Streptococcus thermophilus ST7 in modulating immune competence and antiviral activity

Abstract

To provide use of probiotics, in particular, use of Streptococcus thermophilus ST7 in modulation of immunity.SOLUTION: The present invention discloses use of Streptococcus thermophilus ST7 for modulating immunity, comprising using the Streptococcus thermophilus ST7 as an active ingredient for modulating immunity, where the Streptococcus thermophilus ST7 are deposited under the accession Nos. BCRC911126 and DSM34255. By administering an effective dose of Streptococcus thermophilus ST7 to an individual, an expression level of IL-12p40 on cells can be increased, the gut microbiota can be modulated and a Firmicutes / Bacteroidetes ratio in the intestine is increased to improve immunity and antiviral ability.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of Streptococcus thermophilus ST7 in probiotic applications, particularly in modulating immune competence and antiviral activity. [Background technology]

[0002] In modern society, pathogens that pose a threat to humans include viruses, bacteria, and parasites, among which the most threatening is the outbreak of viruses, such as the coronavirus disease 2019 (Covid-19), which has had an impact on the behavioral patterns of human society.

[0003] The human body's immune system has different specializations. For example, T cell immunity can be regulated into Th1, which has an antiviral tendency, Th2, which is antifungal and antiparasitic, and Treg, which inhibits autoimmune responses. Th1 immune responses contribute to the alleviation of viral symptoms and shorten the duration of illness, and representative antiviral indicators of Th1 are known to be IFN-γ and IL-12. Among these, consuming microorganisms may have the ability to regulate the immune system. The mechanism of promotion is known to be through regulating the bacterial flora, or that microorganisms contain active ingredients that regulate immunity.

[0004] In probiotic health foods, most probiotic supplements are provided to consumers in the form of live bacteria. The main reasons for this are: (1) it is generally believed that only live bacteria have the ability to regulate the bacterial flora; (2) probiotics may contain metabolites of live bacteria as active ingredients that regulate the immune system; and (3) the active ingredients that regulate the immune system may lose their activity during inactivation processing. However, what is worrying is that the number of live probiotic bacteria rapidly declines when stored at room temperature, and the function of the live bacteria after they are converted from live bacteria to dead bacteria is questionable.

[0005] Intestinal cells can detect the presence of viruses in various forms. For example, they use Toll-like receptors (TLRs), a type of pattern recognition receptor, to recognize double-stranded RNA (dsRNA) from viral genomes or dsRNA during viral replication, triggering subsequent immune responses. Polyinosinic acid-polycytidylic acid (hereinafter referred to as poly I:C) is a synthetic dsRNA analog. TLR3 recognizes poly I:C and dsRNA, subsequently activating NF-kB and inducing an inflammatory response. In an animal model of poly I:C-induced intestinal injury, intraperitoneal administration of poly I:C elicited an intestinal immune response, causing severe mucosal damage in the intestine in a TLR3-dependent manner.

[0006] However, in the prior art of probiotics for regulating immune function, there are few probiotics that can simultaneously significantly increase the expression level of IL-12p40 in cells, regulate the intestinal flora, and increase the ratio of Firmicutes to Bacteroidetes (Firmicutes / Bacteroidetes ratio, F / B ratio) in the intestinal tract to improve immune function and enhance antiviral effects. Furthermore, in the prior art of probiotics for regulating immune function, most are complex strains derived from multiple live probiotics, and currently, there are no single strain probiotics in an inactivated state that are used to improve immune function (antiviral ability) and regulate intestinal flora. Summary of the Invention [Problem to be solved by the invention]

[0007] Based on this, the inventors have deeply understood the shortcomings and defects of the prior art, made innovative improvements, and after many years of research, successfully isolated a probiotic that can significantly improve the expression level of IL-12p40 in cells, as well as adjust the intestinal flora and increase the proportion of Firmicutes and Bacteroidetes in the intestinal tract, thereby improving immune function and improving the effectiveness of antiviral capabilities, thereby providing a novel and low-cost health policy for regulating immune function through probiotics. [Means for solving the problem]

[0008] An object of the present invention is to provide a use of Streptococcus thermophilus ST7 in the preparation of a pharmaceutical composition for modulating immune competence, which comprises using Streptococcus thermophilus ST7 as an active ingredient for modulating immune competence, and the accession numbers of Streptococcus thermophilus ST7 are BCRC911126 and DSM34255.

[0009] In one embodiment of the present invention, the Streptococcus thermophilus ST7 is an inactivated bacterium.

[0010] In one embodiment of the present invention, the pharmaceutical composition does not contain any other bacterial species other than Streptococcus thermophilus ST7.

[0011] In one embodiment of the present invention, Streptococcus thermophilus ST7 can be used to increase the expression level of IL-12p40 in cells, and the expression level of IL-12p40 is 400 pg / mL to 1000 pg / mL.

[0012] In one embodiment of the present invention, Streptococcus thermophilus ST7 can be used to adjust the intestinal flora and increase the ratio of Firmicutes and Bacteroidetes in the intestinal tract to improve immune competence.

[0013] In one embodiment of the present invention, Streptococcus thermophilus ST7 can be used to improve antiviral capacity.

[0014] In one embodiment of the present invention, the antiviral ability is to alleviate intestinal damage caused by viruses and to improve antiviral immunity.

[0015] In one embodiment of the present invention, the antiviral capacity is determined by CD4 + T cells, CD8 + The aim is to increase T cell activation.

[0016] In one embodiment of the present invention, the antiviral capacity is determined by CD4 + T cells, CD8 + The goal is to induce T cells to express IFN-γ.

[0017] In one embodiment of the present invention, the dose of Streptococcus thermophilus ST7 is 1×10 6 ~1×10 10 CFU / day. [Effects of the Invention]

[0018] In summary, the live Streptococcus thermophilus ST7 used in this invention can induce greater levels of the antiviral marker IL-12p40 than the same species, with the effect being particularly pronounced after heat inactivation. Surprisingly, in experiments simulating viral infection by intraperitoneal injection of the mouse virus dsRNA mimetic poly I:C, inactivated Streptococcus thermophilus ST7 not only improved the antiviral marker IFN-γ and T cell antiviral activity, but also alleviated intestinal damage and altered the intestinal flora, demonstrating remarkable antiviral potential. [Brief explanation of the drawings]

[0019] [Figure 1]1 is a bar graph comparing the content of IL-12p40, an indicator of antiviral expression, in cells induced with different strains of Streptococcus thermophilus. [Figure 2] FIG. 1 is a diagram showing the animal experiment design of the present invention. [Figure 3] This is a scattergram showing the effect of Streptococcus thermophilus ST7 on body weight change in an animal experiment. [Figure 4] Pathological histology and scattergrams showing the effect of Streptococcus thermophilus ST7 on small intestinal pathology in an animal experiment. [Figure 5] This is a scattergram showing the effect of Streptococcus thermophilus ST7 on the blood IFN-γ content in an animal experiment. [Figure 6] This is a statistical graph showing the effect of Streptococcus thermophilus ST7 on the intestinal flora in an animal experiment. In the figure, panel A shows alpha diversity, and panels B and C show beta diversity by group. [Figure 7] This figure shows microbial taxa with significantly different abundances in the effects of Streptococcus thermophilus ST7 on intestinal bacteria in animal experiments. [Figure 8] This figure shows the abundance ratio of Firmicutes and Bacteroidetes in the effects of Streptococcus thermophilus ST7 on intestinal bacteria in animal experiments. [Figure 9] This figure shows that Streptococcus thermophilus ST7 affects the activation of splenic CD8+ T cells and CD4+ T cells in animal experiments. [Figure 10] This figure shows that Streptococcus thermophilus ST7 affects the expression of IFN-γ in splenic CD8+ T cells and CD4+ T cells in animal experiments. DETAILED DESCRIPTION OF THE INVENTION

[0020] [Term definition]

[0021] In this specification, many technical and scientific terms conventionally used in the biotechnology field are widely used, and in the following description, they are defined as follows, clearly consistent with the scope of the specification and claims and the scope of these terms. Other terms specifically defined below have meanings that are commonly understood by those skilled in the art.

[0022] Unless otherwise specified, all materials used in the present invention are readily available commercially. Streptococcus thermophilus ST7 used in the examples of the present invention is deposited at the Food Industry Development Research Institute, Hsinchu, Taiwan, with accession number BCRC 911126, and at the German Collection of Microorganisms and Cell Cultures (DSMZ), with accession number DSM 34255.

[0023] As used herein, "or," "and," and "and" mean "or / and" unless otherwise specified. Additionally, the terms "comprises" and "includes" are open-ended conjunctions. The preceding paragraphs are general references only and should not be construed as limitations on the subject matter of the present invention.

[0024] In this specification, "%" means "weight percentage (wt%)" unless otherwise specified; a numerical range (e.g., 10% to 11% A) includes both upper and lower limits (i.e., 10%≦A≦11%) unless otherwise specified; when the lower limit of a numerical range (e.g., B less than 0.2% or B equal to or less than 0.2%) is not specifically limited, the lower limit may be 0 (i.e., 0%≦B≦0.2%); the proportional relationship of "weight percentage" of each component may be replaced by the proportional relationship of "parts by weight".

[0025] All numerical values ​​disclosed herein are subject to ±10% standard technique measurement error (standard deviation). The term "about" means ±10%, ±5%, ±2.5%, or ±1% of a given value, i.e., "about 20%" means 20±2%, 20±1%, 20±0.5%, or 20±0.25%.

[0026] As used herein, "modulating immunocompetence" refers specifically or non-exclusively to increasing or decreasing immune response, and can inhibit allergic or autoimmune responses, while simultaneously preserving or even enhancing resistance to foreign invaders or cancer cells.

[0027] As used herein, a "pharmaceutical composition" refers to a solid or liquid composition whose form, concentration, and degree of purity are suitable for administration to a patient and capable of inducing a desired physiological change after administration. A pharmaceutical composition is sterile and / or non-pyrogenic.

[0028] As used herein, the term "individual" refers to any mammalian animal that requires or potentially requires the Streptococcus thermophilus ST7 composition of the present invention, including primates, rodents, pets, laboratory test animals, and domestic wild animals. Examples include, but are not limited to, monkeys, humans, pigs, cattle, sheep, goats, horses, mice, rats, guinea pigs, hamsters, rabbits, felines, and canines. Preferably, the individual is a mouse or a human.

[0029] As used herein, the terms "effective amount," "dosage," and similar terms refer to the amount of a drug used herein to treat, cure, prevent, or ameliorate a disease, disorder, or side effect, or to slow the rate of progression of a disease or disorder. The term also includes within its scope an amount that effectively enhances normal physiological function.

[0030] As used herein, "pharmaceutically acceptable" means that the substance or composition is compatible with the other ingredients of the pharmaceutically acceptable formulation and does not cause progression of symptoms in the patient.

[0031] The pharmaceutical compositions of the present invention can be prepared by combining the active ingredient or composition of the present invention with at least one pharmaceutically acceptable carrier (vehicle) using techniques well known to those skilled in the art, including, but not limited to, solutions, suspensions, powders, tablets, oral tablets, chewing gums, capsules, and other similar oral dosage forms suitable for the present invention.

[0032] As used herein, "pharmaceutically acceptable carrier" includes one or more of the composition types selected from solvents, emulsifiers, suspending agents, disintegrating agents, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, surfactants, and other similar or oral carriers applicable to the present invention.

[0033] If necessary, one or more solubilizing agents, buffering agents, coloring agents, flavoring agents, etc., which are commonly used in the pharmaceutical field, may be added to the composition.

[0034] As used herein, "pharmaceutically acceptable excipient" includes, but is not limited to, at least one of a polymer, a resin, a plasticizer, a filler, a lubricant, a diluent, an adhesive, a disintegrant, a solvent, a co-solvent, a surfactant, a preservative, a sweetener, a flavoring agent, a pharmaceutically acceptable dye or pigment, and a viscosity agent.

[0035] [Streptococcus thermophilus ST7]

[0036] The Streptococcus thermophilus ST7 used in the present invention was deposited at the Food Industry Development Research Institute, Hsinchu, Taiwan on April 25, 2022, with accession number BCRC 911126; and at the German Collection of Microorganisms and Cell Cultures (DSMZ), with accession number DSM34255.

[0037] In one embodiment, Streptococcus thermophilus ST7 may be a fermentation product thereof, for example, a Streptococcus thermophilus ST7 metabolite; in one embodiment, Streptococcus thermophilus ST7 may be a live or inactivated bacterium. However, from the viewpoint of improving immunity, it is preferable that Streptococcus thermophilus ST7 is an inactivated bacterium, and it is more preferable that the Streptococcus thermophilus (heat-killed) used in the present invention is heat-killed.

[0038] In the present invention, Streptococcus thermophilus ST7 functions as an active ingredient for modulating immunocompetence, but the type of active ingredient is not limited. For example, Streptococcus thermophilus ST7 may be combined with one or more other probiotic species or strains to form a probiotic composition, or Streptococcus thermophilus ST7 may be the only active ingredient. In one embodiment, from the perspective of production cost, it is preferred that the active ingredient for modulating immunocompetence contains only Streptococcus thermophilus ST7. That is, when using Streptococcus thermophilus ST7 of the present invention in preparing a pharmaceutical composition for modulating immunocompetence, the pharmaceutical composition preferably does not contain any strains or species other than Streptococcus thermophilus ST7.

[0039] [How to improve your immunity]

[0040] The present invention further discloses a method for enhancing the immunity of Streptococcus thermophilus ST7, which comprises administering an effective amount of Streptococcus thermophilus ST7, having accession numbers BCRC911126 and DSM34255, to an individual in need thereof.

[0041] The method for improving immunity of the present invention involves administering Streptococcus thermophilus ST7 to an individual (e.g., a human individual) to regulate immune function. The method for improving immunity of the present invention improves the expression level of IL-12p40 in cells of the individual, adjusts the intestinal flora (improving the ratio of Firmicutes and Bacteroidetes in the intestinal tract), improves antiviral ability (alleviating intestinal damage caused by viruses and increasing CD4 + T cells, CD8 + Increases T cell activation and CD4 + T cells, CD8 + The method can also be applied to induce immune-mediated immune responses (e.g., induce T cells to express IFN-γ).

[0042] In one embodiment, the expression level of IL-12p40, an indicator of antiviral activity induced by the Streptococcus thermophilus ST7 of the present invention, is about 400 pg / mL to 1000 pg / mL, for example, about 450 pg / mL, 500 pg / mL, 550 pg / mL, 600 pg / mL, 650 pg / mL, 700 pg / mL, 750 pg / mL, 800 pg / mL, 850 pg / mL, 900 pg / mL, 950 pg / mL, and ranges between any two of the foregoing values. The IL-12p40-inducing effect of the Streptococcus thermophilus ST7 of the present invention is superior to that of other Streptococcus thermophilus strains.

[0043] [Administration of Streptococcus thermophilus ST7]

[0044] Suitable routes of administration of the pharmaceutical compositions of the present invention include, but are not limited to, oral, intravenous, rectal, aerosol, intestinal, ocular, pulmonary, transmucosal, transdermal, vaginal, aural, nasal, or topical administration. Examples of intestinal administration include, but are not limited to, intramuscular, subcutaneous, intravenous, intramedullary injection, and intrathecal, intraventricular, intraperitoneal, intralymphatic, or intranasal injection. The dosage form of the pharmaceutical compositions provided by the present invention can be adjusted as needed and is not particularly limited, but is preferably an oral dosage form.

[0045] [Streptococcus thermophilus ST7 dosage]

[0046] In the immune-enhancing method of the present invention, an effective amount of Streptococcus thermophilus ST7 is administered to an individual in need thereof. The details vary depending on the purpose of administration, the individual's health and physical condition, age, taxonomic group (e.g., human, non-human primate, primate, etc.) of the individual, the dosage form of Streptococcus thermophilus ST7, the treating clinician's assessment of the medical condition, and other relevant factors. This amount is expected to fall within a relatively wide range, which can be determined by routine testing.

[0047] In one embodiment, different dosages of Streptococcus thermophilus ST7 may be used as needed, typically about 1 x 10 6 ~1×10 10 CFU / day, e.g., about 5 x 10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 CFU / day, and ranges between any two of the aforementioned values.

[0048] In one embodiment, different dosing cycles of Streptococcus thermophilus ST7 may be employed as needed, typically from about 2 days to 4 months or more, such as about 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 days; or, for example, about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, or more.

[0049] In one embodiment, different dosing frequencies of Streptococcus thermophilus ST7 may be employed as needed, for example, approximately 1-3 times per day, 1-6 times every 2 days, 1-9 times every 3 days, 1-14 times per week, or 1-60 times per month.

[0050] The pharmaceutical composition of the present invention can be further prepared as a food product or health food by adding an edible material. Here, the edible material includes water, fluid milk products, milk, concentrated milk, yogurt, sour milk, frozen yogurt, lactobacillus-fermented beverages, milk powder, ice cream, cheese, cottage cheese, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, jellies, candies, infant formulas, health foods, animal feeds, Chinese herbal medicines, and the like. This includes, but is not limited to, herbals or dietary supplements.

[0051] Below, several examples and comparative examples are provided to illustrate the use of Streptococcus thermophilus ST7 of the present invention in regulating immune competence, but are not intended to limit the present invention, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention.

[0052] The data in each of the following examples were analyzed using Mann-Whitney t tests and one-way ANOVA using Prism software (GraphPad, USA), and the error bars in the results represent the standard error of the mean (SEM). Linear discriminant analysis (LDA) and Linear discriminant analysis effect size (LEfSe) methods were used for bacterial enrichment analysis, and comparative data were tested using Kruskal-Wallis and Wilcoxon. Significant differences were determined when P≦0.05 and logarithmic LDA score ≧2.

[0053] Example 1: Preparation of biomaterials

[0054] Streptococcus thermophilus ST7 can be cultured in a common growth medium, such as a medium containing about 1-2% glucose, about 1-2% peptone, about 0.01-0.08% magnesium sulfate, etc. The other Streptococcus thermophilus strains used in the following examples are readily available to those skilled in the art and do not need to be deposited.

[0055] Example 2: Streptococcus thermophilus ST7 significantly induces the expression of IL-12p40, an indicator of antiviral activity in cells.

[0056] In order to demonstrate that the Streptococcus thermophilus ST7 of the present invention has a superior immunomodulatory effect compared with other Streptococcus thermophilus strains, and whether live or inactivated Streptococcus thermophilus strains affect its immunomodulatory effect, in this example, different strains of Streptococcus thermophilus, such as STI-001, STI-002, STI-003, Ta39, BCRC14086, BCRC13689, and ST7, were selected, and each of the above strains was divided into a live cell group and an inactivated cell group for testing. The specific experimental method is as follows:

[0057] Approximately 50% Brain Heart Infusion broth (BHI broth) was mixed with approximately 50% MRS (DeMan-Rogosa-Sharpe) broth, and the above-mentioned Streptococcus thermophilus strains were cultured in this broth at approximately 37°C. Samples were taken in the early stationary phase, centrifuged, washed with approximately 0.9% NaCl solution, and resuspended in approximately 0.9% NaCl solution to obtain viable cells; the viable cells were inactivated (heat-inactivated) by heating at approximately 70°C for approximately 30 minutes.

[0058] Mouse macrophage J774A.1 cell line (BCRC 60140) purchased from the Bioresource Conservation and Research Center was cultured in Dulbecco's Modified Eagle Medium (DMEM) containing approximately 10% fetal bovine serum at approximately 37°C and 5% CO2. Approximately 4 × 10 4 100 μL of J774A.1 cells were added to a 96-well plate and cultured until approximately 2 × 10 5 Each strain of Streptococcus thermophilus was added at 50 μL / well. A control group consisted of J774A.1 cells alone without any Streptococcus thermophilus. Approximately 24 hours after the addition of Streptococcus thermophilus, the culture supernatant was collected and the IL-12p40 content was measured using an enzyme-linked immunosorbent assay (ELISA) kit.

[0059] Referring to Figure 1, Figure 1 shows the content of IL-12p40, an indicator of cell antiviral expression, induced by different strains of Streptococcus thermophilus. As can be seen from the figure, compared with common strains of Streptococcus thermophilus such as STI-001, STI-002, STI-003, BCRC14086, Ta39, or BCRC13689, the Streptococcus thermophilus ST7 of the present invention can significantly induce the content of IL-12p40, an indicator of cell antiviral expression, thereby indicating that the Streptococcus thermophilus ST7 of the present invention has a better immune regulation effect than other Streptococcus thermophilus.

[0060] As can be seen from FIG. 1, the Streptococcus thermophilus ST7 of the present invention has a significant immune-modulating effect without the need for the addition of other bacterial strains. That is, the present invention provides a novel and low-cost health policy for the immune-modulation of probiotics without the need to include other bacterial strains or species other than Streptococcus thermophilus ST7.

[0061] Furthermore, as can be seen from FIG. 1, in the case of common Streptococcus thermophilus strains such as STI-001, STI-002, STI-003, BCRC14086, Ta39, or BCRC13689, the live bacteria tend to induce macrophage J774A.1 to express antiviral activity and the content of IL-12p40, an indicator of antiviral activity, tends to be higher than that of inactivated bacteria. On the other hand, in the case of the Streptococcus thermophilus ST7 of the present invention, the inactivated bacteria tend to induce macrophage J774A.1 to express antiviral activity. The results showed that the content of IL-12p40, an indicator of antiviral activity that induces IL-1, was significantly higher. This indicates that the Streptococcus thermophilus ST7 of the present invention and common Streptococcus thermophilus strains have very different characteristics in immune regulation. Furthermore, the inactivated Streptococcus thermophilus ST7 of the present invention has a relatively good effect of regulating immune competence. Therefore, the following tests were carried out using the inactivated Streptococcus thermophilus ST7.

[0062] The Streptococcus thermophilus strains other than Streptococcus thermophilus ST7 used in the examples of the present invention are used as comparative examples and should not be used as a basis for evaluating whether these strains (STI-001, STI-002, STI-003, BCRC14086, Ta39, BCRC13689) can be used in the present invention. If necessary, commercially available or other Streptococcus thermophilus strains may be used instead of these strains. The use of commercially available or other Streptococcus thermophilus strains instead of these strains (STI-001, STI-002, STI-003, BCRC14086, Ta39, BCRC13689) as comparative examples does not affect the practice of the present invention.

[0063] Example 3: Animal Experiment Design

[0064] First, approximately 8-week-old C57BL / 6JNar1 male mice (purchased from the National Animal Center) were prepared and housed in a specific-pathogen-free environment. The mice were fed a conventional balanced diet ad libitum for 7 days. Next, referring to Figure 2, which shows the design of the animal experiment of the present invention, as shown in the figure, the day after 7 days of ad libitum feeding on the balanced diet was designated as day 0. The mice were then divided into three groups: a control group, a poly I:C group, and a poly I:C+ST7 group. All three groups of mice were fed a conventional balanced diet ad libitum.

[0065] Referring to Figure 2, in the control group, pure water was administered by gavage from day 0 to day 7, and on day 8, approximately 100 μg / g of phosphate buffered saline (PBS) was intraperitoneally injected. After 2 hours, blood was collected and biochemical analysis was performed. In the poly I:C group, pure water was administered by gavage from day 0 to day 7, and approximately 100 μg / g of poly I:C was intraperitoneally injected on day 8, and after 2 hours, blood was collected and biochemical analysis was performed. In the poly I:C+ST7 group, inactivated Streptococcus thermophilus ST7 was administered by gavage at 107 cells / mouse / day from day 0 to day 7, and approximately 100 μg / g of poly I:C was intraperitoneally injected on day 8. I:C was injected intraperitoneally, and blood was collected 2 hours later for biochemical analysis. On the 9th day, all three groups were weighed, and intestinal tissues (e.g., feces from the small intestine and cecum) were collected and stored at -80°C for bacterial colony analysis. Serum was stored at -80°C and serum IFN-γ content was analyzed using a commercially available ELISA kit.

[0066] Example 4: Weight loss by reducing poly I:C in Streptococcus thermophilus ST7

[0067] Referring to Figure 3, Figure 3 shows that Streptococcus thermophilus ST7 affects the body weight of mice, and the weight loss rate of mice in the control group was significantly lower than that of mice in the control group (n=5). * Expressed in p<0.05 format; As shown in the figure, poly I:C was used to simulate viral infection, and the poly I:C viral infection group caused weight loss (approximately 5%) in mice; on the other hand, the poly I:C + ST7 administration group significantly reduced virus-induced weight loss (approximately 2%), so the Streptococcus thermophilus ST7 of the present invention has the effect of reducing virus-induced weight loss and improving viral symptoms.

[0068] Example 5: Streptococcus thermophilus ST7 alleviates poly I:C-induced small intestinal damage.

[0069] The pathological evaluation of small intestinal tissue was performed as follows: the small intestine was excised, washed with PBS, fixed in formalin solution, dehydrated, embedded, sectioned, and stained with hematoxylin and eosin (H&E stain). The pathological score and villus length were determined by referring to the following technical literature. Int J Clin Exp Pathol 2014,7,4557-4576. PLoS One 2014,9,e110549,doi:10.1371 / journal.pone.0110549.

[0070] Referring to Figure 4, Figure 4 shows that Streptococcus thermophilus ST7 affects the pathology of the small intestine. The upper image in Figure 4 is a pathological photograph of the small intestine section stained with hematoxylin and eosin, the lower left image is the small intestine histopathological score, and the lower right image is the small intestine villus length. n=5. * p<0.05, ** p<0.01; As shown in the figure, the poly I:C virus infection group induced inflammation and villus shortening in the small intestinal tissue of mice, while the poly I:C+ST7 administration group significantly reduced virus-induced small intestinal inflammation and villus shortening. Therefore, the Streptococcus thermophilus ST7 of the present invention has the effect of alleviating intestinal damage caused by virus penetration through TLR3 and improving viral symptoms.

[0071] Example 6: Poly I:C significantly increases IFN-γ expression in Streptococcus thermophilus ST7

[0072] The IFN-γ expression level was analyzed as follows: cells were cultured for 3 hours in RPMI medium containing 100 ng / mL of phorbol myristate acetate, 1 μg / mL of ionophore A23187, and 10% (volume percent) fetal bovine serum (FBS), and then cultured for another 2 hours with 5 μg / mL of brefeldin A. The cells were then stained with a T cell-associated surface marker, and IFN-γ-APC (XMG1.2) or an isotype control was intracellularly stained and analyzed by flow cytometry.

[0073] Referring to Figure 5, Figure 5 shows that Streptococcus thermophilus ST7 affects the IFN-γ content in blood. *** p<0.001; as shown in the figure, the poly I:C virus-infected group stimulated the mice to produce serum IFN-γ; on the other hand, the poly I:C+ST7 administration group significantly increased the virus-induced serum IFN-γ content, therefore, taking the Streptococcus thermophilus ST7 of the present invention can improve antiviral immunity.

[0074] Example 7: Modulation of intestinal flora after poly I:C stimulation with Streptococcus thermophilus ST7

[0075] To clarify the influence of Streptococcus thermophilus ST7 on the intestinal microbiota of mice, this study detected the alpha diversity of the fecal microbiota of each group of mice. Fecal DNA was extracted using the commercially available QIAamp Fast DNA Stool Mini Kit (QiaGen, Germany). The V3-V4 region of bacterial 16S rRNA was amplified using universal primers 341F and 805R on the Illumina Miseq system. Redundant ordinal numbers were removed using cutadapt (v1.12). The filtered sequences were processed using the DADA2 packet (v1.14.1) in R (v3.6.1) software. V3-V4 sequence variation in the samples was estimated using the DADA2 packet, and the frequency of each sequence in each sample was obtained. Bacterial species classification was performed using the SILVA database (v138). Sequence alignment and phylogenetic analysis were performed using DECIPHER (v2.14.0) and phangorn (v2.14.0) software, and colony analysis was performed using phyloseq (v1.30.0) software. Alpha diversity index was calculated to estimate bacterial species richness. Statistical analysis was performed using the Wilcoxon-Mann-Whitney test (α = 0.05). For more detailed test methods, please refer to the following technical paper: Brain SCi 2021, 11, doi:10.3390 / Brainsci11081085.

[0076] Referring to Figure 6, Figure 6 shows that Streptococcus thermophilus ST7 affects the intestinal flora of mice. Samples were collected from the feces of mice in each group to detect the distribution of fecal microbial communities. As can be seen from Figure 6A, the alpha diversity of the microbial communities in each group was similar. However, as can be seen from Figure 6B, compared to the control group of mice, treatment of the virus-infected group with poly I:C significantly changed the distribution of the microbial communities and affected their beta diversity. As can be seen from Figure 6C, the treatment group fed with poly I:C+ST7 of the Streptococcus thermophilus ST7 of the present invention was able to re-change the distribution of the microbial communities.

[0077] Figure 6B shows a principal component analysis of the control, poly I:C-infected, and poly I:C + ST7-treated mice. Significant differences in beta diversity were quantified using a permutational multivariate analysis of variance (vegan::adonis, 1000 permutations) and Betadisper (vegan::Betadisper, 1000 permutations). The Adonis and Betadisper indices yielded P values ​​< 0.05 and P values ​​> 0.05, respectively.

[0078] Referring to Figure 7, Figure 7 shows the effect of Streptococcus thermophilus ST7 on the microbial taxa of the intestinal flora. Using LEfSe analysis, significant taxon differences were defined as LDA scores (log10) ≥ 2, *p<0.05 (defined as p<0.05). As can be seen in Figure 7, the control group was enriched in Actinobacteriota, Bacteroidota, Campylobacterota, and Patescibacteria (Bacillus genus). Compared to the control group, the poly I:C group was enriched in Proteobacteria, Gammaproteobacteria, and Alistipes. Compared to the poly I:C group, the ST7 + poly I:C group was enriched in Firmicutes, Clostridia, and Lachnospirales.

[0079] Referring to Figure 8, Figure 8 shows the abundance ratio of Firmicutes and Bacteroidetes in the intestinal flora of Streptococcus thermophilus ST7. The abundance ratio of Firmicutes and Bacteroidetes is one of the factors used to assess the risk of certain diseases in recent microbial research. In enteritis, a decrease in the abundance ratio of Firmicutes and Bacteroidetes is usually observed. As can be seen from Figure 8, in the virus-infected group treated with poly I:C, the abundance ratio of Firmicutes and Bacteroidetes was reduced. However, in the poly I:C + ST7 treatment group, the administration of Streptococcus thermophilus ST7 significantly increased the abundance ratio of Firmicutes and Bacteroidetes, indicating that the Streptococcus thermophilus ST7 of the present invention can change the intestinal flora and alleviate enteritis.

[0080] From the above, since intestinal microbiota disturbances often occur simultaneously with inflammation and infection, it is generally believed that live probiotic bacteria have the potential to regulate intestinal microbiota and improve intestinal inflammation. However, the results of this study surprisingly showed that heat-inactivated Streptococcus thermophilus ST7 can regulate intestinal microbiota after poly I:C-induced enteritis. That is, the inactivated Streptococcus thermophilus ST7 of the present invention can improve the microbiota imbalance in mice with virus-induced intestinal damage.

[0081] Example 8: Streptococcus thermophilus ST7 induces CD4 + T cells, CD8 + Increases T cell activation

[0082] To demonstrate the effects of the present invention's Streptococcus thermophilus ST7 on immune cells, we performed flow cytometry experiments. Cell surface molecules were stained with specific antibodies and then analyzed using a flow cytometer. Using the following antibodies conjugated to BioLegend fluorescent dyes: CD3-FITC (2C11), CD8α-APC-Cy7 (53-6.7), CD19-PB (6D5), CD4-PE-Cy7 (GK1.5), and CD69-PE (H1.2F3), we detected CD69 expression after poly I:C stimulation to determine whether Streptococcus thermophilus ST7 could regulate T cell activation.

[0083] Referring to Figure 9, Figure 9 shows that Streptococcus thermophilus ST7 induces splenic CD8 + T cells and CD4 + It has been shown to affect T cell activation, n=5, * p<0.05, and as shown in the figure, feeding Streptococcus thermophilus ST7 alone significantly increased the number of CD69+CD8 + T cells and CD69 + CD4 + Although it did not affect the count of T cells, the poly I:C+ST7 treatment group showed a significant increase in CD69 +CD8 + T cells and CD69 + CD4 + Increased T cell counts were observed, and in the presence of poly I:C stimulation, Streptococcus thermophilus ST7 was found to be a splenic CD8 + T cells and CD4 + It has been shown to promote T cell activation, thereby improving immune and antiviral efficacy.

[0084] Example 9: Streptococcus thermophilus ST7 induces CD4 + T cells, CD8 + T cells to express IFN-γ]

[0085] IFN-γ is an important antiviral cellular hormone. To demonstrate the effect of the present invention's Streptococcus thermophilus ST7 on IFN-γ expression, mice in each group were injected with poly I:C or PBS, and their splenic CD4 + T cells and CD8 + Measure the amount of IFN-γ expressed in T cells.

[0086] Referring to Figure 10, Streptococcus thermophilus ST7 induces splenic CD8 + T cells and CD4 + It was shown to affect the expression of IFN-γ in T cells, n=5, * p<0.05, and as shown in the figure, after 2 hours of stimulation with poly I:C, splenic CD8 + T cells and CD4 + Although the expression level of IFN-γ in T cells did not change, what was surprising was that simply administering Streptococcus thermophilus ST7 increased the splenic CD8 + T cells and CD4 + Streptococcus thermophilus ST7 significantly increased the expression of IFN-γ in T cells, and the effect was even stronger in the presence of poly I:C. Regardless of whether or not poly I:C stimulation was performed, Streptococcus thermophilus ST7 significantly increased the expression of splenic CD8 + T cells and CD4 +It promotes the expression of IFN-γ in T cells, thereby improving immune function and enhancing the effectiveness of antiviral capabilities.

[0087] From the above, consuming Streptococcus thermophilus ST7 has been shown to increase the CD4 + T cells and CD8 + It can induce the antiviral capacity of T cells and, when stimulated simultaneously with poly I:C, CD4 + T cells and CD8 + It can increase T cell activation, demonstrating its ability to effectively prevent and alleviate viral infections and associated symptoms.

[0088] The Streptococcus thermophilus ST7 of the present invention has the following characteristics: (1) The induction effect of Streptococcus thermophilus ST7 on IL-12p40 is superior to that of other Streptococcus thermophilus strains. (2) After inactivation, the effect of Streptococcus thermophilus ST7 was enhanced, which is clearly different from other Streptococcus thermophilus strains. (3) Consumption of Streptococcus thermophilus ST7 (especially inactive bacteria) has the ability to regulate the bacterial flora and increase the ratio of Firmicutes to Bacteroidetes in the intestinal tract. (4) Consumption of Streptococcus thermophilus ST7 (especially inactivated bacteria) improves antiviral activity, increases serum IFN-γ expression, and increases CD4 + T cells, CD8 + Induction of T cells to express IFN-γ and CD4 upon viral infection + T cells, CD8 + By improving T cell activation, the antiviral function of T cells can be promoted.

[0089] Although the above detailed description is a specific description of possible embodiments of the present invention, this embodiment does not limit the scope of the claims of the present invention, and any equivalent implementation or modification that does not depart from the spirit of the present invention should be included in the scope of the claims of this application.

[0090] Many of the above effects indeed fully meet the statutory requirements for patentability, i.e., novelty and inventive step. We have filed this application in accordance with the law and hope that your office will approve this invention patent application to encourage invention.

Claims

1. Streptococcus thermophilus ST7 is used in the preparation of a pharmaceutical composition for regulating immunocompetence, wherein the Streptococcus thermophilus ST7 functions as an active ingredient for regulating immunocompetence, and the accession number of the Streptococcus thermophilus ST7 is BCRC911126 or DSM34255.

2. 2. The Streptococcus thermophilus ST7 according to claim 1, wherein the Streptococcus thermophilus ST7 is an inactivated bacterium.

3. The Streptococcus thermophilus ST7 according to claim 1, wherein the pharmaceutical composition does not contain any other bacterial species than Streptococcus thermophilus ST7.

4. The Streptococcus thermophilus ST7 according to claim 1 can be used to adjust the intestinal flora and increase the ratio of the Firmicutes and Bacteroidetes phyla in the intestinal tract, thereby improving immune competence.

5. The Streptococcus thermophilus ST7 according to claim 1, which can be used to improve antiviral ability.

6. The Streptococcus thermophilus ST7 according to claim 5, wherein the antiviral ability is to alleviate intestinal damage caused by viruses and improve antiviral immunity.

7. This antiviral ability is due to the CD4 ＋ T cells, CD8 ＋ The Streptococcus thermophilus ST7 according to claim 5, wherein the activation of T cells of the Streptococcus thermophilus ST7 is increased.

8. The antiviral ability is ＋ T cells, CD8 ＋ 8. The Streptococcus thermophilus ST7 of claim 7, wherein the T cells of the strain are induced to express IFN-γ.

9. The dose of Streptococcus thermophilus ST7 was 1 x 10 ６ ~1 x 10 １０ The Streptococcus thermophilus ST7 according to claim 1, wherein the colony size is 100 CFU / day.

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