CN118178481A - Microbial preparation and preparation method thereof - Google Patents

Abstract

The invention provides a microbial preparation. The microbial preparation comprises active ingredients, wherein the active ingredients are lactobacillus, bifidobacterium, enterococcus faecalis and bacillus cereus, and the water activity of the microbial preparation is 0.03-0.20. The microbial preparation according to the embodiment of the invention has greatly improved stability compared with a microbial preparation of the same dosage form comprising the same microorganism.

Description

Microbial preparation and preparation method thereof

Technical Field

The present invention relates to the field of biological products, and in particular to a microbial preparation and a preparation method thereof.

Background technique

Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the human body and changing the composition of the flora in a certain part of the host. The quality indicators of water activity of multi-linked microecological preparations are controlled differently. Different bacteria are different and have different requirements for conditions. The prior art does not report the optimal conditions for controlling pharmaceutical preparations with different microbial combinations. According to the principles of microbial physiology, it can be seen that the key factor affecting the storage stability of microbial powder is water.

There is an increasing need to find the right technology to improve encapsulation efficiency and viability of probiotics while providing a final product that meets quality requirements. Encapsulation technologies have certain limitations in maintaining the viability of probiotics during processing and in long-term storage. Therefore, it is crucial to find the right system for encapsulation of bacteria without losing their viability. Each element included in the selection process should take into account all aspects to achieve the highest possible microbial viability.

In order to improve the efficacy of drugs, on the basis of commonly used microbial dosage forms, we can also consider developing drug preparations in multiple dosage forms, new pathways and new methods, so as to further reduce side effects and further improve patient compliance with medication. This is also an important aspect of new preparation development research. The transport process of drugs in different dosage forms in the body and the relationship between their blood drug concentration and time are obviously different, and their respective onset time, peak time and intensity of action are also different. How to evaluate the rationality of new drug dosage forms is not only an important aspect of the new drug review process, but also a difficulty for new drug developers in the development process. A basic criterion for judging and evaluating the rationality of new drug dosage forms is that this new dosage form can better serve clinical treatment compared with the original dosage form. As a new drug dosage form research, we should focus on clinical needs and select dosage forms scientifically and objectively.

In addition, one purpose of changing the dosage form is to improve the medication compliance of patients of different age groups. The selection of new dosage forms should be evaluated from multiple aspects such as the method of administration, appearance, shape, size, color, smell, etc. This is especially important in the selection of pediatric dosage forms. On the premise of ensuring the quality and safety of children's medication, the physiological and psychological characteristics of children should be considered as much as possible, and new drug dosage forms that improve children's medication compliance should be selected and developed.

In short, the rationality of the selection of new drug dosage forms and the control of quality indicators should be comprehensively evaluated and analyzed from the perspectives of the drug's physical and chemical properties and biological properties, clinical treatment needs, patient compliance with medication, the pros and cons of existing preparations, market development prospects, etc. Only in this way can we ensure that the dosage form selection and quality indicator control are reasonable and accurate, reduce the waste of drug research resources, and reduce the cost of drug evaluation and supervision. Only in this way can we better play the important role of new drug dosage forms in medical and health practice and promote the comprehensive development of medical science and technology.

However, the prior art does not report on key indicators for quality stability control of microecological preparations for different microbial preparation dosage forms, different types of excipients, ratios between excipients, and preparation stability quality control standards. Therefore, there is still a continuous demand for the improvement of multi-probiotic preparations.

Summary of the invention

This application is based on the inventor's discovery and understanding of the following problems:

The inventors have found that moisture content and water activity have a significant effect on the viability of microorganisms. Water activity (aw) and moisture content will not only affect the viability of microorganisms during the processing of microbial preparations, but also affect the viability of microorganisms during subsequent storage. Studies have reported that if the preparation can maintain stability in a dry state, the water activity must not exceed 0.25 and the moisture content should be 4-7%. However, the water activity cannot be too low. For example, when the water activity is <0.1, the oxidation of membrane lipids will reduce the viability of microorganisms. Through the present invention, the inventors have found that different preparations have different protective effects on microorganisms. The study found that the fundamental reason for the different protective effects is that the different microenvironments formed by preparations prepared with different types of excipients and different excipient ratios affect the storage stability of different bacteria. Different dosage forms of pharmaceutical preparations have their specific preparation technology, scope of application and method of use. After changing the dosage form, due to the different material structures, drug loading forms, drug release mechanisms, administration routes, drug release modes and speeds of various dosage forms, in the process of microbial preparation research, for different microecological dosage forms, it is necessary to screen the excipient formula that can enable the preparation to be stored stably based on the characteristics of the probiotic microorganisms themselves and the characteristics of the preparation prescription process, so as to form a microenvironment that is conducive to the stable storage of different bacteria, thereby achieving the stability of live bacteria in the microecological dosage form during long-term storage.

In the pharmaceutical field, drug powders have a large specific surface area, are easy to disperse and absorb, have a rapid onset of action, are easy to prepare, and have a certain mechanical protective effect on the sore surface when used externally. They are widely used in dentistry, otolaryngology, traumatology, and surgery, and are also suitable for pediatric administration. However, they are easy to absorb moisture, and the chemical activity of drug components is enhanced, which makes them easy to lose and oxidize. In the process of preparing microbial powders, it is necessary to screen the excipient formula that can stabilize the storage of the preparation according to the characteristics of the probiotic microorganisms themselves, and at the same time combine the characteristics of the formulation process to form a microenvironment that is conducive to the stable storage of different bacteria.

Based on the key role of the water activity of the microbial preparation and the influence of different types and proportions of excipients on the stability of the microbial preparation discovered by the above inventors, in the first aspect of the present invention, the present invention proposes a microbial preparation. According to an embodiment of the present invention, the microbial preparation includes an active ingredient, and the active ingredient is Lactobacillus, Bifidobacterium, Enterococcus faecalis and Bacillus cereus, and the water activity of the microbial preparation is 0.03-0.20. According to an embodiment of the present invention, the water activity of the microbial preparation is 0.1-0.15, 0.05-0.1 or 0.15-0.2. Compared with the microbial preparation of the same dosage form including the same microorganism, the microbial preparation according to the embodiment of the present invention has greatly improved stability under normal temperature storage conditions.

According to an embodiment of the present invention, the above-mentioned microbial preparation may further include at least one of the following additional technical features:

According to an embodiment of the present invention, the water activity of the microbial preparation is 0.04-0.20, more preferably, 0.05-0.20.

According to an embodiment of the present invention, the lactobacillus is Lactobacillus acidophilus or Lactobacillus paracasei, and the bifidobacterium is Bifidobacterium infantis or Bifidobacterium animalis subsp. lactis.

According to an embodiment of the present invention, the preservation number of the Lactobacillus paracasei is CGMCC 19077. For detailed information and characteristics of this strain of microorganism, see patent 201911419617.6, application date 2019-12-31, the full text of which is incorporated as part of this application.

According to an embodiment of the present invention, the deposit number of the Enterococcus faecalis is CGMCC No. 19078. For detailed information and characteristics of the strain of microorganism, please refer to patent 201911419590.0, application date is 2019-12-31, and the full text of the patent is introduced as part of this application.

According to the microbial preparation of the embodiment of the present invention, the Lactobacillus paracasei is sensitive to multiple antibiotics, such as penicillins (penicillin, ampicillin, piperacillin), cephalosporins (cefaclor, cefazolin, ceftriaxone, cefuroxime, ceftazidime, cephalothin, cefoperazone), macrolides (erythromycin, azithromycin, clarithromycin), tetracyclines (tetracycline, minocycline, doxycycline), aminoglycosides (spectinomycin), nitrofurans (nitrofurantoin), lincomycins (clindamycin), rifamycins (rifamycin), and styrene. flat), carbapenems (meropenem), chloramphenicol, etc.; it has been proved through experiments that the Lactobacillus paracasei is non-toxic to animals such as mice, and mice survive healthily after taking the strain, and gain weight normally, which meets the quality standard requirements of probiotics; the Lactobacillus paracasei can resist the acidic environment of gastric juice (for example, in an artificial gastric juice environment of pH1.5 to 4.5, the strain survives well); the Lactobacillus paracasei can also resist the bile salt environment in the intestine (for example, at a bile salt concentration of 0.03 to 0.3%, the bacteria survives well). The Lactobacillus paracasei provided by the invention can be colonized in the human body and play a probiotic role. It has a good inhibitory effect on harmful flora in the intestine, thereby playing a role in regulating intestinal microbial flora. In addition, the strain can be stored for a long time under dry and normal temperature conditions, and the strain activity is not lost. Therefore, by formulating the microbial preparation into a solid preparation form, the stable survival time of the microorganism can be extended.

According to the microbial preparation of the embodiment of the present invention, the Enterococcus faecalis meets many indicators of probiotics, so the Enterococcus faecalis can be used as a new type of probiotic. The bacteria are moderately sensitive to norfloxacin, vancomycin and macrolide antibiotics, and are sensitive to commonly used antibiotics (penicillins, cephalosporins, tetracyclines, meropenem, etc.); it has been proved through experiments that the Enterococcus faecalis is not toxic to animals such as mice, and mice survive healthily after taking the strain, and their weight increases normally, which meets the quality standard requirements of probiotics; the Enterococcus faecalis can resist the acidic environment of gastric juice (for example, in an artificial gastric juice environment of pH2.5 to 4.5, the strain survives well); the Enterococcus faecalis can also resist the bile salt environment in the intestine (for example, at a bile salt concentration of 0.03 to 0.3%, the bacteria survive well). The strain can be stored for a long time under dry and normal temperature conditions, and the strain activity is not lost. Therefore, by formulating the microbial preparation into a solid preparation form, the stable survival time of the microorganism can be extended.

According to an embodiment of the present invention, the microbial preparation is in the form of a powder, the water activity of the microbial preparation is 0.05-0.2, and the microbial preparation further comprises an auxiliary material, wherein the auxiliary material comprises at least one selected from a filler, a moisture regulator, a sourness regulator, and a flow aid. The microbial powder according to an embodiment of the present invention has higher stability.

According to the microbial powder of an embodiment of the present invention, the filler includes at least one selected from trehalose, lactose and mannitol.

According to the microbial powder of an embodiment of the present invention, the moisture regulator includes at least one selected from corn starch, soluble starch and potato soluble starch.

According to the microbial powder of the embodiment of the present invention, the moisture regulator is soluble starch. The inventors found that the hygroscopicity of soluble starch from potato is lower than that of soluble starch from corn starch or other sources under different environments, and thus the powder preparation according to the embodiment of the present invention has higher stability.

According to the microbial powder of an embodiment of the present invention, the acidulant includes at least one selected from citric acid and sodium citrate.

According to the microbial powder of the embodiment of the present invention, the glidant is silicon dioxide.

According to the microbial powder of the embodiment of the present invention, the auxiliary material further includes flavor.

According to the microbial powder of the embodiment of the present invention, the filler includes mannitol, and the auxiliary material includes a moisture regulator. The inventors found that if the auxiliary material includes mannitol, the water activity of mannitol is high, and the compatibility with infant bifidobacterium and lactobacillus acidophilus is slightly poor. Therefore, the moisture regulator needs to be added to the auxiliary material to reduce the moisture content so as to improve the compatibility of mannitol with bifidobacterium and lactobacillus.

According to the microbial powder of the embodiment of the present invention, the mass fraction of the flow aid in the microbial powder is not less than 1.0%, preferably, the mass fraction is not less than 1.5%, and more preferably, the mass fraction is 1.5%. The inventors found that the amount of flow aid added significantly affects the fluidity of the powder, and controlling the amount of flow aid added to be not less than 1.5% can significantly improve the fluidity of the powder. At the same time, considering the effect of the flow aid on the taste of the product, choosing 1.5% can further improve the taste of the powder product while ensuring the better fluidity of the product.

According to the microbial powder of the embodiment of the present invention, the mass fraction of the active ingredient in the microbial powder is 1.6-2.6%, preferably, the mass fraction is 2.1%.

According to the microbial powder of the embodiment of the present invention, the mass fraction of the moisture regulator in the microbial powder is 10% to 30%, preferably, the mass fraction is 20%.

According to the microbial powder of the embodiment of the present invention, the mass fraction of the filler in the microbial powder is 70% to 97%, preferably, the mass fraction of the filler in the microbial powder is 74% to 77%.

According to the microbial powder of the embodiment of the present invention, the filler includes lactose and mannitol, thereby further improving the sweetness of the product.

According to the microbial powder of the embodiment of the present invention, the mass ratio of lactose to mannitol is 1:4.

According to the microbial powder of the embodiment of the present invention, the mass fraction of the acidulant in the microbial powder is 0-0.04%, preferably, the mass fraction of the acidulant in the microbial powder is 0.3%-0.40%.

According to the microbial powder of the embodiment of the present invention, the acidulant includes citric acid and sodium citrate.

According to the microbial powder of the embodiment of the present invention, the mass ratio of citric acid to sodium citrate is (7-8):1, preferably, 7.5:1.

In the second aspect of the present invention, the present invention provides a microbial powder. According to an embodiment of the present invention, the water activity of the microbial powder is 0.05-0.2, and the microbial powder comprises: 5 parts by weight of infant bifidobacterium powder, 5 parts by weight of lactobacillus acidophilus powder, 1 part by weight of enterococcus faecalis powder, 1 part by weight of bacillus cereus powder, 100 parts by weight of soluble starch, 383 parts by weight of lactose, and 7.5 parts by weight of silicon dioxide.

In the third aspect of the present invention, the present invention proposes a microbial powder. According to an embodiment of the present invention, the water activity of the microbial powder is 0.05-0.2, and the microbial powder comprises: 5 parts by weight of infant bifidobacterium powder, 5 parts by weight of lactobacillus acidophilus powder, 1 part by weight of enterococcus faecalis powder, 1 part by weight of bacillus cereus powder, 100 parts by weight of soluble starch, 300 parts by weight of mannitol, 75 parts by weight of lactose, 5 parts by weight of silicon dioxide, 1.5 parts by weight of citric acid, 0.2 parts by weight of sodium citrate, and 0.2 parts by weight of flavor.

In a fourth aspect of the present invention, the present invention proposes a microbial powder. According to an embodiment of the present invention, the water activity of the microbial powder is 0.05-0.2, and the microbial powder comprises: 5 parts by weight of infant bifidobacterium powder, 5 parts by weight of lactobacillus acidophilus powder, 1 part by weight of enterococcus faecalis powder, 1 part 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 flavor.

In a fifth aspect of the present invention, the present invention proposes a microbial powder. According to an embodiment of the present invention, the water activity of the microbial powder is 0.05-0.2, and the microbial powder comprises: 5 parts by weight of infant bifidobacterium powder, 5 parts by weight of lactobacillus acidophilus powder, 1 part by weight of enterococcus faecalis powder, 1 part 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 flavor.

The microbial powder according to the embodiment of the present invention has greatly improved stability under normal temperature conditions, saves energy consumption, has strong process operability, and has a good product taste, and is suitable for industrial production.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a microscopic photograph of Lactobacillus paracasei after Gram staining according to Inventive Example 1, with a magnification of 100 times;

Fig. 2 is the effect of different pH values on the viable count of Lactobacillus paracasei according to an embodiment of the invention;

Fig. 3 is the effect of different concentrations of bile salts on the viable count of Lactobacillus paracasei according to an embodiment of the invention;

FIG4 is a graph showing the stable performance of Enterococcus faecalis at pH 2.5-4.5 according to an embodiment of the present invention;

5 is a graph showing the stable performance of Enterococcus faecalis in artificial intestinal fluids with bile salt concentrations of 0.03% and 0.1% according to an embodiment of the present invention;

FIG6 is a diagram showing the structure of the H ⁺ -ATPase gene cluster according to an embodiment of the present invention;

FIG7 is a diagram showing the electrophoresis results of Enterococcus faecalis plasmid according to an embodiment of the present invention;

FIG8 is a diagram showing hemolysis of bacteria on a blood plate according to an embodiment of the present invention;

9 is a graph showing the stability results of Bifidobacterium infantis in the microbial powder preparation prescription 3 according to Example 3 of the present invention at different water activities and 25° C.;

FIG10 is a graph showing the stability results of Lactobacillus acidophilus in the microbial powder preparation formula 3 according to Example 3 of the present invention at different water activities and 25° C.;

FIG11 is a graph showing the stability results of Enterococcus faecalis in the microbial powder preparation prescription 3 according to Example 3 of the present invention at different water activities and 25° C.;

FIG. 12 is a graph showing the stability results of Bacillus cereus in the microbial powder preparation formula 3 according to Example 3 of the present invention at different water activities and 25° C.

FIG. 13 is a graph showing the stability results of the powder prepared by Enterococcus faecalis according to Example 4 of the present invention.

Detailed ways

Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

The present invention will be further explained below in conjunction with specific examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

Example 1: Acquisition and biological activity of Lactobacillus paracasei

1. Isolation, purification and identification of Lactobacillus paracasei

5-10g of fresh feces from healthy infants were collected, placed in a glycerol tube and transferred to a refrigerator with a biological ice pack for later use. In a sterile operating table, transfer 1g of anaerobically stored infant feces sample to 9ml of sterile saline and mix evenly, then dilute 10 times to 1* ^10-8 , and use a pipette to draw 100μL of each of the three gradient dilutions of 1* ^10-6 , 1* ^10-7 , and 1* ^10-8 into the solid culture medium MRS- _CaCO3 , with 3 plates for each dilution. After anaerobic culture at 37℃ for 48 hours, pick the colonies with lactic acid bacteria characteristics on the surface, isolate the single strain and inoculate it into MRS liquid culture medium for expansion culture. The isolated strains were identified from the following aspects:

1.1 Colony and bacterial morphology: The isolated strains were cultured anaerobically at 48℃ and 37℃ on MRS plate medium. The colonies were round, with irregular edges, milky white or grayish white, raised, shiny, opaque, and sticky, with a diameter of 0.6-2mm. After Gram staining of solid culture medium, Gram-positive long and short rods were visible under the microscope, without capsules and spores. The rods can be in short chains, or single, with round ends, and often form short rods after many passages. The smears of liquid culture medium are mainly Gram-positive short rods, which are uniform, as shown in Figure 1.

1.2 Culture characteristics: The isolated strain is a facultative anaerobe and can grow under aerobic conditions, but it grows better under anaerobic culture. The nutritional requirements are general. It can grow in culture medium containing peptone and yeast. Some carbohydrates are conducive to growth, such as glucose. It can also grow well in milk. It can grow at 35-45℃, and the optimal growth temperature is 37℃. It can grow in pH 5.0-8.0, but the optimal pH is 5.5-6.0. Tween-80 and other factors stimulate growth. The isolated strain was cultured anaerobically at 37℃ for 48 hours in MRS liquid culture medium to form granular or flaky precipitation, or turbid growth, and the final pH can be below 4.0.

1.3 Biochemical characteristics

1.3.1 The isolated strain does not produce catalase, does not liquefy gelatin, does not reduce nitrate, and does not produce indole.

1.3.2 The isolated strain can produce acid by fermenting glucose, lactose, maltose, galactose, sucrose, cellobiose, trehalose and mannose.

1.3.3 Mannitol, inulin, ribose and melezitose can be fermented to produce acid or not fermented to produce acid.

1.3.4 Rhamnose, sorbitol, arabinose, mesidic acid, raffinose, sorbitol, etc. do not undergo fermentation and do not produce acid.

1.4 Others: The metabolites of the isolated strains were analyzed by gas-liquid chromatography. MRS medium was used for 48 hours and anaerobic culture was performed at 37°C. Lactic acid was the main product, and a small amount of other acids were produced. However, lactic acid was the dominant product in terms of production. The G+C content in DNA was 37% mol%.

Finally, based on comprehensive experimental analysis of cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequencing, and phsS gene sequence, and with reference to Bergey's Manual of Systematic and Evelutionary Microbiology and relevant research papers in the International Journal of Systematic and Evelutionary Microbiology, the isolated strain was identified as a new type of Lactobacillus paracasei. The inventors deposited the strain with the deposit number CGMCC19077.

1.5 Culture medium formulation

1.5.1 MRS-CaCO ₃ medium formula for separation

Make up to 1 L with distilled water, adjust pH to 6.3±0.1, add 1.5-2.0% agar powder and 2% calcium carbonate.

1.5.2 MRS medium formula for culture

Make up to 1 L with distilled water, adjust pH to 6.3±0.1, and add 1.5-2.0% agar powder.

The specific characteristics of the new strain Lactobacillus paracasei (CGMCC 19077) found by the present invention will be described in detail below.

2. Antibiotic sensitivity, strain stability evaluation and mouse toxicity experiments

2.1 Antibiotic sensitivity: The inventors further used the K-B method to determine the sensitivity of Lactobacillus paracasei isolated in 1 to antibiotics. The inventors found that the bacteria were sensitive to almost all types of antibiotics, such as penicillins (penicillin, ampicillin, piperacillin), cephalosporins (cefaclor, cefazolin, ceftriaxone, cefuroxime, ceftazidime, cephalothin, cefoperazone), macrolides (erythromycin, azithromycin, clarithromycin), tetracyclines (tetracycline, minocycline, doxycycline), aminoglycosides (spectinomycin), nitrofurans (nitrofurantoin), lincomycins (clindamycin), rifamycins (rifampicin), carbapenems (meropenem), chloramphenicol, etc.

2.2 Evaluation of strain stability: The inventors conducted continuous subculture and comparative experiments to examine the morphology, physicochemical and other classification and identification characteristics, and antibiotic sensitivity of the isolated Lactobacillus paracasei in Example 1. It was found that no visible changes were found in the isolated Lactobacillus paracasei after multiple subcultures, indicating that the isolated Lactobacillus paracasei has a stable heritable shape.

2.3 Mouse toxicity experiment: By feeding the isolated bacteria to mice, it was found that all mice survived healthily and gained weight normally, indicating that the isolated Lactobacillus paracasei is non-toxic and highly safe.

3. Acid and bile resistance test of strains

3.1 Strain acid resistance test: Artificial gastric juice was prepared according to the relevant general rules of the fourth volume of the 2015 edition of the Chinese Pharmacopoeia. In short, 16.4 ml of dilute hydrochloric acid was taken, about 800 ml of water and 10 g of pepsin were added, and after shaking, the mixture was diluted with water to 1000 ml to obtain artificial gastric juice. Considering that the bacteria enter the stomach in a liquid state and empty quickly, the survival rate within 3 hours was investigated in artificial gastric juice. Samples were taken and counted at 0 min, 15 min, 30 min, 60 min, 90 min, 120 min, and 180 min respectively. Considering that the pH of the stomach changes (1.5-4.5) after hunger and meals, samples were taken and counted at pH 1.5, 2.5, 3.5, and 4.5 respectively. Considering that the gastric juice is at a pH of about 2.5 in normal state, this study also tested the survival of commercially available Lactobacillus paracasei at a pH of 2.5. The results are shown in Table 1 and Figure 2.

Table 1 Survival of Lactobacillus paracasei in artificial gastric juice

(Note: Lactobacillus paracasei of the present invention: LP; commercially available Lactobacillus paracasei: MLP)

As shown in Table 1 and Figure 2, in 3h, Lactobacillus paracasei of the present invention all shows stable activity at pH 2.5-4.5. Under pH 2.5 condition, it is slower for Lactobacillus paracasei of the present invention to process 3h viable count decline trend under acidic conditions, and viable count of commercially available Lactobacillus paracasei has decreased nearly an order of magnitude compared with initial viable count after viable count of commercially available Lactobacillus paracasei has been processed under acidic conditions for 3h. Thus, it is proved that Lactobacillus paracasei of the present invention is higher than commercially available Lactobacillus paracasei viable count, and Lactobacillus paracasei of the present invention has higher acid resistance.

3.2 Bile salt tolerance test: Artificial intestinal fluid was prepared according to the relevant general rules of the fourth volume of the 2015 edition of the Chinese Pharmacopoeia. In brief, 6.8g of potassium dihydrogen phosphate was taken, 500ml of water was added to dissolve it, and the pH value was adjusted to 6.8 with 0.1mol/L sodium hydroxide solution. Another 10g of pancreatic enzyme was taken, and an appropriate amount of water was added to dissolve it. After mixing the two liquids, the artificial intestinal fluid was diluted to 1000ml. Considering that bacteria enter the small intestine in a liquid state and are emptied quickly, the survival rate within 3 hours was examined in the artificial intestinal fluid. Samples were taken and counted at 0min, 15min, 30min, 60min, 90min, 120min, and 180min respectively. Considering that the bile salt content in the human small intestine fluctuates within the range of 0.03%-0.3%, experiments and sampling and counting were designed under the conditions of 0.03%, 0.1%, 0.2%, and 0.3% bile salt; considering that bile is at about 0.2% in normal conditions, this study also tested the survival of commercially available Lactobacillus paracasei under the extreme bile concentration of about 0.3%. The experimental results are shown in Table 2 and Figure 3.

Table 2 Survival of Lactobacillus paracasei in artificial intestinal fluid

(Note: Lactobacillus paracasei of the present invention: LP; commercially available Lactobacillus paracasei: MLP)

The above data show that the isolated and purified Lactobacillus paracasei has strong acid and bile salt resistance.

Example 2: Acquisition and biological activity of Enterococcus faecalis

1. Isolation, purification and identification of Enterococcus faecalis

5-10g of fresh feces from children were collected, placed in a glycerol tube and transferred to a refrigerator with a biological ice pack for use. In a sterile operating table, 1g of anaerobically stored feces sample from children was transferred to 9mL of sterile saline and mixed evenly, and then diluted 10 times to 1× ^10-8 . 100mL of each of the three gradient dilutions of 1× ^10-6 , 1× ^10-7 , and 1×× ^10-8 was pipetted into the solid culture medium MRS-CaCO3, with ₃ plates for each dilution. After anaerobic culture at 37°C for 48 hours, colonies with lactic acid bacteria characteristics on the surface were picked and inoculated into EC liquid culture medium for expansion culture. Gram staining test, catalase test, and 16SrRNA sequencing were performed to determine that they were Enterococcus faecalis. Among them, MRS-CaCO ₃ peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, Tween 80 1mL, dipotassium hydrogen phosphate 2g, sodium acetate 5g, diammonium citrate 2g, magnesium sulfate heptahydrate 0.5g, manganese sulfate 0.25g, distilled water 1L, pH 6.3±0.1, agar powder 1.5-2.0%, 2% CaCO ₃ ; the formula of EC culture medium is: casein peptone 10.0g, soy peptone 5.0g, glucose 5.0g, yeast extract 5.0g, disodium hydrogen phosphate 4.0g, potassium dihydrogen phosphate 4.0g, agar 15.0g, add water to 1L, pH 7.4±0.1, 25℃.

The specific tests are as follows:

1.1 Colony and bacterial morphology

The colonies are light yellow, round, with neat and slightly raised edges.

The results of fungal morphology examination showed Gram-positive, ovoid cocci, most of which were arranged in pairs or short chains.

1.2 Culture characteristics

Grows well under both anaerobic and aerobic conditions

1.3 Biochemical characteristics

The biochemical reaction characteristics are shown in Table 3.

Table 3 Biochemical reaction characteristics of the tested strains

1.4 API reagent strip identification

The strain to be tested was identified using API 20Strep reagent strips, and a good identification result was obtained, which was Enterococcus faecalis, with an identification value of 99.2%, a T value of 0.99, and no inconsistent items. The reaction results are shown in Table 4.

Table 4 API reaction characteristics of tested strains

1.5 16S rRNA sequence analysis

The 16S rRNA gene sequence of the strain to be tested in this experiment was compared with the sequence in GenBank by running the BLAST program of NCBI, and the strain was identified as Enterococcus faecalis. The classification name of the Enterococcus faecalis is Enterococcus faecalis, and the deposit number is CGMCC No.19078.

Various properties of Enterococcus faecalis (CGMCC No. 19078) will be studied below.

Study on the acid and bile resistance of 2 strains

2.1 Cultivation of strains to be tested

EC medium is used to culture Enterococcus faecalis strains and commercially available Enterococcus faecalis strains. Accurately weigh according to the formula, add distilled water and stir to mix, adjust the pH to 7.4, and sterilize at 121℃ for 20min. Take 2% inoculation amount and inoculate Enterococcus faecalis into the sterilized EC medium, and culture aerobically at 180rpm and 37℃.

2.2 Preparation of artificial gastric juice and intestinal juice

Artificial gastric juice: Take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, shake well, and dilute with water to 1000 mL.

Artificial intestinal fluid, i.e. phosphate buffer (containing pancreatic enzyme, pH 6.8): take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve it, and adjust the pH to 6.8 with 0.1 mol/L sodium hydroxide solution; take another 10 g of pancreatic enzyme, add appropriate amount of water to dissolve it, mix the two solutions, and dilute with water to 1000 mL.

The specific experimental process is as follows:

2.3 Gastrointestinal survival experiment

In artificial gastric juice and artificial intestinal juice, the effects of pH, bile salt concentration and treatment time on the survival rate of Enterococcus faecalis were investigated. Considering that the pH value of gastric juice is about 2.5 in normal state, this study also tested the survival of commercially available Enterococcus faecalis at a pH value of 2.5; considering that bile is about 0.2% in normal state, this study also tested the survival of commercially available Enterococcus faecalis at an extreme bile concentration of about 0.3%. The experimental results are as follows:

(1) Acid resistance

Enterococcus faecalis showed good tolerance to simulated gastric fluid.

Table 5 Acid resistance test of Enterococcus faecalis

(Note: Enterococcus faecalis of the present invention: EC; Commercially available Enterococcus faecalis: MEC)

As shown in Table 5 and FIG. 4 , the Enterococcus faecalis of the present invention exhibits stable activity at pH 2.5-4.5 within 3 hours. Under pH 2.5, the number of viable bacteria of the Enterococcus faecalis of the present invention is almost unchanged after being treated under acidic conditions for 3 hours, and the number of viable bacteria of the commercially available Enterococcus faecalis after being treated under acidic conditions for 3 hours is reduced by about 6 times compared with the initial number of viable bacteria. This proves that the number of viable bacteria of the Enterococcus faecalis of the present invention is higher than that of the commercially available Enterococcus faecalis, and the Enterococcus faecalis of the present invention has higher acid resistance.

(2) Bile salt tolerance

Table 6 Bile salt resistance test of Enterococcus faecalis

(Note: Enterococcus faecalis: EC; Commercially available Enterococcus faecalis: MEC)

As shown in Table 6 and Figure 5, the artificial intestinal fluid with bile salt concentrations of 0.03% and 0.3% has little effect on the survival rate of Enterococcus faecalis; when the bile salt concentration is increased to 0.2%, the survival rate of the bacteria slowly decreases with the treatment time; when the bile salt concentration is 0.3%, the survival rate of Enterococcus faecalis is still about 50% after 1 hour. These results all show that Enterococcus faecalis has good bile salt resistance. The number of live bacteria of commercially available Enterococcus faecalis after being treated with 0.3% bile salt for 3 hours shows a significantly lower trend than the initial number of live bacteria than the Enterococcus faecalis of the present invention. This proves that the number of live bacteria of the Enterococcus faecalis of the present invention is higher than that of commercially available Enterococcus faecalis, and the Enterococcus faecalis of the present invention has a higher bile salt resistance.

2.4 Related characteristic genes

The genome information of Enterococcus faecalis was obtained by next-generation sequencing. Comparative genome analysis with reported strains for acid resistance and choline resistance showed that Enterococcus faecalis has complete genes encoding subunits of H ⁺ -ATPase and two Na ⁺ /H ⁺ antiporters NhaC, one Na ⁺ , K ⁺ , Li ⁺ and Rb ⁺ /H ⁺ antiporter NhaK, and a gene encoding cholylglycine hydrolase. Primers were designed based on the genome information, and the genes encoding H ⁺ -ATPase and cholylglycine hydrolase were obtained.

Primers were designed respectively: getH-ATPase-f, getH-ATPase-r and getCGH-f, getCGH-r.

The strains were activated and cultured, and the genome was extracted from fresh cultures as a template for gene cloning. The cells were lysed using the EasyPureGenomic DNA Kit enzymatic method to extract genomic DNA. The steps are as follows:

① Take 2 mL of overnight cultured bacteria, centrifuge at 10,000 × g for 1 min, and discard the supernatant.

② Centrifuge again and aspirate the supernatant as much as possible.

③ Add 200 μL (20 mg/mL) of lysozyme to the bacteria and resuspend them. Incubate at 37°C with shaking for 60 min. Centrifuge at 10,000 × g for 1 min and discard the supernatant.

④ Add 100 μL LB11 and 20 μL proteinase K, and use a pipette to pipette until the bacteria are completely suspended.

⑤Incubate at 55℃ for 15 minutes.

⑥Add 20 μL RNase A, mix well and let stand at room temperature for 2 minutes.

⑦ Add 400 μL of BB11 added with anhydrous ethanol and vortex for 30 seconds. Add all the solution to the centrifuge column, centrifuge at 10,000×g for 1 minute, and discard the effluent.

⑧Add 500 μL CB11, centrifuge at 10,000×g for 1 min, and discard the flow-through.

⑨Repeat the above steps once.

⑩ Add 500 μL of WB11 to which anhydrous ethanol has been added, centrifuge at 10,000×g for 1 min, and discard the flow-through.

Repeat the above steps once.

Centrifuge at 10,000 × g for 2 min to completely remove residual WB11.

Place the centrifuge column in a clean centrifuge tube, add 50 μL of preheated EB to the center of the column, let stand at room temperature for 2 minutes, centrifuge at 10,000×g for 1 minute, elute the DNA, and draw 3-5 μL of the obtained genomic DNA for agarose gel electrophoresis verification. The remaining DNA is stored at -20℃.

The excellent acid resistance of Enterococcus faecalis (CGMCC No. 19078) is closely related to its H ⁺ -ATPase. By analyzing the H ⁺ -ATPase gene cluster (structure shown in FIG6 ), it was found that the atpD has 84% homology with the atpD of E. hirae ATCC 9790. Its gene sequence is shown in SEQ ID NO:1.

Cholylglycine hydrolase (EC 3.5.1.24) is a hydrolase of enterococci, with a total of 1068 bp, which can catalyze the hydrolysis of glycocholate into bile acid and glycine, and can provide carbon and nitrogen sources for bacteria. Its sequence is shown in SEQ ID NO:2.

Safety study of 3 strains of Enterococcus faecalis

3.1 Drug resistance analysis

The drug-sensitive paper diffusion method was used. Freshly cultured Enterococcus faecalis was diluted to 10 ⁷ cfu/ml with physiological saline, evenly spread on an M17 culture dish, and placed at room temperature for 3-5 minutes before placing drug-sensitive paper. The distance between the centers of the paper discs was no less than 24 mm, and the distance between the edge of the paper disc and the edge of the agar was no less than 15 mm. Inverted culture was placed in a 37°C incubator, and the diameter of the antibacterial ring was measured for 24 hours.

The drug resistance of Enterococcus faecalis strains and their 30th generation strains was determined by the disc diffusion method. The results are shown in Tables 7 and 8 below.

Table 7 Drug resistance results of Enterococcus faecalis

Note: R = resistant; I = intermediate; S = sensitive; -: no inhibition zone;

Table 8 Drug resistance results of Enterococcus faecalis strains passaged 30 generations

Note: R = resistant; I = intermediate; S = sensitive; -: no inhibition zone;

From the above drug resistance results, it can be seen that Enterococcus faecalis and Enterococcus faecalis-30 are sensitive and moderately sensitive to most common antibiotics. The drug resistance results of Enterococcus faecalis and Enterococcus faecalis-30 are consistent, indicating that the passage process has no effect on the drug resistance of Enterococcus faecalis. This proves that the Enterococcus faecalis of the present invention has a low risk of antibiotic transfer and a high safety of the strain.

3.2 Virulence factor analysis

The hemolysin and biofilm formation of Enterococcus faecalis were determined using the methods reported in the literature, and the in vivo safety evaluation in animals was carried out according to the methods of the current edition of the Pharmacopoeia.

(1) Plasmid extraction was performed on the strain, and the results are shown in Figure 7. It shows that the strain does not contain plasmids, indicating that the transfer level of drug-resistant genes is low and the strain is highly safe.

(2) Hemolysin detection

Hemolysin secreted by bacteria can cause erythrocyte lysis or other tissue damage. The production of hemolysin is mainly related to virulence genes such as cylA and cylB. The hemolysis of each group of bacteria on the blood plate is shown in Figure 8. From the hemolytic experiment, it can be seen that there is no hemolytic ring around the Enterococcus faecalis colony of the present invention, the ATCC 29212 strain (hereinafter referred to as the control strain) has a slight hemolytic ring, and there is an obvious hemolytic ring around Staphylococcus aureus. The results show that the hemolytic test of the Enterococcus faecalis strain of the present invention is negative, the strain is non-hemolytic, and the safety is high.

(3) Biofilm assay

According to statistics, 80% of bacterial infections are related to the formation of bacterial biofilms (biofilms for short). The morphology and physiological effects of bacteria in biofilms are different from those of free bacteria. The tolerance to antibiotics can be increased by 10-1000 times, and the resistance to host immune defense is very strong, which is the main cause of bacterial resistance. The ability to form biofilms has a great influence on bacterial virulence.

The results of the experimental detection of biofilm OD values of Enterococcus faecalis are shown in Table 9.

Table 9 Results of Enterococcus faecalis biofilm assay

Experimental Grouping Experiment 1 Experiment 2 Mean Biofilm OD value blank 0.090 0.084 0.087 / Enterococcus faecalis 0.412 0.412 0.412 0.325 Enterococcus faecalis-30 0.298 0.282 0.290 0.203

According to the analysis of tetracycline-resistant Enterococcus faecalis biofilm in the literature, the biofilm-forming ability of Enterococcus faecalis is strongly positive when OD>2, medium when 1<OD<2, and weakly positive when OD<1. The experimental results show that the biofilm-forming ability of Enterococcus faecalis is weakly positive, and the OD values are low, indicating that the biofilm-forming ability of the Enterococcus faecalis of the present invention is weak, the results remain unchanged after 30 generations of bacteria, and the level of antibiotic resistance of the strain is low.

(4) Safety evaluation in animals

After intragastric administration, the mice were in good health. Compared with the saline group, there was no difference in the activity of the mice, and the hair condition was normal. The weight of the mice before and after intragastric administration is shown in Table 10.

Table 10 Changes in mouse body weight before and after oral administration

The results showed that the weight of mice in each group increased, and there was little difference in weight change between the two groups (there was no statistically significant difference between the two groups, p>0.05).

Example 3: Preparation and stability study of microbial powder

1. Auxiliary materials inspection experiment,

The following excipients are listed for investigation:

Fillers: Trehalose, lactose, mannitol

Moisture regulator: corn starch, soluble starch

Acidulant: citric acid

· Glidants: silicon dioxide, silicon dioxide, light anhydrous silicic acid, talc, magnesium stearate Inspection method:

The sample is weighed in the ratio of Bifidobacterium infantis powder: Lactobacillus acidophilus powder: Enterococcus faecalis powder: Bacillus cereus powder: auxiliary materials = 5:5:1:1:500 (the weight ratio of citric acid to silicon dioxide is 50), and the auxiliary materials are dried in advance.

The control group was prepared by weighing Bifidobacterium infantis powder: Lactobacillus acidophilus powder: Enterococcus faecalis powder: Bacillus cereus powder in a ratio of 5:5:1:1.

The aluminum foil bags were directly heat-sealed and placed in a 37°C stabilization box to test the number of viable bacteria at 0, 3, 6, and 10 days.

The results showed that trehalose, lactose, corn starch, soluble starch and silicon dioxide were compatible with the four bacteria, and the number of live bacteria basically did not decrease on the 10th day, maintaining the original order of magnitude; mannitol and citric acid were not compatible with Bifidobacterium infantis and Lactobacillus acidophilus, and had decreased by 1 order of magnitude on the 10th day, but were compatible with Enterococcus faecalis and Bacillus cereus.

2. Excipient Optimization Experiment

This time, mannitol and citric acid were re-examined, and sodium citrate and flavors were also examined together. The examination reduced the moisture by adding soluble starch, proving that the poor compatibility of mannitol and citric acid was caused by the high water activity, not the poor compatibility between the auxiliary materials themselves and the bacteria.

Bifidobacterium infantis powder: Lactobacillus acidophilus powder: Enterococcus faecalis powder: Bacillus cereus powder: first auxiliary material: soluble starch = 5:5:1:1:4:1. Weigh the sample (the first auxiliary material includes citric acid, sodium citrate and flavor), and dry the auxiliary materials in advance.

Bifidobacterium infantis powder: Lactobacillus acidophilus powder: Enterococcus faecalis powder: Bacillus cereus powder: second auxiliary material: soluble starch = 5:5.1:1:40:10. Weigh the sample (the second auxiliary material can be mannitol or soluble starch), and dry the auxiliary materials in advance.

The aluminum foil bags were directly heat-sealed and placed in a 37°C stabilization box to test the number of viable bacteria at 0, 3, 6, and 10 days.

The results showed that citric acid, sodium citrate, essence, mannitol, and soluble starch had good compatibility with the four bacteria. Conclusion: Trehalose, lactose, corn starch, soluble starch, silicon dioxide, citric acid, sodium citrate, essence, and mannitol excipients had good compatibility with the four bacteria of Siliankang. The number of viable bacteria did not decrease on the 10th day, and the original number of viable bacteria was maintained. The excipients were selected according to the requirements of formula and process for formula screening.

3. Formulation screening

3.1 Filler screening and investigation

Firstly, soluble starch and lactose with good compatibility were selected as fillers, and silicon dioxide was selected as a flow aid for screening, and the particle size distribution, water content, and fluidity were used as the evaluation indicators for screening. The screening prescription is shown in Table 11.

Table 11: Microbial powder prescription

1000 bags of prescription (unit: g) Prescription 1 Prescription 2 Prescription 3 Prescription 4 Bifidobacterium infantis powder 5 5 5 5 Lactobacillus acidophilus powder 5 5 5 5 Enterococcus faecalis powder 1 1 1 1 Bacillus cereus powder 1 1 1 1 Soluble starch 150 100 50 lactose 483 333 383 433 Silicon dioxide 5 5 5 5 total 500 500 500 500

Conclusion: The fluidity of the four prescriptions was poor, and the effect of different silicon dioxide ratios on fluidity was investigated in the future. At the same time, the formula contains soluble starch, and the oral granular texture is strong. The powder dosage form is positioned as a children's product. Some excipients for adjusting the taste are added to the formula, including citric acid, sodium citrate, and sweet orange flavor.

3.2 Silica ratio screening

Commonly used glidants for solid preparations include silicon dioxide, light anhydrous silicic acid, talc, magnesium stearate, etc. In this study, silicon dioxide was selected for research. Different proportions of silicon dioxide were added during the experiment, and the optimal addition ratio of silicon dioxide was determined by the flowability index. The results are shown in Table 12.

Table 12: Effect of different proportions of silica addition on fluidity

The experimental results show that when the proportion of silicon dioxide is ≥1.5%, the overall fluidity of the formula is good, and 1.5% silicon dioxide can already meet the fluidity requirements. Considering that the amount of silicon dioxide added has an impact on taste and other aspects, the lowest concentration of 1.5% is selected.

4. Stability test under different conditions

4.1 Formulation information

By studying the types and proportions of raw materials and auxiliary materials of powder preparations, the preparation prescription was finally determined as shown in Table 13.

Table 13: Prescription of microecological preparation powder

4.2 Stability investigation

In the present invention, the inventors simultaneously investigated the stability of the four-combination probiotic preparations at different water activities at two temperature conditions of 4°C and 25°C. The stability investigation data is such as the tablet stability investigation data. The number of live bacteria of the four strains of infant Bifidobacterium, Lactobacillus acidophilus, Enterococcus faecalis and Bacillus cereus at different water activities at 4°C is relatively stable. When stored for 3 months, the water activity has almost no change during the stability investigation. The inventors found in the research process that the stability of the four-combination microbial preparation at 25°C can be achieved by controlling the water activity index, and the stability of the four strains of probiotics can be achieved by controlling the water activity range of different preparation prescriptions, thereby achieving the stability of the storage of the probiotic preparation at room temperature. Formula B was selected for stability investigation, and the stability investigation results of the four bacteria in the microbial powder preparation prescription at different water activities and 25°C are shown in Table 14 and Figures 9 to 12.

Table 14: Results of stability study of four bacteria in microbial powder formulation at 25℃ at different water activities

Results: As can be seen from the table above, when the water activity in the quadruple microbial powder preparation is higher than 0.2, the levels of infant Bifidobacterium and Lactobacillus acidophilus decrease rapidly, and the stability of the two strains is poor. When the water activity is controlled within the range of 0.05-0.2, the stability of the four strains is good, and the quadruple probiotic preparation can be stored at room temperature.

In addition, during this study, the inventors also investigated that when the water activity of the formulation is controlled below 0.05, the excipients used in the formulation need to be pre-treated and dried according to environmental conditions before the powder is prepared, which greatly increases the powder process and energy consumption costs. In addition, in the actual production process, it is necessary to control environmental conditions to prevent the moisture absorption of the raw and excipients of the formulation, and the feasibility of the product preparation process is low, and large-scale production in the production workshop cannot be guaranteed. Therefore, for the microbial powder dosage form, the stability of the formulation with a water activity below 0.05 is no longer investigated.

Example 4: Preparation of Enterococcus faecalis powder (powder)

5.1 Preparation of raw material powder: After activation, primary seed liquid culture, seed tank seed preparation and fermentation, the Enterococcus faecalis working seeds are centrifuged to collect the bacteria, and a freeze-drying protective agent is added to freeze-dry the bacteria to obtain Enterococcus faecalis powder.

5.2 Pretreatment of raw materials and auxiliary materials: According to the weight percentage of the product, Enterococcus faecalis and Bifidobacterium animalis powder, Lactobacillus paracasei powder, Bacillus cereus powder and pharmaceutically acceptable excipients such as lubricants (magnesium stearate, talc, polyethylene glycol 6000, stearic acid, sodium lauryl sulfate/magnesium, sodium stearyl fumarate, glyceryl behenate), glidants (talc, micro-powdered silica gel, silicon dioxide), fillers (skim milk powder, maltodextrin, fructooligosaccharides, pregelatinized starch, lactose, glucose, sucrose, D-mannitol, starch), sweeteners (mannitol, glucose, sucrose, D-mannitol, stevioside, white sugar), etc. are taken for pretreatment.

5.3 Mixing preparation: After passing the auxiliary materials through a 50-200 mesh sieve, add them into a batch mixer and mix evenly. Finally, add the bacterial powder and mix evenly. Discharge the materials after 50 minutes.

5.4 Packaging: The mixed raw and auxiliary materials were transferred to the powder packaging machine for packaging. The viable bacterial count of Enterococcus faecalis in the final product was ≥1×10 ⁷ cfu/g. The stability of Enterococcus faecalis in the finished product was tested at 20℃ and 25℃ respectively to determine the stability of Enterococcus faecalis in the finished product. The results are shown in Table 15 and Figure 13.

Table 15: Changes in the number of viable bacteria in the Enterococcus faecalis composite powder

As can be seen from Table 15 and Figure 13, the powder prepared from Enterococcus faecalis has good stability during the 12-month storage period at 20°C and 25°C.

Through the present invention, the inventors found that different preparations have different protective effects on microorganisms. The study found that the fundamental reason for the different protective effects is that the different microenvironments formed by the preparations prepared by different types of excipients and different excipient ratios affect the storage stability of different bacteria. In the process of microbial preparation research, for different microecological formulations, it is necessary to screen the excipient formula that can stabilize the preparation according to the characteristics of the probiotic microorganisms themselves, combined with the characteristics of the formulation process, to form a microenvironment that is conducive to the stable storage of different bacteria, thereby achieving the stability of live bacteria in the microecological formulation during long-term storage.

In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (9)

1. A microbial preparation, wherein the microbial preparation is in the form of a powder, and is characterized in that the microbial preparation comprises an active ingredient, wherein the active ingredient is lactobacillus, bifidobacterium, enterococcus faecalis and bacillus cereus, the water activity of the microbial preparation is 0.05-0.20, the lactobacillus is Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CGMCC No.19077. 2. The microbial preparation according to claim 1, characterized in that the bifidobacterium is Bifidobacterium infantis or Bifidobacterium animalis subsp. lactis. 3. The microbial preparation according to claim 1, characterized in that the microbial preparation further comprises an auxiliary material. 4. The microbial preparation according to claim 3, characterized in that the auxiliary material comprises at least one selected from a filler, a moisture regulator, a sourness regulator and a flow aid. 5. The microbial preparation according to claim 4, characterized in that the filler comprises at least one selected from trehalose, lactose, mannitol, glucose, sucrose, skimmed milk powder, and fructooligosaccharide; Optionally, the moisture regulator includes at least one selected from corn starch, soluble starch and potato soluble starch; Preferably, the moisture regulator is soluble starch; Optionally, the sour agent includes at least one selected from citric acid and sodium citrate; Optionally, the glidant comprises at least one selected from silicon dioxide, light anhydrous silicic acid, talc and magnesium stearate; Optionally, the auxiliary materials further include flavors. 6. The microbial preparation according to claim 5, characterized in that the filler comprises mannitol and/or the acidulant comprises citric acid, and the auxiliary material comprises a moisture regulator. 7. The microbial preparation according to claim 6, characterized in that the mass fraction of the glidant in the microbial powder is not less than 1.0%, preferably, the mass fraction is not less than 1.5%, and more preferably, the mass fraction is 1.5%. 8. The microbial preparation according to claim 6, characterized in that the mass fraction of the moisture regulator in the microbial powder is 10% to 30%, preferably 20%. Optionally, the mass fraction of the filler in the microbial powder is 70% to 97%, Preferably, the mass fraction of the filler in the microbial powder is 74% to 77%. Preferably, the filler comprises lactose and mannitol, Optionally, the mass ratio of lactose to mannitol is 1:4, Optionally, the mass fraction of the sour agent in the microbial powder is 0 to 0.04%, Preferably, the mass fraction of the acidulant in the microbial powder is 0.3% to 0.40%. Optionally, the acidulant comprises citric acid and sodium citrate, Optionally, the mass ratio of citric acid to sodium citrate is (7-8):1, preferably, 7.5:1. 9. A microbial powder, characterized in that the water activity of the microbial powder is 0.05-0.2, comprising: 5 parts by weight of infant Bifidobacterium powder or animal Bifidobacterium lactis subsp. 5 weight portions of Lactobacillus paracasei, the preservation number of which is CGMCC No.19077, 1 part by weight of Enterococcus faecalis powder, 1 part by weight of Bacillus cereus powder, 100 parts by weight of soluble starch, 383 parts by weight of lactose, 7.5 parts by weight of silicon dioxide; or include: 5 parts by weight of infant Bifidobacterium powder or animal Bifidobacterium lactis subsp. 5 weight portions of Lactobacillus paracasei, the preservation number of which is CGMCC No.19077, 1 part by weight of Enterococcus faecalis powder, 1 part by weight of Bacillus cereus powder, 100 parts by weight of soluble starch, 300 parts by weight of mannitol, 75 parts by weight of lactose, 5 parts by weight of silicon dioxide, 1.5 parts by weight of citric acid, 0.2 parts by weight of sodium citrate, 0.2 parts by weight of flavor; or include: 5 parts by weight of infant Bifidobacterium powder or animal Bifidobacterium lactis subsp. 5 parts by weight of Lactobacillus paracasei, the accession number of which is CGMCC No.19077, 1 part by weight of Enterococcus faecalis powder, 1 part 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 flavor; or include: 5 parts by weight of infant Bifidobacterium powder or animal Bifidobacterium lactis subsp. 5 parts by weight of Lactobacillus paracasei, the accession number of which is CGMCC No.19077, 1 part by weight of Enterococcus faecalis powder, 1 part 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 flavor.