Microbial preparation and method for producing the same

Abstract

This invention provides a microbial preparation. The microbial preparation comprises active ingredients, namely Lactobacillus, Bifidobacterium, Enterococcus faecalis, and Bacillus cereus, and the water activity of the microbial preparation is 0.03-0.45. Compared with microbial preparations of the same dosage form containing the same microorganisms, the microbial preparation according to the embodiments of this invention exhibits significantly improved stability.

Description

Technical Field

[0001] This invention relates to the field of biological products, specifically to a microbial preparation and its preparation method. Background Technology

[0002] Probiotics are a class of live microorganisms that benefit the host by colonizing the host's body and altering the composition of the host's microbial community in a specific area. The quality control of water activity in multi-probiotic formulations varies. According to the principles of microbial physiology, moisture is a key factor affecting the storage stability of microbial powders.

[0003] There is a growing need to find suitable technologies to improve encapsulation efficiency and probiotic viability while delivering end products that meet quality requirements. Encapsulation technologies have limitations in maintaining probiotic viability and long-term preservation during processing. Therefore, finding a suitable system for bacterial encapsulation without compromising their viability is crucial. Every element involved in the selection process should be considered from all aspects to achieve the highest possible microbial viability.

[0004] To improve drug efficacy, in addition to commonly used microbial dosage forms, further development of drug formulations can be considered in various dosage forms, new pathways, and new methods. This can further reduce side effects and improve patient compliance, which is an important aspect of new formulation development research. Different dosage forms exhibit significantly different transport processes in the body and varying blood drug concentrations over time, resulting in differences in onset time, peak effect time, and potency. Evaluating the rationality of new drug dosage forms is not only a crucial aspect of the new drug review process but also a challenge for new drug developers. A fundamental criterion for judging the rationality of a new drug dosage form is that it better serves clinical treatment compared to the original dosage form. Research on new drug dosage forms should closely focus on clinical needs, selecting dosage forms scientifically and objectively.

[0005] Another purpose of changing dosage forms is to improve medication adherence in patients of different ages. The selection of new dosage forms should be evaluated from multiple aspects, including method of administration, appearance, shape, size, color, and smell. This is especially important in the selection of dosage forms for pediatric medications. While ensuring the quality and safety of pediatric medications, the physiological and psychological characteristics of children should be considered as much as possible when selecting and developing new drug dosage forms that improve medication adherence in children.

[0006] In summary, the rationality of selecting new drug dosage forms and controlling quality indicators should be comprehensively evaluated and analyzed from various aspects, including the drug's physicochemical and biological properties, clinical treatment needs, patient compliance, a trade-off between the advantages and disadvantages of existing formulations, and market development prospects. Only in this way can we ensure the rationality and accuracy of dosage form selection and quality indicator control, reduce the waste of drug research resources, lower drug evaluation and regulatory costs, and better leverage the important role of new drug dosage forms in medical and health practice, thereby promoting the comprehensive development of pharmaceutical science and technology.

[0007] However, current technologies lack reports on key indicators for quality stability control of probiotic preparations, addressing different dosage forms, excipient types, excipient ratios, and formulation stability quality control standards. Therefore, there remains a continuous need for improvements in multi-probiotic formulations. Summary of the Invention

[0008] This invention provides a microbial preparation comprising one or more active components selected from Lactobacillus, Bifidobacterium, Enterococcus faecalis, or Bacillus cereus, wherein the water activity of the microbial preparation is 0.03-0.45; wherein the specific water activity value is selected from any point value or range value within the range of 0.03-0.45, and the specific point value can be 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, or 0.40.

[0009] Preferably, the water activity of the microbial preparation can be 0.04-0.40, 0.05-0.35, 0.05-0.1, 0.05-0.20, 0.15-0.35, 0.25-0.35, 0.15-0.25, or 0.15-0.2.

[0010] Compared to other microbial preparations of the same type containing the same microbial components, the microbial preparation of this invention exhibits significantly improved stability under ambient temperature storage conditions. The inventors' research revealed that moisture content and water activity have a significant impact on the survival ability of microorganisms. Water activity (aw) and moisture content not only affect the survival ability of microorganisms during the processing of the microbial preparation but also during subsequent storage. Therefore, through extensive experiments, the inventors found that when the water activity of the microbial preparation is between 0.03 and 0.45, the Lactobacillus, Bifidobacterium, Enterococcus faecalis, or Bacillus cereus in the preparation exhibit higher stability.

[0011] Optionally, the above-mentioned microbial preparation further includes at least one of the following additional technical features:

[0012] Optionally, the microbial preparation is a solid or liquid preparation.

[0013] Optionally, the dosage form of the solid preparation is a powder, granule, tablet, capsule or drop pill.

[0014] On the one hand, when the microbial preparation is a microbial powder, the water activity of the microbial powder is 0.05-0.20.

[0015] Optionally, the microbial powder further includes excipients.

[0016] Optionally, the excipients are selected from at least one of fillers, moisture regulators, acidulants, and flow aids.

[0017] Optionally, the moisture regulator is selected from at least one of corn starch, corn soluble starch, and potato soluble starch.

[0018] Preferably, the moisture regulator is potato soluble starch. The inventors have discovered that potato soluble starch can better regulate the water activity of microbial preparations, resulting in microbial preparations (especially microbial powders) with better viable cell stability. Furthermore, when moisture content is reduced, corn starch and corn soluble starch tend to gelatinize, producing more black spots, while potato soluble starch produces fewer black spots. Potato soluble starch is also easier to handle when preparing microbial preparations.

[0019] Optionally, the acidulant includes at least one selected from citric acid and sodium citrate.

[0020] Optionally, the moisture regulator in the microbial powder has a mass fraction of 10% to 30%, preferably 20%.

[0021] Optionally, the filler is selected from at least one of pregelatinized starch, trehalose, lactose, mannitol, glucose, sucrose, skim milk powder, dextrin, calcium sulfate, dicalcium phosphate, calcium carbonate, microcrystalline cellulose, maltodextrin, and fructooligosaccharides.

[0022] Preferably, the filler is selected from at least one of trehalose, lactose, mannitol, glucose, sucrose, skim milk powder, and fructooligosaccharides.

[0023] Optionally, the acidulant includes at least one selected from citric acid and sodium citrate.

[0024] Optionally, the flow aid comprises at least one selected from silica, light anhydrous silica, talc, stearic acid, micronized silica, sodium / magnesium dodecyl sulfate, sodium stearate fumarate, glyceryl behenate, and magnesium stearate.

[0025] Preferably, the flow aid comprises at least one selected from silica, light anhydrous silica, talc and magnesium stearate, more preferably silica.

[0026] Optionally, the filler includes mannitol and / or the acidulant includes citric acid, and the excipient includes a moisture regulator. The inventors have found that if the excipient includes mannitol and / or citric acid, its compatibility with *Bifidobacterium animalis* subsp. *lactobacter* and *Lactobacillus paracasei* is slightly poor. Therefore, a moisture regulator needs to be added to the excipient to reduce moisture content and improve the compatibility of mannitol and / or citric acid with *Bifidobacterium animalis* subsp. *lactobacter* and *Lactobacillus*.

[0027] Preferably, the filler includes mannitol, and the excipient includes a moisture regulator. The inventors have discovered that if the excipient includes mannitol, the high water activity of mannitol results in slightly poor compatibility with *Bifidobacterium animalis* subsp. *lactobacter* and *Lactobacillus paracasei*. Therefore, a moisture regulator needs to be added to the excipient to reduce moisture content and improve the compatibility of mannitol with *Bifidobacterium animalis* subsp. *lactobacter* and *Lactobacillus*.

[0028] Optionally, the excipients may further include flavorings.

[0029] Optionally, the filler has a mass fraction of 70% to 97% in the microbial powder, preferably 74% to 77%.

[0030] Optionally, the filler includes lactose and mannitol.

[0031] Optionally, the flow aid in the microbial powder has a mass fraction of not less than 1.0%, preferably not less than 1.5%, and more preferably 1.5%. The inventors have found that the amount of flow aid added significantly affects the flowability of microbial preparations. Controlling the amount of flow aid added to not less than 1.5% can significantly improve the flowability of microbial preparations (especially microbial powders). Furthermore, considering the impact of the flow aid on the product's taste, choosing 1.5% can further improve the taste of the microbial preparation (especially microbial powder) while ensuring good flowability.

[0032] Optionally, the mass ratio of lactose to mannitol is 1:4.

[0033] Optionally, the acidulant in the microbial powder has a mass fraction of 0-0.40%, preferably 0.3%-0.40%.

[0034] Optionally, the acidulant includes citric acid and sodium citrate.

[0035] Optionally, the mass ratio of citric acid to sodium citrate is (7-8):1, preferably 7.5:1.

[0036] Optionally, the Bifidobacterium includes Bifidobacterium animalis or Bifidobacterium infantis.

[0037] Optionally, the Bifidobacterium is Bifidobacterium lactis subsp. animalis.

[0038] Optionally, the accession number of the said Bifidobacterium lactis subsp. is CGMCC No. 19079.

[0039] It should be noted that detailed information and characteristics of *Bifidobacterium animalis* subsp. *lactis* with accession number CGMCC No. 19079 can be found in patent 202011640744.1, filed on December 31, 2020, the entire contents of which are incorporated herein by reference. The accession number of *Bifidobacterium animalis* subsp. *lactis* is CGMCC NO. 19079, and its taxonomical name is *Bifidobacterium animalis* subsp. *lactis*. It was deposited on December 4, 2019, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Optionally, the lactobacillus includes *Lactobacillus paracasei* or *Lactobacillus acidophilus*.

[0040] Optionally, the Lactobacillus paracasei has the accession number CGMCC No. 19077.

[0041] It should be noted that the accession number of the *Lactobacillus paracasei* is CGMCC No. 19077. Detailed information and characteristics of this microorganism can be found in patent 201911419617.6, filed on December 31, 2019, the entire contents of which are incorporated herein by reference. The *Lactobacillus paracasei*, with accession number CGMCC No. 19077, is classified as *Lactobacillus paracasei* and was deposited on December 4, 2019, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0042] On the other hand, when the microbial preparation is a microbial tablet, the water activity of the microbial tablet is 0.15-0.35.

[0043] Optionally, the microbial tablets further include excipients.

[0044] Optionally, the excipient is selected from at least one of fillers, moisture regulators, and flow aids.

[0045] Optionally, the moisture regulator is selected from at least one of corn starch, corn soluble starch, and potato soluble starch.

[0046] Preferably, the moisture regulator is corn starch.

[0047] Optionally, the moisture regulator in the microbial tablet has a mass fraction of 10% to 30%, preferably 10% to 20%.

[0048] Optionally, the filler is selected from at least one of pregelatinized starch, trehalose, lactose, mannitol, glucose, sucrose, skim milk powder, dextrin, calcium sulfate, dicalcium phosphate, calcium carbonate, microcrystalline cellulose, maltodextrin, and fructooligosaccharides.

[0049] Preferably, the filler includes microcrystalline cellulose, lactose, and sucrose.

[0050] Optionally, the filler has a mass fraction of 70% to 97% in the microbial powder.

[0051] Optionally, the sucrose in the microbial tablets has a mass fraction of 15-25%.

[0052] Optionally, the lactose in the microbial tablet has a mass fraction of 12-18%, preferably 12-13%.

[0053] Optionally, the microcrystalline cellulose in the microbial tablet has a mass fraction of 45-55%, preferably 49-50.5%.

[0054] Optionally, the flow aid comprises at least one selected from silica, light anhydrous silica, talc, stearic acid, micronized silica, sodium / magnesium dodecyl sulfate, sodium stearate fumarate, glyceryl behenate, and magnesium stearate.

[0055] Preferably, the gliding agent comprises at least one selected from stearic acid, magnesium stearate, micronized silica gel, sodium / magnesium dodecyl sulfate, talc, sodium stearate fumarate, and glyceryl behenate, more preferably magnesium stearate.

[0056] Optionally, the excipients may further include flavorings.

[0057] Optionally, the Bifidobacterium includes Bifidobacterium animalis or Bifidobacterium infantis.

[0058] Optionally, the Bifidobacterium is Bifidobacterium lactis subsp. animalis.

[0059] Optionally, the accession number of the said Bifidobacterium lactis subsp. is CGMCC No. 19079.

[0060] It should be noted that detailed information and characteristics of Bifidobacterium animalis subsp. lactis with accession number CGMCC No.19079 can be found in patent 202011640744.1, filed on December 31, 2020, which is incorporated in its entirety as part of this application.

[0061] Optionally, the lactobacillus includes Lactobacillus paracasei or Lactobacillus acidophilus.

[0062] Optionally, the Lactobacillus paracasei has the accession number CGMCC No. 19077.

[0063] It should be noted that the accession number of the Lactobacillus paracasei is CGMCC No. 19077. Detailed information and characteristics of this microorganism can be found in patent 201911419617.6, filed on 2019-12-31, the entire contents of which are incorporated herein by reference.

[0064] In a second aspect, the present invention provides a microbial powder. Optionally, the water activity of the microbial powder is 0.05-0.2, and the microbial powder comprises: 1-10 parts by weight of *Bifidobacterium animalis* subsp. *lactobacter* powder with accession number CGMCC No. 19079, 1-10 parts by weight of *Lactobacillus paracasei* powder, 0.5-5 parts by weight of *Enterococcus faecalis* powder, 0.5-5 parts by weight of *Bacillus cereus* powder, 100 parts by weight of corn soluble starch, 383 parts by weight of lactose, and 7.5 parts by weight of silicon dioxide.

[0065] Optionally, the number of viable bacteria in the Bifidobacterium animalis subsp. lactis powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0066] Optionally, the viable count of the Lactobacillus paracasei powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0067] Optionally, the number of viable bacteria in the Enterococcus faecalis powder is not less than 1×10⁻⁶. 6 CFU / g.

[0068] Optionally, the number of viable bacteria in the Bacillus cereus powder is not less than 1 × 10⁻⁶. 5 CFU / g.

[0069] In a third aspect, the present invention provides a microbial powder. Optionally, the water activity of the microbial powder is 0.05-0.2, and the microbial powder comprises: 1-10 parts by weight of *Bifidobacterium animalis* subsp. *lactobacter* powder with accession number CGMCC No. 19079, 1-10 parts by weight of *Lactobacillus paracasei* powder, 0.5-5 parts by weight of *Enterococcus faecalis* powder, 0.5-5 parts by weight of *Bacillus cereus* powder, 100 parts by weight of corn soluble starch, 300 parts by weight of mannitol, 75 parts by weight of lactose, 7.5 parts by weight of silicon dioxide, 1.5 parts by weight of citric acid, 0.2 parts by weight of sodium citrate, and 4 parts by weight of flavoring.

[0070] Optionally, the number of viable bacteria in the Bifidobacterium animalis subsp. lactis powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0071] Optionally, the viable count of the Lactobacillus paracasei powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0072] Optionally, the number of viable bacteria in the Enterococcus faecalis powder is not less than 1×10⁻⁶. 6 CFU / g.

[0073] Optionally, the number of viable bacteria in the Bacillus cereus powder is not less than 1 × 10⁻⁶. 5 CFU / g.

[0074] In a fourth aspect, the present invention provides a microbial powder. Optionally, the water activity of the microbial powder is 0.05-0.2, and the microbial powder comprises: 1-10 parts by weight of *Bifidobacterium animalis* subsp. *lactobacter* powder with accession number CGMCC No. 19079, 1-10 parts by weight of *Lactobacillus paracasei* powder, 0.5-5 parts by weight of *Enterococcus faecalis* powder, 0.5-5 parts by weight of *Bacillus cereus* powder, 100 parts by weight of corn starch, 300 parts by weight of mannitol, 75 parts by weight of lactose, 7.5 parts by weight of silicon dioxide, 1.5 parts by weight of citric acid, 0.2 parts by weight of sodium citrate, and 4 parts by weight of flavoring.

[0075] Optionally, the number of viable bacteria in the Bifidobacterium animalis subsp. lactis powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0076] Optionally, the viable count of the Lactobacillus paracasei powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0077] Optionally, the number of viable bacteria in the Enterococcus faecalis powder is not less than 1×10⁻⁶. 6 CFU / g.

[0078] Optionally, the number of viable bacteria in the Bacillus cereus powder is not less than 1 × 10⁻⁶. 5 CFU / g.

[0079] In a fifth aspect, the present invention provides a microbial powder. Optionally, the water activity of the microbial powder is 0.05-0.2, and the microbial powder comprises: 1-10 parts by weight of *Bifidobacterium animalis* subsp. *lactobacter* powder with accession number CGMCC No. 19079, 1-10 parts by weight of *Lactobacillus paracasei* powder, 0.5-5 parts by weight of *Enterococcus faecalis* powder, 0.5-5 parts by weight of *Bacillus cereus* powder, 100 parts by weight of potato soluble starch, 300 parts by weight of mannitol, 75 parts by weight of lactose, 7.5 parts by weight of silicon dioxide, 1.5 parts by weight of citric acid, 0.2 parts by weight of sodium citrate, and 4 parts by weight of flavoring.

[0080] Optionally, the number of viable bacteria in the Bifidobacterium animalis subsp. lactis powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0081] Optionally, the viable count of the Lactobacillus paracasei powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0082] Optionally, the number of viable bacteria in the Enterococcus faecalis powder is not less than 1×10⁻⁶. 6 CFU / g.

[0083] Optionally, the number of viable bacteria in the Bacillus cereus powder is not less than 1 × 10⁻⁶. 5 CFU / g.

[0084] In a sixth aspect, the present invention provides a microbial tablet. Optionally, the microbial tablet has a water activity of 0.15-0.35 and comprises: 5-10 parts by weight of Bifidobacterium infantis powder or Bifidobacterium animalis subsp. lactis powder, 5-10 parts by weight of Lactobacillus acidophilus powder or Lactobacillus paracasei powder, 0.5-5 parts by weight of Enterococcus faecalis powder, 0.5-5 parts by weight of Bacillus cereus powder, 500 parts by weight of microcrystalline cellulose, 129 parts by weight of lactose, 195 parts by weight of sucrose, 156 parts by weight of corn starch, and 5 parts by weight of magnesium stearate; wherein the Bifidobacterium animalis subsp. lactis powder has the accession number CGMCC No. 19079.

[0085] Optionally, the number of live bacteria in the Bifidobacterium animalis subsp. lactis powder or Bifidobacterium infantis powder is not less than 1×10⁻⁶. 6 CFU / g.

[0086] Optionally, the viable count in the *Lactobacillus paracasei* powder or *Lactobacillus acidophilus* powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0087] Optionally, the number of viable bacteria in the Enterococcus faecalis powder is not less than 1×10⁻⁶. 6CFU / g.

[0088] Optionally, the number of viable bacteria in the Bacillus cereus powder is not less than 1 × 10⁻⁶. 5 CFU / g.

[0089] The microbial powder described in the embodiments of the present invention has greatly improved stability under normal temperature conditions, saves energy, has strong process operability, and the product has a good taste, making it suitable for industrial production.

[0090] In a seventh aspect, the present invention provides a microbial tablet. Optionally, the microbial tablet has a water activity of 0.15-0.35 and comprises: 5-10 parts by weight of *Bifidobacterium animalis* subsp. *lactobacter* powder, 5-10 parts by weight of *Lactobacillus paracasei* powder, 0.5-5 parts by weight of *Enterococcus faecalis* powder, 0.5-5 parts by weight of *Bacillus cereus* powder, 500 parts by weight of microcrystalline cellulose, 129 parts by weight of lactose, 195 parts by weight of sucrose, 156 parts by weight of corn starch, and 5 parts by weight of magnesium stearate; wherein the *Bifidobacterium animalis* subsp. *lactobacter* powder has the accession number CGMCC No. 19079.

[0091] Optionally, the number of viable bacteria in the Bifidobacterium animalis subsp. lactis powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0092] Optionally, the viable count of the Lactobacillus paracasei powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0093] Optionally, the number of viable bacteria in the Enterococcus faecalis powder is not less than 1×10⁻⁶. 6 CFU / g.

[0094] Optionally, the number of viable bacteria in the Bacillus cereus powder is not less than 1 × 10⁻⁶. 5 CFU / g.

[0095] The microbial powder described in the embodiments of the present invention has greatly improved stability under normal temperature conditions, saves energy, has strong process operability, and the product has a good taste, making it suitable for industrial production.

[0096] In an eighth aspect, the present invention provides a microbial tablet. Optionally, the microbial tablet has a water activity of 0.15-0.35 and comprises: 5-10 parts by weight of Bifidobacterium infantis powder, 5-10 parts by weight of Lactobacillus acidophilus powder, 0.5-5 parts by weight of Enterococcus faecalis powder, 0.5-5 parts by weight of Bacillus cereus powder, 500 parts by weight of microcrystalline cellulose, 129 parts by weight of lactose, 195 parts by weight of sucrose, 156 parts by weight of corn starch, and 5 parts by weight of magnesium stearate; wherein the Bifidobacterium animalis subsp. lactis powder has the accession number CGMCC No. 19079.

[0097] Optionally, the number of live bacteria in the Bifidobacterium infantis powder is not less than 1×10⁻⁶. 6 CFU / g.

[0098] Optionally, the number of viable bacteria in the Lactobacillus acidophilus powder is not less than 1 × 10⁻⁶. 6 CFU / g.

[0099] Optionally, the number of viable bacteria in the Enterococcus faecalis powder is not less than 1×10⁻⁶. 6 CFU / g.

[0100] Optionally, the number of viable bacteria in the Bacillus cereus powder is not less than 1 × 10⁻⁶. 5 CFU / g.

[0101] Beneficial effects:

[0102] In this invention, by using a moisture regulator to adjust the water activity of the microbial powder, the water activity of the microbial powder is controlled between 0.05 and 0.20, which can improve the stability of probiotics in the microbial powder. Furthermore, the stability of the obtained microbial powder under normal temperature conditions is greatly improved, saving energy consumption, with strong process operability, and the product has a good taste, making it suitable for industrial production.

[0103] In this invention, by controlling the water activity of the microbial tablets between 0.15 and 0.35, the stability of probiotics in the microbial tablets can be improved; moreover, the stability of the obtained microbial tablets under room temperature conditions is greatly improved, saving energy consumption, with strong process operability, and the product has a good taste, making it suitable for industrial production. Attached Figure Description

[0104] Figure 1 This is the stability result of Bifidobacterium lactis subsp. animalis in microbial powder formulation B according to Example 1 of the present invention under different water activities and 25°C conditions;

[0105] Figure 2 These are the stability results of Lactobacillus paracasei in the microbial powder formulation B according to Example 1 of the present invention under different water activities and 25°C conditions;

[0106] Figure 3 These are the stability results of Enterococcus faecalis in the microbial powder formulation B according to Example 1 of the present invention under different water activities and 25°C conditions;

[0107] Figure 4 This is the stability result of Bacillus cereus in microbial powder formulation B according to Example 1 of the present invention under different water activities and 25°C conditions;

[0108] Figure 5 This is the stability result of Bifidobacterium infantis in the microbial tablet 1 of Example 2 of the present invention under different water activities and 25°C conditions;

[0109] Figure 6 This is the stability result of Lactobacillus acidophilus in the microbial tablet 1 of Example 2 of the present invention under different water activities and 25°C conditions;

[0110] Figure 7 This is the stability result of Enterococcus faecalis in the microbial tablet 1 of Example 2 of the present invention under different water activity conditions and at 25°C;

[0111] Figure 8 These are the stability results of Bacillus cereus in the microbial tablet 1 of Example 2 of the present invention under different water activities and 25°C conditions;

[0112] Figure 9 This is the stability result of Bifidobacterium lactis subsp. animalis in the microbial tablet 2 of Example 3 of the present invention under different water activities and 25°C conditions;

[0113] Figure 10 These are the stability results of Lactobacillus paracasei in the microbial tablet 2 of Example 3 of the present invention under different water activities and 25°C conditions;

[0114] Figure 11 These are the stability results of Enterococcus faecalis in the microbial tablet 2 of Example 3 of the present invention under different water activity conditions and at 25°C;

[0115] Figure 12 The results show the stability of Bacillus cereus in the microbial tablet 2 of Example 3 of the present invention under different water activities and 25°C conditions. Detailed Implementation

[0116] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0117] Microbial preparations

[0118] In some embodiments, this invention provides a microbial preparation comprising active ingredients, namely Lactobacillus, Bifidobacterium, Enterococcus faecalis, and Bacillus cereus, wherein the water activity of the microbial preparation is 0.03-0.45; wherein the specific water activity value is selected from any point or range within the range of 0.03-0.45, and the specific point value can be 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, or 0.40. Compared with microbial preparations of the same type comprising the same microbial components, the microbial preparation of this invention exhibits significantly improved stability under ambient temperature storage conditions. The inventors have discovered that moisture content and water activity have a significant impact on the survival ability of microorganisms; water activity (aw) and moisture content not only affect the survival ability of microorganisms during the processing of the microbial preparation but also affect their survival ability during subsequent storage. Therefore, through extensive experiments, the inventors discovered that when the water activity of the microbial preparation is 0.03-0.45, the stability of Lactobacillus, Bifidobacterium, Enterococcus faecalis, or Bacillus cereus in the microbial preparation is relatively high.

[0119] In some embodiments, the water activity of the microbial preparation can be 0.04-0.40, 0.05-0.35, 0.05-0.1, 0.05-0.20, 0.15-0.35, 0.25-0.35, 0.15-0.25, or 0.15-0.2. Compared to microbial preparations of the same dosage form containing the same microorganisms, the microbial preparations according to embodiments of the present invention exhibit significantly improved stability under ambient temperature storage conditions.

[0120] In some embodiments, the water activity of the microbial preparation is 0.04-0.40, preferably 0.05-0.35, more preferably 0.05-0.20 or 0.15-0.35.

[0121] In some embodiments, the microbial preparation includes a solid or liquid formulation.

[0122] In some embodiments, the dosage form of the solid dosage form includes powder, granules, tablets, capsules, or pellets.

[0123] In some embodiments, when the microbial preparation is a microbial powder, the water activity of the microbial powder is 0.05-0.20. The inventors have discovered through extensive experiments that when the water activity of the microbial powder is 0.05-0.20, the microorganisms in the powder exhibit high stability.

[0124] In some embodiments, when the microbial preparation is a microbial tablet, the water activity of the microbial tablet is 0.15-0.35. The inventors have discovered through extensive experimentation that when the water activity of the microbial tablet is 0.05-0.20, the microbial stability of the tablet is high.

[0125] In some embodiments, the microbial preparation further includes excipients, said excipients being selected from at least one of fillers, moisture regulators, acidulants, and flow aids.

[0126] In some embodiments, the filler includes at least one selected from trehalose, lactose, mannitol, glucose, sucrose, skim milk powder, dextrin, calcium sulfate, dicalcium phosphate, calcium carbonate, microcrystalline cellulose, maltodextrin, and fructooligosaccharides.

[0127] In some embodiments, the filler includes at least one selected from trehalose, lactose, and mannitol.

[0128] In some embodiments, the moisture regulator includes at least one selected from corn starch, corn soluble starch, and potato soluble starch.

[0129] In some embodiments, the moisture regulator is potato soluble starch. The inventors have found that potato soluble starch can better regulate the water activity of microbial preparations, resulting in microbial preparations (especially microbial powders) with better viable cell stability. Furthermore, when moisture is reduced, corn starch and corn soluble starch tend to gelatinize easily, producing more black spots, while potato soluble starch produces fewer black spots. Potato soluble starch is also easier to handle when preparing microbial preparations.

[0130] In some embodiments, the acidulant includes at least one selected from citric acid and sodium citrate.

[0131] In some embodiments, the flow aid comprises at least one selected from silica, light anhydrous silica, talc, stearic acid, micronized silica, sodium / magnesium dodecyl sulfate, sodium stearate fumarate, glyceryl behenate, and magnesium stearate.

[0132] In some embodiments, the flow aid is silica. The inventors have discovered through extensive experimentation that silica has better compatibility with microorganisms compared to other flow aids, and the resulting microbial preparations have better flowability.

[0133] In some implementations, the excipients further include flavorings.

[0134] In some embodiments, the filler includes mannitol and / or the acidulant includes citric acid, and the excipient includes a moisture regulator. The inventors have found that if the excipient includes mannitol and / or citric acid, its compatibility with *Bifidobacterium animalis* subsp. *lactobacter* and *Lactobacillus paracasei* is slightly poor. Therefore, a moisture regulator needs to be added to the excipient to reduce moisture content and improve the compatibility of mannitol and / or citric acid with *Bifidobacterium animalis* subsp. *lactobacter* and *Lactobacillus*.

[0135] In some embodiments, the filler includes mannitol, and the excipient includes a moisture regulator. The inventors have found that if the excipient includes mannitol, its high water activity results in slightly poor compatibility with *Bifidobacterium animalis* subsp. *lactospirum* and *Lactobacillus paracasei*. Therefore, a moisture regulator needs to be added to the excipient to reduce moisture content and improve the compatibility of mannitol with *Bifidobacterium animalis* subsp. *lactospirum* and *Lactobacillus*.

[0136] In some embodiments, the moisture regulator is present in the microbial preparation at a mass fraction of 10% to 30%, preferably 20%. This results in a microbial preparation with better performance.

[0137] In some embodiments, the filler has a mass fraction of 70% to 97% in the microbial preparation, preferably 74% to 77% by mass. This results in a microbial preparation with better performance.

[0138] In some implementations, the Bifidobacterium comprises Bifidobacterium animalis or Bifidobacterium infantis.

[0139] In some embodiments, the animal bifidobacterium is Bifidobacterium lactis subsp. animalis.

[0140] In some embodiments, the accession number of the *Bifidobacterium animalis* subsp. *lactum* is CGMCC No. 19079. Detailed information and characteristics of this microorganism can be found in patent 202011640744.1, filed on December 31, 2020, which is incorporated in its entirety as part of this application.

[0141] In some implementations, the lactobacillus is Lactobacillus paracasei.

[0142] In some embodiments, the Lactobacillus paracasei is accessed under the number CGMCC 19077. Detailed information and characteristics of this microorganism can be found in patent 201911419617.6, filed on December 31, 2019, the entire contents of which are incorporated herein by reference.

[0143] The present invention will be further explained and described below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0144] It should be noted that the water activity in the following embodiments of the present invention was detected using a temperature-controlled water activity meter (Novasina LabMaster-aw, Switzerland).

[0145] Example 1: Preparation and stability study of microbial powder

[0146] In this embodiment, *Bifidobacterium animalis* subsp. *lactamase* with accession number CGMCC No. 19079 and *Lactobacillus paracasei* with accession number CGMCC No. 19077 were selected for the following experiments; *Enterococcus faecalis* powder and *Bacillus cereus* powder can be any commercially available or isolated bacteria, wherein the *Enterococcus faecalis* powder and *Bacillus cereus* powder in this embodiment are from Hangzhou Yuanda Biopharmaceutical Co., Ltd.

[0147] 1. Experiment on excipients

[0148] The commonly used excipients in microbial powders were examined, and the following excipients were investigated respectively:

[0149] Fillers: Trehalose, lactose, mannitol;

[0150] Moisture regulators: corn starch, corn soluble starch, potato soluble starch;

[0151] Acidulant: Citric acid;

[0152] Flow aids: silica, light anhydrous silica, talc, magnesium stearate;

[0153] Examination methods:

[0154] The weight ratio of *Bifidobacterium animalis* subsp. lactis powder: *Lactobacillus paracasei* powder: *Enterococcus faecalis* powder: *Bacillus cereus* powder: excipients is 5:5:1:1:500. The excipients are dried in advance. When the excipients are citric acid and silica, the weight ratio of *Bifidobacterium animalis* subsp. lactis powder: *Lactobacillus paracasei* powder: *Enterococcus faecalis* powder: *Bacillus cereus* powder: excipients is 5:5:1:1:50.

[0155] The weight ratio of Bifidobacterium animalis subsp. lactis powder, Lactobacillus paracasei powder, Enterococcus faecalis powder, and Bacillus cereus powder was 5:5:1:1, and these were weighed to form a control group.

[0156] The aluminum foil bags were directly heat-sealed and placed in a 37°C stability chamber to test the viable bacterial count at 0, 3, 6, and 10 days.

[0157] The results showed that trehalose, lactose, corn starch, corn soluble starch, and silicon dioxide were well compatible with the four types of bacteria, and the number of viable bacteria did not decrease significantly on day 10, maintaining the original order of magnitude. Mannitol and citric acid were not well compatible with Bifidobacterium animalis subsp. lactis and Lactobacillus paracasei, and their numbers decreased by one order of magnitude on day 10, but they were well compatible with Enterococcus faecalis and Bacillus cereus.

[0158] 2. Excipient optimization experiment

[0159] This study re-examined mannitol and citric acid, along with sodium citrate and flavoring. The study investigated the effects of adding corn soluble starch to reduce moisture content, and separately examined mannitol, citric acid, sodium citrate, and flavoring. The results showed that the poor compatibility of mannitol and citric acid was due to their high water activity, not due to poor compatibility between the excipients themselves and the bacteria.

[0160] Bifidobacterium animalis subsp. lactis powder: Lactobacillus paracasei powder: Enterococcus faecalis powder: Bacillus cereus powder: first excipient: corn soluble starch = 5:5:1:1:4:1. The sample was weighed and the excipients were dried in advance. Three groups of experiments were conducted. The first excipients in the three groups of experiments were citric acid, sodium citrate and flavoring, respectively.

[0161] The sample was weighed in the following ratio: Bifidobacterium animalis subsp. lactis powder: Lactobacillus paracasei powder: Enterococcus faecalis powder: Bacillus cereus powder: second excipient: corn soluble starch = 5:5.1:1:40:10. The excipients were dried in advance. Two sets of experiments were conducted, and the second excipients in the two sets of experiments were mannitol and corn soluble starch, respectively.

[0162] The aluminum foil bags were directly heat-sealed and placed in a 37°C stability chamber to test the viable bacterial count at 0, 3, 6, and 10 days.

[0163] The results showed that trehalose, lactose, corn starch, corn soluble starch, silicon dioxide, citric acid, sodium citrate, flavoring, and mannitol excipients were compatible with the four bacteria, and the viable bacterial count did not decrease significantly on day 10, maintaining the original order of magnitude. Excipients were screened for formulation based on the requirements of the formula and process.

[0164] Both corn starch and corn soluble starch can be used as moisture regulators to control product moisture content. Compatibility studies have shown that they are compatible with four types of bacteria. Since powders are generally taken orally, the solubility and clarity of the material in water are more important. Corn soluble starch has better solubility and clarity in water than corn starch, so corn soluble starch was selected during the formulation screening process.

[0165] 3. Formula screening

[0166] 3.1 Screening and evaluation of fillers

[0167] First, we selected compatible corn soluble starch and lactose as fillers and silica as a flow aid for screening, using particle size distribution, moisture content, and flowability as evaluation indicators. The screening formulations are shown in Table 1.

[0168] Table 1: Prescriptions for Microbial Powders

[0169] Bifidobacterium animalis subspecies of lactobacillus powder 5 5 5 5 Lactobacillus paracasei powder 5 5 5 5 Enterococcus faecalis powder 1 1 1 1 Bacillus cereus powder 1 1 1 1 Corn soluble starch 150 100 50 lactose 483 333 383 433 silicon dioxide 5 5 5 5 total 500 500 500 500

[0170] Conclusion: All four formulations exhibited poor flowability. The key factor affecting the flowability of the formulation was the proportion of silica added. Further investigation will be conducted to examine the impact of different silica proportions on flowability. Additionally, the formulations contain corn soluble starch, resulting in a strong granular texture upon oral administration. Therefore, the powder dosage form is positioned as a children's product. Adding excipients to the formulation to improve taste, including citric acid, sodium citrate, and sweet orange flavoring, is recommended.

[0171] 3.2 Screening based on silica ratio

[0172] Commonly used flow aids for solid dosage forms include silica, light anhydrous silicic acid, talc, and magnesium stearate. Compatibility studies with four types of bacteria revealed that silica exhibited better compatibility and flowability compared to other flow aids. Therefore, silica was selected for this study. Different proportions of silica were added during the experiment, and the optimal addition ratio was determined using flowability indicators. The results are shown in Table 2.

[0173] Table 2: Effect of different proportions of silica addition on flowability

[0174]

[0175] The experimental results show that when the proportion of silica is ≥1.5%, the overall fluidity of the formula is good. 1.5% silica is sufficient to meet the fluidity requirements. Considering that the amount of silica added has an impact on taste and other aspects, the minimum concentration of 1.5% was selected.

[0176] 4. Stability test under different conditions

[0177] 4.1 Formulation Information

[0178] Through research on the types and proportions of raw materials and excipients for microbial powders, the final formulation was determined as shown in Table 3 below.

[0179] Table 3: Prescriptions for Microecological Preparations in Powder Formulation

[0180]

[0181]

[0182] Corn soluble starch is widely used in the market, but other sources of soluble starch also exist. Potato soluble starch allows for lower moisture control. Research has shown that corn soluble starch, when moisture is controlled at high temperatures, is prone to developing more black spots when moisture levels are low, affecting the properties of the microbial dispersant. However, when using potato soluble starch, black spots are less likely to appear when moisture levels are low, without affecting the properties of the microbial dispersant. Therefore, potato soluble starch allows for a lower controllable water activity range. Studies of formulations A, B, and C show that formulation B has a more easily adjustable water activity, allowing for lower control and thus better viable cell stability.

[0183] In this invention, the inventors simultaneously investigated the stability of microbial powders at different water activities under two temperature conditions: 4℃ and 25℃. The results showed that at 4℃, the viable counts of four strains—Bifidobacterium animalis subsp. lactis, Lactobacillus paracasei, Enterococcus faecalis, and Bacillus cereus—were relatively stable under different water activities. After three months of storage, the water activity remained almost unchanged during the stability study period. The inventors discovered that by controlling the water activity index, the stability of the microbial powder at 25℃ could be achieved. Furthermore, by controlling the water activity range of different formulations, the stability of the four probiotics in the microbial powder could be achieved, thus ensuring the stability of the microbial powder during storage at room temperature. Table 4 and... Figures 1-4 The stability data of formulation B are presented, namely the stability results of the four bacteria in formulation B at different water activities and 25°C.

[0184] Table 4: Stability test results of four bacteria in the microbial powder formulation at 25℃ under different water activities.

[0185]

[0186]

[0187] Results: As shown in the table above, when the water activity in the microbial powder formulation is higher than 0.2, the levels of *Bifidobacterium animalis* subsp. *lactobacter*, *Lactobacillus paracasei*, and *Enterococcus faecalis* all decrease rapidly, indicating poor stability of these three bacteria. When the water activity is controlled within the range of 0.05-0.2, the stability of all four bacteria is good, enabling the microbial powder to be stored at room temperature.

[0188] Furthermore, during this study, the inventors also investigated the conditions under which the water activity in the microbial powder formulation was controlled below 0.05. Before preparing the microbial powder, the excipients used in the formulation need to be pre-treated and dried according to environmental conditions, significantly increasing the processing time and energy costs. In actual production, environmental conditions need to be controlled to prevent moisture absorption by the raw materials and excipients in the microbial powder formulation, resulting in low feasibility of the product preparation process and making large-scale production in a production workshop impossible. Therefore, for microbial powder dosage forms, the stability of formulations with a water activity below 0.05 was no longer investigated.

[0189] Example 2: Preparation and stability study of microbial tablet 1

[0190] The Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis powder, and Bacillus cereus powder in this invention can be any commercially available or isolated bacteria. In this embodiment, the Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis powder, and Bacillus cereus powder are sourced from Hangzhou Yuanda Biopharmaceutical Co., Ltd.

[0191] Preparation process: A direct powder compression process was adopted. Four types of bacterial powder were passed through a 60-mesh sieve and collected for later use; sucrose was pulverized and passed through a 60-mesh sieve for later use. Then, microcrystalline cellulose, corn starch, sucrose, lactose, and the mixed bacterial powder were sequentially added to a mixer and mixed for 25 minutes. Magnesium stearate was then added and mixed for 5 minutes. The final mixture was compressed into tablets using a shallow concave circular punch, and the tablet weight was adjusted to within the range of 500±25 mg.

[0192] Table 5: Prescription for Microbial Tablet 1

[0193] Infant Bifidobacterium powder 35 <![CDATA[The viable count is not less than 1×10 6 CFU / g]]> Lactobacillus acidophilus powder 35 <![CDATA[The viable count is not less than 1×10 6 CFU / g]]> Enterococcus faecalis powder 5 <![CDATA[The viable count is not less than 1×10 6 CFU / g]]> Bacillus cereus powder 5 <![CDATA[The viable count is not less than 1×10 5 CFU / g]]> microcrystalline cellulose 2500 sucrose 976 corn starch 780 lactose 645 magnesium stearate 25 Total 5006

[0194] Experiments revealed that the four bacteria in the microbial tablet formulation exhibited the best stability when the water activity was controlled within the range of 0.15-0.35. The results are shown in Table 5. Figures 5-8 As shown.

[0195] In this invention, the inventors simultaneously investigated the stability of microbial tablets at different water activities under two temperature conditions: 4℃ and 25℃. At 4℃, the viable counts of four bacteria—Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis, and Bacillus cereus—were relatively stable at different water activities, and the water activity remained almost unchanged during the stability study period after 3 months of storage. The inventors discovered that by controlling the water activity index, the stability of the microbial tablets at 25℃ could be achieved, and by controlling the water activity range of the microbial tablets, the stability of the four bacteria could be achieved, thus ensuring the stability of the microbial tablets during storage at room temperature. The stability results of the four bacteria in the microbial tablets at different water activities and 25℃ are shown in Table 6 below:

[0196] Table 6: Stability test results of four bacteria in microbial tablet 1 at different water activities and 25°C

[0197]

[0198] Results: As shown in the table above, when the water activity is higher than 0.35, the viable counts of Bifidobacterium infantis, Lactobacillus acidophilus, and Enterococcus faecalis, except for Bacillus cereus, decrease rapidly, indicating poor stability of these three bacteria. When the water activity is controlled within the range of 0.15-0.35, the stability of all four bacteria is good, enabling the storage of this microbial tablet at room temperature.

[0199] In addition, during this study, the inventors also investigated that when the water activity of the formulation was controlled below 0.15, the compressibility of the microbial tablets deteriorated during the tableting process, failing to meet the tableting requirements. Therefore, for the microbial tablet dosage form, the stability of the formulation with a water activity below 0.15 was no longer investigated.

[0200] Example 3: Preparation and stability study of microbial tablet 2

[0201] In this embodiment, *Bifidobacterium animalis* subsp. *lactamase* with accession number CGMCC No. 19079 and *Lactobacillus paracasei* with accession number CGMCC No. 19077 were selected for the following experiments; *Enterococcus faecalis* powder and *Bacillus cereus* powder can be any commercially available or isolated bacteria, wherein the *Enterococcus faecalis* powder and *Bacillus cereus* powder in this embodiment are from Hangzhou Yuanda Biopharmaceutical Co., Ltd.

[0202] Formulation preparation method:

[0203] 1) Raw material pretreatment: Pass the raw materials through a 50-200 mesh sieve;

[0204] 2) Mixing preparation: Sucrose powder and starch are sieved through a 50-mesh sieve and then added to a batch mixer. Microcrystalline cellulose and lactose are added to the batch mixer in sequence. Finally, a mixture of four freeze-dried bacterial powders is added. After the four freeze-dried bacterial powders and excipients are mixed in the batch mixer for 25 minutes, magnesium stearate is added to the batch mixer and mixed for 5 minutes before the mixture is discharged.

[0205] 3) Molding: The sample mixed in step 2) is shaped and packaged to obtain the finished product. The formulation of the microbial tablets is shown in Table 7 below:

[0206] Table 7: Prescription for Microbial Tablet 2

[0207] Bifidobacterium animalis subsp. lactis powder 35 <![CDATA[The viable count is not less than 1×10 6 CFU / g]]> Lactobacillus paracasei powder 35 <![CDATA[The viable count is not less than 1×10 6 CFU / g]]> Enterococcus faecalis powder 5 <![CDATA[The viable count is not less than 1×10 6 CFU / g]]> Bacillus cereus powder 5 <![CDATA[The viable count is not less than 1×10 5 CFU / g]]> microcrystalline cellulose 2500 sucrose 976 corn starch 780 lactose 645 magnesium stearate 25 Total 5006

[0208] Experiments revealed that the four bacteria showed the best stability when the water activity of the microbial tablet formulation was controlled within the range of 0.15-0.35. The results are shown in Table 7. Figures 9-12 As shown.

[0209] In this invention, the inventors simultaneously investigated the stability of microbial tablets at different water activities under two temperature conditions: 4℃ and 25℃. At 4℃, the viable counts of four bacteria—Bifidobacterium animalis subsp. lactis, Lactobacillus paracasei, Enterococcus faecalis, and Bacillus cereus—were relatively stable at different water activities, and the water activity remained almost unchanged during the stability study period after 3 months of storage. The inventors discovered that by controlling the water activity index, the stability of microbial tablets at 25℃ can be achieved. By controlling the water activity range of the microbial tablet formulation, the stability of the four bacteria can be achieved, thus ensuring the stability of the microbial tablets during storage at room temperature. The stability results of the four bacteria in the microbial tablet formulation at different water activities and 25℃ are shown in Table 8 below:

[0210] Table 8: Stability test results of four bacteria in microbial tablet 2 at different water activities and 25℃

[0211]

[0212]

[0213] Results: As shown in the table above, when the water activity is higher than 0.35, the viable counts of *Bifidobacterium animalis* subsp. *lactobacter* and *Lactobacillus paracasei* decrease rapidly, except for *Enterococcus faecalis* and *Bacillus cereus*, indicating poor stability of these two bacteria. When the water activity is controlled within the range of 0.15-0.35, the stability of all four bacteria is good, enabling the preservation of microbial tablets at room temperature.

[0214] In addition, during this study, the inventors also investigated that when the water activity of the microbial tablet formulation was controlled below 0.15, the compressibility of the microbial tablets deteriorated during the tableting process, failing to meet the tableting requirements. Therefore, for microbial tablet dosage forms, the stability of formulations with water activity below 0.15 was no longer investigated.

[0215] Through this invention, the inventors discovered that different formulations have varying protective effects on microorganisms. Research revealed that the fundamental reason for these differences lies in the varying microenvironments created by different types and ratios of excipients, which influence the storage stability of different bacteria. In the research of microbial formulations, for different microecological dosage forms, it is necessary to screen excipient formulations that enable stable storage of the formulation, based on the characteristics of the probiotics themselves and combined with the formulation's process characteristics. This creates a microenvironment conducive to the stable storage of different bacteria, thereby achieving the stability of live bacteria in the microbial dosage form during long-term storage.

[0216] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0217] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0218] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A microbial preparation, characterized in that, Includes active ingredients, said active ingredients being Lactobacillus, Bifidobacterium, and Enterococcus faecalis (…). Enterococcus faecalis ) and Bacillus cereus ( Bacillus cereus The microbial preparation is a microbial powder or a microbial tablet; When the microbial preparation is a microbial powder, the water activity of the microbial powder is 0.05-0.20; When the microbial preparation is a microbial tablet, the water activity of the microbial tablet is 0.15-0.35; The Bifidobacterium is Bifidobacterium lactis subsp. animalis (Bifidobacterium Bifidobacterium animalis subsp. lactis The accession number of the animal Bifidobacterium lactis subsp. is CGMCC No. 19079.

2. The microbial preparation according to claim 1, characterized in that, When the microbial preparation is a microbial powder, the microbial powder further includes excipients; the excipients are selected from at least one of fillers, moisture regulators, acidulants, and flow aids.

3. The microbial preparation according to claim 2, characterized in that... The moisture regulator is selected from at least one of corn starch and potato soluble starch.

4. The microbial preparation according to claim 2, characterized in that, The moisture regulator is potato soluble starch.

5. The microbial preparation according to claim 2, characterized in that, The moisture regulator has a mass fraction of 10% to 30% in the microbial powder.

6. The microbial preparation according to claim 2, characterized in that, The moisture regulator has a mass fraction of 20% in the microbial powder.

7. The microbial preparation according to claim 2, characterized in that, The filler is selected from at least one of pregelatinized starch, trehalose, lactose, mannitol, glucose, sucrose, skim milk powder, dextrin, calcium sulfate, dicalcium phosphate, calcium carbonate, microcrystalline cellulose, and sucrose oligosaccharides.

8. The microbial preparation according to claim 2, characterized in that, The filler is selected from at least one of trehalose, lactose, mannitol, glucose, sucrose, skim milk powder, and fructooligosaccharides.

9. The microbial preparation according to claim 2, characterized in that, The acidulant includes at least one selected from citric acid and sodium citrate.

10. The microbial preparation according to claim 2, characterized in that, The flow aid comprises at least one of the following: silica, light anhydrous silica, talc, stearic acid, micronized silica, sodium / magnesium dodecyl sulfate, sodium stearate fumarate, glyceryl behenate, and magnesium stearate.

11. The microbial preparation according to claim 2, characterized in that, The flow aid comprises at least one selected from silica, light anhydrous silica, talc, and magnesium stearate.

12. The microbial preparation according to claim 2, characterized in that, The filler includes mannitol and / or the acidulant includes citric acid, and the excipients include a moisture regulator.

13. The microbial preparation according to claim 2, characterized in that, The excipients further include flavorings.

14. The microbial preparation according to claim 2, characterized in that, The filler has a mass fraction of 70% to 97% in the microbial powder.

15. The microbial preparation according to claim 2, characterized in that, The filler has a mass fraction of 74% to 77% in the microbial powder.

16. The microbial preparation according to claim 2, characterized in that, The filler includes lactose and mannitol.

17. The microbial preparation according to claim 16, characterized in that, The mass ratio of lactose to mannitol is 1:

4.

18. The microbial preparation according to claim 2, characterized in that, The mass fraction of the glidant in the microbial powder is not less than 1.0%.

19. The microbial preparation according to claim 2, characterized in that, The flow aid in the microbial powder has a mass fraction of not less than 1.5%.

20. The microbial preparation according to claim 2, characterized in that, The flow aid has a mass fraction of 1.5% in the microbial powder.

21. The microbial preparation according to claim 2, characterized in that, The acidulant has a mass fraction of 0-0.40% in the microbial powder.

22. The microbial preparation according to claim 2, characterized in that, The acidulant has a mass fraction of 0.3% to 0.40% in the microbial powder.

23. The microbial preparation according to claim 2, characterized in that, The acidulant includes citric acid and sodium citrate; The mass ratio of citric acid to sodium citrate is (7~8):

1.

24. The microbial preparation according to claim 23, characterized in that, The mass ratio of citric acid to sodium citrate is 7.5:

1.

25. The microbial preparation according to claim 2, characterized in that, The lactobacilli include Lactobacillus paracasei ( Lactobacillus paracasei ) or Lactobacillus acidophilus ( Lactobacillus acidophilus ).

26. The microbial preparation according to claim 25, characterized in that, The Lactobacillus paracasei has the accession number CGMCC No. 19077.

27. The microbial preparation according to claim 1, characterized in that, When the microbial preparation is a microbial tablet... The microbial tablets further include excipients; The excipients are selected from at least one of fillers, moisture regulators, and flow aids.

28. The microbial preparation according to claim 27, characterized in that, The moisture regulator is selected from at least one of corn starch and potato soluble starch.

29. The microbial preparation according to claim 27, characterized in that, The moisture regulator is corn starch.

30. The microbial preparation according to claim 27, characterized in that, The moisture regulator has a mass fraction of 10% to 30% in the microbial tablets.

31. The microbial preparation according to claim 27, characterized in that, The moisture regulator has a mass fraction of 10% to 20% in the microbial tablets.

32. The microbial preparation according to claim 27, characterized in that, The filler is selected from at least one of pregelatinized starch, trehalose, lactose, mannitol, glucose, sucrose, skim milk powder, dextrin, calcium sulfate, dicalcium phosphate, calcium carbonate, microcrystalline cellulose, and sucrose oligosaccharides.

33. The microbial preparation according to claim 27, characterized in that, The filler includes microcrystalline cellulose, lactose, and sucrose.

34. The microbial preparation according to claim 27, characterized in that, The filler has a mass fraction of 70% to 97% in the microbial tablets.

35. The microbial preparation according to claim 33, characterized in that, The sucrose in the microbial tablets has a mass fraction of 15-25%.

36. The microbial preparation according to claim 33, characterized in that, The lactose in the microbial tablets has a mass fraction of 12-18%.

37. The microbial preparation according to claim 33, characterized in that, The lactose in the microbial tablets has a mass fraction of 12-13%.

38. The microbial preparation according to claim 33, characterized in that, The microcrystalline cellulose in the microbial tablets has a mass fraction of 45-55%.

39. The microbial preparation according to claim 33, characterized in that, The microcrystalline cellulose in the microbial tablets has a mass fraction of 49-50.5%.

40. The microbial preparation according to claim 27, characterized in that, The flow aid comprises at least one of the following: silica, light anhydrous silica, talc, stearic acid, micronized silica, sodium / magnesium dodecyl sulfate, sodium stearate fumarate, glyceryl behenate, and magnesium stearate.

41. The microbial preparation according to claim 27, characterized in that, The gliding agent comprises at least one selected from stearic acid, magnesium stearate, micronized silica gel, sodium / magnesium dodecyl sulfate, talc, sodium stearate fumarate, and glyceryl behenate.

42. The microbial preparation according to claim 27, characterized in that, The excipients further include flavorings.

43. The microbial preparation according to claim 27, characterized in that, The lactobacilli include Lactobacillus paracasei or Lactobacillus acidophilus.

44. The microbial preparation according to claim 43, characterized in that, The Lactobacillus paracasei has the accession number CGMCC No. 19077.

45. A microbial powder, characterized in that, The water activity of the microbial powder is 0.05-0.2, comprising: 1-10 parts by weight of Bifidobacterium animalis subsp. lactis powder with accession number CGMCC No. 19079. 1-10 parts by weight of Lactobacillus paracasei powder, 0.5-5 parts by weight of Enterococcus faecalis powder, 0.5-5 parts by weight of Bacillus cereus powder, 100 parts by weight of corn soluble starch, 383 parts by weight of lactose, 7.5 parts by weight of silicon dioxide; or, include: 1-10 parts by weight of Bifidobacterium animalis subsp. lactis powder with accession number CGMCC No. 19079. 1-10 parts by weight of Lactobacillus paracasei powder, 0.5-5 parts by weight of Enterococcus faecalis powder, 0.5-5 parts by weight of Bacillus cereus powder, 100 parts by weight of corn soluble starch, 300 parts by weight of mannitol, 75 parts by weight of lactose, 7.5 parts by weight of silicon dioxide, 1.5 parts by weight of citric acid, 0.2 parts by weight of sodium citrate, 4 parts by weight of fragrance; or, include: 1-10 parts by weight of Bifidobacterium animalis subsp. lactis powder with accession number CGMCC No. 19079. 1-10 parts by weight of Lactobacillus paracasei powder, 0.5-5 parts by weight of Enterococcus faecalis powder, 0.5-5 parts by weight of Bacillus cereus powder, 100 parts by weight of corn starch, 300 parts by weight of mannitol, 75 parts by weight of lactose, 7.5 parts by weight of silicon dioxide, 1.5 parts by weight of citric acid, 0.2 parts by weight of sodium citrate, 4 parts by weight of fragrance; or, include: 1-10 parts by weight of Bifidobacterium animalis subsp. lactis powder with accession number CGMCC No. 19079. 1-10 parts by weight of Lactobacillus paracasei powder, 0.5-5 parts by weight of Enterococcus faecalis powder, 0.5-5 parts by weight of Bacillus cereus powder, 100 parts by weight of potato soluble starch 300 parts by weight of mannitol, 75 parts by weight of lactose, 7.5 parts by weight of silicon dioxide, 1.5 parts by weight of citric acid, 0.2 parts by weight of sodium citrate, 4 parts by weight of fragrance.

46. ​​The microbial powder according to claim 45, characterized in that, The viable bacterial count in the animal Bifidobacterium lactis bacterial powder is not less than 1 x 10 6 CFU / g; The viable count of Lactobacillus paracasei powder is not less than 1×10⁻⁶. 6 CFU / g; The viable bacteria count in the Enterococcus faecalis powder is not less than 1×10⁻⁶. 6 CFU / g; The viable count of the Bacillus cereus powder is not less than 1×10⁻⁶. 5 CFU / g.

47. A microbial tablet, characterized in that, The water activity of the microbial tablets is 0.15-0.35, comprising: 5-10 parts by weight of *Bifidobacterium animalis* subsp. *lactobacter* powder, wherein the preservation number of the *Bifidobacterium animalis* subsp. *lactobacter* powder is CGMCC No. 19079. 5-10 parts by weight of Lactobacillus acidophilus powder or Lactobacillus paracasei powder, 0.5-5 parts by weight of Enterococcus faecalis powder, 0.5-5 parts by weight of Bacillus cereus powder, 500 parts by weight of microcrystalline cellulose, 129 parts by weight of lactose, 195 parts by weight of sucrose 156 parts by weight of corn starch, 5 parts by weight of magnesium stearate.

48. The microbial tablet according to claim 47, characterized in that, The viable bacteria count in the Bifidobacterium lactis subsp. animalis powder is not less than 1×10⁻⁶. 6 CFU / g; The viable count of the Lactobacillus paracasei powder or Lactobacillus acidophilus powder is not less than 1×10⁻⁶. 6 CFU / g; The viable bacteria count in the Enterococcus faecalis powder is not less than 1×10⁻⁶. 6 CFU / g; The viable count of the Bacillus cereus powder is not less than 1×10⁻⁶. 5 CFU / g.

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