Probiotic combinations and uses thereof for aiding defecation, anti-inflammation or boosting immunity

Abstract

A probiotic composition, which is composed of a yeast flora and a lactic acid bacteria flora, wherein the yeast flora is Kluyveromyces marxianus and Saccharomyces cerevisiae, the lactic acid bacteria flora is Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii and Lactobacillus plantarum, and the ratio of the number of the yeast flora to the lactic acid bacteria flora is 1:10-100.

Description

Technical Field

[0001] This invention relates to a probiotic blend, and more particularly to a probiotic blend and its use in preparing compositions for gastrointestinal health and enhancing immunity. Background Technology

[0002] Modern lifestyles often involve staying up late or lack of sleep due to various factors, preventing the body from resting and recuperating in time. This reduces the efficiency of the immune system, weakens immunity, and makes the body more susceptible to colds, allergies, and infections as it struggles to fight off external pathogens. Furthermore, the body cannot eliminate free radicals in a timely manner, leading to an excessive accumulation of free radicals that can cause inflammation and premature aging.

[0003] The gastrointestinal tract can also be considered the body's most powerful immune organ. Because it contains trillions of gut bacteria that participate in metabolism and absorption, and has a large distribution of neurons, it is often called the "second brain." Furthermore, gastrointestinal cancers consistently rank high among the top ten causes of cancer death, with colorectal cancer long holding the top spot.

[0004] Consequently, in recent years, probiotic products catering to various requirements have continuously emerged on the market. Kephir (or Kefir) is a natural yogurt originating from the Caucasus region and is considered one of the oldest traditional probiotic foods. Studies show that the strains contained in kephir can promote human health, such as reducing lactose intolerance symptoms, stimulating the immune system, lowering cholesterol, and fighting cancer. However, natural kephir contains many edible and safe strains that are not currently permitted by regulations. Furthermore, there is no scientific evidence to support the efficacy of the strains and their proportions in existing kephir products on the market. Therefore, developing scientifically based and highly effective probiotic products is essential.

[0005] To address the aforementioned issues, those skilled in the art urgently need to develop scientifically based and highly effective probiotic products to benefit the vast population with such needs. Summary of the Invention

[0006] In view of this, the present invention provides a probiotic combination and provides the use of this probiotic combination in preparing compositions for gastrointestinal health care and immune enhancement.

[0007] In some embodiments, a probiotic combination is provided, wherein the probiotic combination comprises a yeast community and a lactic acid bacteria community, wherein the yeast community is Kluyveromyces marxianus and Saccharomyces cerevisiae, and the lactic acid bacteria community comprises Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactobacillus plantarum, and the ratio of the yeast community to the lactic acid bacteria community is 1:10 to 100.

[0008] In some embodiments, a probiotic blend is provided, wherein the probiotic blend comprises a yeast community and a lactic acid bacteria community, wherein the yeast community is Kluyveromyces martensii and Saccharomyces cerevisiae, and the lactic acid bacteria community comprises Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus bulgaricus and Lactobacillus plantarum, and the ratio of the yeast community to the lactic acid bacteria community is 1:60.

[0009] In some embodiments, a probiotic blend is used to prepare a composition for enhancing antioxidant capacity, wherein the probiotic blend comprises a yeast community and a lactic acid bacteria community, wherein the yeast community is *Kluyveromyces martensii* and *Saccharomyces cerevisiae*, and the lactic acid bacteria community comprises *Leuconostoc mesenteroides* subsp. *enteroides*, *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum*, and the ratio of the yeast community to the lactic acid bacteria community is 1:10 to 100. In some embodiments, the ratio of the yeast community to the lactic acid bacteria community in the probiotic blend is 1:60.

[0010] In some embodiments, the probiotic combination enhances antioxidant capacity by increasing total antioxidant capacity.

[0011] In some embodiments, the probiotic combination enhances antioxidant capacity by increasing sulfur-containing compounds (f-Thiols) and / or GST-RBC.

[0012] In some embodiments, a probiotic blend is used to prepare a composition for enhancing and / or regulating immunity, wherein the probiotic blend comprises a yeast community and a lactic acid bacteria community, wherein the yeast community is *Kluyveromyces martensii* and *Saccharomyces cerevisiae*, and the lactic acid bacteria community comprises *Leuconostoc mesenteroides* subsp. *enteroides*, *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum*, and the ratio of the yeast community to the lactic acid bacteria community is 1:10 to 100. In some embodiments, the ratio of the yeast community to the lactic acid bacteria community in the probiotic blend is 1:60.

[0013] In some embodiments, the probiotic combination system enhances and / or regulates immunity by increasing the cytotoxicity of natural killer cells.

[0014] In some embodiments, the probiotic combination system enhances and / or modulates immunity by increasing the phagocytic capacity of neutrophils.

[0015] In some embodiments, the probiotic combination system enhances and / or regulates immunity by increasing the concentration of IL-10 in the blood.

[0016] In some embodiments, the probiotic combination system enhances and / or regulates immunity by increasing total glutathione (t-GSH) in the body.

[0017] In some embodiments, a probiotic blend is used to prepare a composition for improving gastrointestinal health, wherein the probiotic blend comprises a yeast community and a lactic acid bacteria community, wherein the yeast community is *Kluyveromyces martensii* and *Saccharomyces cerevisiae*, and the lactic acid bacteria community comprises *Leuconostoc mesenteroides* subsp. *enteroides*, *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum*, and the ratio of the yeast community to the lactic acid bacteria community is 1:10 to 100. In some embodiments, the ratio of the yeast community to the lactic acid bacteria community in the probiotic blend is 1:60.

[0018] In some embodiments, probiotic combinations improve gastrointestinal health by modulating the gut microbiota.

[0019] In some embodiments, the probiotic combination improves gastrointestinal health by relieving gastrointestinal discomfort and / or aiding in bowel movements.

[0020] In some embodiments, the effective dose of the aforementioned composition is 100 mg / day.

[0021] In some embodiments, the aforementioned composition is a pharmaceutical composition, a food composition, a beverage composition, or a nutritional supplement composition.

[0022] In summary, the probiotic combination of any embodiment can enhance antioxidant capacity, enhance and / or regulate immunity, or improve gastrointestinal health. In other words, the probiotic combination of any embodiment is suitable for preparing compositions that enhance antioxidant capacity, enhance and / or regulate immunity, or improve gastrointestinal health. In other words, the aforementioned composition has the function of enhancing antioxidant capacity, enhancing and / or regulating immunity, or improving gastrointestinal health. In some embodiments, the probiotic combination or the composition obtained therefrom may also have one or more of the following functions: enhancing total antioxidant capacity, enhancing sulfur-containing compounds (f-Thiols), enhancing GST-RBC, enhancing the cytotoxic ability of natural killer cells, enhancing the phagocytic capacity of neutrophils, enhancing the concentration of IL-10 in the blood, enhancing the total glutathione (t-GSH) in the body, regulating the gut microbiota, relieving gastrointestinal discomfort, and aiding in defecation.

[0023] Therefore, the probiotic combination of any embodiment is obtained by analyzing the bacterial flora contained in natural kefir, selecting edible and safe strains listed in positive lists from various countries, and combining the proportions of these edible and safe strains. The probiotic combination of any embodiment has been confirmed through simultaneous cell experiments and efficacy verification to use strains with proportions closest to kefir and with the same or even better efficacy, making it a rare probiotic product on the market with actual and solid scientific research. Attached Figure Description

[0024] Figure 1 This is a bar chart showing the results of cell experiments on the relative toxicity of natural killer cells.

[0025] Figure 2 This is a bar chart showing the results of cell experiments on the relative phagocytic capacity of neutrophils.

[0026] Figure 3 This is a bar chart showing the results of the human trial of total antioxidant capacity (TAC) at weeks 0 and 4.

[0027] Figure 4 This is a bar chart showing the results of human trials on glutathione S-transferase (GST-RBC) levels at weeks 0 and 4.

[0028] Figure 5 This is a bar chart showing the concentrations of sulfur-containing compounds (f-Thiols) in human trials during weeks 0 and 4.

[0029] Figure 6 This is a bar chart showing the results of human trials on IL-10 concentrations at weeks 0 and 4.

[0030] Figure 7 This is a bar chart showing the results of human trials on total glutathione (t-GSH) concentration in the body at weeks 0 and 4.

[0031] Figure 8 This is a bar chart showing the results of human trials on TNF-α concentrations at weeks 0 and 4.

[0032] Figure 9 This is a graph showing the questionnaire results regarding the severity of gastrointestinal discomfort in weeks 0 and 4.

[0033] Figure 10 This is a graph showing the results of the questionnaire regarding the number of bowel movements in weeks 0 and 4.

[0034] Preservation of biological materials

[0035] Candida albicans Y03 is deposited at the German Center for the Preservation of Microbial Cultures (Germany); November 18, 2021; accession number DSM 34088.

[0036] Saccharomyces cerevisiae TCI907 is deposited at the German Collection of Microorganisms (Germany); March 27, 2020; accession number DSM 33480.

[0037] Leuconostoc mesenteroides TCI007 is deposited at the German Center for the Depository of Microorganisms (Germany); April 17, 2020; accession number DSM 33502.

[0038] Lactococcus lactis TCI807 is deposited at the German Center for the Depository of Microorganisms (Germany); November 18, 2021; accession number DSM 34085.

[0039] Lactobacillus acidophilus TCI800 is deposited at the German Center for the Depository of Microorganisms (Germany); November 18, 2021; accession number DSM 34084.

[0040] Lactobacillus bulgaricus TCI808 is deposited at the German Center for the Depository of Microorganisms (Germany); November 18, 2021; accession number DSM 34086.

[0041] Lactobacillus embryonic stem cell TCI999 is deposited at the German Center for the Depository of Microorganisms (Germany); May 2, 2019; accession number DSM 33106. Detailed Implementation

[0042] The following describes some specific implementations of this case. Without departing from the spirit of this case, it can be implemented in many different forms, and the scope of protection should not be limited to the conditions specifically stated in the specification.

[0043] In some embodiments, a probiotic combination is provided, wherein the probiotic combination comprises a yeast community and a lactic acid bacteria community, wherein the yeast community is Kluyveromyces marxianus and Saccharomyces cerevisiae, and the lactic acid bacteria community comprises Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii, and Lactobacillus plantarum, and the ratio of the yeast community to the lactic acid bacteria community is 1:10 to 100.

[0044] In some embodiments, a probiotic blend is provided, wherein the probiotic blend comprises a yeast community and a lactic acid bacteria community, wherein the yeast community is Kluyveromyces martensii and Saccharomyces cerevisiae, and the lactic acid bacteria community comprises Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus bulgaricus and Lactobacillus plantarum, and the ratio of the yeast community to the lactic acid bacteria community is 1:60.

[0045] In some embodiments, a probiotic blend is provided, wherein the probiotic blend comprises *Kluyveromyces martensii*, *Saccharomyces cerevisiae*, *Leuconostoc mesenteroides* subsp. *enteroides*, *Lactococcus lactis* subsp. *lactocortisone*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum*. The weight ratio of the bacterial powders of *Kluyveromyces martensii*, *Saccharomyces cerevisiae*, *Leuconostoc mesenteroides* subsp. *enteroides*, *Lactococcus lactis* subsp. *lactocortisone*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum* in this probiotic blend is 2-6:1-2:1-2:1-2:1-2:1-2:1-2:1-2. For example, the weight ratio of the bacterial powders of *Kluyveromyces martensii*, *Saccharomyces cerevisiae*, *Leuconostoc mesenteroides* subsp. *enteroides*, *Lactococcus lactis* subsp. *lactocortisone*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum* in the probiotic blend is 4:1:1:1:1:1:1.

[0046] In some embodiments, Kluyveromyces marxianus is Kluyveromyces marxianus Y03, isolated from milk, and deposited at DSMZ (Germany Center for Culture Collections of Microorganisms, Germany) with accession number DSM 34088.

[0047] In some embodiments, the brewer's yeast (Saccharomyces cerevisiae) is Saccharomyces cerevisiae TCI907, isolated from the human intestine, deposited at DSMZ (Germany), with accession number DSM 33480.

[0048] In some embodiments, the subsp. mesenteroides of Leuconostoc mesenteroides is Leuconostoc mesenteroides TCI007, isolated from figs, deposited in DSMZ (Germany), with accession number DSM 33502.

[0049] In some embodiments, Lactococcus lactis subsp. lactis is Lactococcus lactis TCI807, isolated from waterfall water, deposited in DSMZ (Germany), and the accession number is DSM 34085.

[0050] In some embodiments, Lactobacillus acidophilus is Lactobacillus acidophilus TCI800, isolated from the human intestine, deposited in DSMZ (Germany), with accession number DSM 34084.

[0051] In some embodiments, Lactobacillus delbrueckii is Lactobacillus delbrueckii subsp. Bulgaricus TCI808, isolated from the human intestine, deposited at DSMZ (Germany), with accession number DSM 34086.

[0052] In some embodiments, Lactobacillus plantarum is Lactobacillus plantarum TCI999, isolated from the ancient Chinese medical text "Chinese Ancient Medical Book - Immortal's Elixir", deposited in DSMZ (Germany) with accession number DSM 33106.

[0053] In some embodiments, the bacterial powders of Kluyveromyces martensii, Saccharomyces cerevisiae, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillus plantarum are all produced by TCI Biotech Inc.

[0054] In some embodiments, the aforementioned bacterial powder preparation process involves preparing a culture medium, sterilizing and cooling the medium, and then planting the aforementioned bacterial strains in the medium for fermentation. After fermentation, the strains are centrifuged at ultrahigh speed to obtain the fermented bacterial strains. The fermented bacterial strains are then encapsulated or coated and freeze-dried to obtain freeze-dried bacterial strains. Next, the freeze-dried bacterial strains are ground, sieved, packaged, and stored in a freezer to obtain the bacterial powder.

[0055] In some embodiments, the probiotic combination is prepared under a constant temperature and humidity environment, with a preparation temperature <25°C and humidity <60%. For example, the preparation temperature of the probiotic combination is 23±2°C and the humidity is 50±10%.

[0056] In some embodiments, the storage temperature of the bacterial powder and probiotic combination is -18±2℃.

[0057] In some embodiments, the mixing time of the bacterial powder is 15 to 20 minutes. This mixing time ensures that the bacterial powder is mixed evenly and that the number of live bacteria is preserved to the maximum extent, resulting in the best quality probiotic blend. Since probiotics mainly survive in a facultative anaerobic manner, if the mixing time is too long, it will not only lead to the absorption of moisture from the air (increased water activity) and thus cause contamination by other bacteria, but also cause a decrease in the number of live bacteria due to prolonged exposure to oxygen. If the mixing time is too short, the bacterial powder itself will not mix evenly due to its viscosity.

[0058] In some embodiments, after the probiotic powder is thoroughly mixed, samples are taken for testing to confirm that the probiotic combination is free of clumping. Uneven mixing of the probiotic powder will result in inconsistent levels of each bacterial species when the end consumer ingests the prescribed amount, or even in severe cases, only a single bacterial species. Furthermore, clumping of the probiotic powder indicates excessive humidity, which can easily lead to contamination by other bacteria. Therefore, sampling and testing to confirm that the probiotic combination is free of clumping is to ensure that the consumer ingests the prescribed amount of probiotic powder with the same bacterial species content and to ensure that the humidity is not too high, eliminating the possibility of contamination by other bacteria.

[0059] In some embodiments, after the bacterial powder is mixed evenly, samples are taken for testing to confirm that the moisture content of the probiotic combination is <5%. Studies have shown that water activity can increase pathogen contamination and even reduce the survival rate and acid production capacity of probiotics. In addition, excessive moisture content can cause the bacterial powder to clump, making it difficult for subsequent factory production processes. Therefore, based on production experience, the moisture content must be <5%.

[0060] In some embodiments, after the probiotic powder is thoroughly mixed, samples are taken for testing to confirm that the heavy metal content of the probiotic blend meets the specifications. The testing method follows the international standard MOHW Food No. 1021950329 Announced, and analysis is performed using ICP / OES.

[0061] In some embodiments, after the bacterial powder is mixed evenly, samples are taken for testing to confirm that the number of live bacteria in the probiotic combination meets the specifications. The testing method refers to the microbial testing method, and the number of aerobic bacteria, Salmonella, Pseudomonas aeruginosa, and lactic acid bacteria is determined using specified culture media.

[0062] In some embodiments, when the probiotic combination consists of yeast and lactic acid bacteria, this probiotic combination has the ability to enhance antioxidant capacity. In other words, when the probiotic combination is administered to an individual, it can enhance that individual's antioxidant capacity. Therefore, the probiotic combination is suitable for preparing compositions that enhance antioxidant capacity. In this case, the yeast is *Kluyveromyces martensii* and *Saccharomyces cerevisiae*, the lactic acid bacteria are *Leuconostoc mesenteroides* subsp. *enteroides*, *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum*, and the ratio of yeast to lactic acid bacteria in this probiotic combination is 1:10 to 100. In some embodiments, the ratio of yeast to lactic acid bacteria in this probiotic combination is 1:60.

[0063] In some embodiments, the aforementioned probiotic combination has the ability to enhance total antioxidant capacity, enhance sulfur-containing compounds (f-Thiols), enhance GST-RBC, or any combination thereof. In other words, when administered to an individual, the probiotic combination can enhance total antioxidant capacity, enhance sulfur-containing compounds (f-Thiols), enhance GST-RBC, or any combination thereof. Therefore, the probiotic combination is suitable for preparing compositions that enhance total antioxidant capacity, enhance sulfur-containing compounds (f-Thiols), enhance GST-RBC, or any combination thereof.

[0064] In some embodiments, when the probiotic combination consists of yeast and lactic acid bacteria, the probiotic combination has the ability to enhance and / or regulate immunity. In other words, when the probiotic combination is administered to an individual, it can enhance and / or regulate the individual's immunity. Therefore, the probiotic combination is suitable for preparing compositions that enhance and / or regulate immunity. In this case, the yeast is *Kluyveromyces martensii* and *Saccharomyces cerevisiae*, the lactic acid bacteria are *Leuconostoc mesenteroides* subsp. *mesenteroides*, *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum*, and the ratio of yeast to lactic acid bacteria in the probiotic combination is 1:10 to 100. In some embodiments, the ratio of yeast to lactic acid bacteria in the probiotic combination is 1:60.

[0065] In some embodiments, the aforementioned probiotic combination has the ability to enhance the cytotoxicity of natural killer cells, enhance the phagocytic capacity of neutrophils, increase the blood concentration of IL-10, increase the total glutathione (t-GSH) in vivo, or any combination thereof. In other words, when administered to an individual, the probiotic combination can enhance the cytotoxicity of natural killer cells, enhance the phagocytic capacity of neutrophils, increase the blood concentration of IL-10, increase the total glutathione (t-GSH) in vivo, or any combination thereof. Therefore, the probiotic combination is suitable for preparing compositions that enhance the cytotoxicity of natural killer cells, enhance the phagocytic capacity of neutrophils, increase the blood concentration of IL-10, increase the total glutathione (t-GSH) in vivo, or any combination thereof.

[0066] In some embodiments, when the probiotic combination consists of yeast and lactic acid bacteria, this probiotic combination has the ability to improve gastrointestinal health. In other words, when the probiotic combination is administered to an individual, it can improve that individual's gastrointestinal health. Therefore, the probiotic combination is suitable for preparing compositions that improve gastrointestinal health. In this case, the yeast is *Kluyveromyces martensii* and *Saccharomyces cerevisiae*, the lactic acid bacteria are *Leuconostoc mesenteroides* subsp. *mesenteroides*, *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum*, and the ratio of yeast to lactic acid bacteria in this probiotic combination is 1:10 to 100. In some embodiments, the ratio of yeast to lactic acid bacteria in this probiotic combination is 1:60.

[0067] In some embodiments, the aforementioned probiotic combination has the ability to regulate the gut microbiota, relieve gastrointestinal discomfort, aid defecation, or any combination thereof. In other words, when administered to an individual, the probiotic combination can regulate the gut microbiota, relieve gastrointestinal discomfort, aid defecation, or any combination thereof. Therefore, the probiotic combination is suitable for preparing compositions that regulate the gut microbiota, relieve gastrointestinal discomfort, aid defecation, or any combination thereof.

[0068] In some embodiments, the aforementioned individual may be a person.

[0069] In some embodiments, the effective dose of the aforementioned composition is 100 mg / day.

[0070] In some embodiments, the aforementioned probiotic combination has the ability to enhance antioxidant capacity, enhance and / or regulate immunity, or improve gastrointestinal health. Therefore, the aforementioned probiotic combination can be used to prepare compositions that have the ability to enhance antioxidant capacity, enhance and / or regulate immunity, or improve gastrointestinal health.

[0071] In some embodiments, the resulting composition may be a pharmaceutical composition, a food composition, a beverage composition, or a nutritional supplement composition.

[0072] In some embodiments, when the aforementioned composition is a pharmaceutical composition, the pharmaceutical composition comprises an effective amount of a probiotic combination. This pharmaceutical composition can be manufactured using techniques well known to those skilled in the art into dosage forms suitable for enteral, parenterally, orally, or topically administration.

[0073] In some embodiments, the dosage form for oral or enteral administration may be, but is not limited to, tablets, troche, lozenges, pills, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, or similar substances.

[0074] In some embodiments, the dosage form for non-enteral or local administration may be, but is not limited to, an injection [e.g., a sterile aqueous solution or dispersion], a sterile powder, an external preparation, or the like.

[0075] In some embodiments, the administration method of the injectable may be, but is not limited to, intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, or intralesional injection.

[0076] In some embodiments, the pharmaceutical composition containing an effective amount of the probiotic combination may further comprise a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technologies. In some embodiments, the pharmaceutically acceptable carrier may be one or more of the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The type and quantity of carrier selected fall within the scope of expertise and routine practice of those skilled in the art. Among them, the solvents that can be used as pharmaceutically acceptable carriers are water, normal saline, phosphate buffered saline (PBS), or an alcohol-containing aqueous solution.

[0077] In some embodiments, a pharmaceutical composition containing an effective amount of a probiotic combination can be manufactured using techniques known to those skilled in the art as a topical preparation suitable for application to the skin. This includes, but is not limited to, emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions, salves, and bandages.

[0078] In some embodiments, when the aforementioned pharmaceutical composition is an external preparation, the pharmaceutical composition may be prepared by mixing an effective amount of a probiotic combination with a base known to those skilled in the art.

[0079] In some embodiments, the substrate may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons [such as petroleum jelly and white petrolatum], waxes [such as paraffin and yellow wax], preserving agents, antioxidants, surfactants, absorption enhancers, stabilizing agents, gelling agents [such as Carbopol® 974P, microcrystalline cellulose, and carboxymethylcellulose], active agents, humectants, odor absorbers, fragrances, pH adjusting agents, and chelating agents. Agents, emulsifiers, occlusive agents, softeners, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants, etc. The selection and quantity of these additives fall within the scope of the professional competence and routine techniques of those skilled in the art.

[0080] In some embodiments, when the aforementioned composition is a food composition, beverage composition, or nutritional supplement composition, the food composition, beverage composition, or nutritional supplement composition contains an effective amount of a probiotic combination. The food composition, beverage composition, or nutritional supplement composition may be in the form of powder, granules, solution, colloid, or paste.

[0081] In some embodiments, a food composition, beverage composition, or nutritional supplement composition containing a probiotic combination may be a food product or a food additive.

[0082] In some embodiments, the food composition, beverage composition, or nutritional supplement composition containing a probiotic combination may be beverages, fermented foods, bakery products, health foods, or dietary supplements. In some embodiments, the food composition, beverage composition, or nutritional supplement composition containing a probiotic combination may further include an adjuvant. For example, the adjuvant may be maltodextrin, malic acid, sucralose, citric acid, fruit flavoring, honey flavoring, steviol glycosides, or combinations thereof. The type and quantity of the carrier selected fall within the scope of professional competence and routine practice of those skilled in the art.

[0083] In some embodiments, the food composition, beverage composition, or nutritional supplement composition containing a probiotic combination may be a seasoning, sweetener, flavoring, pH adjuster, emulsifier, colorant, or stabilizer, etc.

[0084] Unless otherwise specified, the "%" symbol in the following examples refers to weight percentage.

[0085] Unless otherwise specified, the experimental procedures in the following examples were performed at room temperature (approximately 25°C) and atmospheric pressure (1 atm).

[0086] Example 1: Preparation of bacterial powder

[0087] I. Raw materials:

[0088] 1. Kluyveromyces marxianus, specifically Kluyveromyces marxianus Y03 (this strain is deposited in DSMZ (Germany), with accession number DSM 34088).

[0089] 2. Saccharomyces cerevisiae, specifically Saccharomyces cerevisiae TCI907 (this strain is deposited in DSMZ (Germany), with accession number DSM 33480).

[0090] 3. Leuconostoc mesenteroides subsp. mesenteroides, specifically Leuconostoc mesenteroides TCI007 (this bacterium is deposited in DSMZ (Germany), with accession number DSM 33502).

[0091] 4. Lactococcus lactis subsp. lactis, specifically Lactococcus lactis TCI807 (deposited in DSMZ (Germany), with accession number DSM 34085).

[0092] 5. Lactobacillus acidophilus, specifically Lactobacillus acidophilus TCI800 (deposited in DSMZ (Germany), accession number DSM 34084).

[0093] 6. Lactobacillus delbrueckii, specifically Lactobacillus delbrueckii subsp. Bulgaricus TCI808 (this bacterium is deposited in DSMZ (Germany), with accession number DSM 34086).

[0094] 7. Lactobacillus plantarum, specifically Lactobacillus plantarum TCI999 (this bacterium is deposited in DSMZ (Germany), with accession number DSM 33106).

[0095] 8. Culture medium: BD Difco™ Lactobacilli MRS Broth, purchased from BD.

[0096] II. Preparation process:

[0097] 1. Inoculate strains 1 to 7 (i.e., Kluyveromyces martensii, Saccharomyces cerevisiae, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Lactobacillus bulgaricus and Lactobacillus plantarum) at a concentration of 10% into culture flasks containing culture medium, and ferment each strain at 37°C for 16 to 18 hours.

[0098] 2. After fermentation, each strain was subjected to ultra-high speed centrifugation, and the supernatant was removed.

[0099] 3. Embed or coat each strain at room temperature for 5-10 minutes.

[0100] 4. After embedding or coating, freeze-dry each strain at -80℃ for 24 hours.

[0101] 5. After freeze-drying, each strain is ground and sieved through a 300-mesh sieve, then packaged and stored in a freezer to obtain the bacterial powder for each strain. The bacterial powders for each strain are: *Candida marxianus* Y03, *Saccharomyces cerevisiae* TCI907, *Leuconostoc mesenteroides* TCI007, *Lactococcus lactis* TCI807, *Lactobacillus acidophilus* TCI800, *Lactobacillus delbrueckii* subsp. Bulgaricus TCI808, and *Lactobacillus plantarum* TCI999. The freezer temperature is set at -18±2℃.

[0102] Example 2: Preparation of probiotic combinations

[0103] I. Raw materials:

[0104] 1. The Kluyveromyces marxianus powder prepared in Example 1 is Kluyveromyces marxianus Y03 powder.

[0105] 2. The brewer's yeast (Saccharomyces cerevisiae) powder obtained in Example 1 is brewer's yeast (Saccharomyces cerevisiae) TCI907 powder.

[0106] 3. The Leuconostoc mesenteroides subsp. mesenteroides bacterial powder prepared in Example 1 is Leuconostoc mesenteroides TCI007 bacterial powder.

[0107] 4. The Lactococcus lactis subsp. lactis bacterial powder obtained in Example 1 is Lactococcus lactis TCI807 bacterial powder.

[0108] 5. The Lactobacillus acidophilus powder prepared in Example 1 is Lactobacillus acidophilus TCI800 powder.

[0109] 6. The Lactobacillus delbrueckii bacterial powder obtained in Example 1 is Lactobacillus delbrueckii subsp. Bulgaricus TCI808 bacterial powder.

[0110] 7. The Lactobacillus plantarum powder obtained in Example 1 is Lactobacillus plantarum TCI999 powder.

[0111] II. Preparation process:

[0112] 1. The bacterial powders (raw materials 1 to 7, namely, *Kluyveromyces martensii* powder, *Saccharomyces cerevisiae* powder, *Leuconostoc mesenteroides* subsp. *enteroides* powder, *Lactococcus lactis* subsp. *lactolaccos* powder, *Lactobacillus acidophilus* powder, *Lactobacillus bulgaricus* powder, and *Lactobacillus plantarum* powder) stored in the freezer are sieved through a 30-mesh sieve under a constant temperature and humidity environment (temperature 23±2℃ and humidity 50±10%) to obtain uniformly sized bacterial powders. The freezer temperature is set at -18±2℃.

[0113] 2. Add the uniformly granulated bacterial powder to the mixing tank according to the following weight ratio: *Kluyveromyces martensii* : *Saccharomyces cerevisiae* : *Leuconostoc mesenteroides* subsp. *enteroides* : *Lactococcus lactis* subsp. *lactocortisone* : *Lactobacillus acidophilus* : *Lactobacillus bulgaricus* : *Lactobacillus plantarum* = 4 : 1 : 1 : 1 : 1 : 1 : 1. The bacterial count ratio of *Kluyveromyces martensii* : *Saccharomyces cerevisiae* : *Leuconostoc mesenteroides* subsp. *enteroides* : *Lactococcus lactis* subsp. *lactocortisone* : *Lactobacillus acidophilus* : *Lactobacillus bulgaricus* : *Lactobacillus plantarum* is 4 : 100 : 100 : 10 : 1 : 100. The bacterial counts of the strains added in this example are shown in Table 1.

[0114] Table 1

[0115]

[0116] 3. Turn on the mixing tank and set the speed, continuously stir and mix for 15 minutes to obtain a uniformly mixed bacterial powder, which is a probiotic combination of one embodiment.

[0117] 4. Sampling (probiotic combination) testing to confirm that the probiotic combination is free of clumping, and that the moisture content is <5%, heavy metal content, and live bacteria count all meet specifications. Specifically, a live bacteria count meeting specifications means a total lactic acid bacteria count ≥ 1 x 10⁻⁶. 10 cfu / g, and total yeast count ≥ 1 x 10⁻⁶ 8 cfu / g.

[0118] 5. After filling the probiotic combination into the aluminum bag, vacuum-seal the bag, seal it, and transfer it to a freezer for storage to facilitate subsequent testing. The freezer temperature is set at -18±2℃.

[0119] Example 3: Experiment on the natural killer cytotoxic ability of probiotic combinations

[0120] I. Materials and Instruments:

[0121] 1. Cell line: Human leukemia cells, purchased from ATCC (American Type Culture Collection), cell number CRL-3344, hereinafter referred to as K562 cells.

[0122] 2. Cell culture medium: RPMI (Roswell Park Memorial Institute, purchased from Gibco, product number 22400089), with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028) and 1% antibiotic (purchased from Gibco, product number 15240-062).

[0123] 3. Calcein AM, purchased from Thermo, product number C3099.

[0124] 4. Propidium Iodide, purchased from BD Biosciences, product number 556463.

[0125] 5. Flow cytometer, purchased from BD, model Accuri™ C6 Plus.

[0126] II. Test Procedure:

[0127] 1. The following experiment consists of four experimental groups: blank group, K562 group, control group, and experimental group. Each group is performed in triplicate (meaning each group has three wells).

[0128] 2. Purify human blood to obtain PBMCs (peripheral blood mononuclear cells, containing approximately 10% natural killer cells), resuspend PBMCs in cell culture medium, add Calcein AM (chromosome volume ratio 1:5000), and let stand for 5 minutes for staining.

[0129] 3. After staining, centrifuge the cells to remove the supernatant, then wash the cells three to five times with PBS to remove excess Calcein AM and obtain stained PBMCs.

[0130] 4. Divide the stained PBMCs into 1x10 wells. 6 Cells were seeded in the wells of the blank group, control group, and experimental group in a V-bottom 96-well plate.

[0131] 5. Pack K562 cells at 5 x 10⁻⁶ cells per well. 4 Cells were seeded in the wells of the K562 group, control group, and experimental group in a V-bottom 96-well plate.

[0132] 6. Add 0.0625% (v / v) of the probiotic combination prepared in Example 1 to the wells of the experimental group.

[0133] 7. Add Propidium Iodide (chromosome volume ratio 1:200) to the wells of the control group and K562 group, and confirm the distribution range of cells in the scatter plot using flow cytometry. Select the PBMC and K562 populations in the scatter plot for subsequent red fluorescence (PE-A) signal quantification experiments. The red fluorescence signal values ​​of the control group and K562 group are thus obtained.

[0134] 8. Transfer the control group and experimental group to an incubator and incubate at 37°C for 6 hours. Then, add Propidium Iodide (chromosome volume ratio 1:200) to the wells of the control group and experimental group, and analyze and quantify the red fluorescence signal using flow cytometry. The red fluorescence signal values ​​of the control group and experimental group are thus obtained.

[0135] III. Experimental Results:

[0136] The relative natural killer cell cytotoxicity of all groups was calculated using the following formula: Relative natural killer cell cytotoxicity (%) = (red fluorescence signal value of each group / red fluorescence signal value of the blank group) × 100%.

[0137] Statistically significant differences in measurement results between the control group and other groups, and between the control group and other groups, were obtained using the Student's t-test. In the graph, "*" indicates a p-value less than 0.05 compared to the control group, "**" indicates a p-value less than 0.01 compared to the control group, and "***" indicates a p-value less than 0.001 compared to the control group; in the graph, "#" indicates a p-value less than 0.05 compared to the control group, "##" indicates a p-value less than 0.01 compared to the control group, and "###" indicates a p-value less than 0.001 compared to the control group.

[0138] Natural killer cells (NK cells) are a natural army within the immune system. They don't require special training; upon encountering virus-infected cells, they activate automatically, rapidly lysing enemy cells and preventing the virus from surviving and replicating in the body. Therefore, we analyzed the cytotoxic capacity of natural killer cells over 6 hours using PI / Calcein-AM staining and flow cytometry to evaluate whether the probiotic combination could promote the killing of leukemia cancer cells by natural killer cells. Please refer to [link / reference]. Figure 1 The natural killer cells in the control group existed alone and were not treated with samples; therefore, the results of the control group represent the performance of natural killer cells under normal physiological metabolic conditions. Thus, with the relative natural killer cell cytotoxicity of the control group set at 100%, the relative natural killer cell cytotoxicity of the control group was 109%, while the relative natural killer cell cytotoxicity of the experimental group was 148%. In other words, compared to the control group, when natural killer cells were co-cultured with K562 cells, the K562 cells induced the cytotoxicity of the natural killer cells, increasing the relative natural killer cell cytotoxicity of the control group by approximately 9%. Compared to the control group, when natural killer cells in the experimental group were co-cultured with K562 cells and supplemented with probiotics, the relative natural killer cell cytotoxicity of the experimental group was significantly increased by approximately 48%. Compared to the control group, when natural killer cells in the experimental group were co-cultured with K562 cells and supplemented with probiotics, the relative natural killer cell cytotoxicity of the experimental group was significantly increased by approximately 39%.

[0139] Therefore, it can be seen that probiotic combinations can significantly enhance the cytotoxic ability of natural killer cells and improve innate immunity. Probiotic combinations can significantly enhance the cytotoxic ability of natural killer cells, increasing their ability to kill cancer cells, virus-infected cells, cancerous cells, and aging or stressed cells. Furthermore, they can enhance the early detection and elimination of abnormal cells, preventing their growth in the body and thus preventing the formation of cancer.

[0140] Example 4: Neutrophilic phagocytic capacity test of probiotic combinations

[0141] I. Materials and Instruments:

[0142] 1. Cell culture medium: RPMI (Roswell Park Memorial Institute, purchased from Gibco, product number 22400089), with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028) and 1% antibiotic (purchased from Gibco, product number 15240-062).

[0143] 2. Fluorescent microbeads, SPHEROTM Fluorescent Yellow Particles, purchased from Spheroteth, product number FH-2052-2.

[0144] 3. Flow cytometer, purchased from BD, model Accuri™ C6 Plus.

[0145] II. Test Procedure:

[0146] 1. Purify peripheral blood to obtain neutrophils, and distribute them at a rate of 1 × 10⁻⁶ per well. 6 Cells were seeded in 6-well culture dishes. Neutrophils were divided into three experimental groups: a blank control group, a control group, and an experimental group. Each group was performed in triplicate (meaning each group had three wells).

[0147] 2. Add 0.0625% (v / v) of the probiotic combination prepared in Example 1 to the wells of the experimental group.

[0148] 3. After adding 0.5% fluorescent microbeads to the wells of the control group and experimental group, all groups were incubated at 37°C for 4 hours.

[0149] 4. After collecting cells from each group, the cells were washed with PBS and then resuspended in PBS. Next, flow cytometry was used to analyze and quantify the FITC fluorescence signal of each group. The fluorescence signal values ​​for each group were then obtained.

[0150] III. Experimental Results:

[0151] The relative neutrophil phagocytic capacity of all groups was calculated using the following formula: Relative neutrophil phagocytic capacity (%) = (fluorescence signal value of each group / fluorescence signal value of the blank group) × 100%.

[0152] Statistically significant differences in measurement results between the control group and other groups, and between the control group and other groups, were obtained using Student's t-test statistical analysis. In the graph, "*" represents a p-value less than 0.05 compared to the control group, "**" represents a p-value less than 0.01 compared to the control group, and "***" represents a p-value less than 0.001 compared to the control group; in the graph, "#" represents a p-value less than 0.05 compared to the control group, "##" represents a p-value less than 0.01 compared to the control group, and "###" represents a p-value less than 0.001 compared to the control group.

[0153] Neutrophils are the most abundant type of white blood cell in human blood and are the primary non-specific phagocytic cells. These cells can immediately eliminate invaders upon detection. Besides macrophages, they are the most important phagocytic cells in the human body after bacterial infection. Neutrophils play a crucial role in the non-specific immune system. Therefore, neutrophils were isolated from peripheral blood and co-reacted with fluorescent particles for 4 hours. The fluorescence performance of the neutrophils was analyzed to evaluate whether the probiotic combination could enhance the phagocytic capacity of neutrophils. Please refer to [link to relevant documentation]. Figure 2 The neutrophils in the blank group existed independently and were not treated with samples; therefore, the results of the blank group represent the performance of neutrophils under normal physiological metabolic conditions. Thus, with the relative neutrophil phagocytic capacity of the blank group set at 100%, the relative neutrophil phagocytic capacity of the control group was 115.9%, while that of the experimental group was 129.7%. In other words, compared to the blank group, the neutrophils in the control group already possessed a certain neutrophil phagocytic capacity, and the relative neutrophil phagocytic capacity of the control group was significantly increased by approximately 15.9%. Compared to the blank group, the relative neutrophil phagocytic capacity of the experimental group significantly increased by approximately 29.7% after the addition of the probiotic combination. Compared to the control group, the relative neutrophil phagocytic capacity of the experimental group significantly increased by approximately 13.8% after the addition of the probiotic combination.

[0154] Therefore, it can be seen that probiotic combinations can significantly enhance the phagocytic capacity of neutrophils, improve innate immunity, and boost the non-specific immune system. Probiotic combinations can also significantly enhance the phagocytic capacity of neutrophils and improve the ability to clear pathogens and kill bacteria.

[0155] Example 5: Human trials of probiotic combinations - determination of antioxidant capacity

[0156] I. Test Procedure:

[0157] Six adult subjects with weakened immunity, gastrointestinal discomfort, or constipation were given one probiotic combination capsule (i.e., a capsule containing 100 mg of the probiotic combination prepared in Example 1) daily for four weeks (i.e., 28 days).

[0158] Blood samples were drawn from subjects before starting treatment (week 0) and after 28 days of treatment (week 4) to measure changes in total antioxidant capacity (TAC), glutathione S-transferase (GST-RBC) levels, and sulfur-containing compound (f-Thiols) concentrations in the blood before and after taking the probiotic combination containing one example.

[0159] In this embodiment, the total antioxidant capacity (TAC), glutathione S-transferase (GST-RBC) levels, and sulfur-containing compound (f-Thiols) concentrations in the subjects' blood were measured by Li-Jen Laboratory (Taiwan, China) in accordance with blood testing standards.

[0160] It should be noted that the statistically significant difference between the results of week 0 and week 4 was obtained through Student's t-test statistical analysis. In the graph, "*" indicates that the p-value is less than 0.05 compared with week 0, "**" indicates that the p-value is less than 0.01 compared with week 0, and "***" indicates that the p-value is less than 0.001 compared with week 0.

[0161] II. Experimental Results:

[0162] Please see Figure 3 At week 0, the subjects' total antioxidant capacity (TAC) was approximately 0.75 mmol / L, increasing to approximately 0.82 mmol / L at week 4. Therefore, setting the relative TAC at week 0 as 100% and converting the week 4 measurements to relative TAC, we find that the subjects' relative TAC increased to approximately 110.1% at week 4, an increase of 10.1%. Total Antioxidant Capacity (TAC) measures the overall antioxidant capacity of non-enzymatic antioxidants in plasma, providing a more accurate reflection of systemic antioxidant capacity than measuring a single antioxidant. A higher TAC value indicates higher antioxidant capacity in the body. Thus, the probiotic combination in any embodiment can enhance the body's antioxidant capacity, helping to resist free radical-induced damage.

[0163] Please see Figure 4At week 0, the subjects' GST-RBC content was approximately 6.36 U / g-Hb, which increased to approximately 6.72 U / g-Hb at week 4. Therefore, setting the relative GST-RBC content at week 0 as 100%, and converting the week 4 measurements to relative GST-RBC content, we find that the subjects' relative GST-RBC content at week 4 increased to approximately 105.7%, an increase of 5.7%. This indicates that the probiotic combination in any embodiment can increase glutathione S-transferase, thereby enhancing antioxidant and immune function, reducing hydrogen peroxide in the body to water and oxygen, and reducing lipid peroxides to harmless products, thus lowering oxidative stress in the body.

[0164] Please see Figure 5 The f-Thiols concentration of the subjects at week 0 was approximately 231.8 μg / mL, and increased to approximately 257.0 μg / mL at week 4. Therefore, setting the relative f-Thiols concentration at week 0 as 100%, and converting the measurements at week 4 into relative f-Thiols concentrations, we find that the relative f-Thiols concentration at week 4 increased to approximately 110.9%, an increase of 10.9%. This indicates that the probiotic combination in any embodiment can increase the concentration of sulfur-containing compounds. Sulfur-containing compounds are excellent antioxidants, highly sensitive to free radicals, and can mitigate the damage from reactive oxygen species (ROS), thus having detoxifying and liver-strengthening effects.

[0165] Antioxidant capacity can influence the body's immune response. Enhancing the body's antioxidant capacity helps resist various free radical damage and protect the immune system. After taking the probiotic combination of one embodiment for 4 weeks, the subjects' total antioxidant capacity increased by 10.1%. Furthermore, various antioxidant indicators in the blood (glutathione S-transferase and sulfur-containing compounds) all increased, with glutathione S-transferase and sulfur-containing compounds increasing by 5.7% and 10.9%, respectively. This indicates that the probiotic combination of any embodiment can strengthen the body's antioxidant capacity and help boost immunity.

[0166] Example 6: Human trials of probiotic combinations - assay of immune capacity

[0167] I. Test Procedure:

[0168] Six adult subjects with weakened immunity, gastrointestinal discomfort, or constipation were given one probiotic combination capsule (i.e., a capsule containing 100 mg of the probiotic combination prepared in Example 1) daily for four weeks (i.e., 28 days).

[0169] Blood samples were drawn from subjects before starting treatment (week 0) and after 28 days of treatment (week 4) to measure changes in blood IL-10 concentration, total glutathione (t-GSH) concentration, and TNF-α concentration before and after taking the probiotic combination containing one example.

[0170] In this embodiment, the concentrations of IL-10 in the subjects' blood, total glutathione (t-GSH), and TNF-α in their bodies were measured by Li-Jen Laboratory (Taiwan, China).

[0171] It should be noted that the statistically significant difference between the measurement results in week 0 and week 4 was obtained through Student's t-test statistical analysis. In the graph, "*" indicates that the p-value is less than 0.05 compared with week 0, "**" indicates that the p-value is less than 0.01 compared with week 0, and "***" indicates that the p-value is less than 0.001 compared with week 0.

[0172] II. Experimental Results:

[0173] Please see Figure 6 At week 0, the subjects' IL-10 concentration was approximately 0.064 pg / mL, which increased to approximately 0.196 pg / mL by week 4, an increase of 206.3%. IL-10 is an anti-inflammatory cytokine, initially thought to have immunosuppressive effects, but recent studies have shown that IL-10 can also promote the body's immune response and activate natural killer cells. The presence of IL-10 can regulate the body's immune response and enhance the cytotoxic ability of natural killer cells, with this enhancement increasing with increasing IL-10 concentration, indicating a more active immune system. Therefore, the probiotic combination in any of the embodiments can increase IL-10 concentration, indicating that it has an immunomodulatory effect.

[0174] Please see Figure 7At week 0, the subjects' t-GSH concentration was approximately 1384.3 μmol / L, which increased to approximately 1609.0 μmol / L at week 4. Therefore, setting the relative t-GSH concentration at week 0 as 100% and converting the week 4 measurements to relative t-GSH concentrations, we find that the relative t-GSH concentration at week 4 significantly increased to approximately 116.2%, a significant increase of 16.2%. Maintaining the normal functioning of the immune system requires the full activation and differentiation of immune cells. GSH can activate lymphocytes, differentiating and proliferating T and B lymphocytes, thereby killing viruses or cancer cells. Furthermore, existing literature indicates that GSH depletion increases the incidence of infection. Further research has shown that GSH has been proven to effectively inhibit viral replication in patients with human immunodeficiency virus (HIV) and tuberculosis (TB), and also has beneficial effects on other pulmonary infectious diseases. Therefore, GSH is also considered as an adjunctive therapy for COVID-19. Therefore, it can be seen that the probiotic combination of any embodiment can increase total glutathione in the body, thereby enhancing immunity.

[0175] Please see Figure 8 At week 0, the subjects' TNF-α concentration was approximately 0.810 pg / mL, which decreased to approximately 0.253 pg / mL by week 4, a significant reduction of 68.8%. TNF-α is a pro-inflammatory cytokine involved in many inflammatory responses in the body; higher blood TNF-α concentrations indicate more severe inflammatory responses. Therefore, the probiotic combination of any embodiment possesses anti-inflammatory potential, reducing blood TNF-α concentrations and thus lowering inflammatory responses in the body.

[0176] Example 7: Human Trial of Probiotic Combinations - Perception Questionnaire Survey

[0177] I. Test Procedure:

[0178] Six adult subjects with weakened immunity, gastrointestinal discomfort, or constipation were given one probiotic combination capsule (i.e., a capsule containing 100 mg of the probiotic combination prepared in Example 1) daily for four weeks (i.e., 28 days).

[0179] Subjects completed a self-assessment questionnaire before starting treatment (week 0) and after 28 days of treatment (week 4). The self-assessment questionnaire focused on gastrointestinal discomfort, including nausea, bloating, belching and / or hiccups, and bowel movement frequency.

[0180] II. Experimental Results:

[0181] Please see Figure 9 . Figure 9This study illustrates the changes in the severity of gastrointestinal discomfort experienced by subjects taking the probiotic combination containing one embodiment at weeks 0 and 4. Self-assessments of gastrointestinal discomfort by the six subjects before taking the probiotic combination (week 0) were considered as 100%. Gastrointestinal discomfort included nausea, bloating, and belching and / or hiccups. After four weeks of continuous use, the severity of nausea was 90.9%; the severity of bloating was 95.5%; and the severity of belching and / or hiccups was 81.8%. In other words, compared to before taking the probiotic combination of one embodiment (week 0), continuous use of capsules containing 100 mg of the probiotic combination of one embodiment for 4 weeks reduced nausea by 9.1% in these subjects; continuous use of capsules containing 100 mg of the probiotic combination of one embodiment for 4 weeks reduced bloating by 4.5% in these subjects; and continuous use of capsules containing 100 mg of the probiotic combination of one embodiment for 4 weeks reduced belching and / or hiccups by 18.2% in these subjects. In other words, the severity of nausea, bloating, belching, and / or hiccups was reduced, and the probiotic combination of one embodiment has the effect of relieving and improving gastrointestinal discomfort. Therefore, it can be concluded that the probiotic combination of any embodiment can reduce the severity of nausea, bloating, belching, and / or hiccups in subjects, and has the effect of relieving and improving gastrointestinal discomfort.

[0182] Please see Figure 10 . Figure 10 The study shows the changes in bowel movement frequency in subjects taking the probiotic combination containing one embodiment at weeks 0 and 4. Before taking the probiotic combination (week 0), the six subjects self-assessed an average of 4.3 bowel movements. After four weeks of continuous use, the average number of bowel movements increased to 7. In other words, compared to before taking the probiotic combination (week 0), four weeks of continuous use of capsules containing 100 mg of the probiotic combination from one embodiment increased the average number of bowel movements by 2.7, an increase of 62.8%. This indicates a reduction in the severity of inability to defecate or constipation. Therefore, the probiotic combination of any embodiment has a laxative effect.

[0183] Example 8: Human trials of probiotic combinations - intestinal flora detection

[0184] I. Test Procedure:

[0185] Six adult subjects with weakened immunity, gastrointestinal discomfort, or constipation were given one probiotic combination capsule (i.e., a capsule containing 100 mg of the probiotic combination prepared in Example 1) daily for four weeks (i.e., 28 days).

[0186] Sample (stool) collection: Intestinal flora analysis was performed on subjects before starting treatment (week 0) and after 28 days of treatment (week 4). For the intestinal flora analysis, subjects affixed clean stool collection bags to the toilet seat for subsequent sampling. Before sampling, subjects emptied their bladders to avoid sample contamination and placed an appropriate amount of sample into a sampling tube containing preservation fluid. The sample was then sent to Turs Biotechnology Co., Ltd. for sequencing analysis.

[0187] The following ordinal analysis process was provided by Turs Biotechnology Co., Ltd.

[0188] Sample sequencing: Based on the Illumina MiSeq sequencing platform, paired-end sequencing was used, and species annotation and abundance analysis were performed through read splicing filtering and OUT (Operational Taxonomic Units) clustering. This not only revealed the species composition of the samples, but also explored the differences between samples through alpha diversity analysis and beta diversity analysis.

[0189] Sample ordination data analysis:

[0190] (a) Ordinal data processing

[0191] According to the barcode sequence and PCR amplification primer sequence, the data of each sample were separated from the data after the machine. After removing the barcode and primer sequence, the reads of each sample were spliced ​​using FLASH (http: / / ccb.jhu.edu / software / FLASH / ) (minimum overlap length 10bp; maximum mismatch rate of overlap region 0.1). The spliced ​​sequence is called Raw Tags. Then, the Raw Tags were subjected to quality filtering to obtain Clean Tags, and chimeric sequences were further removed to obtain the final Effective Tags for subsequent analysis. The sequence processing used Qiime's Tags quality control process (http: / / qiime.org / scripts / split_libraries_fastq.html). The filtering conditions are as follows: (1) Tags truncation: Tags with three consecutive bases Q<19 were truncated from the first low-quality base site. (2) Tags length filtering: After the tags were truncated, Tags with consecutive high-quality bases less than 75% of the tag length were further filtered out. Chimeric sequence processing uses the UCHIME algorithm (http: / / www.drive5.com / usearch / manual / uchime_algo.html) and databases (Gold Database (16S); http: / / drive5.com / uchime / uchime_download.html / unite Database (ITS); https: / / unite.ut.ee / repository.php) to detect chimeric sequences. After removing chimeric sequences, Effective Tags are obtained.

[0192] (b) Sample complexity analysis (Alpha Diversity)

[0193] Qiime was used to calculate the Observed-species, Shannon, Simpson, ACE, Chao1, PD_whole_tree, and Goods_coverage indices. R was used to plot the Rarefaction Curve, Rank Abundance Curve, species accumulation curve, and to perform Alpha diversity intergroup difference analysis. Alpha diversity index intergroup difference analysis employed both parametric and nonparametric tests. For only two groups, t-tests and Wilcoxon tests were used; for more than two groups, Tukey's test and Kruskal's post-hoc test were used. Links describing the Alpha diversity indices are as follows:

[0194] Calculate the community richness index:

[0195] (1) Chao1 estimator

[0196] (2) ACE estimator

[0197] Calculate the community diversity index:

[0198] (1) Shannon index

[0199] (2) Simpson index

[0200] Ordered Depth Index:

[0201] (1) Good's coverage

[0202] Phylogenetic diversity index:

[0203] (1) PD_whole_tree index

[0204] II. Experimental Results:

[0205] (1) The probiotic combination of any embodiment improves the balance of bacterial flora.

[0206] Recent studies have shown that the Firmicutes / Bacteroidetes ratio (F / B ratio) is widely considered to have a significant impact on maintaining normal gut stability. An increase or decrease in the F / B ratio is considered a gut dysbiosis, with the former typically observed in obesity and the latter in inflammatory bowel disease (IBD). Therefore, the closer the F / B ratio is to 1, the more balanced the gut microbiome, and vice versa. In other words, the closer the F / B ratio is to 1, the less likely obesity is to occur.

[0207] Before taking the probiotic capsule combination (week 0), the six subjects had a Firmicutes abundance of 31% and a Bacteroidetes abundance of 59% in their gut microbiota, with an F / B ratio of 0.53. After four weeks of continuous use, the Firmicutes abundance increased to 54% and the Bacteroidetes abundance to 42%, with an F / B ratio of 1.29. In other words, compared to before taking the probiotic capsule combination (week 0), four weeks of continuous use of capsules containing 100 mg of the probiotic combination increased the Firmicutes abundance in the gut microbiota of these subjects by 23%; and four weeks of continuous use of capsules containing 100 mg of the probiotic combination decreased the Bacteroidetes abundance in the gut microbiota of these subjects by 17%, with the F / B ratio closer to 1. This resulted in a more balanced gut microbiota, improved gut microbiota balance, and a reduced risk of obesity.

[0208] The lactic acid bacteria in the probiotic combination belong to the Firmicutes phylum, which helps increase short-chain fatty acids in the gut, provide energy to intestinal cells, and promote digestion and metabolism. The lactic acid bacteria refer to *Leuconostoc mesenteroides* subsp. *mesenteroides*, *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus acidophilus*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum* in any of the probiotic combinations in any embodiment.

[0209] (2) The probiotic combination in any embodiment reduces pathogens.

[0210] Proteobacteria is a major phylum of bacteria and is the group to which most human pathogens are classified. Therefore, the fewer Proteobacteria there are, the healthier the gut is, and vice versa.

[0211] Before taking the probiotic combination capsules (week 0), the abundance of Proteobacteria in the gut microbiota of the six subjects was 9%. After four weeks of continuous use, the abundance of Proteobacteria in their gut microbiota was 3%. In other words, compared to before taking the probiotic combination capsules (week 0), continuous use of capsules containing 100 mg of probiotic combination for four weeks reduced the abundance of Proteobacteria in the gut microbiota of these subjects by 6%, resulting in a healthier gut. Therefore, it can be concluded that the probiotic combination of any embodiment can reduce the abundance of Proteobacteria and reduce intestinal pathogens.

[0212] (3) The probiotic combination of any embodiment enhances the richness of the gut anti-inflammatory bacterial community.

[0213] Recent studies have shown that *Faecalibacterium* can produce butyric acid to supply the intestines. Butyric acid is the most preferred and primary nutrient source for intestinal epithelial cells, which can maintain normal intestinal function and metabolism, inhibit intestinal inflammation, help repair the intestinal mucosa, and strengthen immune regulation.

[0214] Before taking the probiotic combination capsules (week 0), the abundance of *Clostridium tenuifolium* in the gut microbiota of the six subjects was 9.6%. After four weeks of continuous use, the abundance of *Clostridium tenuifolium* in their gut microbiota increased to 10.7%. In other words, compared to before taking the probiotic combination capsules (week 0), continuous use of capsules containing 100 mg of probiotic combination for four weeks increased the abundance of *Clostridium tenuifolium* in the gut microbiota of these subjects, maintained normal intestinal function and metabolism, inhibited intestinal inflammation, helped repair the intestinal mucosa, and thus strengthened immune regulation.

[0215] (4) The probiotic combination of any embodiment enhances the species diversity of the gut microbiota.

[0216] The greater the diversity of gut microbiota, the easier it is to maintain a stable ecological balance among the microbiota, and this ecological balance is closely related to human health. Improving the diversity of gut microbiota species can, in turn, improve the body's immune regulation capabilities.

[0217] Before taking the probiotic combination capsules (week 0), the gut microbiota species diversity of the six subjects was 90.4%. After four weeks of continuous use, the gut microbiota species diversity was 92.1%. In other words, compared to before taking the probiotic combination capsules (week 0), continuous use of capsules containing 100 mg of probiotics for four weeks improved the gut microbiota species diversity of these subjects, maintained the ecological balance of the gut microbiota, improved the body's immune regulation capacity, and thus promoted better health and improved gastrointestinal health. Therefore, the probiotic combination of any embodiment can regulate the gut microbiota, increase gut microbiota species diversity, maintain the ecological balance of the gut microbiota, improve the body's immune regulation capacity, and thus promote better health and improved gastrointestinal health.

[0218] In summary, the probiotic combination of any embodiment can be used to prepare a composition that enhances antioxidant capacity, improves and / or regulates immunity, or improves gastrointestinal health. In other words, the aforementioned composition has the function of enhancing antioxidant capacity, improving and / or regulating immunity, or improving gastrointestinal health. In some embodiments, the composition obtained from the probiotic combination also has one or more of the following functions: enhancing total antioxidant capacity, enhancing sulfur-containing compounds (f-Thiols), enhancing GST-RBC, enhancing the cytotoxic ability of natural killer cells, enhancing the phagocytic capacity of neutrophils, enhancing the concentration of IL-10 in the blood, enhancing the total glutathione (t-GSH) in the body, regulating the gut microbiota, relieving gastrointestinal discomfort, and / or aiding in defecation.

[0219] Of course, the present invention may have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and modifications according to the present invention, but these corresponding changes and modifications should all fall within the protection scope of the claims of the present invention.

Claims

1. The use of a probiotic blend in the preparation of a composition for enhancing and / or regulating immunity, characterized in that, The probiotic combination consists of yeast and lactic acid bacteria. The yeast species are *Kluyveromyces marxianus* Y03 (DSM 34088) and *Saccharomyces cerevisiae* TCI907 (DSM 33480). The lactic acid bacteria species are *Leuconostoc mesenteroides* TCI007 (DSM 33502), *Lactococcus lactis* TCI807 (DSM 34085), *Lactobacillus acidophilus* TCI800 (DSM 34084), and *Lactobacillus delbrueckii subsp. Bulgaricus* (DSM 34086). The Lactobacillus plantarum TCI999 with accession number DSM 33106, and the ratio of the yeast and lactic acid bacteria are 1:10~100. The C. plantarum TCI999, L. plantarum TCI907, L. plantarum TCI907, L. plantarum TCI807, L. lactococcus TCI800, L. acidophilus TCI800, L. bulgaricus TCI808, and L. plantarum TCI999 are deposited at the German Microbiological Culture Collection Center.

2. The use as described in claim 1, characterized in that, The probiotic combination enhances and / or regulates immunity by increasing the cytotoxicity of natural killer cells.

3. The use as described in claim 1, characterized in that, The probiotic combination achieves the enhancement and / or regulation of immunity by increasing the phagocytic capacity of neutrophils.

4. The use as described in claim 1, characterized in that, The probiotic combination achieves the enhancement and / or regulation of immunity by increasing the concentration of IL-10 in the blood.

5. The use as described in claim 1, characterized in that, The probiotic combination achieves the enhancement and / or regulation of immunity by increasing total glutathione t-GSH in the body.

6. The use as described in any one of claims 1 to 5, characterized in that, The effective dose of the composition is 100 mg / day.

7. The use as described in any one of claims 1 to 5, characterized in that, The composition is a pharmaceutical composition or a food composition.

8. Use of a probiotic blend in the preparation of a composition to aid bowel movements, characterized in that, The probiotic combination consists of yeast and lactic acid bacteria. The yeast species are *Kluyveromyces marxianus* Y03 (DSM 34088) and *Saccharomyces cerevisiae* TCI907 (DSM 33480). The lactic acid bacteria species are *Leuconostoc mesenteroides* TCI007 (DSM 33502), *Lactococcus lactis* TCI807 (DSM 34085), *Lactobacillus acidophilus* TCI800 (DSM 34084), and *Lactobacillus delbrueckii subsp. Bulgaricus* (DSM 34086). The Lactobacillus plantarum TCI999 with accession number DSM 33106, and the ratio of the yeast and lactic acid bacteria are 1:10~100. The C. plantarum TCI999, L. plantarum TCI907, L. plantarum TCI907, L. plantarum TCI807, L. lactococcus TCI800, L. acidophilus TCI800, L. bulgaricus TCI808, and L. plantarum TCI999 are deposited at the German Microbiological Culture Collection Center.

9. The use as described in claim 8, characterized in that, The probiotic combination has the ability to regulate the gut microbiota.

10. The use as described in claim 8, characterized in that, The probiotic combination has the ability to relieve gastrointestinal discomfort.

11. The use as described in any one of claims 8 to 10, characterized in that, The effective dose of the composition is 100 mg / day.

12. The use as described in any one of claims 8 to 10, characterized in that, The composition is a pharmaceutical composition or a food composition.

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