A strain of Staphylococcus epidermidis derived from human milk and its application

Abstract

The present invention discloses a strain of Staphylococcus epidermidis derived from breast milk and its application. The present invention provides a strain of Staphylococcus epidermidis ( Staphylococcus epidermidis ) strain, wherein the deposit number of the strain is CGMCC No.31507. The present invention provides Staphylococcus epidermidis ( Staphylococcus epidermidis ) strains have good intestinal adaptability, safety and stability, and can inhibit intestinal pathogens and improve the immune response of host immune cells.

Description

Technical Field

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

[0002] Staphylococcus epidermidis ( 表皮葡萄球菌 ) 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: 表皮葡萄球菌 : A differential trait of the fecal microbiota of breast-fed infants) records that Staphylococcus epidermidis is the predominant bacterial species in the intestines of breastfed infants, and its presence is a characteristic difference in the intestinal microbiota between breastfed and formula-fed infants. Previous research on S. epidermidis has primarily focused on its pathogenic mechanisms and corresponding prevention and treatment strategies under specific conditions (such as impaired immune system, impaired skin barrier, and placement of artificial joints or catheters). With the advent of multidisciplinary approaches and diverse technologies, researchers have gained new insights into the properties of S. epidermidis, but research into its potential host health benefits remains in its infancy.

[0003] Some literature and patents indicate that Staphylococcus epidermidis can promote wound healing and improve skin barrier function. For example, the literature (Journal of Cellular and Molecular Medicine 2019: 葡萄球菌属 表皮的role in the skin microenvironment) discloses that Staphylococcus epidermidis can induce epithelial regeneration of the skin after injury and accelerate wound closure. Similarly, the Chinese invention patent application with publication number CN113730648A (application number 202111038989.1) discloses a hydrogel combined with Staphylococcus epidermidis that can be used for rapid repair of superficial skin injuries and reduce scarring. 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 a good anti-inflammatory effect, significantly reducing the expression level of the 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] Furthermore, Chinese invention patent application publication number CN104745501A (application number 201310752880.3) discloses Staphylococcus epidermidis and its use in the production of fermented meat products. Meat products fermented with Staphylococcus epidermidis in this patent possess excellent flavor, stable quality, no odor, and good color development. Similarly, Chinese invention patent application publication number CN101717741A (application number 200910264536.3) discloses meat products fermented with Staphylococcus epidermidis inoculated into raw meat and then fermented to maturity. Compared to traditional fermented meat products such as dry-cured ham, this method shortens the processing time of fermented pork products by nearly five months, significantly improves the sensory quality of the product, and offers a richer ham flavor and aroma. A Chinese invention patent application, publication number CN108823124A (application number 201810603967.7), discloses a strain of Staphylococcus epidermidis isolated from the nasal cavity of a healthy individual and colonized with the bacteria. The strain exhibits broad-spectrum antimicrobial activity against common Gram-positive, drug-resistant bacteria such as Staphylococcus aureus, Streptococcus pneumoniae, and Micrococcus luteus. This suggests that Staphylococcus epidermidis has potential probiotic properties and potential applications as an edible beneficial bacterium.

[0005] For edible probiotics to exert their probiotic effects, survival within the host's digestive tract, reaching the colon, and successfully colonizing intestinal epithelial cells are crucial. A short reproduction cycle (generation time), specific carbon source utilization, and strong adhesion to intestinal epithelial cells contribute to their colonization potential. Furthermore, maintaining strong antibiotic susceptibility and low pathogenicity (virulence genes, pathogen-host interactions, secretion systems, etc.) are essential for probiotics to function.

[0006] In recent years, people's health awareness has continued to grow, and the coronavirus crisis has driven unprecedented interest in immunity-related products. Consequently, the role of probiotics in inhibiting intestinal pathogens, maintaining a stable intestinal microbiome, and enhancing 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 to study its intestinal adaptability, safety and probiotic properties has scientific value and practical significance for the development and application of Staphylococcus epidermidis. Summary of the Invention

[0008] Problem to be solved by the invention

[0009] The technical problem to be solved by the present invention is to provide a new strain of Staphylococcus epidermidis isolated from breast milk ( 表皮葡萄球菌 ) and its applications.

[0010] Solution for solving the problem

[0011] To solve the above problems, in a first aspect of the present invention, a Staphylococcus epidermidis ( 表皮葡萄球菌 ) strain, wherein the strain is Staphylococcus epidermidis FeiHeS06, and the deposit number of the strain is CGMCC No. 31507. Specifically, the present invention provides a strain of Staphylococcus epidermidis ( 表皮葡萄球菌 ) FeiHeS06, the Staphylococcus epidermidis FeiHeS06 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.31507 and the deposit date of August 1, 2024.

[0012] In a second aspect of the present invention, a culture is provided, wherein the culture is obtained by culturing the strain according to the first aspect of the present invention.

[0013] In a third aspect of the present invention, a microbial preparation is provided, wherein the microbial preparation comprises the strain according to the first aspect of the present invention or the culture according to the second aspect of the present invention.

[0014] In a fourth aspect of the present invention, a starter culture is provided, wherein the starter culture comprises the strain according to the first aspect of the present invention, the culture according to the second aspect of the present invention, or the microbial preparation according to the third aspect of the present invention.

[0015] In the fifth aspect of the present invention, a product is provided, wherein the product comprises the strain described in the first aspect of the present invention, the culture described in the second aspect of the present invention, or the microbial preparation described in the third aspect of the present invention, or the product is obtained by fermentation using the starter described in the fourth aspect of the present invention.

[0016] In the sixth aspect of the present invention, there is provided a 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.

[0017] In some embodiments, the helping to regulate intestinal flora comprises inhibiting at least one of Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes.

[0018] 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.

[0019] In the seventh aspect of the present invention, provided is 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, or the microbial preparation described in the third aspect of the present invention in the preparation of a drug that helps regulate intestinal flora or helps enhance immunity.

[0020] In some embodiments, the helping to regulate intestinal flora comprises inhibiting at least one of Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes.

[0021] 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.

[0022] Effects of the invention

[0023] The present invention provides a strain of Staphylococcus epidermidis ( 表皮葡萄球菌 )FeiHeS06, this Staphylococcus epidermidis ( 表皮葡萄球菌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:

[0024] (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.

[0025] (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 were different. The generation time of the Staphylococcus epidermidis FeiHeS06 of the present invention was shorter. When the strain was co-cultured with HT-29 cells in DMEM medium for 4 hours, the cell adhesion rate of the Staphylococcus epidermidis FeiHeS06 of the present invention was the highest. In MRS liquid culture medium with oligofructose or oligogalactose as carbon source, the Staphylococcus epidermidis FeiHeS06 of the present invention was able to utilize these two common oligosaccharides and had a stronger ability to utilize oligofructose.

[0026] (3) Determination of antibiotic resistance of probiotics is an important part of its safety assessment. When the minimum inhibitory concentration (MIC) of 14 common antibiotics was determined 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 comparable to that of the control probiotic strain, and its sensitivity to rifampicin and clindamycin was the strongest among the five strains. Analysis of the whole genome data (virulence genes, secretion system, prophage, and interaction with the host) of the Staphylococcus epidermidis FeiHeS06 of the present invention showed that the virulence-related genes carried by the Staphylococcus epidermidis FeiHeS06 of the present invention were mainly involved in metabolism, synthesis of extracellular components, and provision of adhesion. There were no significant toxin-producing genes. The genes related to host interaction were mainly related to phenotypes with weakened virulence or no effect on pathogenicity. It only had the common non-toxic Sec-SRP type secretion system and no prophage. The possibility of horizontal transfer of drug-resistant genes to other intestinal bacteria in the intestine was low, and its pathogenicity was low, and its safety was good.

[0027] (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 Continuously monitoring its growth revealed that the Staphylococcus epidermidis FeiHeS06 strain of the present invention had a relatively fast growth rate and a reasonable biomass (the mass of the bacterial sludge produced during the stable growth phase) when it reached the stable growth phase. The present Staphylococcus epidermidis FeiHeS06 strain and a control probiotic strain were inoculated into a series of MRS culture media with varying osmotic pressures, prepared by adding varying amounts of NaCl. The present Staphylococcus epidermidis FeiHeS06 strain exhibited a relatively high osmotic pressure at which its growth rate began to be inhibited and at which its growth was completely inhibited, indicating a strong osmotic pressure tolerance. Osmotic pressure affects intracellular water activity, cell membrane permeability, and the structure of macromolecules such as proteins, thereby influencing the structure, physiological function, and growth and reproduction of microbial cells.

[0028] (5) The organic acids produced by probiotics can lower 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 cultivation. During 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 a significant inhibition zone, regulate intestinal flora, and promote intestinal health.

[0029] (6) Macrophages are important immune effector cells differentiated from monocytes and play a key role in innate and acquired immune responses. Activating macrophages RAW264.7 and adding LPS to induce an inflammatory response in macrophages, the supernatant of the Staphylococcus epidermidis FeiHeS06 of the present invention promoted the production of anti-inflammatory factors (IL-10) by macrophages RAW264.7 and reduced the secretion of inflammatory factors (TNF-α) and inflammatory mediators (NO) by macrophages RAW264.7.

[0030] Therefore, the Staphylococcus epidermidis FeiHeS06 of the present invention has great application prospects in the preparation of products (such as medicines) that improve host intestinal health and regulate immune responses with good intestinal adaptability, host safety and production stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 :Staphylococcus epidermidis( 表皮葡萄球菌 ) Characteristic colony morphology of FeiHeS06 on MRS solid medium.

[0032] Figures 2A - 2B :The changes of viable bacterial counts of Staphylococcus epidermidis FeiHeS06 and control probiotic strains before and after culture in simulated gastric fluid and simulated intestinal fluid. Figure 2A To simulate gastric fluid treatment, Figure 2B To simulate intestinal fluid treatment.

[0033] 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).

[0034] 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).

[0035] Figure 5 Distribution of minimum inhibitory concentrations (MICs) for Staphylococcus epidermidis FeiHeS06 and a control probiotic strain against 14 common antibiotics. Darker blocks represent higher MICs for the antibiotic. Colors represent the results of normalization within each column; the values ​​above the blocks are the actual MICs for the antibiotics.

[0036] Figure 6: Growth patterns of Staphylococcus epidermidis FeiHeS06 and control probiotic strains in MRS liquid culture medium.

[0037] Figure 7 : The bacterial sludge mass (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).

[0038] Figures 8A - 8E :Osmotic pressure tolerance of Staphylococcus epidermidis FeiHeS06 and control probiotic strains. Figure 8A :Staphylococcus epidermidis FeiHeS06 osmotic pressure resistance, Figure 8B : Staphylococcus epidermidis MRJSWX39M8 osmotic pressure resistance, Figure 8C :Lactobacillus gasseri MRJSWX2L1 osmotic pressure resistance, Figure 8D : Bifidobacterium longum subspecies infantis MRJSWX29M3 osmotic pressure resistance, Figure 8E : Osmotic pressure resistance of Bifidobacterium animalis subsp. lactis BB12.

[0039] Figure 9 : Changes in culture medium pH before and after culture of Staphylococcus epidermidis FeiHeS06 and control probiotics.

[0040] Figures 10A - 10C :The regulatory ability of Staphylococcus epidermidis FeiHeS06 and control probiotic strains on LPS-induced inflammatory response in macrophages RAW264.7. Figure 10A The ability of different strains to regulate the anti-inflammatory factor IL-10 in macrophage RAW264.7 cells. Figure 10B The regulatory ability of different strains on the inflammatory factor TNF-α of macrophage RAW264.7, Figure 10C The different strains have significant differences in their ability to regulate the production of inflammatory mediator NO in macrophages 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 macrophages RAW264.7. P <0.05).

[0041] 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. DETAILED DESCRIPTION

[0042] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0043] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0044] Unless otherwise stated, the units used in this specification are 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.

[0045] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0046] References throughout this specification to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the particular 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 be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0047] In this specification, a numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0048] Biomaterial Deposit

[0049] A strain of Staphylococcus epidermidis ( 表皮葡萄球菌 ) FeiHeS06, taxonomically named Staphylococcus epidermidis 表皮葡萄球菌 , was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (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.

[0050] strain

[0051] In some aspects, the present invention provides a strain of Staphylococcus epidermidis ( 表皮葡萄球菌) FeiHeS06, the Staphylococcus epidermidis FeiHeS06 is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC No. 31507 and the deposit date being August 1, 2024.

[0052] The Staphylococcus epidermidis ( 表皮葡萄球菌 FeiHeS06 was derived from breast milk collected from healthy lactating women in Wuxi, Jiangsu Province. Sequencing analysis revealed that the strain had a 100% similarity to the nucleic acid sequence of Staphylococcus epidermidis, a species of Staphylococcus. The strain was named Staphylococcus epidermidis ( 表皮葡萄球菌 ) FeiHeS06, whose original number in the laboratory was CCFM1371.

[0053] The Staphylococcus epidermidis ( 表皮葡萄球菌 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 shown).

[0054] In some embodiments of the present invention, the Staphylococcus epidermidis ( 表皮葡萄球菌 ) FeiHeS06 has good tolerance in simulated gastric fluid and simulated intestinal fluid environments, and has a strong ability to survive in the gastrointestinal tract.

[0055] In some embodiments of the present invention, the Staphylococcus epidermidis ( 表皮葡萄球菌 ) 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), and a stronger ability to utilize oligofructose, and has stronger intestinal adaptability.

[0056] In some embodiments of the present invention, the Staphylococcus epidermidis ( 表皮葡萄球菌 ) 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 provision of adhesion, with no significant toxin-producing genes. Genes related to host interaction were mainly associated with phenotypes of weakened virulence or no effect on pathogenicity. It only has the common non-toxic Sec-SRP type secretion system and no prophage. The risk of resistance gene transfer is small, its potential pathogenicity and toxicity are low, and its safety is good.

[0057] In some embodiments of the present invention, the Staphylococcus epidermidis ( 表皮葡萄球菌 ) FeiHeS06 has a fast growth rate, a decent biomass in the stable period, a strong osmotic pressure tolerance, and good stability.

[0058] In some embodiments of the present invention, the Staphylococcus epidermidis ( 表皮葡萄球菌 ) FeiHeS06 produces acid, and the organic acid produced can reduce the intestinal pH and inhibit the growth of intestinal pathogenic microorganisms. The Staphylococcus epidermidis ( 表皮葡萄球菌 ) FeiHeS06 can antagonize the growth of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes, regulate intestinal flora and promote intestinal health.

[0059] In some embodiments of the present invention, the Staphylococcus epidermidis ( 表皮葡萄球菌 ) 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 they play a key role in innate and acquired immune responses.

[0060] culture

[0061] In some aspects, the present invention provides a culture, wherein the culture is obtained by culturing the above-mentioned Staphylococcus epidermidis ( 表皮葡萄球菌 ) obtained by FeiHeS06.

[0062] For the purposes of this invention, the term "culture" refers to any liquid or solid product (at least a portion of the material within a culture vessel, such as the supernatant) containing a microbial population following artificial inoculation and cultivation. This refers to a product obtained by growing and / or amplifying microorganisms. This product may be a biologically pure culture of microorganisms, contain a certain amount of culture medium, metabolites, or other components produced during the culture process, or be the supernatant after centrifugation to remove cells.

[0063] In some embodiments, the culture is a supernatant (cell-free supernatant) obtained by centrifuging a product obtained by growing and / or expanding a microorganism.

[0064] In some embodiments, the culturing is performed by the following steps:

[0065] (1) Staphylococcus epidermidis ( 表皮葡萄球菌 ) FeiHeS06 was streaked onto MRS solid medium and a single colony was obtained;

[0066] (2) picking a single colony and inoculating it into MRS liquid culture medium, culturing it for activation, and continuously activating it for at least one generation, for example, two generations, to obtain an activated liquid;

[0067] Optionally, (3) the activation solution is inoculated into MRS liquid culture medium at an inoculum volume of about 2% (v / v), and cultured to a stationary phase to obtain a culture solution;

[0068] (4) Centrifuging the activation solution obtained in step (2) or the culture solution obtained in step (3) to obtain a supernatant.

[0069] Optionally, (5) the supernatant is collected and filtered to obtain a cell-free supernatant for later use.

[0070] In some specific embodiments, the MRS solid culture medium includes 10 g / L peptone, 10 grams / liter (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 milliliter / liter (mL / L) Tween 80, and 20 g / L agar.

[0071] 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.

[0072] Microbial agents

[0073] In some aspects, the present invention also provides a method comprising the above-mentioned Staphylococcus epidermidis ( 葡萄球菌属 表皮的 ) FeiHeS06 or the above-mentioned culture microbial preparation.

[0074] 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.

[0075] Furthermore, the microbial preparation may further comprise a protective agent (such as a freeze-drying protective agent), including but not limited to skim milk powder, trehalose, sodium glutamate and / or glycerol, etc.; preferably, the protective agent is skim milk powder.

[0076] In other embodiments, the microbial preparation of the present invention is semi-solid, for example, it can be bacterial sludge. The bacterial sludge refers to bacterial cells containing a small amount of water.

[0077] In other embodiments, the microbial preparation of the present invention is a liquid, for example, a suspension or a culture (supernatant). Furthermore, the microbial preparation may further comprise a solvent, including but not limited to water or culture medium.

[0078] Preparation method of microbial preparation

[0079] In some embodiments of the present invention, the method for preparing the microbial preparation comprises the steps of: 表皮葡萄球菌 ) Steps of inoculating FeiHeS06 into appropriate culture medium for cultivation.

[0080] In some embodiments, the method further comprises the steps of centrifuging the culture fluid obtained by the cultivation to collect a bacterial sludge, and mixing the bacterial sludge with a protective agent (e.g., a lyoprotectant). Centrifugation can increase bacterial cell concentration and shorten lyophilization time. In some embodiments, after the mixing step, a freeze-drying step is also included. Freeze-drying facilitates the transportation and storage of the microbial preparation. The addition of a protective agent minimizes bacterial cell death or loss of activity during the freeze-drying process, thereby improving the fermentation efficiency of the microbial preparation.

[0081] In some specific embodiments, the method for preparing the microbial preparation of the present invention comprises the following steps:

[0082] The above-mentioned Staphylococcus epidermidis ( 表皮葡萄球菌 ) FeiHeS06 is inoculated into a suitable culture medium to obtain a culture solution;

[0083] The culture solution was centrifuged to obtain bacterial cells;

[0084] After washing the bacteria, resuspend them in a freeze-drying protective agent to obtain a resuspension;

[0085] The resuspension is freeze-dried by a vacuum freezing method to obtain a microbial preparation.

[0086] 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.

[0087] 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 a skim milk powder aqueous solution.

[0088] 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 or 7.0.

[0089] Fermentation Agents

[0090] In some aspects, the present invention also provides a method comprising the above-mentioned Staphylococcus epidermidis ( 葡萄球菌属 表皮的 ) FeiHeS06, the above-mentioned culture, the above-mentioned microbial preparation or the starter of the microbial preparation prepared by the above-mentioned preparation method.

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

[0092] In the present invention, the lactic acid bacteria used in the starter are not particularly limited as long as they do not hinder the effects of the present invention, and may be either animal-derived or plant-derived.

[0093] Preferred lactic acid bacteria of the present invention include Lactobacillus ( 乳杆菌属 ) bacteria, Streptococcus ( 链球菌属 ) bacteria, Lactococcus ( 乳球菌属 ) bacteria, Enterococcus ( 肠球菌属 )genus, Leuconostoc ( 明串珠菌属 ) 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 enumerate the combination of the genus Lactobacillus, the combination of the genus Lactobacillus and the genus 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., and also can suitably use commercially available products.

[0094] Examples of Streptococcus bacteria include Streptococcus thermophilus ( 嗜热链球菌 )wait.

[0095] Preferred examples of the combination of Lactobacillus bacteria and Streptococcus bacteria include Lactobacillus bulgaricus and Streptococcus thermophilus.

[0096] product

[0097] The present invention further provides a product comprising the above-mentioned Staphylococcus epidermidis ( 表皮葡萄球菌 ) 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.

[0098] 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:

[0099] Contains milk ingredients or protein ingredients, wherein the milk ingredients include dairy products including fresh milk, milk powder, whey protein or cheese derived from fresh cow (sheep) milk; the protein ingredients can come from plant proteins, such as soy protein, peanut protein, etc.

[0100] Plants or plant extracts, including fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, rubber trees, grapes, pears, cherries, blueberries, blackberries, black currants, cranberries, raspberries, melons, emblica chinensis, and bilberries, 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, japonica, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, millet, buckwheat, soybeans, broad beans, peas, mung beans, adzuki beans, and kidney beans, or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricots, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, and ginkgo nuts, or their extracts; coffee or its extracts.

[0101] Animal ingredients, including meat products from beef, lamb, fish or poultry.

[0102] 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 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.

[0103] 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, isomalto-oligosaccharides, soybean polysaccharides, cyclodextrin, resistant dextrin or soybean fiber, etc.).

[0104] 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.

[0105] Any acceptable excipients include, but are not limited to, solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible colorings, etc.

[0106] The present invention does not specifically limit the specific types of the above products.

[0107] In other embodiments, the product is a pharmaceutical.

[0108] In some embodiments of the present invention, the drug contains the above-mentioned Staphylococcus epidermidis ( 葡萄球菌属 表皮的 ) 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.

[0109] In some embodiments of the present invention, the drug carrier comprises a microcapsule, a microsphere, a nanoparticle and / or a liposome.

[0110] In some embodiments of the present invention, the pharmaceutical excipient comprises an excipient and / or an additive.

[0111] In some embodiments of the present invention, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0112] In some embodiments of the present invention, the additive comprises a solubilizer, a co-solvent, a co-solvent and / or a preservative.

[0113] In some embodiments of the present invention, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0114] In other embodiments, the product is a non-food product.

[0115] In other embodiments, the product is a non-nutraceutical food.

[0116] In other embodiments, the product is non-drug.

[0117] For different product categories, the present invention does not specifically limit the specific form of the product. For example, it can be in powder form or liquid form.

[0118] The present invention does not specifically limit the target population of the product. For example, the product can be used for infants, children, teenagers or adults.

[0119] 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.

[0120] Helps regulate intestinal flora

[0121] The present invention provides Staphylococcus epidermidis ( 表皮葡萄球菌 ) 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.

[0122] Therefore, the Staphylococcus epidermidis provided by the present invention ( 表皮葡萄球菌 The microbial agent prepared by the FeiHeS06 culture, microbial preparation, and preparation method can be used to prepare a product that helps regulate intestinal flora. The product of the present invention also helps regulate intestinal flora.

[0123] The present invention does not specifically limit the specific product categories that help regulate intestinal flora. In some embodiments, the purpose of regulating intestinal flora is not to prevent and / or treat diseases. For example, when there is a disorder in the intestinal flora, or when intestinal pathogenic microorganisms (Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes) are present but not to the extent that they cause disease, or when there is a disorder in the intestinal flora or an increase in intestinal pathogenic microorganisms that is not caused by disease, the use of the Staphylococcus epidermidis provided by the present invention ( 表皮葡萄球菌 ) FeiHeS06, culture, microbial preparations, microbial agents prepared by preparation methods, etc.

[0124] In other embodiments, the product is a drug. In some specific embodiments, the drug is used to inhibit enteric pathogens.

[0125] Uses that help enhance immunity and regulate immune response

[0126] The present invention provides Staphylococcus epidermidis ( 表皮葡萄球菌 ) 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.

[0127] Therefore, the Staphylococcus epidermidis provided by the present invention ( 表皮葡萄球菌 The microbial preparation prepared by the FeiHeS06 culture, microbial preparation, and preparation method can be used to prepare products that help enhance immunity and regulate immune responses. The products of the present invention also help enhance immunity and regulate immune responses.

[0128] The present invention does not particularly limit the specific categories of products that help enhance immunity and regulate immune response.

[0129] Furthermore, in some embodiments, the purpose of helping to enhance immunity and regulate immune response is not to prevent and / or treat diseases, for example, when immunity is reduced or inflammatory factor levels are changed, the Staphylococcus epidermidis provided by the present invention is used. 表皮葡萄球菌 ) FeiHeS06, culture, microbial preparation, microbial agent prepared by preparation method, etc.

[0130] In other embodiments, the product is a drug. In some specific embodiments, the drug is used for anti-infection, anti-inflammatory, etc.

[0131] In some embodiments, the anti-infection comprises inhibiting the growth of enteric pathogenic microorganisms. In some embodiments, the anti-infection comprises antagonizing the growth of Staphylococcus aureus, Escherichia coli and / or Listeria monocytogenes.

[0132] 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

[0133] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0134] The Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, and Bifidobacterium longum subsp. infantis MRJSWX29M3 involved in the following examples are all strains screened and isolated from breast milk during the same period and are deposited in the proprietary strain bank of the Biotechnology Center of the School of Food Science and Engineering of Jiangnan University. The Bifidobacterium animalis subsp. lactis BB12 involved in the following examples is a commercially available probiotic strain purchased from Chr. Hansen A / S, Denmark.

[0135] The culture medium involved in the following examples is as follows:

[0136] MRS solid 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, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.25 g / L, Tween 80 1 mL / L, agar 20 g / L.

[0137] MRS liquid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.25 g / L, Tween 80 1 mL / L.

[0138] The culture of Bifidobacterium requires the addition of 0.5 g / L L-cysteine ​​to MRS solid medium or MRS liquid medium.

[0139] The detection methods involved in the following embodiments are as follows:

[0140] Detection method of viable bacteria count: adopt the national standard "GB 4789.2-2022 National Food Safety Standard Food Microbiology Examination Determination of Total Colony Count".

[0141] The preparation method of the probiotic bacteria involved in the following examples is as follows:

[0142] Streak Staphylococcus epidermidis on MRS solid culture medium and 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 it 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 it at 37℃ for 18 hours to obtain a bacterial solution; centrifuge the bacterial solution at 5000 revolutions per minute (r / min) for 15 minutes (min), discard the supernatant culture medium, and obtain Staphylococcus epidermidis cells.

[0143] The cultivation steps for Lactobacillus are the same as those for Staphylococcus epidermidis. The cultivation steps for Bifidobacterium are the same as those for MRS solid medium or MRS liquid medium supplemented with 0.5 g / L L-cysteine ​​and cultured at 37°C under anaerobic conditions.

[0144] The preparation methods of the simulated gastric fluid and simulated intestinal fluid involved in the following examples are as follows:

[0145] Simulated gastric fluid: Adjust the pH of saline to 3.0 with HCl. Add pepsin (1:10,000) to sterile saline (0.9% w / v) at pH 3.0 to a final concentration of 3 g / L. Filter through a 0.22 μm sterile filter and prepare immediately.

[0146] Simulated intestinal fluid: Adjust the pH of saline to 8.0 with NaOH; dissolve trypsin (1:250) in sterile saline (0.9% w / v) at pH 8.0 to a final concentration of 1 g / L; add bile salts to a concentration of 0.3% w / v; filter through a 0.22 micron (μm) sterile filter membrane and prepare immediately.

[0147] The methods for HT-29 cell recovery, passaging, plating and culture termination involved in the following examples are as follows:

[0148] Cell recovery: Preheat DMEM complete medium (with fetal bovine serum and double-antibody) in a 37°C water bath. Remove the frozen cells and quickly thaw them in a 37°C water bath. Transfer all the liquid in the cryotube into a 15-mL centrifuge tube containing 5 mL of complete medium and centrifuge (1000 rpm, 5 min). After centrifugation, discard the culture medium, add 1 mL of complete medium to resuspend the cells, transfer them to a culture dish containing 9 mL of complete medium, and culture them in a 37°C 5% CO2 incubator. Change the culture medium every other day.

[0149] Cell passaging: When cells have grown to 80% of the culture dish, wash the cell surface twice with PBS. Then, add 1 mL of trypsin for 2 min. After digestion is complete (cell morphology shrinks and can be detached from the dish wall by pipetting), stop the digestion with 5 mL of complete medium. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge (1000 rpm, 5 min). Discard the supernatant and add 2 mL of complete medium to mix the cells by pipetting. Pipette 1 mL of cell suspension and 9 mL of complete medium into two culture dishes respectively, remove bubbles, mix thoroughly, and culture in a 37°C 5% CO2 incubator.

[0150] Cell plating: wait until the number of cells reaches 10 6 Repeat the cell passaging steps, wash the culture dish with PBS, trypsinize, terminate the digestion, centrifuge and discard the supernatant, then add 12 mL of complete medium to resuspend; add 500 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°C 5% CO2 incubator.

[0151] Terminate 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.

[0152] Example 1: Staphylococcus epidermidis ( 表皮葡萄球菌 ) Screening, identification and cultivation of FeiHeS06

[0153] The specific steps are as follows:

[0154] 1. Breast milk sample collection

[0155] Healthy breastfeeding women from Wuxi City, Jiangsu Province were recruited according to the following criteria: (1) aged 25–35 years; (2) natural childbirth; (3) infants aged 1–6 months at the time of breast milk collection; (4) full-term childbirth, healthy infants, and no hospitalization experience during the neonatal period; (5) self-assessed good health and not taking any medications; and (6) no gestational diabetes, gestational hypertension, or other diseases during pregnancy.

[0156] Breast milk collection: The mother washed her hands, put on disposable latex gloves, and wiped the collection side with a wet wipe. The first 2 mL of milk was discarded and the remaining milk was collected directly into a sampling tube. The sampling tube was pre-filled with sterile 60% v / v glycerol, with a milk:glycerol volume ratio of 1:1, for a final glycerol concentration of 30% v / v. The tube was then tightly capped, sealed with parafilm, and labeled with the name and collection date. Breast milk samples were placed on ice and transported to the laboratory within 4 hours for storage at -80°C.

[0157] 2. Staphylococcus epidermidis ( 表皮葡萄球菌 ) Acquisition of FeiHeS06

[0158] 1. Screening

[0159] The breast milk sample obtained in step 1 was inoculated into MRS liquid culture medium at a 20% inoculum volume and pre-cultured 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 sample was taken from each of the dilution gradients of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 The dilutions were plated on MRS solid medium at pH = 7.0 and cultured at 37°C for 48 h. The colony morphology was observed and recorded.

[0160] Colonies of different morphologies on MRS solid medium were picked and streaked to separate them. After culturing at 37°C for 48 h, single colonies of different morphologies on MRS solid medium were picked and streaked to separate them again until pure single colonies with consistent morphology were obtained.

[0161] Pure colonies on MRS solid medium were picked and inoculated into 5 mL of MRS liquid medium and cultured at 37°C for 18 h;

[0162] 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.

[0163] 2. Identification

[0164] The isolated strains were amplified by 16S rDNA PCR, and the PCR products were sent to Genwi (Suzhou) Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the NCBI standard Nucleotide BLAST. The result was Staphylococcus epidermidis, and the strain was named Staphylococcus epidermidis FeiHeS06. The sequencing results of the 16S rDNA of the strain were as follows:

[0165]

[0166] 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.

[0167] 3. Cultivation

[0168] The above-mentioned Staphylococcus epidermidis FeiHeS06 was inoculated onto MRS solid medium and cultured at 37°C for 48 h. The colonies were observed. The colonies were found to be grayish-white, round, smooth, flat, and opaque, with a diameter of 1 to 2 mm (see Figure 1 The pH of the blank culture medium and the culture medium with bacteria was measured with a pH meter before and after cultivation. 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 a microplate reader, and cultured at 37°C for 48 hours. During the cultivation process, the OD value of the culture medium was measured every 1 hour. 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.

[0169] Example 2: Staphylococcus epidermidis ( 表皮葡萄球菌 ) Tolerance of FeiHeS06 in simulated gastric and intestinal fluids

[0170] The specific steps are as follows:

[0171] (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 were streaked onto MRS solid culture medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid culture medium and cultured at 37°C for 18 h for activation. Two generations of activation were performed to obtain activation liquid.

[0172] Bifidobacterium should be cultured in MRS solid medium or MRS liquid medium supplemented with 0.5 g / L L-cysteine ​​and placed under anaerobic conditions at 37°C. Other steps are the same.

[0173] (2) The activation solution was inoculated at a 2% v / v inoculum into 5 mL of MRS liquid medium (Bifidobacteria were inoculated into MRS liquid medium supplemented with 0.5 g / L L-cysteine) and cultured until the stationary phase. The cells were collected by centrifugation at 5000 g for 10 min in two centrifuge tubes, the supernatant was discarded, and the cells were washed twice with physiological saline. The cells were 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 physiological saline. After incubation at 37°C for 3 h, the viable bacteria were counted on the plate. Two replicates were made for each dilution. The experimental results of the two treatment methods were compared and analyzed. The logarithmic decrease of the colony count after treatment with simulated gastric juice was less than 3 compared with that of the 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 JFood Microbiol. 2012 Feb 1;153(1-2):216-22.; Wang Chuan, Zhang Chaowu, Sun Xiaofan, et al. Preliminary study on the acid and bile salt resistance of lactobacilli from human stomach and intestine [J]. Modern Preventive Medicine, 2006, (10):1792-1794.).

[0174] (3) The activated liquid was inoculated at a volume of 2% v / v into 5 mL of MRS liquid medium (Bifidobacteria were inoculated into MRS liquid medium supplemented with 0.5 g / L L-cysteine) and cultured until the stable phase. The bacteria were collected in two centrifuge tubes by centrifugation at 5000 g for 10 min, the supernatant was discarded, and the bacteria were washed twice with physiological saline, centrifuged, and the supernatant was poured out. The bacteria in one centrifuge tube were resuspended in simulated intestinal fluid and vortexed to mix evenly. The bacteria in the other centrifuge tube were resuspended in an equal volume of physiological saline. After incubation at 37°C for 4 h, the live bacteria were counted on the plate, and two parallel plates 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 one, and the number of live bacteria was still above 10 6 CFU / mL (colony-forming units per milliliter) or above indicates good intestinal fluid tolerance.

[0175] 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 5 strains reached 10 when they were cultured to the stable stage without being treated with simulated gastric fluid. 8 ~10 9The CFU / mL values ​​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 had good tolerance to gastric juice.

[0176] Tolerance of 5 strains of bacteria to simulated intestinal fluid Figure 2B As shown in the figure, the number of viable bacteria of the 5 strains reached 10 when they were cultured to the stable stage without being treated with simulated intestinal fluid. 8 ~10 9 CFU / mL; after 4 h of treatment with simulated intestinal fluid, there were significant differences in intestinal fluid tolerance among different bacterial species and strains; the logarithmic decrease values ​​of the viable counts of Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 were 0.46, 2.37, 2.67, 2.17, and 2.67, respectively; these results showed that Staphylococcus epidermidis FeiHeS06 had the best tolerance to intestinal fluid and maintained a 10 8 The number of viable bacteria of Staphylococcus epidermidis MRJSWX39M8, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 was reduced to 10 6 CFU / mL; Lactobacillus gasseri MRJSWX2L1 has the worst tolerance to simulated intestinal fluid, with the number of viable bacteria ranging from 10 8 CFU / mL decreased to 10 5 CFU / mL.

[0177] Example 3: Staphylococcus epidermidis ( 表皮葡萄球菌 ) Colonization potential of FeiHeS06 (generation time, intestinal epithelial cell adhesion ability, oligosaccharide utilization ability)

[0178] The specific steps are as follows:

[0179] (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 were streaked onto MRS solid culture medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid culture medium and cultured at 37°C for 18 h for activation. Two generations of activation were performed to obtain activation liquid.

[0180] Bifidobacterium should be cultured in MRS solid medium or MRS liquid medium supplemented with 0.5 g / L L-cysteine ​​and placed under anaerobic conditions at 37°C. Other steps are the same.

[0181] (2) Generation time determination: Take 1 mL of activation solution and place it in a sterile centrifuge tube. Centrifuge at 5000 r / min for 15 min, then discard the upper culture medium to obtain bacterial sludge. Resuspend the bacterial sludge with 1 mL of sterile saline and inoculate it into MRS liquid culture medium at a 2% (v / v) inoculum. Place the tube in a microplate reader and culture 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 By plotting the values ​​against the corresponding culture time, we can create a growth curve for the strain under specific conditions. Bifidobacteria require anaerobic culture at 37°C in MRS liquid medium supplemented with 0.5 g / L L-cysteine. All other steps are the same.

[0182] (3) Determination of intestinal epithelial cell adhesion ability: The activated solution was inoculated into MRS liquid culture medium at a volume 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) of bacterial liquid; 980 μL of bacterial liquid 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 still at 37°C in the dark for 15 min, centrifuged (8000 r / min, 4°C, 10 min), and then washed three times with PBS to remove excess fluorescent dye, and then the cells were resuspended in 1 mL of DMEM basal medium; HT-29 cells (purchased from the National Model and Specialty Experimental Cell Resource Bank, catalog number: TCHu103) were revived, passaged and plated in advance; 12-well cell plate monolayer HT-29 cells were washed twice with PBS, and 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 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 pipetting 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 transferred to a 96-well plate and the fluorescence OD was measured. 488 The adhesion rate of the strain to intestinal epithelial cells was calculated.

[0183] (4) Determination of oligosaccharide utilization ability: replace the glucose in the MRS medium with an equal amount of oligofructose or oligogalactose, and add bromocresol purple as an indicator (0.0075% w / v); first add 200 μL of MRS liquid medium with oligofructose or oligogalactose as the carbon source to a 96-well plate; then add 2 μL of the strain activation solution that was centrifuged and the supernatant was discarded and resuspended in physiological 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 used as the positive control, the culture medium without bacterial solution was used as the negative control 1, and the bacterial solution + MRS medium without carbon source with bromocresol purple was used as the negative control 2.

[0184] 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. 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.

[0185] Depend on Figure 3It can be seen that the generation time of the five bacterial strains in the logarithmic growth phase is different. 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.

[0186] Adhesion ability of the five bacterial strains to HT-29 cells Figure 4 As shown in the results, S. epidermidis FeiHeS06 had the highest adhesion rate, reaching 28.69%, followed by Lactobacillus gasseri MRJSWX2L1 at 22.86%, S. 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 S. epidermidis FeiHeS06 was approximately 3.5 times higher than that of the same strain, S. epidermidis MRJSWX39M8, and 4-6 times higher than that of both Bifidobacterium strains. These results indicate that S. epidermidis FeiHeS06 has excellent adhesion to intestinal epithelial cells and has the potential to successfully colonize the host intestine.

[0187] Table 1 shows the ability of the five bacterial strains to utilize fructooligosaccharides and galacto-oligosaccharides. These five strains produce acid during their metabolism, which lowers the pH of the culture medium when growing on fructooligosaccharides or galacto-oligosaccharides. This characteristic can be exploited by using bromocresol purple as an indicator (color change range: pH 5.2-6.8), which changes from purple to yellow under acidic conditions. By observing the color change of the culture medium surrounding the colonies, it can be determined whether the strains are utilizing the target carbon source (fructooligosaccharides or galacto-oligosaccharides) for metabolism.

[0188] Table 1: Utilization ability of 5 bacterial strains on oligofructose and oligogalactose

[0189]

[0190] 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.

[0191] As shown in Table 1, all five strains can utilize both fructooligosaccharides and galacto-oligosaccharides. Among them, Staphylococcus epidermidis FeiHeS06 was more efficient at utilizing fructooligosaccharides than Lactobacillus gasseri MRJSWX2L1. This ability to utilize oligosaccharides can enhance the colonization of strains in the intestine and provide formulation ideas for the combined use of oligosaccharides and probiotic strains.

[0192] Example 4: Staphylococcus epidermidis ( 表皮葡萄球菌) Antibiotic resistance and potential pathogenicity of FeiHeS06

[0193] The specific steps are as follows:

[0194] (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 were streaked onto MRS solid culture medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid culture medium and cultured at 37°C for 18 h for activation. Two generations of activation were performed to obtain activation liquid.

[0195] Bifidobacterium should be cultured in MRS solid medium or MRS liquid medium supplemented with 0.5 g / L L-cysteine ​​and placed under anaerobic conditions at 37°C. Other steps are the same.

[0196] (2) Antibiotic resistance determination (minimum inhibitory concentration MIC test): 14 commonly used antibiotics were selected and first dissolved in a small amount of water or anhydrous ethanol or methanol as a solvent, sterilized by filtering through a 0.22 μm filter membrane, and then diluted with sterile water to prepare a stock solution for use; before the test, 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, with 100 μL per well, and incubated at 37°C for 48 hours. The OD value was then measured using a microplate reader to check for bacterial growth. The last column of the 96-well plate served as a positive control, containing only the bacterial suspension and sterile water without antibiotics. The first column served as a negative control, containing only MRS liquid medium and sterile water without antibiotics or bacterial strains.

[0197] Table 2: Antibiotic names and dilution concentration ranges

[0198]

[0199] (3) Analysis of potential pathogenicity or drug resistance gene transferability: Staphylococcus epidermidis FeiHeS06 bacteria were collected and sent to Meiji (Shanghai) 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.

[0200] The results showed that the minimum inhibitory concentration (MIC) of the five strains to 14 common antibiotics was as follows: Figure 5 As shown in Figure 2, the antibacterial mechanisms of antibiotics primarily include hindering bacterial cell wall synthesis, inhibiting protein synthesis, affecting nucleic acid metabolism, and affecting folate metabolism, causing them to lose their ability to grow and reproduce normally, thereby inhibiting or killing them. MIC refers to the lowest drug concentration of a chemical or biological agent that inhibits bacterial growth in culture. It is the lowest antibiotic concentration at which no visible growth is observed when performing a continuous concentration gradient dilution drug susceptibility test according to standard operating procedures at a specific incubation temperature and time.

[0201] 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, 1024 μg / 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.

[0202] Virulent factors are substances derived from microorganisms that promote their own infection and the induction of specific host diseases. By comparing the Virulence Factor Database (VFDB), an annotation overview of the virulence factor genes of Staphylococcus epidermidis FeiHeS06 was obtained and statistically analyzed. Table 3 shows that S. epidermidis FeiHeS06 has homologous sequences to 24 (≥3) known virulence factors, similar to other probiotic strains analyzed in previous studies. Most of these genes encode proteins 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 differ from the currently reported major virulence factors of S. epidermidis (PIA, Bap / Bhp, PGA, Clpxp, etc.).

[0203] The Pathogen-Host Interaction Database (PHI) contains experimentally validated entries primarily from fungal, oomycete, and bacterial pathogens, infecting hosts including animals, plants, fungi, and insects. By comparing the PHI database, we obtained information on pathogen-host interaction genes in the genome of Staphylococcus epidermidis FeiHeS06. As shown in Table 4, genes associated with pathogen-host interactions (analyzed only for primates) in the FeiHeS06 genome are primarily associated with phenotypes that reduce virulence or do not affect pathogenicity.

[0204] Secretion systems are often found in bacterial genomes, which are used to release or even transport DNA, proteins, or DNA / protein complexes to the extracellular space or even to the interior of host cells. 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 secretion system. The Sec-SRP secretion system itself is not toxic. This system is a widespread protein transport mechanism in bacteria. Its main function is to direct and transport newly synthesized proteins from the cytoplasm to specific locations on the cell membrane, outside the cell, or inside the cell. This process is crucial for the normal function and structure of the cell.

[0205] Phage genomes integrated into bacterial genomes are called prophages. Prophage sequences often contain functional genes, such as antibiotic resistance and virulence genes, that enhance bacterial adaptability or confer pathogenicity. Using Phage_Finder software, prophage prediction for the genome of Staphylococcus epidermidis FeiHeS06 revealed the absence of prophage sequences, indicating a low risk of horizontal transfer of antibiotic resistance and virulence genes and a high safety profile.

[0206] Overall, the virulence-related genes carried by S. epidermidis FeiHeS06 are primarily involved in metabolism, synthesis of extracellular components, and adhesion. No significant toxin-producing genes are present. Genes associated with host interactions are primarily associated with phenotypes that attenuate virulence or do not affect pathogenicity. The strain possesses only the common, non-toxic Sec-SRP secretion system. Combined with antibiotic susceptibility (MIC) testing, S. epidermidis FeiHeS06 exhibits low antibiotic resistance. Furthermore, the strain lacks prophages, minimizing the risk of resistance gene transfer. These findings suggest that S. epidermidis FeiHeS06 is relatively safe.

[0207] Table 3: Prediction analysis of virulence factor-related genes in the genome of Staphylococcus epidermidis FeiHeS06

[0208]

[0209]

[0210] Table 4: Prediction analysis of pathogen-host interaction-related genes in the genome of Staphylococcus epidermidis FeiHeS06

[0211]

[0212] Example 5: Staphylococcus epidermidis ( 表皮葡萄球菌 ) Stability of FeiHeS06 (growth rate, maximum biomass, osmotic pressure tolerance)

[0213] The specific steps are as follows:

[0214] (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 were streaked onto MRS solid culture medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid culture medium and cultured at 37°C for 18 h for activation. Two generations of activation were performed to obtain activation liquid.

[0215] Bifidobacterium should be cultured in MRS solid medium or MRS liquid medium supplemented with 0.5 g / L L-cysteine ​​and placed under anaerobic conditions at 37°C. Other steps are the same.

[0216] (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 a microplate reader and culture 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 By plotting the values ​​against the corresponding culture time, we can draw the growth curve of the strain under certain conditions (lag phase, logarithmic growth phase, stationary phase, and decay phase). Bifidobacteria should be cultured in MRS liquid medium supplemented with 0.5 g / L L-cysteine ​​and kept under anaerobic conditions at 37°C. All other steps are the same.

[0217] (3) Maximum biomass: Inoculate the activated liquid into 1 L of MRS liquid culture medium at a 2% (v / v) inoculation rate 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.

[0218] For the cultivation of Bifidobacterium, 0.5 g / L L-cysteine ​​was added to MRS liquid medium and cultured under anaerobic conditions at 37°C. Other steps were the same.

[0219] (4) Determination of osmotic pressure tolerance: 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) inoculum 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.

[0220] For the cultivation of Bifidobacterium, 0.5 g / L L-cysteine ​​was added to MRS liquid medium and cultured under anaerobic conditions at 37°C. Other steps were the same.

[0221] The results showed that the growth curves of the five strains were as follows: Figure 6As shown in the figure, Staphylococcus epidermidis FeiHeS06 enters the logarithmic growth phase at around 5-6 hours and reaches the growth stable phase at 12-13 hours. Its growth pattern is similar to that of Staphylococcus epidermidis MRJSWX39M8, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12. However, the OD value of Staphylococcus epidermidis FeiHeS06 is higher than that of Staphylococcus epidermidis FeiHeS06 when it reaches the stable phase. 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.

[0222] Depend on Figure 7 It can be seen that when the stable period is reached, the bacterial sludge volume of Staphylococcus epidermidis FeiHeS06 is comparable 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.

[0223] In addition, by Figures 8A to 8E It can be seen that the osmotic pressure when the growth rate of Staphylococcus epidermidis FeiHeS06 begins to be inhibited is 1500 mOsm / kg, and the osmotic pressure when the growth rate is completely inhibited is 2400 mOsm / kg; the osmotic pressure when the growth rate of Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis subsp. MRJSWX29M3, and Bifidobacterium animalis lactis subsp. BB12 begins to be inhibited is 900 mOsm / kg, 900 mOsm / kg, 1500 mOsm / kg, 1500 mOsm / kg; the osmotic pressure when the growth rate of Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum infantis subsp. MRJSWX29M3, and Bifidobacterium animalis lactis subsp. BB12 is completely inhibited is 1500 mOsm / kg, 1500 mOsm / kg, 2400 mOsm / kg mOsm / kg, 2400 mOsm / kg; the osmotic pressure tolerance of Staphylococcus epidermidis FeiHeS06 was comparable to that of Bifidobacterium longum subsp. infantis MRJSWX29M3 and Bifidobacterium animalis subsp. lactis BB12, and was significantly better than that of Staphylococcus epidermidis MRJSWX39M8 of the same species.

[0224] Example 6: Staphylococcus epidermidis ( 表皮葡萄球菌 ) Acid production and pathogen antagonism of FeiHeS06

[0225] The specific steps are as follows:

[0226] (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 were streaked onto MRS solid culture medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid culture medium and cultured at 37°C for 18 h for activation. Two generations of activation were performed to obtain activation liquid.

[0227] Bifidobacterium should be cultured in MRS solid medium or MRS liquid medium supplemented with 0.5 g / L L-cysteine ​​and placed under anaerobic conditions at 37°C. Other steps are the same.

[0228] (2) Acid production capacity: The activation solution was inoculated into MRS liquid culture medium at a rate of 2% (v / v), and cultured at 37°C for 18 h until the growth stationary phase; the initial pH value and the pH value during the stationary phase of the culture medium were measured.

[0229] (3) Antagonistic ability 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 period, and use a pipette to draw 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; draw 100 μL of pathogenic bacteria onto an MRS solid plate and evenly spread it 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 cell-free supernatant of the four probiotic strains to the other four wells; incubate the plate upright for 48 h, take out the plate to observe the inhibition zone and measure it.

[0230] The results showed that the pH changes during the growth of the five bacterial strains were as follows: Figure 9 All five strains were able to produce acid, reducing the culture medium pH from an initial 7.0 to 3.5–5.0. The endpoint pH of the culture medium for Staphylococcus epidermidis FeiHeS06 was 4.79, indicating inferior acid production compared to Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 (all three strains had an endpoint pH of 3.7–3.8). This may be because Bifidobacteria and Lactobacilli belong to the lactic acid bacteria category and generally have stronger acid production abilities. The organic acids produced by these strains upon entry into the host intestine can lower the pH of the intestinal microecological environment, inhibiting the growth of pathogens and regulating the intestinal flora.

[0231] As shown in Table 5, there are great differences in the ability of probiotics to inhibit 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 pathogens 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.

[0232] Table 5: Inhibitory ability of five bacterial strains against pathogenic bacteria (Staphylococcus aureus, Salmonella, Escherichia coli and Listeria monocytogenes)

[0233]

[0234] Note: " / " indicates that the strain has no antagonistic effect on the growth of the pathogen. The data in the table are the inhibition zone sizes of the probiotic strains against the growth of pathogens, in mm.

[0235] Example 7: Staphylococcus epidermidis ( 表皮葡萄球菌 ) Regulatory effect of FeiHeS06 on lipopolysaccharide (LPS)-induced inflammatory response in macrophage RAW264.7 cells

[0236] The specific steps are as follows:

[0237] (1) Staphylococcus epidermidis FeiHeS06, Staphylococcus epidermidis MRJSWX39M8, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis BB12 were streaked onto MRS solid culture medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid culture medium and cultured at 37°C for 18 h for activation. Two generations of activation were performed to obtain activation liquid.

[0238] Bifidobacterium should be cultured in MRS solid medium or MRS liquid medium supplemented with 0.5 g / L L-cysteine ​​and placed under anaerobic conditions at 37°C. Other steps are the same.

[0239] (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.

[0240] The strain activation solution was centrifuged at 10,000 r / min for 15 min to obtain the supernatant;

[0241] In the experiment, the 5 Cell number RAW264.7 cells were seeded in 96-well plates (200 μL of culture medium was added to each well), stimulated with 100 ng / mL (ng / mL) LPS, and cultured for 24 h to establish a cell inflammation model;

[0242] In the probiotic intervention group, each well contained 200 μL of culture medium (160 μL culture medium + 40 μL strain supernatant). Cells were also stimulated with 100 ng / mL LPS and cultured for 24 h to evaluate the regulatory effect of probiotics on the LPS-induced inflammatory response of RAW264.7 cells.

[0243] The cell culture medium was obtained by centrifugation, and the levels of IL-10, TNF-α, and NO were determined according to the kit instructions. The levels of cytokines IL-10 and TNF-α were determined using enzyme-linked immunosorbent assay (ELISA). The total amount of the inflammatory mediator NO was obtained by converting nitrite to nitrite using Griess reagent based on the principle that nitrate reductase reduces nitrate to nitrite. The kits used to determine the levels of cytokines IL-10 and TNF-α were purchased from Shanghai ELISA Biotechnology Co., Ltd., and the kit used to determine the total amount of NO was purchased from Beyotime Biotechnology Co., Ltd.

[0244] The results showed that the ability of the five bacterial strains to regulate LPS-induced inflammatory response in macrophages RAW264.7 cells was as follows: Figures 10A - 10C As shown. LPS stimulated macrophages to produce an inflammatory response. Among the five bacterial strains, Staphylococcus epidermidis FeiHeS06 produced the highest level of anti-inflammatory factor IL-10 in macrophages, with the concentration in the cell supernatant reaching 224.81 pg / mL (pg / mL), demonstrating good anti-inflammatory ability ( Figure 10A Compared with the LPS model group, intervention with Staphylococcus epidermidis FeiHeS06, Lactobacillus gasseri MRJSWX2L1, Bifidobacterium longum subsp. infantis MRJSWX29M3, and Bifidobacterium animalis subsp. lactis 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 that differentiate from monocytes and play a key role in both innate and adaptive immune responses. This result suggests that Staphylococcus epidermidis FeiHeS06 has excellent immunomodulatory activity.

[0245] Example 8: Staphylococcus epidermidis ( 表皮葡萄球菌Application of FeiHeS06

[0246] Staphylococcus epidermidis FeiHeS06 can be used to prepare bacterial powder. The specific preparation process of bacterial powder is as follows:

[0247] Staphylococcus epidermidis FeiHeS06 was streaked onto 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. Two generations of activation were performed to obtain an activated solution. The activated solution was inoculated into the medium at a 2% (v / v) inoculum 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 slurry. The bacterial slurry was washed three times with physiological saline and resuspended in a lyophilization protectant (the mass ratio of lyophilization protectant to bacterial cells was 2:1) to a concentration of 1×10 10 CFU / mL to obtain a bacterial suspension; the bacterial suspension was incubated at 37°C for 60 minutes and then freeze-dried to obtain Staphylococcus epidermidis FeiHeS06 bacterial powder;

[0248] The culture medium is prepared by dissolving 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone, and 0.3% yeast extract in 87.7% water based on the total weight of the culture medium, and then adjusting the pH to 7.0 to obtain the culture medium;

[0249] The freeze-drying protective agent comprises: 130 g / L skim milk powder aqueous solution.

[0250] Example 9: Staphylococcus epidermidis ( 表皮葡萄球菌 Application of FeiHeS06

[0251] Staphylococcus epidermidis FeiHeS06 can be used to prepare capsule products. The specific preparation process of capsule products is as follows:

[0252] Staphylococcus epidermidis FeiHeS06 was streaked onto 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. Two generations of activation were performed to obtain an activated solution. The activated solution was inoculated into the medium at a 2% (v / v) inoculum 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 slurry. The bacterial slurry was washed three times with physiological saline and resuspended in a lyophilization protectant (the mass ratio of lyophilization protectant to bacterial cells 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 9CFU / mL, stirring thoroughly to uniformly disperse the cells of Staphylococcus epidermidis FeiHeS06 in the sodium alginate solution to obtain a mixed solution; extruding the mixed solution into a 20 g / L calcium chloride solution to form micelles; allowing the formed micelles to solidify for 30 minutes, filtering and collecting the micelles; freeze-drying the collected micelles for 48 hours to obtain a powder; and filling the powder into a pharmaceutical capsule to obtain a capsule product;

[0253] The culture medium is prepared by dissolving 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in 87.7% water based on the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0254] The freeze-drying protective agent comprises: 130 g / L skim milk powder aqueous solution.

[0255] Example 10: Staphylococcus epidermidis ( 表皮葡萄球菌 Application of FeiHeS06

[0256] Staphylococcus epidermidis FeiHeS06 can be used to prepare fermented milk. The specific preparation process of fermented milk is as follows:

[0257] Staphylococcus epidermidis FeiHeS06 was streaked onto 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. Two generations of activation were performed to obtain an activated solution. The activated solution was inoculated into the medium at a 2% (v / v) inoculum 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 slurry. The bacterial slurry was washed three times with physiological saline and resuspended in a lyophilization protectant (the mass ratio of lyophilization protectant to bacterial cells was 2:1) to a concentration of 1×10 10 CFU / mL to obtain a bacterial suspension; the bacterial suspension was incubated at 37°C for 60 minutes and then freeze-dried to obtain Staphylococcus epidermidis FeiHeS06 bacterial powder;

[0258] The culture medium is prepared by dissolving 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone, and 0.3% yeast extract in 87.7% water based on the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium;

[0259] The freeze-drying protective agent comprises: 130 g / L skim milk powder aqueous solution.

[0260] 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 at 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 inoculum rate of 0.03% (v / v), and the mixture was fermented at 35°C for 16 h to obtain fermented milk; the fermented milk was placed at 42°C for 4 h to coagulate, and then refrigerated at 4°C for 24 h to after-ripen to obtain a fermented milk product.

[0261] Example 11: Staphylococcus epidermidis ( 表皮葡萄球菌 Application of FeiHeS06

[0262] Staphylococcus epidermidis FeiHeS06 can be used to prepare fermented pork. The specific preparation process of fermented pork is as follows:

[0263] Staphylococcus epidermidis FeiHeS06 was streaked onto 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. Two generations of activation were performed to obtain an activated solution. The activated solution was inoculated into the medium at a 2% (v / v) inoculum 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 slurry. The bacterial slurry was washed three times with physiological saline and resuspended to a concentration of 1×10 10 CFU / mL, bacterial suspension was obtained;

[0264] Cut the pork into 1.0-1.5 kg pieces and rapidly cool them at 5-8°C for 14-18 h. Add salt (2% w / w) to the pre-cooled pork pieces and pre-marinate them at 2.0-5.0°C 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°C and 65%-75% humidity for 7-10 d. Soak the twice-marinated pork pieces in 5°C water for 10 h to wash off the surface salt, rinse with new water, and air-dry them at 10-15°C and 70%-95% humidity for 3-5 d. Adjust the density of the Staphylococcus epidermidis FeiHeS06 bacterial solution to 10 7 ~10 8CFU / mL, and 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℃ and humidity of 65%-80% for 60 days, and the conditions in the late stage of maturation were 20-25℃ and humidity of 60%-75% for about 60 days; the fermented pork blocks were trimmed and aseptically vacuum-packed to obtain pork fermented with Staphylococcus epidermidis FeiHeS06.

[0265] Example 12: Staphylococcus epidermidis ( 表皮葡萄球菌 Application of FeiHeS06

[0266] Staphylococcus epidermidis FeiHeS06 can be used to prepare fermented surimi. The specific preparation process of fermented surimi is as follows:

[0267] Staphylococcus epidermidis FeiHeS06 was streaked onto 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. Two generations of activation were performed to obtain an activated solution. The activated solution was inoculated into the medium at a 2% (v / v) inoculum 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 slurry. The bacterial slurry was washed three times with physiological saline and resuspended in a lyophilization protectant (the mass ratio of lyophilization protectant to bacterial cells was 2:1) to a concentration of 1×10 10 CFU / mL to obtain a bacterial suspension; the bacterial suspension was incubated at 37°C for 60 minutes and then freeze-dried to obtain Staphylococcus epidermidis FeiHeS06 bacterial powder;

[0268] The black carp was killed, scaled, skinned, headed, tailed, and visceral removed, then cleaned and 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 inoculum size of 10 7 CFU / g surimi, mixed evenly; the first stage fermentation conditions are temperature 30°C, relative humidity 90%, and fermentation to pH 5.2-5.5; the second stage fermentation conditions are temperature 15°C, relative humidity 80%, and fermentation to pH 4.6-4.8; after fermentation is completed, vacuum packaging is performed to obtain surimi fermented with Staphylococcus epidermidis FeiHeS06.

[0269] In summary, the present invention discloses a strain of Staphylococcus epidermidis derived from human milk and its application in pharmaceutical production. 表皮葡萄球菌) 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 fluid and simulated intestinal fluid, 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 epidermidis Staphylococcus 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 epidermidis Staphylococcus FeiHeS06 of the present invention is sensitive to common antibiotics, especially kanamycin and rifampicin, and the virulence-related genes it carries are mainly involved in metabolism, synthesis of extracellular components and providing adhesion, without significant toxin-producing genes, and the genes related to host interaction are mainly related to phenotypes with weakened virulence or no effect on pathogenicity, and it only has a common non-toxic Sec-SRP type secretion system, and does not have a 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 Staphylococcus epidermidis FeiHeS06 of the present invention has a fast growth rate, and the biomass is still 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 cells to produce the anti-inflammatory factor IL-10, and significantly reduce the secretion of the inflammatory factor TNF-α and the inflammatory mediator NO. Therefore, this Staphylococcus epidermidis FeiHeS06 has great application prospects in the preparation of products (such as medicines) that improve host intestinal health, regulate immune responses and enhance immunity with good intestinal adaptability, host safety and production stability.

[0270] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0271] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled 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 microbial preparation, wherein: The microbial preparation comprises the strain according to claim 1.

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

4. Use of the strain according to claim 1 or the microbial preparation according to claim 2 in the preparation of a drug that helps regulate intestinal flora or enhance immunity; in, The helping to regulate intestinal flora is to inhibit at least one of Staphylococcus aureus, Escherichia coli and Listeria monocytogenes; The method of helping to enhance immunity is 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.

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