264.7, and promoted the production of anti-inflammatory cytokine IL-10 by macrophages RAW264.7. Macrophages are important immune effector cells differentiated from monocytes, which play a key role in innate and acquired immune responses.

[0118] Therefore, the Staphylococcus epidermidis provided by the present invention ( Staphylococcus epidermidis ) FeiHeS06, culture, microbial preparation, preparation method The microbial preparation prepared can be used to prepare products that help enhance immunity and regulate immune response. The product of the present invention also helps enhance immunity and regulate immune response.

[0119] The present invention does not particularly limit the specific categories of products that help enhance immunity and regulate immune response.

[0120] Furthermore, in some embodiments, the purpose of helping to enhance immunity and regulating immune response is not to prevent and / or treat diseases, for example, when immunity is reduced and inflammatory factor levels are changed but not caused by diseases, the Staphylococcus epidermidis provided by the present invention ( Staphylococcus epidermidis )FeiHeS06, culture, microbial preparation, microbial preparation prepared by preparation method, etc. In some embodiments, the product that helps to enhance immunity and regulate immune response is a food. In some embodiments, the product that helps to enhance immunity and regulate immune response is a health food. In some embodiments, the product that helps to enhance immunity and regulate immune response is a probiotic product.

[0121] In other embodiments, the product is a drug. In some specific embodiments, the drug is used for anti-infection, anti-inflammatory, etc.

[0122] 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.

[0123] In some specific embodiments, the anti-inflammatory effect includes reducing the levels of inflammatory factor TNF-α and inflammatory mediator NO, and promoting the production of anti-inflammatory cytokine IL-10. Example

[0124] 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.

[0125] The Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, and Bifidobacterium longum infantis subspecies MRJSWX29M3 involved in the following embodiments are strains screened and isolated from breast milk during the same period and are preserved in the proprietary strain bank of the Biotechnology Center of the School of Food Science and Engineering of Jiangnan University. The animal Bifidobacterium lactis subspecies BB12 involved in the following embodiments is a commercially available probiotic strain purchased from Chr. Hansen A / S, Denmark.

[0126] The culture medium involved in the following examples is as follows: MRS solid culture 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, agar 20 g / L.

[0127] MRS liquid culture 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.

[0128] The culture of Bifidobacterium requires the addition of 0.5 g / L L-cysteine ​​in MRS solid medium or MRS liquid medium.

[0129] The detection methods involved in the following embodiments are as follows: Detection method of viable bacteria count: adopt the national standard "GB 4789.2-2022 National Food Safety Standard Food Microbiology Examination Total Colony Count Determination".

[0130] The preparation method of the probiotic bacteria involved in the following embodiments is as follows: Streak Staphylococcus epidermidis on MRS solid culture medium, culture at 37 degrees Celsius (℃) for 48 hours (h) to obtain a single colony; pick a single colony and inoculate it into 5 milliliters (mL) of MRS liquid culture medium, culture at 37℃ for 18 hours for activation, and activate it for two generations to obtain an activated solution; inoculate the activated solution into MRS liquid culture medium at an inoculum volume of 2% (v / v), culture 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 Staphylococcus epidermidis cells.

[0131] The cultivation steps of Lactobacillus are the same as those of Staphylococcus epidermidis. The cultivation of Bifidobacterium requires the addition of 0.5 g / L L-cysteine ​​to MRS solid medium or MRS liquid medium and the culture under anaerobic conditions at 37°C. The other steps are the same.

[0132] The preparation methods of the simulated gastric fluid and simulated intestinal fluid involved in the following embodiments are as follows: Simulated gastric fluid: adjust the pH of saline to 3.0 with HCl; add pepsin (1:10000) to sterile saline (0.9% w / v) with a pH of 3.0 to a final concentration of 3 g / L; filter with a 0.22 μm sterile filter membrane and prepare for use.

[0133] Simulated intestinal fluid: adjust the pH of saline to 8.0 with NaOH; dissolve trypsin (1:250) in sterile saline (0.9% w / v) with a pH of 8.0 to a final concentration of 1 g / L; add bile salts to a concentration of 0.3% w / v; filter with a 0.22 micron (μm) sterile filter membrane and prepare for use immediately.

[0134] The methods for HT-29 cell recovery, passaging, plating and terminating culture involved in the following examples are as follows: Cell recovery: Preheat DMEM complete medium (with fetal bovine serum and double antibody) in a 37℃ water bath, take out the frozen cells and quickly thaw them in a 37℃ water bath, transfer all the liquid in the cryopreservation tube into a 15mL centrifuge tube containing 5mL complete medium, and centrifuge (1000 r / min, 5min); after centrifugation, pour out the culture medium, add 1mL complete medium to resuspend, transfer to a culture dish with 9mL complete medium, and culture in a 37℃ 5% CO2 incubator, changing the culture medium every other day.

[0135] Cell passaging: When the cells have grown to 80% of the culture dish, wash the cell surface with PBS twice; then add 1 mL of trypsin for 2 min. After digestion is complete (the cell morphology shrinks and can be blown off the dish wall), stop digestion with 5 mL of complete culture medium; transfer the cell suspension to a 15 mL centrifuge tube, centrifuge (1000 r / min, 5 min), discard the supernatant, add 2 mL of complete culture medium and blow to mix the cells; respectively aspirate 1 mL of cell suspension and 9 mL of complete culture medium and add them to 2 culture dishes, remove bubbles, mix well, and culture in a 37°C 5% CO2 incubator.

[0136] Cell plating: When the number of cells grows to 10 6Repeat the cell passaging steps, wash the culture dish with PBS, digest with trypsin, terminate digestion, centrifuge and discard the supernatant, then add 12 mL of complete medium to resuspend; add 500 microliters (µL) of cell suspension and 1.5 mL of complete medium to each well of the 12-well plate, mix well, and culture in a 37℃ 5% CO2 incubator.

[0137] Termination of cell co-culture: Wash the 12-well plate monolayer cells three times with PBS, add 250 μL trypsin-EDTA to each well and place in a 5% CO2 incubator for 10 min; add 250 μL complete culture medium to terminate digestion.

[0138] Example 1: Staphylococcus epidermidis ( Staphylococcus epidermidis ) Screening, strain identification and cultivation of FeiHeS06 The specific steps are as follows: 1. Collection of breast milk samples Healthy breastfeeding women from Wuxi City, Jiangsu Province were recruited according to the following conditions: (1) 25-35 years old; (2) natural childbirth; (3) infants were 1-6 months old when breast milk was collected; (4) full-term childbirth, healthy infants, and no hospitalization experience in the neonatal period; (5) self-assessed good health and not taking any medications; (6) no gestational diabetes, gestational hypertension or other diseases during pregnancy.

[0139] Breast milk collection: The mother washes her hands, puts on disposable latex gloves, and wipes the breast on the collection side with a wet wipe. The first 2 mL of milk is discarded and not collected. After that, the milk is directly collected and put into the sampling tube. The sampling tube is pre-filled with sterile 60% v / v glycerol. The volume ratio of milk to glycerol is 1:1. The final glycerol concentration in the sampling tube is 30% v / v. Then, the lid is tightly closed, the sealing film is sealed, and the name and collection time are marked. The breast milk sample is placed on ice, transported to the laboratory within 4 hours, and stored in a -80℃ refrigerator.

[0140] 2. Staphylococcus epidermidis ( Staphylococcus epidermidis ) Acquisition of FeiHeS06 1. Screening The breast milk sample obtained in step 1 was inoculated with 20% inoculum in MRS liquid culture medium for pre-culture for 48 h. After pre-culture, 0.5 mL of the sample was mixed and added to 4.5 mL of sterile saline for gradient dilution. Then 100 μL of the dilution gradient of 10 was taken. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The dilution was plated on MRS solid medium with pH = 7.0, cultured at 37°C for 48 h, and the colony morphology was observed and recorded; Pick colonies of different morphologies on the MRS solid medium for streaking separation. After culturing at 37°C for 48 h, pick single colonies of different morphologies on the MRS solid medium for streaking separation again until pure single colonies with consistent morphology are obtained. The pure colonies on the MRS solid medium were picked and inoculated into 5 mL of MRS liquid medium and cultured at 37°C for 18 h; Take 1 mL of bacterial solution in a sterile centrifuge tube, centrifuge at 5000 r / min for 15 min, discard the upper culture medium, resuspend the bacterial mud in 30% glycerol solution and store it at -80℃ to obtain the strain.

[0141] 2. Identification The isolated strains were amplified by 16S rDNA PCR, and the PCR products were sent to GeneWeichi (Suzhou) Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the nucleic acid sequence in NCBI standard Nucleotide BLAST. The result was Staphylococcus epidermidis, named Staphylococcus epidermidis FeiHeS06. The sequencing results of the 16SrDNA of the strain were: During the same period, three breast milk-derived strains were screened and named: Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, and Bifidobacterium longum subsp. infantis MRJSWX29M3.

[0142] 3. Cultivation The above-mentioned Staphylococcus epidermidis FeiHeS06 was inoculated into MRS solid medium and cultured at 37°C for 48 h, and then its colonies were observed. It was found that its colonies were grayish white round protrusions with a smooth, flat, opaque surface and a diameter of 1-2 mm (see Figure 1 ). The pH of the blank culture medium and the culture medium with bacteria was measured by a pH meter before and after cultivation, and it was found that Staphylococcus epidermidis FeiHeS06 produced acid during the cultivation process. Staphylococcus epidermidis FeiHeS06 was inoculated into MRS liquid culture medium, placed in an ELISA instrument, and cultured at 37°C for 48 h. During the cultivation process, the OD of the culture medium was measured every 1 h. 600 It was found that Staphylococcus epidermidis FeiHeS06 entered the logarithmic growth phase after 5-6 h of culture and reached the stable growth phase after 12-13 h.

[0143] Example 2: Staphylococcus epidermidis ( Staphylococcus epidermidis )Tolerance of FeiHeS06 in simulated gastric fluid and simulated intestinal fluid The specific steps are as follows: (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis MRJSWX29M3, and Bifidobacterium animalis lactis BB12 were streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. Activation was repeated for two generations to obtain activation solution.

[0144] Bifidobacterium needs to be cultured in MRS solid medium or MRS liquid medium with 0.5 g / L L-cysteine ​​added and placed under anaerobic conditions at 37°C. Other steps are the same.

[0145] (2) The activation solution was inoculated at 2% v / v in 5 mL of MRS liquid medium (Bifidobacteria were inoculated in MRS liquid medium supplemented with 0.5 g / L L-cysteine) and cultured until the stable phase. The cells were collected in two centrifuge tubes by centrifugation at 5000 g for 10 min, the supernatant was discarded, and the cells were washed twice with saline solution, centrifuged, and the supernatant was discarded. The cells in one centrifuge tube were resuspended in simulated gastric fluid and vortexed to mix evenly. The cells in the other centrifuge tube were resuspended in an equal volume of saline solution. After culturing at 37°C for 3 h, the live bacteria were counted on the plate, and two replicates were made for each dilution. The experimental results of the two treatment methods were compared and analyzed. The colony count after simulated gastric juice treatment decreased by less than 3 logarithmically compared with that of untreated bacteria, indicating that the bacteria had good tolerance to gastric juice (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.).

[0146] (3) The activated liquid was inoculated at a volume of 2% v / v in 5 mL of MRS liquid culture medium (Bifidobacteria were inoculated in MRS liquid culture medium supplemented with 0.5 g / L L-cysteine) and cultured until the stable phase. The cells were collected in two centrifuge tubes by centrifugation at 5000 g for 10 min, the supernatant was discarded, the cells were washed twice with physiological saline, centrifuged, the supernatant was discarded, the cells in one centrifuge tube were resuspended in simulated intestinal fluid, vortexed to mix evenly, and the cells in the other centrifuge tube were resuspended in an equal volume of physiological saline. After culturing at 37°C for 4 h, the live bacteria were counted on the plate, and two parallels were made for each dilution. The experimental results of the two treatments were compared and analyzed. The colony count after the simulated intestinal fluid treatment decreased by less than 3 logarithmically compared with the untreated cells, and the live bacteria count was still above 10. 6 CFU / mL (colony-forming units per Milliliter) or above indicates good tolerance to intestinal fluid.

[0147] The results showed that the tolerance of the five strains to simulated gastric fluid was as follows: Figure 2A As shown in the figure, the number of viable bacteria of the five bacterial strains that were not treated with simulated gastric fluid reached 10 8 ~10 9CFU / mL, there were differences among different species and strains. The CFU / mL of Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 were 2.20×10 8 CFU / mL, 7.35×10 8 CFU / mL, 2.35×10 8 CFU / mL, 4.65×10 8 CFU / mL, 1.35×10 9 CFU / mL; after 3 h of treatment with simulated gastric juice, the number of viable bacteria decreased to a certain extent, but the logarithm of the colony count after treatment with simulated gastric juice was less than 3 compared with that of the untreated strains, indicating that the five strains of bacteria had good tolerance to gastric juice.

[0148] Tolerance of 5 strains of bacteria to simulated intestinal fluid Figure 2B As shown in the figure, the number of viable bacteria of the five bacterial strains that were not treated with simulated intestinal fluid reached 10 8 ~10 9 CFU / mL; after being treated with simulated intestinal fluid for 4 h, the intestinal fluid tolerance of different species and strains was significantly different; the logarithmic decrease values ​​of the viable counts of Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis subsp. MRJSWX29M3, and Bifidobacterium animalis lactis subsp. BB12 were 0.46, 2.37, 2.67, 2.17, and 2.67, respectively; this result shows that Staphylococcus epidermidis FeiHeS06 has the best tolerance to intestinal fluid and still maintains 10 8 CFU / mL of viable bacteria; while the viable bacteria counts of Staphylococcus epidermidis MRJSWX39M8, Bifidobacterium longum infantis subsp. MRJSWX29M3, and Bifidobacterium animalis lactis subsp. BB12 were reduced to 10 6 CFU / mL; Lactobacillus gasseri MRJSWX2L1 had the worst tolerance to simulated intestinal fluid, with the number of live bacteria ranging from 10 8 CFU / mL decreased to 10 5 CFU / mL.

[0149] Example 3: Staphylococcus epidermidis ( Staphylococcus epidermidis ) Colonization potential of FeiHeS06 (generation time, intestinal epithelial cell adhesion ability, oligosaccharide utilization ability) The specific steps are as follows: (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis MRJSWX29M3, and Bifidobacterium animalis lactis BB12 were streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. Activation was repeated for two generations to obtain activation solution.

[0150] Bifidobacterium needs to be cultured in MRS solid medium or MRS liquid medium with 0.5 g / L L-cysteine ​​added and placed under anaerobic conditions at 37°C. Other steps are the same.

[0151] (2) Generation time determination: Take 1 mL of activation solution in a sterile centrifuge tube, centrifuge at 5000 r / min for 15 min, discard the upper culture medium to obtain bacterial sludge, resuspend the bacterial sludge with 1 mL of sterile saline, and then inoculate it into MRS liquid culture medium at a 2% (v / v) inoculum. Place it in an ELISA reader and culture it at 37°C for 48 h. During the culture process, measure the OD value of the culture medium every 1 hour. 600 According to the measured OD 600 The growth curve of the strain under certain conditions can be drawn by plotting the value and the corresponding culture time. Bifidobacterium needs to be cultured in MRS liquid medium with 0.5 g / L L-cysteine ​​added and placed under anaerobic conditions at 37°C. The other steps are the same.

[0152] (3) Determination of intestinal epithelial cell adhesion ability: The activation solution was inoculated into MRS liquid culture medium at a rate of 2% (v / v), cultured at 37°C for 24 h, and the cells were collected by centrifugation and resuspended in PBS to obtain a certain concentration (10 8CFU / mL); 980 μL of bacterial solution was drawn into a brown centrifuge tube, and 20 μL of cFDA-SE stock solution (fluorescent probe) was added to make the final concentration of 20 μmol / L, and the tube was kept at 37℃ in the dark for 15 min, centrifuged (8000r / min, 4℃, 10 min), and then washed 3 times with PBS to remove excess fluorescent dye, and then the bacteria were resuspended in 1 mL of DMEM basal culture medium; HT-29 cells (purchased from the National Model and Characteristic Experimental Cell Resource Bank, Catalog No.: TCHu103) were revived, passaged and plated in advance; 12-well cell plate monolayer HT-29 cells were washed twice with PBS, 1 mL of DMEM basal culture medium and 1 mL of bacterial suspension were added to each well, and co-cultured in the dark for 4 h; at the same time, 1 mL of bacterial suspension resuspended in DMEM basal culture medium with fluorescent dye was prepared and added to a brown centrifuge tube, and placed in a cell culture incubator in the dark for 4 h as a control; after terminating the co-culture, the solution in each well was collected by blowing into a brown centrifuge tube and centrifuged (1000 The cells were centrifuged at 400 rpm for 5 min and the supernatant was discarded. The cells were resuspended in DMEM basal medium. The control group was also centrifuged and the supernatant was discarded. The cells were resuspended in DMEM basal medium. 200 μL of the suspension of the control group and the co-culture group was pipetted into a 96-well plate and the fluorescence OD was measured. 488 The adhesion rate of the strain to intestinal epithelial cells was calculated.

[0153] (4) Determination of oligosaccharide utilization ability: replace the glucose in the MRS medium with an equal amount of fructo-oligosaccharide or galacto-oligosaccharide, and add bromocresol purple as an indicator (0.0075% w / v); first add 200 μL of MRS liquid medium with fructo-oligosaccharide or galacto-oligosaccharide as the carbon source to a 96-well plate; then add 2 μL of the strain activation solution that was centrifuged and discarded with normal saline; culture for 24 h, and observe and record the color change of the culture medium every 2 h; the bacterial solution + ordinary MRS medium with bromocresol purple was the positive control, the medium without bacterial solution was the negative control 1, and the bacterial solution + MRS medium without carbon source with bromocresol purple was the negative control 2.

[0154] The results showed that the generation time of the five strains was as follows: Figure 3 As shown; where generation time refers to the time required for a cell to divide once; the shorter the generation time, the faster the growth rate; the generation time of each strain in the logarithmic phase can reflect the speed of its growth rate, and the formula for logarithmic generation time is as follows: generation time = (t2-t1) / 3.322 (OD2-OD1); where t2 is the time at the end of the logarithmic growth phase, t1 is the time just entering the logarithmic growth phase, and OD2 and OD1 are the absorbance values ​​of the bacterial suspension measured at t2 and t1.

[0155] Depend on Figure 3It can be seen that there are differences in the generation time of the five bacterial strains in the logarithmic growth phase. The generation time of Staphylococcus epidermidis FeiHeS06 is shorter than that of Lactobacillus gasseri MRJSWX2L1, and is comparable to that of Staphylococcus epidermidis MRJSWX39M8, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12.

[0156] The adhesion ability of the five bacterial strains to HT-29 cells Figure 4 As shown in the figure, the adhesion rate of Staphylococcus epidermidis FeiHeS06 was the highest, reaching 28.69%, followed by Lactobacillus gasseri MRJSWX2L1 at 22.86%, Staphylococcus epidermidis MRJSWX39M8 at 8.42%, Bifidobacterium longum subsp. infantis MRJSWX29M3 at 6.72%, and Bifidobacterium animalis subsp. lactis BB12 at 5.72%; the adhesion ability of Staphylococcus epidermidis FeiHeS06 was about 3.5 times higher than that of Staphylococcus epidermidis MRJSWX39M8 of the same strain, and 4 to 6 times higher than that of the two strains of Bifidobacterium. This result shows that Staphylococcus epidermidis FeiHeS06 has good adhesion ability to intestinal epithelial cells and has the potential to successfully colonize after entering the host intestine.

[0157] The utilization capacity of the five strains of fructo-oligosaccharides and galacto-oligosaccharides is shown in Table 1. The metabolic process of these five strains will produce acid, and when using fructo-oligosaccharides or galacto-oligosaccharides for growth, the pH of the culture medium system will decrease; based on this characteristic, bromocresol purple can be used as an indicator (color change range pH 5.2-6.8), which will change from purple to yellow under acidic conditions; by observing the color change of the culture medium around the colony, it can be determined whether the strain has utilized the target carbon source (fructo-oligosaccharides or galacto-oligosaccharides) for metabolism.

[0158] Table 1: Utilization ability of 5 bacterial strains on oligofructose and oligogalactose

[0159] Note: If Figure 11 As shown, the colors of bromocresol purple indicator corresponding to the number of "+" are, from left to right: ++++, +++, ++, +, control; the more "+" there are, the stronger the strain's ability to utilize this sugar.

[0160] As shown in Table 1, all five strains can utilize oligofructose and galacto-oligosaccharide, among which the utilization ability of Staphylococcus epidermidis FeiHeS06 for oligofructose is better than that of Lactobacillus gasseri MRJSWX2L1. The ability to utilize oligosaccharides can improve the colonization ability of strains in the intestine, and also provide formulation ideas for the application of synbiotic preparations of oligosaccharides and probiotic strains.

[0161] Example 4: Staphylococcus epidermidis ( Staphylococcus epidermidis ) Antibiotic resistance and potential pathogenicity of FeiHeS06 The specific steps are as follows: (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis MRJSWX29M3, and Bifidobacterium animalis lactis BB12 were streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. Activation was repeated for two generations to obtain activation solution.

[0162] Bifidobacterium needs to be cultured in MRS solid medium or MRS liquid medium with 0.5 g / L L-cysteine ​​added and placed under anaerobic conditions at 37°C. Other steps are the same.

[0163] (2) Antibiotic resistance determination (minimum inhibitory concentration (MIC) experiment): 14 commonly used antibiotics were selected and first dissolved in a small amount of water or anhydrous ethanol or methanol as a solvent. After sterilization through a 0.22 μm filter membrane, the stock solution was fixed to volume with sterile water for use. Before the experiment, the antibiotics were diluted with sterile water according to the effective concentration range of different antibiotics (as shown in Table 2). Different concentrations of antibiotic solutions were added to a sterile 96-well plate, 100 μL per well (since 100 μL of bacterial solution and antibiotic solution were added to the 96-well plate, in order to make the antibiotics in the well plate reach an effective concentration, the antibiotic concentration in each well should be doubled when the antibiotics were diluted). The bacterial suspension diluted with MRS liquid culture medium (10 6 CFU / mL) were also added to a sterile 96-well plate, 100 μL per well, and cultured at 37°C for 48 h; then the OD value was measured using an ELISA reader to check whether the strain grew. The last column of the 96-well plate was a positive control, with no antibiotics, only bacterial suspension and sterile water, and the first column was a negative control, with no antibiotics or strains, only MRS liquid culture medium and sterile water.

[0164] Table 2: Antibiotic names and dilution concentration ranges

[0165] (3) Analysis of potential pathogenicity or drug-resistant gene transferability: Staphylococcus epidermidis FeiHeS06 cells were collected and sent to Shanghai Meiji Biotechnology Co., Ltd. for whole genome sequencing. Based on the genomic data obtained by sequencing, the number and distribution of potential pathogenic genes (virulence genes, secretion system, pathogen-host interaction) and prophages were predicted and analyzed.

[0166] The results showed that the minimum inhibitory concentration (MIC) of the five strains to 14 common antibiotics was as follows: Figure 5As shown in the figure, the mechanism of antibiotic inhibition mainly includes hindering the synthesis of bacterial cell walls, inhibiting protein synthesis, affecting nucleic acid metabolism, affecting folic acid metabolism, etc., making them lose their normal growth and reproduction ability and achieving the effect of inhibition or killing. MIC refers to the lowest drug concentration of chemical or biological agents that inhibits the growth of bacteria in the culture medium. It is the lowest antibiotic concentration at which no visible growth can be observed at a specific culture temperature and time when conducting a continuous concentration gradient dilution drug sensitivity test according to standard operating procedures.

[0167] Depend on Figure 5 It can be seen that the MIC values ​​of the five strains to 14 common antibiotics vary greatly. The MIC values ​​of Staphylococcus epidermidis FeiHeS06 to kanamycin and rifampicin are the smallest among the five strains, which are 256 micrograms / ml (μg / mL) and 1 μg / mL, respectively, showing the strongest sensitivity; the MIC values ​​of kanamycin in Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis subsp. MRJSWX29M3, and Bifidobacterium animalis lactis subsp. BB12 are 1024 μg / mL, >1024 μg / mL, 1024μg / mL, and >1024 μg / mL, respectively, which are 4 times or more than that of Staphylococcus epidermidis FeiHeS06; the MIC values ​​of rifampicin in Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis subsp. MRJSWX29M3, and Bifidobacterium animalis lactis subsp. BB12 are 8 μg / mL, >64 μg / mL, 16 μg / mL, >64 μg / mL, which were 8 to 64 times or more than that of Staphylococcus epidermidis FeiHeS06; among 12 of the 14 antibiotics, Staphylococcus epidermidis FeiHeS06 did not show the maximum MIC value, indicating that its sensitivity to most of the tested antibiotics was in the middle range of the five strains.

[0168] Substances derived from microorganisms that promote microbial infection and specific host diseases are called virulent factors. By comparing the Virulence Factor Database (VFDB), the annotation profile of the virulence factor genes of Staphylococcus epidermidis FeiHeS06 was obtained and statistically analyzed. As shown in Table 3, Staphylococcus epidermidis FeiHeS06 has 24 (≥3) homologous sequences of known virulence factors, which is similar to other probiotic strains analyzed in previous studies. Most of the genes encode proteins that are mainly involved in the synthesis of capsular polysaccharides (capsule), metal ion transport (heme uptake ABC transport system (HitABC)) and carbohydrate degradation (lipo-oligosaccharide (LOS)). These genes are different from the most important virulence factors of Staphylococcus epidermidis reported so far (PIA, Bap / Bhp, PGA, Clpxp, etc.).

[0169] The contents of the pathogen-host interaction database (PHI) have been experimentally verified and are mainly derived from fungal, oomycete and bacterial pathogens, and the infected hosts include animals, plants, fungi and insects. By comparing the PHI database, the information of pathogen-host interaction-related genes contained in the genome of Staphylococcus epidermidis FeiHeS06 was obtained. As shown in Table 4, the pathogen-host interaction-related genes (only those related to primates were analyzed) in the genome of Staphylococcus epidermidis FeiHeS06 are mainly related to the phenotypes with reduced virulence or no effect on pathogenicity.

[0170] Secretion systems are often found in bacterial genomes, which are used to discharge or even transport DNA, proteins or DNA / protein complexes to the extracellular space or even to the host cell interior. Secretion systems are often key structures for pathogens to exert virulence. The genome of Staphylococcus epidermidis FeiHeS06 contains 12 genes encoding secretion systems (including yidC 2、 secE , secY 2、 secA 2、 secG , SecDF , yajC , ftsY and ffh ), all belong to the Sec-SRP type secretion system. The Sec-SRP type secretion system itself is not toxic. This system is a protein transport mechanism widely present in bacteria. Its main function is to guide and transport newly synthesized proteins from the cytoplasm to specific parts of the cell membrane, extracellular or intracellular. This process is crucial for the normal function and structure of the cell.

[0171] The phage genome integrated into the bacterial genome is called a prophage. There are often some functional genes on the prophage sequence, such as antibiotic resistance genes, virulence genes, etc., which enhance the adaptability of bacteria to the environment or make bacteria pathogenic. The prophage prediction of the FeiHeS06 genome of Staphylococcus epidermidis using Phage_Finder software was found to be absent in the prophage sequence, indicating that the risk of horizontal transfer of antibiotic resistance genes, virulence genes and other genes is low and the safety is relatively high.

[0172] In general, the virulence-related genes carried by Staphylococcus epidermidis FeiHeS06 are mainly involved in metabolism, synthesis of extracellular components and adhesion, and there are no significant toxin-producing genes. Genes related to host interaction are mainly related to phenotypes with reduced virulence or no effect on pathogenicity, and only have the common non-toxic Sec-SRP type secretion system. Combined with the antibiotic sensitivity (MIC) experiment, it can be seen that Staphylococcus epidermidis FeiHeS06 has low resistance to antibiotics. In addition, Staphylococcus epidermidis FeiHeS06 does not have prophages, and the risk of resistance gene transfer is low. These analysis results show that Staphylococcus epidermidis FeiHeS06 is safe.

[0173] Table 3: Prediction analysis of virulence factor-related genes in the genome of Staphylococcus epidermidis FeiHeS06

[0174] Table 4: Prediction analysis of genes related to pathogen-host interaction in the genome of Staphylococcus epidermidis FeiHeS06

[0175] Example 5: Staphylococcus epidermidis ( Staphylococcus epidermidis )Stability of FeiHeS06 (growth rate, maximum biomass, osmotic pressure tolerance) The specific steps are as follows: (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis MRJSWX29M3, and Bifidobacterium animalis lactis BB12 were streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. Activation was repeated for two generations to obtain activation solution.

[0176] Bifidobacterium needs to be cultured in MRS solid medium or MRS liquid medium with 0.5 g / L L-cysteine ​​added and placed under anaerobic conditions at 37°C. Other steps are the same.

[0177] (2) Growth curve determination: Take 1 mL of activation solution in a sterile centrifuge tube, centrifuge at 5000 r / min for 15 min, discard the upper culture medium to obtain bacterial sludge, resuspend the bacterial sludge with 1 mL of sterile saline, and then inoculate it into MRS liquid culture medium at a 2% (v / v) inoculation volume. Place it in an ELISA reader and culture it at 37°C for 48 h. During the culture process, measure the OD value of the culture medium every 1 hour. 600 According to the measured OD 600By plotting the value against the corresponding culture time, the growth curve of the strain under certain conditions (lag phase, logarithmic growth phase, stable phase and decay phase) can be drawn. Bifidobacterium needs to be added with 0.5 g / L L-cysteine ​​in MRS liquid medium and cultured under anaerobic conditions at 37°C. The other steps are the same.

[0178] (3) Maximum biomass: Inoculate the activated solution at a 2% (v / v) inoculum into 1 L of MRS liquid culture medium and culture at 37°C for 18 h until the growth stationary phase to obtain a bacterial solution. Centrifuge the bacterial solution at 5000 r / min for 15 min, discard the upper culture medium, obtain the bacterial slurry, and weigh it.

[0179] The culture of bifidobacteria required the addition of 0.5 g / L L-cysteine ​​to MRS liquid medium and culture at 37°C under anaerobic conditions. The other steps were the same.

[0180] (4) Osmotic pressure tolerance test: NaCl was added to the MRS liquid culture medium to make the initial osmotic pressure of the culture medium 350 (MRS control), 600, 700, 800, 900, 1200, 1500, 2400, 2700, 3000, and 3300 milliosmole (mOsm) / kg, respectively, and the pH of the culture medium was adjusted to 7.0; the activation solution was inoculated at a 2% (v / v) inoculation rate into the MRS liquid culture medium under different osmotic pressure conditions, and cultured at 37°C for 18 h. The OD of the culture medium was measured every 2 hours. 600 ; Draw a growth curve and determine the osmotic pressure at which the growth rate begins to be inhibited and the osmotic pressure at which growth is completely inhibited.

[0181] The culture of bifidobacteria required the addition of 0.5 g / L L-cysteine ​​to MRS liquid medium and culture at 37°C under anaerobic conditions. The other steps were the same.

[0182] The results showed that the growth curves of the five strains were as follows: Figure 6 As shown in the figure. Staphylococcus epidermidis FeiHeS06 entered the logarithmic growth phase at around 5-6 h and reached the growth stable phase at 12-13 h. Its growth pattern was similar to that of Staphylococcus epidermidis MRJSWX39M8, Bifidobacterium longum infantis subsp. MRJSWX29M3, and Bifidobacterium animalis lactis subsp. BB12. However, the OD of Staphylococcus epidermidis FeiHeS06 was 600 The absorbance value is greater than that of Bifidobacterium animalis subsp. lactis BB12 and Lactobacillus gasseri MRJSWX2L1, and the number of bacteria is greater.

[0183] Depend on Figure 7It can be seen that when reaching the stable period, the bacterial sludge volume of Staphylococcus epidermidis FeiHeS06 is equivalent to that of Lactobacillus gasseri MRJSWX2L1 and Bifidobacterium animalis subsp. lactis BB12, reaching a bacterial sludge volume of more than 5 g / L culture medium.

[0184] In addition, by Figures 8A to 8E It can be seen that the osmotic pressure was 1500 mOsm / kg when the growth rate of Staphylococcus epidermidis FeiHeS06 began to be inhibited, and the osmotic pressure was 2400 mOsm / kg when the growth rate was completely inhibited; the osmotic pressures of Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis subspecies MRJSWX29M3, and Bifidobacterium animalis lactis subspecies BB12 began to be inhibited, and were 900 mOsm / kg, 900 mOsm / kg, 1500 mOsm / kg, and 1500 mOsm / kg, respectively; the osmotic pressures of Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis subspecies MRJSWX29M3, and Bifidobacterium animalis lactis subspecies BB12 were 1500 mOsm / kg, 1500 mOsm / kg, and 2400 mOsm / kg, respectively. mOsm / kg, 2400 mOsm / kg; the osmotic pressure tolerance of Staphylococcus epidermidis FeiHeS06 was comparable to that of Bifidobacterium longum infantis subspecies MRJSWX29M3 and Bifidobacterium animalis lactis subspecies BB12, and was significantly superior to that of Staphylococcus epidermidis MRJSWX39M8 of the same species.

[0185] Example 6: Staphylococcus epidermidis ( Staphylococcus epidermidis )Acid production and pathogen antagonism of FeiHeS06 The specific steps are as follows: (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis MRJSWX29M3, and Bifidobacterium animalis lactis BB12 were streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. Activation was repeated for two generations to obtain activation solution.

[0186] Bifidobacterium needs to be cultured in MRS solid medium or MRS liquid medium with 0.5 g / L L-cysteine ​​added and placed under anaerobic conditions at 37°C. Other steps are the same.

[0187] (2) Acid production capacity: Inoculate the activation solution into MRS liquid culture medium at a rate of 2% (v / v), and culture at 37°C for 18 h until the growth stationary phase; measure the initial pH value and the pH value during the stationary phase of the culture medium.

[0188] (3) Antagonism against pathogenic bacteria: Activate pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Salmonella, Listeria monocytogenes) and subculture them twice for later use; inoculate the activated solution of probiotic strains into MRS liquid culture medium at a 2% (v / v) inoculation volume, culture to the stable phase, pipette the bacterial solution into a centrifuge tube, centrifuge at 8000 r / min for 20 min, collect the supernatant and filter it through a 0.22 μm filter membrane to obtain the cell-free supernatant for later use; pipette 100 μL of pathogenic bacteria onto an MRS solid plate and spread it evenly until there is no water mark, use a hole puncher to punch 5 wells on the plate, add MRS culture medium to the middle well as a blank control, and add 100 μL of the cell-free supernatant of the four probiotic strains to the remaining four wells; incubate the plates upright for 48 h, take out the plates, observe the inhibition zone and measure.

[0189] The results showed that the pH changes during the growth of the five bacterial strains were as follows: Figure 9 All five strains can produce acid, and the pH of the culture medium decreases from the initial 7.0 to 3.5-5.0; the end point pH of the culture medium of Staphylococcus epidermidis FeiHeS06 is 4.79, and its acid production ability is not as good as that of Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis subsp. MRJSWX29M3, and Bifidobacterium animalis lactis subsp. BB12 (the end point pH of the culture medium of the three strains is 3.7-3.8), which may be because Bifidobacterium and Lactobacillus belong to the category of lactic acid bacteria and generally have strong acid production ability; the organic acids produced by the strains entering the host intestine can reduce the pH of the intestinal microecological environment, inhibit the growth of pathogenic bacteria, and regulate the intestinal flora.

[0190] As can be seen from Table 5, there are great differences in the inhibitory ability of probiotics on the growth of pathogenic bacteria; Staphylococcus epidermidis FeiHeS06 has the strongest antibacterial ability and can inhibit the growth of three pathogenic bacteria: Staphylococcus aureus, Escherichia coli and Listeria monocytogenes; Staphylococcus epidermidis MRJSWX39M8 of the same strain has no inhibitory effect on the growth of the four pathogenic bacteria tested; Lactobacillus gasseri MRJSWX2L1 can inhibit the growth of two pathogenic bacteria, Salmonella and Listeria monocytogenes, and Bifidobacterium longum subspecies infantis MRJSWX29M3 can inhibit the growth of two pathogenic bacteria, Salmonella and Escherichia coli.

[0191] Table 5: Inhibitory ability of the five bacterial strains against pathogenic bacteria (Staphylococcus aureus, Salmonella, Escherichia coli and Listeria monocytogenes)

[0192] Note: " / " indicates that the strain has no antagonistic effect on the growth of the pathogen. The data in the table are the size of the inhibition zone of the probiotic strain against the growth of pathogenic bacteria, in mm.

[0193] Example 7: Staphylococcus epidermidis ( Staphylococcus epidermidis)Regulatory effect of FeiHeS06 on inflammatory response of RAW264.7 macrophages induced by lipopolysaccharide (LPS) The specific steps are as follows: (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis MRJSWX29M3, and Bifidobacterium animalis lactis BB12 were streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. Activation was repeated for two generations to obtain activation solution.

[0194] Bifidobacterium needs to be cultured in MRS solid medium or MRS liquid medium with 0.5 g / L L-cysteine ​​added and placed under anaerobic conditions at 37°C. Other steps are the same.

[0195] (2) RAW264.7 cells were cultured in DMEM complete medium in a 37°C 5% CO2 incubator. RAW264.7 cells adhered to the wall and their morphology was observed under an inverted microscope. The medium was changed every day. When the density of adherent cells reached 70-80%, they were passaged at a ratio of 1:3. The strain activation solution was centrifuged at 10000 r / min for 15 min to obtain the supernatant; In the experiment, the cells were digested with trypsin and then 5 Cell number RAW264.7 cells were seeded in 96-well plates (200 µL of culture medium was added to each well), and the cells were stimulated with 100 ng / mL (ng / mL) LPS and cultured for 24 h to construct a cell inflammation model; The probiotic intervention group also had a system of 200 µL per well (160 µL culture medium + 40 µL strain supernatant). The cells were also stimulated with 100 ng / mL LPS and co-cultured for 24 h to evaluate the regulatory effect of probiotics on LPS-induced inflammatory response of RAW264.7 cells. The cell culture medium was obtained by centrifugation, and the levels of IL-10, TNF-α and NO were determined according to the instructions of the kit. The levels of cytokines IL-10 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA). The total amount of inflammatory mediator NO was obtained by conversion of nitrite determination using Griess reagent based on the principle that nitrate reductase reduces nitrate to nitrite. The kits used for the determination of cytokine IL-10 and TNF-α levels were purchased from Shanghai ELISA Biotechnology Co., Ltd., and the kit used for the determination of total NO was purchased from Beyotime Biotechnology Co., Ltd.

[0196] The results showed that the ability of the five bacterial strains to regulate LPS-induced inflammatory response in macrophages RAW264.7 cells was Figures 10A - 10C As shown. LPS stimulated macrophages to produce an inflammatory response. Among the five bacterial strains, the level of anti-inflammatory factor IL-10 produced by macrophages under the intervention of Staphylococcus epidermidis FeiHeS06 was the highest, with a concentration of 224.81 pg / mL in the cell supernatant, showing good anti-inflammatory ability ( Figure 10A ). Compared with the LPS modeling group, intervention with Staphylococcus epidermidis FeiHeS06, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis subsp. MRJSWX29M3, and Bifidobacterium animalis lactis subsp. BB12 significantly reduced the level of inflammatory factor TNF-α secreted by macrophages, while Staphylococcus epidermidis MRJSWX39M8 had no such effect ( Figure 10B In addition, Staphylococcus epidermidis FeiHeS06 and Bifidobacterium animalis subsp. lactis BB12 significantly reduced the level of inflammatory mediator NO secreted by macrophages ( Figure 10C ). Macrophages are important immune effector cells differentiated from monocytes, which play a key role in innate and acquired immune responses. This result shows that Staphylococcus epidermidis FeiHeS06 has good immunomodulatory activity.

[0197] Example 8: Staphylococcus epidermidis ( Staphylococcus epidermidis )Application of FeiHeS06 Staphylococcus epidermidis FeiHeS06 can be used to prepare bacterial powder. The specific preparation process of bacterial powder is as follows: Staphylococcus epidermidis FeiHeS06 was streaked on MRS solid medium and cultured at 37°C for 48 h to obtain a single colony; a single colony was picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation, and activated for two generations to obtain an activated solution; the activated solution was inoculated into the medium at an inoculum volume of 2% (v / v), and cultured 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 a bacterial sludge; the bacterial sludge was washed three times with physiological saline and resuspended with a lyophilization protective agent (the mass ratio of lyophilization protective agent to bacterial body was 2:1) to a concentration of 1×10 10 CFU / mL, and obtain a bacterial suspension; the bacterial suspension was incubated at a temperature of 37°C for 60 minutes and then freeze-dried to obtain Staphylococcus epidermidis FeiHeS06 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 is adjusted to 7.0 to obtain the culture medium; The ingredients of the freeze-drying protective agent include: 130 g / L skim milk powder aqueous solution.

[0198] Example 9: Staphylococcus epidermidis ( Staphylococcus epidermidis )Application of FeiHeS06 Staphylococcus epidermidis FeiHeS06 can be used to prepare capsule products. The specific preparation process of capsule products is as follows: Staphylococcus epidermidis FeiHeS06 was streaked on MRS solid medium and cultured at 37°C for 48 h to obtain a single colony; a single colony was picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation, and activated for two generations to obtain an activated solution; the activated solution was inoculated into the medium at an inoculum volume of 2% (v / v), and cultured 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 a bacterial sludge; the bacterial sludge was washed three times with physiological saline and resuspended with a lyophilization protective agent (the mass ratio of lyophilization protective agent to bacterial body was 2:1) to a concentration of 1×10 10 CFU / mL, and a bacterial suspension was obtained; the bacterial suspension was added to a 30 g / L sodium alginate solution to a concentration of 2×10 9 CFU / mL, stirring sufficiently to make the cells of Staphylococcus epidermidis FeiHeS06 evenly dispersed in the sodium alginate solution to obtain a mixed solution; squeezing the mixed solution into a calcium chloride solution with a concentration of 20 g / L to form colloid particles; after the formed colloid particles were statically solidified for 30 minutes, filtering and collecting the colloid particles; freeze-drying the collected colloid particles for 48 hours to obtain a powder; filling the powder into a medicinal capsule to obtain a capsule product; 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 is adjusted to 6.8 to obtain the culture medium.

[0199] The ingredients of the freeze-drying protective agent include: 130 g / L skim milk powder aqueous solution.

[0200] Example 10: Staphylococcus epidermidis ( Staphylococcus epidermidis )Application of FeiHeS06 Staphylococcus epidermidis FeiHeS06 can be used to prepare fermented milk. The specific preparation process of fermented milk is as follows: Staphylococcus epidermidis FeiHeS06 was streaked on MRS solid medium and cultured at 37°C for 48 h to obtain a single colony; a single colony was picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation, and activated for two generations to obtain an activated solution; the activated solution was inoculated into the medium at an inoculum volume of 2% (v / v), and cultured 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 a bacterial sludge; the bacterial sludge was washed three times with physiological saline and resuspended with a lyophilization protective agent (the mass ratio of lyophilization protective agent to bacterial body was 2:1) to a concentration of 1×10 10 CFU / mL, and obtain a bacterial suspension; the bacterial suspension was incubated at a temperature of 37°C for 60 minutes and then freeze-dried to obtain Staphylococcus epidermidis FeiHeS06 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 ingredients of the freeze-drying protective agent include: 130 g / L skim milk powder aqueous solution.

[0201] Staphylococcus epidermidis FeiHeS06 powder 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.

[0202] Example 11: Staphylococcus epidermidis ( Staphylococcus epidermidis )Application of FeiHeS06 Staphylococcus epidermidis FeiHeS06 can be used to prepare fermented pork. The specific preparation process of fermented pork is as follows: Staphylococcus epidermidis FeiHeS06 was streaked on MRS solid medium and cultured at 37°C for 48 h to obtain a single colony; a single colony was picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation, and activated for two generations to obtain an activated solution; the activated solution was inoculated into the medium at an inoculum volume of 2% (v / v), and cultured 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 a bacterial sludge; the bacterial sludge was washed three times with physiological saline and resuspended to a concentration of 1×10 10CFU / mL, and bacterial suspension was obtained; Cut the pork into 1.0-1.5 kg pieces and place them at 5-8℃ for rapid cooling for 14-18 h; add salt (2% w / w) to the pre-cooled pork pieces and pre-marinate them at 2.0-5.0℃ and 75%-95% humidity for 2-4 d; add salt (6% w / w) to the pre-marinated pork pieces and marinate them at 2.5-4.5℃ and 65%-75% humidity for 7-10 d; soak the pork pieces in 5℃ water for 10 h after the second marination, wash off the surface salt, rinse with water, and air-dry them at 10-15℃ and 70%-95% humidity for 3-5 d; adjust the density of the Staphylococcus epidermidis FeiHeS06 bacterial solution to 10 7 ~10 8 CFU / mL, the bacterial liquid was inoculated into the air-dried pork blocks at an inoculum size of 45-55 mL / kg by internal injection and surface smearing, respectively; the inoculated pork blocks were hung in a constant temperature and humidity chamber for fermentation and maturation, the conditions in the early stage of maturation were 15-20℃, humidity was 65%-80%, and fermentation was 60 d, and the conditions in the late stage of maturation were 20-25℃, humidity was 60%-75%, and fermentation was about 60 d; the fermented pork blocks were trimmed and aseptically vacuum packed to obtain Staphylococcus epidermidis FeiHeS06 fermented pork.

[0203] Example 12: Staphylococcus epidermidis ( Staphylococcus epidermidis )Application of FeiHeS06 Staphylococcus epidermidis FeiHeS06 can be used to prepare fermented fish paste. The specific preparation process of fermented fish paste is as follows: Staphylococcus epidermidis FeiHeS06 was streaked on MRS solid medium and cultured at 37°C for 48 h to obtain a single colony; a single colony was picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation, and activated for two generations to obtain an activated solution; the activated solution was inoculated into the medium at an inoculum volume of 2% (v / v), and cultured 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 a bacterial sludge; the bacterial sludge was washed three times with physiological saline and resuspended with a lyophilization protective agent (the mass ratio of lyophilization protective agent to bacterial body was 2:1) to a concentration of 1×10 10 CFU / mL, and obtain a bacterial suspension; the bacterial suspension was incubated at a temperature of 37°C for 60 minutes and then freeze-dried to obtain Staphylococcus epidermidis FeiHeS06 bacterial powder; The black carp was killed, scaled, skinned, headed, tailed and visceral removed, and the meat was minced. 3% w / w starch, 1.5% w / w sucrose, 2% w / w glucose and 2% w / w salt were added at low temperature (5-10°C) and the mixture was chopped for 20 minutes. Staphylococcus epidermidis FeiHeS06 was added to the chopped fish paste at an inoculation rate of 107 CFU / g surimi, mixed evenly; the first stage fermentation conditions are temperature 30°C, relative humidity 90%, fermentation to pH 5.2-5.5; the second stage fermentation conditions are temperature 15°C, relative humidity 80%, fermentation to pH 4.6-4.8; after fermentation is completed, vacuum packaging is obtained to obtain Staphylococcus epidermidis FeiHeS06 fermented surimi.

[0204] In summary, the present invention discloses a strain of Staphylococcus epidermidis derived from breast milk and its application in the production of food, food additives and medicines. Staphylococcus epidermidis ) FeiHeS06 has good intestinal adaptability, safety and stability and can inhibit intestinal pathogens and regulate the immune response of host immune cells, which is specifically reflected in: (1) after being treated with simulated gastric juice and simulated intestinal juice, the death of Staphylococcus epidermidis FeiHeS06 cells of the present invention is small, and the number of viable bacteria is still 10 6 CFU / mL or more; (2) the Staphylococcus epidermidis FeiHeS06 of the present invention has a shorter generation time, a higher intestinal epithelial cell adhesion ability, and can also utilize oligofructose and oligogalactose; (3) the Staphylococcus epidermidis FeiHeS06 of the present invention is sensitive to common antibiotics, especially to kanamycin and rifampicin, and the virulence-related genes it carries are mainly involved in metabolism, synthesis of extracellular components, and providing adhesion. There are no significant toxin-producing genes, and the genes related to host interaction are mainly related to the phenotype of weakened virulence or no effect on pathogenicity, and it only has the common non-toxic Sec-SRP type secretion system, and there is no prophage, and the resistance gene is horizontally transferred to other intestinal bacteria in the intestine. The possibility is low, the pathogenicity is low, and the safety is good; (4) The growth rate of the Staphylococcus epidermidis FeiHeS06 of the present invention is fast, the biomass is acceptable when the growth reaches the stable period, and it has a strong osmotic pressure tolerance; (5) The Staphylococcus epidermidis FeiHeS06 of the present invention produces acid during the production process, which can reduce the pH of the growth environment and antagonize the growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes; (6) The Staphylococcus epidermidis FeiHeS06 of the present invention can regulate the inflammatory response of macrophages RAW264.7 induced by lipopolysaccharide (LPS), significantly promote the production of anti-inflammatory factor IL-10 by cells, and significantly reduce the secretion of inflammatory factor TNF-α and inflammatory mediator NO. Therefore, this Staphylococcus epidermidis FeiHeS06 has great application prospects in the preparation of products (such as food, medicine, functional food or health care products, etc.) that have good intestinal adaptability, host safety and production stability to improve host intestinal health, regulate immune response and enhance immunity.

[0205] 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.

[0206] 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 Staphylococcus epidermidis ( Staphylococcus epidermidis ) strains, among which, The strain is Staphylococcus epidermidis ( Staphylococcus epidermidis ) FeiHeS06, the deposit number of the strain is CGMCC No.31507.

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

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

4. A leavening agent, wherein The fermentation agent comprises the strain according to claim 1, the culture according to claim 2 or the microbial preparation according to claim 3.

5. A product, wherein The product comprises the strain according to claim 1, the culture according to claim 2 or the microbial preparation according to claim 3, or the product is obtained by fermentation using the starter according to claim 4.

6. The product according to claim 5, wherein: The product is a probiotic product.

7. The product according to claim 5, wherein: The product is a food or a food additive.

8. The product according to claim 7, wherein The food includes nutritional food, functional food or health food.

9. Use of the strain according to claim 1, the culture according to claim 2, the microbial preparation according to claim 3, or the product according to any one of claims 5 to 8 in helping to regulate intestinal flora or help enhance immunity.

10. The use according to claim 9, wherein The helping to regulate intestinal flora includes inhibiting at least one of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes.

11. The use according to claim 9, wherein The method of helping to enhance immunity includes at least one of promoting macrophages to produce anti-inflammatory factor IL-10, reducing the secretion of macrophage inflammatory factor TNF-α, and reducing the secretion of inflammatory mediator NO.

12. Use of the strain according to claim 1, the culture according to claim 2, or the microbial preparation according to claim 3 in preparing a product that helps regulate intestinal flora or helps enhance immunity.

13. The use according to claim 12, wherein The helping to regulate intestinal flora includes inhibiting at least one of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes.

14. The use according to claim 12, wherein The method of helping to enhance immunity includes at least one of promoting macrophages to produce anti-inflammatory factor IL-10, reducing the secretion of macrophage inflammatory factor TNF-α, and reducing the secretion of inflammatory mediator NO.

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