Bacillus subtilis MG-D587 of high-yield digestive enzyme and application of bacillus subtilis MG-D587

Abstract

The invention relates to the field of microorganisms, in particular to bacillus subtilis MG-D587 for high-yield digestive enzyme and application of the bacillus subtilis MG-D587. The preservation number of the bacillus subtilis MG-D587 is CGMCC (China General Microbiological Culture Collection Center) No.34354, the preservation time of the bacillus subtilis MG-D587 is 2025-4-25, and the bacillus subtilis MG-D587 is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. The bacillus subtilis MG-D587 is separated from a healthy human body and has the capacity of producing protease, amylase, lipase and cellulase, the activity of the amylase and the activity of the cellulase are remarkably higher than those of positive strains, and it is indicated that the bacillus subtilis MG-D587 has the huge potential of promoting food digestion and absorption and improving intestinal health.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a high-yield digestive enzyme Bacillus subtilis MG-D587 and its uses. Background Technology

[0002] Bacillus subtilis has become a hot topic in probiotic development due to its ability to form highly resistant spores and its tolerance to high temperatures and the gastrointestinal environment. However, existing Bacillus subtilis preparations still have the following limitations: Functional limitation: Most strains focus only on antibacterial or enzyme production functions, making it difficult to simultaneously meet multiple needs such as intestinal colonization, digestive enzyme secretion, and immune regulation. Insufficient survival rate and stability: Some strains have low survival rates in gastric acid and bile salt environments, or their activity is easily lost during processing, affecting the actual application effect.

[0003] Currently, several Bacillus subtilis strains have been developed and applied in probiotic products. For example, the DE111 strain has been clinically validated to colonize the small intestine and regulate the gut microbiota, but its function is mainly focused on immune support, with limited effect on promoting digestive enzyme synthesis. Therefore, developing a new Bacillus subtilis strain that combines highly efficient enzyme production, excellent intestinal adaptability, and stable genetic traits is of great value in overcoming the technical bottlenecks of current probiotic preparations and achieving systemic improvement in digestive function. Attached Figure Description

[0004] Figure 1 The colony morphology of MG-D 587 in this invention is shown. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a high-yield digestive enzyme Bacillus subtilis MG-D587 and its uses, in order to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention provides Bacillus subtilis (… Bacillus subtle MG-D587, with accession number CGMCC No.34354, deposited on April 25, 2025, is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing.

[0007] Preferably, the Bacillus subtilis MG-D587 comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID NO.1.

[0008] The present invention also provides a microbial agent containing the aforementioned Bacillus subtilis MG-D587.

[0009] The present invention also provides the use of the aforementioned Bacillus subtilis MG-D587 or the aforementioned bacterial agent in the preparation of probiotic products.

[0010] As described above, the Bacillus subtilis MG-D587, which produces a high amount of digestive enzymes, and its uses according to the present invention have the following beneficial effects: In this invention, Bacillus subtilis MG-D587 was isolated from healthy human bodies and has the ability to produce protease, amylase, lipase and cellulase. Moreover, the activities of amylase and cellulase are significantly higher than those of positive strains, indicating that Bacillus subtilis MG-D587 has great potential to promote food digestion and absorption and improve intestinal health. Detailed Implementation

[0011] This application provides Bacillus subtilis ( Bacillus subtilis MG-D587, with accession number CGMCCNo.34354, deposited on April 25, 2025, is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing.

[0012] In some specific embodiments, the Bacillus subtilis MG-D587 comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID NO. 1.

[0013] In some specific embodiments, the Bacillus subtilis MG-D587 comprises a DNA fragment with a nucleotide sequence having a similarity of more than 90% to SEQ ID NO. 1. Specifically, the Bacillus subtilis MG-D587 comprises a DNA fragment with a nucleotide sequence having a similarity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more to SEQ ID NO. 1. Preferably, the Bacillus subtilis MG-D587 comprises a DNA fragment with a nucleotide sequence having a similarity of more than 99% to SEQ ID NO. 1.

[0014] In some embodiments, the Bacillus subtilis MG-D587 contains two or more digestive enzymes. Specifically, the digestive enzymes are selected from one or more of amylase, cellulase, protease, or lipase. Preferably, the digestive enzymes are amylase, lipase, and / or cellulase.

[0015] The present invention also provides a microbial agent containing the aforementioned Bacillus subtilis MG-D587.

[0016] In some specific embodiments, the microbial agent further contains one or more of the following: Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus paracasei, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium lactis, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Streptococcus salivarius, Saccharomyces cerevisiae and Saccharomyces boulardii, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus clausti, or Bacillus coagulans.

[0017] Further, the *Indiana* strain is selected from *Indiana* HU36; the *Licheniformis* strain may be selected from *Licheniformis* CGMCC1256; and / or, the *Clausii* strain is selected from *Clausii* OC, *Clausii* NR, *Clausii* SIN, or *Clausii* T; and / or, the *Coagulant* strain is selected from *Clausii* BC30, *Clausii* SNZ 1969, or *Clausii* Unique IS-2.

[0018] In some specific embodiments, the microbial agent further includes one or more of the following: prebiotics, vitamins, minerals, amino acids, polypeptides, emulsifiers, thickeners, sweeteners, flavorings, stabilizers, or preservatives.

[0019] Furthermore, the prebiotic may be selected from one or more of fructooligosaccharides, inulin, galactooligosaccharides, or polydextrose.

[0020] In some specific embodiments, the concentration of Bacillus subtilis MG-D587 in the bacterial agent can be at least 1.10. 9 CFU / g, at least 2.10 9 CFU / g, at least 3.10 9 CFU / g, at least 4.10 9 CFU / g, at least 5.10 9 CFU / g, at least 6.10 9 CFU / g, at least 7.10 9 CFU / g, at least 8.10 9 CFU / g, at least 9.10 9 CFU / g, at least 1.10 10 CFU / g, at least 2.10 10 CFU / g, at least 3.10 10 CFU / g, at least 4.10 10 CFU / g, at least 5.10 10 CFU / g, at least 6.10 10CFU / g, at least 7.10 10 CFU / g, at least 8.10 10 CFU / g, at least 9.10 10 CFU / g, or at least 1.10 11 CFU / g or higher.

[0021] The present invention also provides the use of the aforementioned Bacillus subtilis MG-D587 or the aforementioned bacterial agent in the preparation of probiotic products.

[0022] In some specific embodiments, the probiotic product is a product having one or more of the following functions: 1) Promotes digestion; 2) Improve the gut microenvironment; 3) Enhance immune function; 4) Relieves constipation.

[0023] In some specific embodiments, the probiotic product is a pharmaceutical, health product, or food.

[0024] In some specific embodiments, the dosage form of the probiotic product may be selected from one or more of the following: capsules, tablets, powders, granules, oral liquids, drops, gummies, compressed candies, solid beverages, or fermented dairy products.

[0025] When the probiotic product is a pharmaceutical product, it contains pharmaceutically acceptable excipients. Specifically, these excipients include various excipients and diluents that are not essential active ingredients and do not cause excessive toxicity after administration. The excipients include sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also contain other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. When the excipients are used in an aqueous solution for injection, they are selected from physiological saline, glucose isotonic solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sugar alcohol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG) and / or nonionic surfactants (Tween 80 or HCO-50).

[0026] In this invention, the term "similarity" is defined as the percentage of identical nucleotide residues in a candidate nucleotide sequence to a reference nucleotide sequence after aligning nucleotide sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage nucleotide sequence similarity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0027] In this invention, the term "vitamin" refers to a class of trace organic substances that humans and animals must obtain from food to maintain normal physiological functions. Common examples may be selected from one or more of vitamin A, B vitamins, vitamin C, vitamin D, vitamin E, or vitamin K.

[0028] In this invention, the term "mineral" refers to an inorganic element essential to the human body, which often exists in the form of metal salts, such as calcium salts, phosphates, potassium salts, sodium salts, magnesium salts, iron salts, zinc salts, copper salts, iodates, or selenates.

[0029] In this invention, the term "probiotics" refers to live microorganisms that, when administered in adequate amounts, confer health benefits to the host, such as restoring or improving the composition of the gut microbiota. They are often provided as dietary supplements containing potentially beneficial bacteria or yeasts and are widely used in foods, including dairy products and probiotic-fortified foods.

[0030] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0031] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0032] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0033] Example 1 Enzyme activity of Bacillus subtilis MG-D587

[0034] Target bacterium: Bacillus subtilis MG-D587 was isolated from the intestine of a healthy human and identified as Bacillus subtilis by 16S rDNA after three passages in LB medium.

[0035] Positive bacteria: Bacillus subtilis HU-58, obtained from the digestive aid product MEGA SporeBiotics; another positive strain is Bacillus subtilis DE111. ® These two strains have a strong ability to produce digestive enzymes, and therefore were selected as positive screening bacteria.

[0036] 2. Basic information about the target bacteria 2.1 Source and culture of strains Intestinal metabolites from healthy humans were collected, diluted, and spread onto LB solid medium. After aerobic incubation at 37°C for 24 hours, single colonies were picked for identification and purified by streaking on LB solid medium. After purification three times, the culture was inoculated into LB broth and incubated at 37°C and 180 rpm for 16 hours. 60% glycerol was added and the culture was dispensed into cryovials and stored at -80°C.

[0037] 2.2 Strain morphology MG-D 587 on LB medium is characterized by grayish-white edges, a milky-white center, irregular flattened shape, clear edges, and a dry surface. Figure 1 ).

[0038] 2.3 Strain sequence After 16S rRNA identification of MG-D 587, BLAST alignment using NCBI showed that this strain shared 100% homology with Bacillus subtilis. The 16S rRNA sequence (SEQ ID No. 1) is as follows: > 3. Industrialization of bacterial powder production 3.1 Fermentation medium The mixture consists of 10-20g glucose, 10g peptone, 5g yeast extract, 2-3g dipotassium hydrogen phosphate, and 0.5-1g magnesium sulfate, diluted with distilled water to a final volume of 1000mL. The pH is adjusted to 7.0-7.5.

[0039] 3.2 Fermentation process The fermentation medium was sterilized at 121℃ for 15 min, cooled, and then inoculated with 5% seed culture. It was then incubated aerobically at 37℃ for 24 h, and the bacterial cells were collected. The viable cell count in the fermentation broth reached 1.3 × 10⁻⁶. 9 CFU / mL.

[0040] 3.3 Preparation of bacterial powder The bacterial cells were resuspended in a mixture of 10% skim milk and 10% sucrose as a freeze-drying protectant, and then freeze-dried to obtain bacterial powder with a viable count of up to 2.6 × 10⁻⁶. 11 CFU / g.

[0041] 4. Determination of four digestive enzymes Strain activation Remove the cryovial from the -80℃ freezer, take 2% bacterial suspension, inoculate it onto LB liquid medium, and anaerobic culture at 37℃ for 18-24 hours. Resuspend the suspension with physiological saline and adjust the OD600 of the bacterial suspension to 1.0 for later use.

[0042] LB liquid medium consists of 10g peptone, 5g yeast extract, and 10g sodium chloride, diluted to 1000mL with distilled water, and the pH is adjusted to 7.0-7.2.

[0043] Protease assay Screening culture medium 1% skim milk solid culture medium: 10 g skim milk powder, 20 g agar, 1 L distilled water, sterilized at 105℃ for 15 min.

[0044] Filtering methods After resuspending the activated bacterial solution, 10 μL of the bacterial solution was added dropwise to 1% skim milk solid medium. After incubation at 37°C for 48 h, the diameter of the transparent hydrolysis zone (D) around the colony and the diameter of the colony (d) were measured. The enzyme activity was expressed as the ratio of the diameter of the transparent hydrolysis zone D to the diameter of the colony d, D / d.

[0045] Amylase assay Screening culture medium Soluble starch solid culture medium: 10 g soluble starch, 1.0 g dipotassium hydrogen phosphate, 1.0 g magnesium sulfate, 1.0 g sodium chloride, 2.0 g ammonium sulfate, 2.0 g calcium carbonate, 0.001 g ferrous sulfate, 0.001 g manganese chloride, 0.001 g zinc sulfate, 20 g agar, 1 L distilled water, sterilized at 121℃ for 20 min.

[0046] Filtering methods After resuspending the activated bacterial solution, 10 μL of the bacterial solution was added dropwise to a soluble starch solid medium. After incubation at 37°C for 48 h, 2 mL of iodine solution was added to the plate and shaken well. After color development, the transparent hydrolysis zone (D) around the colony and the colony diameter (d) were measured. The enzyme activity was expressed as the ratio of the transparent hydrolysis zone diameter D to the colony diameter d, D / d.

[0047] Lipase assay Screening culture medium Tributyric acid glyceride solid culture medium: 10 g tributyric acid glyceride, 5.0 g casein peptone, 3.0 g yeast extract, 20 g agar, 1 L distilled water, sonicated for 60 min, then sterilized at 121℃ for 20 min.

[0048] Filtering methods After resuspending the activated bacterial solution, 10 μL of the bacterial solution was added dropwise to glyceryl tartrate solid medium. After incubation at 37°C for 48 h, the diameter of the transparent hydrolysis zone (D) around the colony and the diameter of the colony (d) were measured. The enzyme activity was expressed as the ratio of the diameter of the transparent hydrolysis zone D to the diameter of the colony d, D / d.

[0049] Cellulase assay Screening culture medium Sodium carboxymethyl cellulose solid culture medium: 2.0 g sodium nitrate, 1.0 g dipotassium hydrogen phosphate, 0.5 g potassium chloride, 0.5 g magnesium sulfate, 0.01 g ferrous sulfate, 10 g carboxymethyl cellulose, 20 g agar, 1 L distilled water. Sonicate for 30 min, then sterilize at 115℃ for 15 min.

[0050] Filtering methods After resuspending the activated bacterial solution, 10 μL of the bacterial solution was added dropwise to sodium tricarboxymethyl cellulose solid medium and incubated at 37°C for 48 h. The plate was then stained with 0.1% Congo red solution for 15 min and the stain was discarded. An appropriate amount of 1M sodium chloride solution was added to decolorize the plate for 15 min. The transparent hydrolysis zone (D) around the colony and the colony diameter (d) were measured. The enzyme activity was expressed as the ratio of the transparent hydrolysis zone diameter D to the colony zone diameter d, D / d.

[0051] Results Analysis The digestive enzyme activity of Bacillus subtilis MG-D587 was determined using different selection media. The results are shown in Table 1. Bacillus subtilis MG-D587 exhibits the ability to produce protease, amylase, lipase, and cellulase. The abundance of digestive enzymes was similar to that of positive strains HU-58 and DE111. ® The results were similar, and the amylase and cellulase were significantly superior to HU-58 and DE111. ® The lipase was not significantly different from HU-58 and was superior to DE111. ® The presence of abundant enzymes with excellent enzyme activity indicates that Bacillus subtilis MG-D587 has great potential to promote food digestion and absorption and improve gut health.

[0052] Table 1. Ability of Bacillus MG-D587 to produce different digestive enzymes

[0053] Note: Different letters represent significant differences between different strains. p <0.05) Example 2: In vitro food digestion assay

[0054] Bacterial cell preparation Culture medium: 25 g maltodextrin, 5.0 g yeast extract, 20 g peptone, 2.0 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 1000 mL pure water. Adjust pH to 6.8.

[0055] Take 5% activated bacterial solution, inoculate it into bacterial culture medium, and incubate at 37°C in a shaker for 18-24 hours. Resuspend the culture in physiological saline and adjust the viable count to 1×10⁻⁶. 9 CFU / mL, take 6 mL, centrifuge, discard the supernatant, and keep the bacterial sludge for later use.

[0056] Preparation of digestive enzyme solutions Protease-producing culture medium: 20 g skim milk, 1000 mL pure water, sterilized at 105℃ for 15 min.

[0057] Amylase-producing culture medium: 10 g soluble starch, 15 g peptone, 5.0 g yeast extract, 0.45 g dipotassium hydrogen phosphate, 0.2 g magnesium sulfate, 5.0 g sodium chloride, 2.0 g sodium carbonate, 1000 mL pure water, sterilized at 121℃ for 20 min.

[0058] Lipase-producing culture medium: 10 g tretinoin, 10 g glucose, 10 g peptone, 3.0 g yeast extract, 2.0 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 1.0 g ammonium sulfate, 1000 mL pure water, sterilized at 121℃ for 20 min.

[0059] Cellulase-producing culture medium: 10 g sodium carboxymethyl cellulose, 2.0 g sodium nitrate, 1.0 g potassium chloride, 1.0 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.01 g ferrous sulfate, 1000 mL pure water, sterilized at 115℃ for 30 min.

[0060] Take 5% of each activated bacterial solution and inoculate it into four different digestive enzyme-producing culture media. Incubate at 37°C in a shaker for 48 hours. Centrifuge 6 mL of the culture and collect the supernatant for later use.

[0061] Food digestion assay Preparation of in vitro food digestion samples Protein food: 120 g skim milk, 1000 mL pure water, sterilized at 105℃ for 15 min.

[0062] Starchy foods: 100 g of rice flour, 1000 mL of pure water, sterilize at 100℃ for 15 min.

[0063] Fatty foods: 170 g olive oil, 20 g emulsifier, 1 g whey protein, 1000 mL pure water, sterilize at 105℃ for 15 min.

[0064] Cellulose-rich foods: 70 g of oat dietary fiber, 1000 mL of pure water, sterilized at 105℃ for 15 min.

[0065] Take 100 g of each food, add 6 mL of bacterial cells and corresponding enzyme solution, mix well, incubate at 37°C on a shaker for 4 hours, remove, and stop digestion by magnetic stirring at 95°C for 5 min.

[0066] Protein digestion assay Protein samples were precipitated with TCA, the supernatant was discarded, and the protein content was determined by the Kjeldahl method according to GB 5009.5-2016 National Food Safety Standard. The protein digestibility was expressed as the ratio of digested protein content to undigested protein content.

[0067] Starch digestion assay Starch content was determined by enzymatic hydrolysis according to the national food safety standard GB 5009.9-2023, and starch digestibility was expressed as the ratio of digested starch content to undigested starch content.

[0068] fat digestion assay Triglyceride content was determined using the Beyotime triglyceride assay kit, and fat digestibility was expressed as the ratio of digested triglyceride content to undigested triglyceride content.

[0069] Cellulose digestion assay Dietary fiber content was determined according to the national food safety standard GB 5009.9-2023, and cellulose digestibility was expressed as the ratio of digestible dietary fiber content to undigested dietary fiber content.

[0070] In vitro food digestion The in vitro food digestibility of Bacillus subtilis MG-D587 was determined by measuring the protein, starch, fat, and cellulose content. The results are shown in Table 2. The results indicate that Bacillus subtilis MG-D587 possesses the ability to digest protein, starch, fat, and cellulose. Its protein digestibility is not significantly different from that of Bacillus subtilis DE111® and is significantly superior to that of Bacillus subtilis HU-58. Furthermore, the fat and cellulose digestibility of this strain are significantly better than those of the two positive strains. This demonstrates that Bacillus subtilis MG-D587 has great potential to promote the digestion and absorption of protein, fat, and cellulose, improve indigestion, and promote intestinal health.

[0071] Table 2. Digestibility of Bacillus subtilis MG-D587 food

[0072] Note: Different letters represent significant differences between different strains, and ac is labeled from largest to smallest. p < 0.05) The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. Bacillus subtilis ( Bacillus subtilis MG-D587, with accession number CGMCC No.34354.

2. The Bacillus subtilis MG-D587 according to claim 1, characterized in that, The Bacillus subtilis MG-D587 contains a DNA fragment with a nucleotide sequence as shown in SEQ ID NO.

1.

3. The Bacillus subtilis MG-D587 according to claim 1, characterized in that, The Bacillus subtilis MG-D587 contains two or more digestive enzymes; preferably, the digestive enzymes are selected from one or more of amylase, cellulase, protease or lipase; more preferably, the digestive enzymes are amylase, lipase and / or cellulase.

4. A microbial agent, characterized in that, The bacterial agent contains Bacillus subtilis MG-D587 as described in any one of claims 1-3.

5. The microbial agent according to claim 4, characterized in that, The microbial agent also contains one or more of the following: Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus paracasei, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium lactis, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Streptococcus salivarius, Saccharomyces cerevisiae and Saccharomyces boulardii, Bacillus amyloliquefaciens, Bacillus indicum, Bacillus licheniformis, Bacillus clausti, or Bacillus coagulans.

6. The microbial agent according to claim 4, characterized in that, The microbial agent also contains one or more of the following: prebiotics, vitamins, minerals, amino acids, polypeptides, emulsifiers, thickeners, sweeteners, flavorings, stabilizers, or preservatives.

7. The microbial agent according to claim 4, characterized in that, The concentration of Bacillus subtilis MG-D587 in the bacterial agent is at least 1.

10. 9 CFU / g, at least 2.10 9 CFU / g, at least 3.10 9 CFU / g, at least 4.10 9 CFU / g, at least 5.10 9 CFU / g, at least 6.10 9 CFU / g, at least 7.10 9 CFU / g, at least 8.10 9 CFU / g, at least 9.10 9 CFU / g, at least 1.10 10 CFU / g, at least 2.10 10 CFU / g, at least 3.10 10 CFU / g, at least 4.10 10 CFU / g, at least 5.10 10 CFU / g, at least 6.10 10 CFU / g, at least 7.10 10 CFU / g, at least 8.10 10 CFU / g, at least 9.10 10 CFU / g, or at least 1.10 11 CFU / g or higher.

8. Use of Bacillus subtilis MG-D587 according to any one of claims 1-3 or the bacterial agent according to any one of claims 4-7 in the preparation of probiotic products.

9. The use according to claim 8, characterized in that, The probiotic product is a product that has one or more of the following functions: 1) Promotes digestion; 2) Improve the gut microenvironment; 3) Enhance immune function; 4) Relieves constipation.

10. The use according to claim 8, characterized in that, The probiotic product also includes one or more of the following characteristics: 1) The probiotic product is a pharmaceutical, health product, or food; preferably, when the probiotic product is a pharmaceutical, the probiotic product contains pharmaceutically acceptable excipients; 2) The dosage form of the probiotic product is selected from one or more of the following: capsules, tablets, powders, granules, oral liquids, drops, soft candies, compressed candies, solid beverages, or fermented dairy products.

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