Application and method of bacillus cereus PE-Z and traditional Chinese medicine composition fermented by bacillus cereus PE-Z in improvement of animal immunity and improvement of intestinal flora

Abstract

The invention relates to the technical field of fermentation of traditional Chinese medicine compositions, in particular to application of a traditional Chinese medicine composition fermented by bacillus cereus PE-Z to improvement of animal immunity and improvement of intestinal flora and a method. According to the invention, a microbial bacillus cereus strain PE-Z suitable for fermentation of the traditional Chinese medicine composition disclosed by the invention is screened out; the traditional Chinese medicine composition is developed for improving the animal immunity, the traditional Chinese medicine composition is prepared from radix astragali, rhizoma atractylodis macrocephalae, red dates, poria cocos, pericarpium citri reticulatae, fructus crataegi, fructus mume, radix glycyrrhizae, rhizoma polygonati and / or malt, and after the traditional Chinese medicine composition is fermented through bacillus cereus PE-Z, the animal immunity can be remarkably improved; the richness of AKK bacteria and lactobacillus in the intestinal tract is obviously improved; after the traditional Chinese medicine composition is subjected to PE-Z fermentation, the polysaccharide content can be remarkably increased, an optimal culture medium is obtained after orthogonal optimization, and the fermentation process is optimized.

Description

Technical Field

[0001] This invention relates to the field of fermentation of traditional Chinese medicine compositions, and particularly to the application and methods of Bacillus cereus PE-Z and its fermented traditional Chinese medicine compositions in enhancing animal immunity and improving intestinal flora. Background Art

[0002] The overuse of antibiotics in the current livestock industry has led to a series of problems: imbalance of animal gastrointestinal flora, increase of drug-resistant bacteria, significant decline in animal immunity, and frequent outbreaks of infectious diseases. At the same time, antibiotic residues in meat, eggs and milk directly threaten human health, making the development of antibiotic alternatives an urgent need for the livestock industry.

[0003] While traditional Chinese medicine (TCM) possesses the effects of enhancing immunity and resisting diseases, it suffers from drawbacks such as slow efficacy, long treatment duration, and low utilization rate of effective components, limiting its efficient application in the aquaculture industry. TCM fermentation technology offers a new approach to addressing these issues. The core advantage of TCM fermentation technology lies in the powerful extracellular enzyme secretion capabilities of microorganisms, including but not limited to proteases, amylases, and cellulases. The enzyme functions secreted by these strains include, but are not limited to: (1) promoting the release of effective components and enhancing efficacy through the appropriate fermentation of specific microorganisms with TCM. (2) Some specific microorganisms also exhibit another important capability during fermentation—the ability to synthesize their own polysaccharides. These strains can directly ferment and synthesize their own unique extracellular polysaccharides (EPS) using small molecule carbohydrates (such as monosaccharides and oligosaccharides) produced after decoction or enzymatic hydrolysis of TCM as precursors, constituting another important source of increased total polysaccharide content in TCM extracts (i.e., the "incremental effect"). (3) The protease, amylase and cellulase secreted by the strain can work together with the endogenous enzymes produced by the livestock and poultry to digest and decompose the intestinal chyme, thereby improving the feed conversion rate.

[0004] However, these effects are highly dependent on the specific microbial strains used and their unique metabolic pathways and enzyme system configurations. Strain specificity is extremely prominent in this process, becoming a key limiting factor and screening difficulty. This mainly presents the following technical defects: (1) Nutritional adaptability differences: Not all strains can effectively utilize the nutrients in specific Chinese medicine raw materials for growth and proliferation. The composition of Chinese medicine matrix is ​​complex and may lack certain specific factors necessary for the growth of some strains, resulting in the inability of the cells to grow normally and the inability to effectively start fermentation; (2) Antibacterial challenge: Many traditional Chinese medicines themselves contain natural antibacterial and bactericidal active ingredients, which make them unable to survive or severely limited in the Chinese medicine environment; (3) Risk of non-synergistic or even negative effects: If unsuitable strains are selected for fermentation, not only will the above-mentioned enzymatic hydrolysis and synthetic advantages be unable to increase the polysaccharide content, but they may even competitively consume or destroy the original polysaccharide components in the Chinese medicine matrix, ultimately leading to a decrease in polysaccharide extraction instead of an increase.

[0005] Therefore, for the development of the technology of fermenting traditional Chinese medicine with microorganisms, the key prerequisite and core step for success is to screen out fermentation strains that are highly compatible with the target traditional Chinese medicine composition. The selected target microorganisms must have strong adaptability, strong enzymatic hydrolysis ability, and strong directional synthesis ability in the target traditional Chinese medicine composition to ensure that the comprehensive effect after final fermentation is a significant increase in polysaccharides and a significant increase in animal immunity.

[0006] In short, scientifically and rigorously screening out fermentation strains that are highly compatible with the target traditional Chinese medicine composition and determining the significant clinical effects of the fermented traditional Chinese medicine composition through animal efficacy tests are the key foundation and decisive link for transforming the technology of fermenting traditional Chinese medicine with microorganisms from theoretical potential into practical application. Summary of the Invention

[0007] In view of the above, it is necessary for us to scientifically and rigorously screen out fermentation strains that are highly compatible with the target traditional Chinese medicine composition, improve the precipitation amount of active ingredients and polysaccharide content of traditional Chinese medicine after fermentation, and clarify its significant effects of enhancing immunity and improving intestinal flora through animal efficacy tests.

[0008] To achieve the above object, the technical solution adopted in the present invention is:

[0009] Bacillus cereus strain PE-Z, its preservation information is: Bacillus cereus PE-Z, its taxonomic name is: Bacillus cereus, the Chinese taxonomic name is: Bacillus cereus, and the preservation number is GDMCC NO: 66471; this strain is preserved in the Guangdong Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the preservation date is June 6, 2025.

[0010] The present invention also includes a fermented traditional Chinese medicine composition containing the above-mentioned Bacillus cereus strain PE-Z.

[0011] Furthermore, the components of the fermented traditional Chinese medicine composition are composed of 15 - 20 parts of Astragalus membranaceus, 10 - 15 parts of Atractylodes macrocephala, 15 - 20 parts of Chinese date, 10 - 15 parts of Poria cocos, 10 - 15 parts of Citrus reticulata Blanco, 10 - 15 parts of Crataegus pinnatifida, 5 - 8 parts of Prunus mume, and 2 - 5 parts of Glycyrrhiza uralensis Fisch.

[0012] The present invention also includes the application of the above-mentioned fermented traditional Chinese medicine composition in increasing the intestinal bacterium Akkermansia muciniphila.

[0013] Furthermore, the fermented traditional Chinese medicine composition comprises 15-20 parts of Astragalus membranaceus, 10-15 parts of Atractylodes macrocephala, 15-20 parts of jujube, 10-15 parts of Poria cocos, 10-15 parts of dried tangerine peel, 10-15 parts of hawthorn, 5-8 parts of dried plum, 2-5 parts of licorice, 10-15 parts of Polygonatum sibiricum, and 10-15 parts of malt.

[0014] The present invention also includes the application of the fermented traditional Chinese medicine composition in enhancing animal immunity.

[0015] The present invention also includes the application of the Bacillus cereus strain PE-Z in increasing the polysaccharide content of fermented traditional Chinese medicine compositions.

[0016] The present invention also includes a method for preparing the fermented traditional Chinese medicine composition, the method comprising:

[0017] (1) After pulverizing the Chinese herbal composition, place it in a Chinese herbal decoction pot, add 8-10 times the weight of the herbal material in water and soak for 30 minutes, boil for 1 hour and take the filtered decoction. The dregs are then decocted a second time with 8-10 times the weight of the herbal material. The two decoctions are combined and concentrated to 0.5g / mL.

[0018] (2) Inoculate the seed liquid of Bacillus cereus strain PE-Z into the Chinese medicine broth culture medium, place it in a constant temperature shaker at 160 rpm / min, and ferment it at 37℃ for 48 h.

[0019] Furthermore, the fermentation conditions for the herbal broth culture medium are as follows: fermentation time 2 days, liquid volume percentage 20%, inoculum volume percentage 4%, and rotation speed 160 rpm / min.

[0020] Furthermore, the herbal broth culture medium consists of 40% by volume broth culture medium and 60% by volume herbal rotary evaporation liquid.

[0021] The present invention has the following beneficial effects:

[0022] 1. Through extensive strain screening, this invention has identified Bacillus cereus strain PE-Z as a suitable fermentation microorganism for the traditional Chinese medicine composition of this application. The traditional Chinese medicine composition of this application is developed to enhance animal immunity. The composition consists of Astragalus membranaceus, Atractylodes macrocephala, jujube, Poria cocos, tangerine peel, hawthorn, dried plum, licorice, Polygonatum sibiricum and / or malt. This composition can enhance animal immunity. After fermentation by strain PE-Z, the content of polysaccharides in the traditional Chinese medicine can be significantly increased. Furthermore, the optimal culture medium and fermentation process were obtained through orthogonal optimization. The decoction of the traditional Chinese medicine composition, after fermentation by the strain, can significantly enhance animal immunity and improve intestinal flora.

[0023] 2. The traditional Chinese medicine composition of this application addresses the problems of insufficient animal immunity and intestinal flora disorder caused by antibiotic abuse in the current livestock industry. It develops a basic formula with tonifying Qi as the primary focus and digestion as an adjunct. The basic formula uses Astragalus membranaceus (Huang Qi) as the principal herb to tonify Qi, Atractylodes macrocephala (Bai Zhu) to strengthen the spleen and dry dampness, jujube (Hong Zao) to nourish blood and calm the mind, and Poria cocos (Fu Ling) to strengthen the spleen and eliminate dampness as assistant herbs, enhancing the spleen-tonifying and lung-strengthening functions of Astragalus membranaceus. Adjunct herbs such as Citrus reticulata (Chen Pi), Crataegus pinnatifida (Shan Zha), and Prunus mume (Wu Mei) are added to assist the principal and assistant herbs in regulating Qi and promoting digestion, thus achieving the effect of tonifying Qi and strengthening the spleen while simultaneously eliminating food stagnation. After fermentation with Bacillus cereus PE-Z, this basic formula significantly improves the spleen index and significantly increases the abundance of AKK bacteria and lactic acid bacteria in the intestines.

[0024] 3. Furthermore, this application has developed an optimal formula through techniques such as "adding flavors, subtracting flavors, and substitutions": Polygonatum and malt are added to the basic formula. Polygonatum enhances the depth of tonification from the perspective of replenishing qi and nourishing yin, and together with Astragalus, they form a "dual tonification of qi and yin." The added malt effectively eliminates various food stagnation caused by rice, noodles, meat, and eggs. Combined with hawthorn, it not only enhances the elimination of stagnation caused by greasy meat but also expands the scope of elimination for rice and noodle stagnation. The addition of dried tangerine peel regulates qi, promoting circulation and aiding tonification, achieving a balance of medicinal properties where "tonification is accompanied by circulation, and tonification does not cause stagnation." This constructs an efficacy system of "strengthening the body's resistance and consolidating the foundation, combining tonification and circulation," giving the formula the effects of "strengthening the spleen + replenishing qi and nourishing yin + aiding digestion." This optimal formula, after fermentation with Bacillus cereus PE-Z, significantly increased serum IgG levels. Attached Figure Description

[0025] Figure 1 The figure shows the comparison results of protease production by three Bacillus strains.

[0026] Figure 2 This is a diagram showing the species composition of the gut microbiota at the phylum level in mice.

[0027] Figure 3 This is a diagram showing the species composition of the gut microbiota at the genus level in mice.

[0028] Figure 4 This figure shows the metagenomic sequencing differences in gut microbiota between the treatment groups and the control group (using MetagenomeSeq analysis). Figure A shows the significant differences between the traditional Chinese medicine group and the control group; B shows the significant differences between the fermented traditional Chinese medicine group and the control group; C shows the significant differences between the probiotic group and the control group; D shows the significant differences between Bacillus cereus and the control group; and E shows the significant differences between the modified formula group and the control group.

[0029] Figure 5 This is a standard curve for glucose.

[0030] Figure 6 This is a graph showing the screening results of the culture medium.

[0031] Figure 7It is the single-factor result graph of fermentation time.

[0032] Figure 8 It is the single-factor result graph of inoculum size.

[0033] Figure 9 It is the single-factor result graph of liquid loading volume.

[0034] Figure 10 It is the single-factor result graph of rotation speed.

[0035] Biological material preservation information

[0036] The strain information preserved in this application is: Bacillus cereus PE-Z, its taxonomic name is: Bacillus cereus, and its Chinese taxonomic name is: Bacillus cereus. The preservation number is GDMCC No: 66471; this strain is preserved in the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The preservation date is June 6, 2025. Specific implementation manners

[0037] The present invention will be further described below in conjunction with the drawings, examples and tests, but it should not be construed as a limitation to the present invention:

[0038] Example 1

[0039] This example is the identification and safety detection of Bacillus cereus strain PE-Z, specifically as follows:

[0040] 1. Identification of Bacillus cereus strain PE-Z: The Bacillus cereus strain PE-Z of this application was isolated and preserved by the Animal Pharmacy Laboratory of the College of Animal Science, Jiangxi Agricultural University. It was identified as Bacillus cereus by 16S rDNA sequencing (Shanghai Personal Biotechnology Co., Ltd.) and combined with biochemical identification. Among them, the sequence obtained by amplifying the 16S rDNA sequence of this strain is shown in Sequence Table SEQ ID NO.1. The preservation number of this strain is GDMCC NO: 66471; this strain is preserved in the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences. The preservation date is June 6, 2025.

[0041] 2. To detect the safety of strain PE-Z, the following technical solutions are adopted in this example:

[0042] (1) Rabbit intestinal ligation test: The rabbit intestinal ligation test observes whether injecting the supernatant of Bacillus cereus PE-Z culture into ligated intestinal loops in rabbits induces typical enterotoxin symptoms such as intestinal fluid accumulation and intestinal congestion, thereby determining whether the bacterium produces enterotoxins. Three healthy rabbits were selected, anesthetized after fasting for 24 hours, and the ileum was removed through abdominal surgery. Each rabbit's ileum was ligated into four 5-7 cm long intestinal loops (intervals of about 2 cm) with cotton thread. Two intestinal loops from each rabbit were randomly selected and injected with the supernatant of Bacillus cereus PE-Z, while the other two were injected with an equal volume of physiological saline as a control. The abdominal cavity was then sutured. After the operation, the rabbits were placed in a warm environment, and anesthetized and opened again after 18-24 hours. The congestion and edema of the intestinal loops were observed, and the intestinal fluid volume was collected and measured (as fluid volume / intestinal loop length). If the fluid volume in the experimental group was significantly higher than that in the control group (usually ≥1 mL / cm) and the intestinal congestion was obvious, it suggested that the bacterium might produce enterotoxins. The results are shown in Table 1:

[0043] Table 1 Rabbit intestinal ligation test

[0044]

[0045] As shown in Table 1, the intestinal ligation experiment in rabbits revealed no congestion or edema in the intestinal loops and intestinal walls after injection of Bacillus cereus PE-Z supernatant; the amount of intestinal fluid was not significantly different from the control group. This indicates that Bacillus cereus PE-Z does not produce enterotoxins that could induce intestinal mucosal inflammation (congestion) or excessive intestinal fluid secretion in rabbits, demonstrating intestinal safety.

[0046] (2) 14-day subacute toxicity experiment: Twenty healthy Kunming mice (20±2g each) were selected and divided into two groups: a blank control group and a PE-Z group, with 10 mice in each group. The PE-Z group was administered a gavage concentration of 2×10⁻⁶. 8 Mice were administered 0.5 mL of CFU / mL Bacillus cereus PE-Z bacterial suspension twice daily. The control group received 0.5 mL of physiological saline orally in the morning and afternoon, for 14 consecutive days. The health status of the mice was observed daily, and the number of deaths was recorded. Results showed that all mice administered Bacillus cereus PE-Z via gavage were in good spirits and behaved no differently from the control group; no mice died. This indicates that mice ingesting this strain via the digestive tract at a dose far exceeding the actual possible exposure level did not experience central nervous system or organ damage, and the strain did not induce any abnormal reactions, demonstrating good safety.

[0047] Example 2

[0048] This embodiment studies the screening of Bacillus strains that promote the enhancement of polysaccharides in fermented traditional Chinese medicine:

[0049] 1. Experimental strain:

[0050] ① Bacillus subtilis YVK-M1 (isolated from the probiotic product Yiweikang), abbreviated as YWK-M1, was isolated, identified and preserved by the Animal Pharmacy Laboratory of the College of Animal Science, Jiangxi Agricultural University.

[0051] ② Bacillus subtilis PigCec-M2 (isolated from pig cecal feces), abbreviated as PC-M2, was isolated, identified and preserved by the Animal Pharmacy Laboratory of the College of Animal Science, Jiangxi Agricultural University.

[0052] ③ The preserved strain of Bacillus cereus in Example 1, strain PE-Z (abbreviated as PE-Z strain).

[0053] 2. Enzyme production capacity of the experimental strains

[0054] This experiment used the enzyme-producing clear zone method to compare the enzyme-producing abilities (protease and amylase) of three Bacillus strains isolated by our research group. The core indicator of the enzyme-producing clear zone method is the ratio of the clear zone diameter to the colony diameter (referred to as the "clear zone-to-colony ratio"), which can objectively quantify the enzyme-producing ability and relative enzyme activity of microorganisms. A higher ratio generally indicates a stronger enzyme-producing ability of the strain. The results are shown in Table 2:

[0055] Table 2. Ratio of enzyme-producing bacteria in three Bacillus strains (diameter of clear zone / colony diameter)

[0056]

[0057] Note: In the table, different capital letters under the data indicate that the differences in the same column are extremely significant (p < 0.01), and the same capital letters under the data indicate that the differences in the same column are not significant (p > 0.05). The same applies to the following tables.

[0058] From Table 2 and Figure 1 It was found that the protease production of Bacillus cereus PE-Z was significantly higher than that of Bacillus subtilis YWK-M1 and Bacillus subtilis PC-M2 (p < 0.01); the amylase production of Bacillus cereus PE-Z and Bacillus subtilis YWK-M1 did not differ significantly (p > 0.05). The amylase production of Bacillus subtilis PC-M2 was significantly higher than that of Bacillus subtilis YWK-M1 and Bacillus cereus PE-Z (p < 0.01).

[0059] 3. Comparison of polysaccharide content in the decoction of fermented traditional Chinese medicine compositions by experimental strains:

[0060] (1) Self-created Chinese medicine formula "Astragalus, Jujube and Atractylodes Decoction" (hereinafter referred to as the basic Chinese medicine formula): Astragalus 15 parts, Jujube 20 parts, Atractylodes macrocephala 10 parts, Tangerine peel 10 parts, Hawthorn 10 parts, Poria cocos 10 parts, Prunus mume 5 parts, Licorice 2 parts.

[0061] In this traditional Chinese medicine composition, the principal herb is Astragalus membranaceus, which tonifies Qi, strengthens the exterior, raises Yang, and enhances the body's immunity to resist external pathogens. The assistant herb, Atractylodes macrocephala, strengthens the spleen, dries dampness, and assists Astragalus membranaceus in tonifying Qi; jujubes tonify the middle Jiao, replenish Qi, nourish blood, and calm the mind; Poria cocos strengthens the spleen and eliminates dampness, assisting Atractylodes macrocephala in enhancing its spleen-strengthening effect. The adjuvant herbs are Citrus reticulata peel, hawthorn, and dried plum. Citrus reticulata peel regulates Qi and strengthens the spleen; hawthorn promotes digestion and eliminates food stagnation; dried plum astringes the lungs and intestines, assisting the principal and assistant herbs in regulating Qi, promoting digestion, and preventing the herbs from being too warming and drying. The guiding herb is Glycyrrhiza uralensis, which harmonizes the various herbs, allowing them to work synergistically.

[0062] All of the aforementioned herbs are rich in polysaccharides, such as astragalus polysaccharide, jujube polysaccharide, atractylodes polysaccharide, and hawthorn polysaccharide. Polysaccharides have effects such as enhancing immunity and anti-oxidation. Under the formulation of this invention, all herbs are used together, and the effective components are extracted by decoction to prepare a traditional Chinese medicine liquid. This formula not only leverages the immune-boosting effect of qi-tonifying herbs, but also utilizes the assistant and adjuvant herbs to resolve dampness and promote digestion, forming a synergistic mechanism of "tonifying to strengthen the body and promoting digestion to improve circulation," thereby enhancing immunity.

[0063] (2) Fermentation method: After crushing each medicinal material, soak it in 8-10 times its volume of water for 30 minutes, then decoct for 1 hour. After filtering, add 8-10 times its volume of water to the dregs and decoct for another hour. Combine the two decoctions and concentrate by rotary evaporation to a concentration of 0.5 g / mL of raw medicinal material. Prepare a culture medium by mixing 60% of the 0.5 g / mL basic formula solution with 40% ordinary nutrient broth and sterilize it in an autoclave at 121℃ for 30 minutes. Adjust the seed cultures of the three Bacillus strains YWK-M1, PC-M2, and PE-Z to 5×10⁻⁶. 7 Inoculate 3% of the herbal nutrient culture medium and incubate at 37℃ and 160 rpm / min for 48 h. Perform three replicates.

[0064] (3) Detection of polysaccharide content in the fermentation base liquid: The change in polysaccharide content before and after fermentation was detected using the anthrone-sulfuric acid method. The regression equation of the glucose standard curve was Y = 6.0214x + 0.0101, r 2 =0.9931, and the results are shown in Table 3:

[0065] Table 3 Comparison of polysaccharide content before and after fermentation of basic Chinese herbal formulas

[0066]

[0067] As shown in Table 3, Bacillus cereus PE-Z significantly increased the polysaccharide content of the basic traditional Chinese medicine formula, increasing it by 35.86% compared to before fermentation. After fermentation of the basic formula with Bacillus cereus PE-Z, the polysaccharide content was significantly higher than that of the Bacillus subtilis YWK-M1 and PC-M2 groups (p<0.01).

[0068] Example 3

[0069] This example is an experimental study on how fermented Chinese medicine broth can improve gut microbiota and enhance immunity.

[0070] 1. Strain: The preserved strain Bacillus cereus strain PE-Z (abbreviated as PE-Z strain) in Example 1.

[0071] 2. Activation medium: Broth medium, with the composition (g / L): peptone 10 g, beef extract powder 3 g, sodium chloride 5 g, made up to 1 L.

[0072] 3. Experimental animals: 60 SPF-grade KM male mice, with a body weight of 18 ± 2 g, purchased from Changsha Tianqin Biotechnology Co., Ltd. (License number: SCXK(Xiang)2022-0011).

[0073] 4. Reagents: Total superoxide dismutase (T-SOD) kit, malondialdehyde (MDA) kit, glutathione (GSH) kit, immunoglobulin G (IgG) kit.

[0074] 5. Test method:

[0075] (1) Traditional Chinese medicine composition: ① Basic formula in Example 1: "Qi-Zao-Zhu-Ling Decoction" ② Formula with added ingredients 1: Add dolichos lablab and endothelium corneum gigeriae galli to "Qi-Zao-Zhu-Ling Decoction": astragalus membranaceus 15 parts, atractylodes macrocephala 10 parts, red dates 20 parts, poria cocos 10 parts, dried tangerine peel 10 parts, hawthorn 10 parts, smoked plum 5 parts, licorice root 2 parts, endothelium corneum gigeriae galli 10 parts and dolichos lablab 10 parts. The two added herbs in this prescription: endothelium corneum gigeriae galli and dolichos lablab. Among them, endothelium corneum gigeriae galli is sweet in taste and neutral in nature, and belongs to the spleen, stomach, small intestine and bladder meridians, with the functions of strengthening the stomach and promoting digestion, containing gastrin and digestive enzymes (good at digesting various types of food stagnation such as rice, noodles, meat and eggs). After being combined with hawthorn in the formula, it not only enhances the digestive ability of greasy food stagnation, but also expands the scope of digestion of rice and noodle food stagnation. Dolichos lablab has the effects of strengthening the spleen and removing dampness without harming the stomach, relieving summer heat and detoxifying and immunizing, belongs to the spleen and stomach meridians, and has a sweet and slightly warm nature; both herbs mainly function to strengthen the spleen and promote digestion, and play a digestive function in the prescription.

[0076] (2) Prepare the traditional Chinese medicine liquid from the traditional Chinese medicine composition in (1) according to the following method: Grind the traditional Chinese medicine herbs together into powder, pour it into a decocting pot, add 8 - 10 times the amount of distilled water and decoct for 1 h. After filtering out the decoction, add 8 - 10 times the amount of distilled water again and decoct for 1 h, and filter out the decoction and make up to a concentration of 0.082 g / mL of crude drug.

[0077] (3) Preparation of Bacillus cereus PE-Z seed liquid: Take out the preservation bottle of Bacillus cereus PE-Z from the -80°C refrigerator, cultivate single colonies by streak plate method, pick single colonies, inoculate them into 20 mL of nutrient broth and culture overnight, and then perform subculture for 13 h to obtain the PE-Z seed liquid (>10 8 ), which is the initial seed liquid, and dilute it to 5×10 7 as the inoculation seed liquid before fermentation.

[0078] (4) Preparation of the basic fermentation formula: The decoction of the basic formula was rotary evaporated to a concentration of 0.5 g / mL. The prepared Bacillus cereus PE-Z seed culture was diluted with broth to a concentration of 5 × 10⁻⁶ g / mL. 7 CFU / mL. The fermentation medium was prepared and sterilized according to the ratio of 60% rotary evaporation of traditional Chinese medicine and 40% ordinary broth. The inoculum was 4% and the liquid volume was 20%. It was cultured at 37℃ on a shaker at 160 rpm / min for 48 h.

[0079] (5) Animal grouping and treatment: After 3 days of acclimatization, mice were randomly divided into 6 groups according to body weight: blank control group (C1), basic formula group (C2), fermented basic formula group (C3), Astragalus polysaccharide group (C4), Bacillus cereus group (C5), and modified formula 1 group (basic formula plus white hyacinth bean and chicken gizzard lining, C6), with 10 mice in each group. The administration period was from 6 PM to 8 AM the following day. During this period, each group was given the corresponding drugs according to Table 4. At 8 AM the following day, the water was replaced with clean water for the mice to drink freely. The administration method was continuous administration for 5 days, followed by a 2-day break, and then continuous administration for another 5 days. Sampling was performed after 12 hours of fasting but not water restriction. The mouse room temperature was 25±2℃, and the humidity was 55±3%, with a 12-hour diurnal cycle. The bedding was changed every 2 days. Details are shown in Table 4.

[0080] Table 4. Grouping and Treatment of Experimental Animals

[0081]

[0082] (6) Sample collection: After the drug administration cycle ended, mice were fasted for 12 hours but allowed free access to water. Blood was collected from the eyeballs of the mice, and the serum was collected by centrifugation and stored at -80℃. The mice were euthanized by cervical dislocation, the spleen was removed and weighed, the liver was placed in cryovials and flash-frozen in liquid nitrogen, the ileum was fixed and preserved in 4% paraformaldehyde, and the feces from the cecum were placed in cryovials and flash-frozen in liquid nitrogen. Finally, all cryovials in liquid nitrogen were transferred to a -80℃ freezer for unified storage for subsequent related experiments.

[0083] (7) Test indicators:

[0084] ① Antioxidant function assay: Serum T-SOD and MDA levels and liver GSH content were detected using the corresponding ELISA kits (Nanjing Jiancheng Biotechnology Co., Ltd.). All procedures were strictly performed in accordance with the kit instructions.

[0085] ② Spleen index determination in mice: Spleen index = spleen weight (mg) / mouse body weight (g).

[0086] ③ Serum immunoglobulin IgG content (mg / mL) determination: The IgG ELISA kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd.) was used for detection.

[0087] ④ 16S rRNA sequencing of mouse fecal samples: To compare the effects of each drug group on the gut microbiota, fecal samples from 3 mice in each group were randomly selected for 16S rRNA sequencing. The sequencing work was completed by Shanghai Paisenno Biotechnology Co., Ltd.

[0088] (8) Statistical Analysis: SPSS 27.0 software was used for data processing and statistical analysis. The LSD (Least Significant Difference) method and Duncan's multiple comparisons were used to perform statistical analysis on the experimental data. All experimental data are expressed as mean ± standard deviation (Mean ± SD).

[0089] 6. The test results are as follows:

[0090] (1) The antioxidant function was determined as shown in Table 5:

[0091] Table 5 Antioxidant indices of mice in each treatment group

[0092]

[0093] Note: If the lowercase letters of the same superscript are the same in the same column, the difference is not significant (p>0.05); if the lowercase letters of the same superscript are different in the same column, the difference is significant (p<0.05). The same applies to the following table.

[0094] As shown in Table 5, serum MDA levels in mice in the fermentation-based formula group and the Bacillus cereus group were significantly lower than those in the blank control group (p<0.05), while liver GSH levels in mice in the fermentation-based formula group, the Bacillus cereus group, and the probiotic group were significantly higher than those in the blank control group (p<0.05). However, the three antioxidant indicators of the basic formula alone showed no significant difference compared to the blank group (p>0.05). This indicates that both the fermentation-based formula and Bacillus cereus PE-Z possess significant antioxidant activity.

[0095] (2) The specific effects of each drug administration group on the immunity of mice are shown in Table 6:

[0096] Table 6 Comparison of spleen index and serum IgG in mice of different treatment groups

[0097]

[0098]

[0099] Note: If the lowercase letters of the same column headings are the same, the difference is not significant (p>0.05); if the lowercase letters of the same column headings are different, the difference is significant (p<0.05).

[0100] As shown in Table 6, among all the treatment groups, only the spleen index of the fermented basic formula group was significantly higher than that of the blank control group (p<0.05), while the spleen index of the simple basic formula and the simple Bacillus cereus PE-Z bacterial solution groups were not significantly different from those of the blank group (p>0.05). This indicates that the Bacillus cereus PE-Z fermented basic formula significantly improved the efficacy of the drug (p<0.05). The serum IgG content of the fermented basic formula was 13.6% higher than that of the blank control group, which was the highest among all treatment groups, but the difference from the blank group was not significant (p>0.05). This may be because the fermented basic formula promoted the proliferation of B cells in the spleen, but they had not yet fully differentiated into plasma cells to secrete IgG.

[0101] In this embodiment, the basic formula consists of 15-20 parts of Astragalus membranaceus, 10-15 parts of Atractylodes macrocephala, 15-20 parts of jujube, 10-15 parts of Poria cocos, 10-15 parts of tangerine peel, 10-15 parts of hawthorn, 5-8 parts of dried plum, and 2-5 parts of licorice. After fermentation with PE-Z bacteria, all of them can achieve the desired effect. Within this range, the spleen index of the fermented basic formula group is 3.45-5.41 mg / g, which is significantly higher than that of the blank group.

[0102] (3) The effects of the fermentation base formula on the intestinal flora are detailed in Tables 7-8 and 8. Figures 2-4 As shown:

[0103] The species composition of the gut microbiota at the phylum level in each group of mice is shown in Table 7, and the species composition of the gut microbiota at the genus level in each group of mice is shown in Table 8.

[0104] Table 7. Proportion of gut microbiota species at the phylum level in each group of mice

[0105]

[0106] As shown in Table 7 and Figure 2 As shown, the gut microbiota of the basic fermentation formula group exhibited characteristic changes at the phylum level: the abundance of Verrucomicrobia was significantly increased (see [reference]). Figure 2 The percentages of Verrucomicrobia in the control group were 0.91%, the basic formula was 0.005%, the fermented basic formula was 11.13%, the probiotic group was 0.001%, the Bacillus cereus PE-Z group was 0.001%, and the flavored formula group was 0.26%. This shows that the fermented basic formula can significantly increase the abundance of Verrucomicrobia.

[0107] Table 8. Proportion of gut microbiota species at the genus level in each group of mice

[0108]

[0109] As shown in Table 8 and Figure 3The results showed that the gut microbiota of the basic fermentation formula exhibited characteristic changes at the genus level: the abundance of the genus Akkermansia (AKK) under the phylum Verrucomicrobia was significantly increased (see [reference]). Figure 3 The percentages of AKK bacteria were 0.91% in the blank control group, 0.005% in the basic formula, 11.13% in the fermented basic formula, 0.001% in the probiotic group, 0% in the Bacillus cereus PE-Z group, and 0.26% in the flavored formula 1 group.

[0110] Based on the above experimental data, we can see that, apart from the basic fermentation formula, other treatment groups, including the basic formula alone or Bacillus cereus PE-Z alone, could not increase the abundance of AKK bacteria in the gut. However, the basic formula fermented with Bacillus cereus PE-Z significantly increased the abundance of AKK bacteria in the gut. In this invention, the polysaccharide content was significantly increased after fermentation of the basic formula with Bacillus cereus, and it is speculated that this increase in polysaccharide content may be one of the reasons for the increased abundance of AKK bacteria in the gut. In addition, while specifically increasing the abundance of AKK bacteria, the fermentation formula group also synergistically increased the abundance of Lactobacillus, indicating that its regulatory effect on the gut microbiota is comprehensive and synergistic.

[0111] Comparison of metagenomic sequences of gut microbiota between the drug-treated groups and the blank control group (using MetagenomeSeq analysis) as follows: Figure 4 As shown, Figure 4 In the table, A represents the difference analysis between the basic formula group and the control group; B represents the difference analysis between the fermented basic formula group and the control group; C represents the difference analysis between the probiotic group and the control group; D represents the difference analysis between Bacillus cereus and the control group; and E represents the difference analysis between the flavored formula group and the control group.

[0112] As shown in the figure, the results of MetagenomeSeq analysis of the gut microbiota between the drug-treated groups and the blank control group were obtained by sequencing the 16S rRNA gene of mouse fecal samples. Figure 4 As shown, analysis based on ASV / OTU levels revealed that, compared to the blank control group, the abundance of *Akkermansia*, *Lactobacillus*, *Lactococcus*, *Enterococcus*, and *Staphylococcus* was significantly upregulated in the basic fermentation formula group (P<0.05). The abundance of *Oscillospira* and *Ruminococcus* was significantly upregulated in the *Bacillus cereus* PE-Z group (P<0.05), while the abundance of *Oscillospira*, *Ruminococcus*, and *Bacillus* was significantly upregulated in the basic formula group (P<0.05).

[0113] As a recognized potential probiotic in the gut, Akkermansia muciniphila is an important target for the treatment of microbial diseases. Its core functions include maintaining the integrity of the intestinal barrier, enhancing the intestinal barrier function by promoting the generation and repair of the intestinal mucus layer, and reducing the invasion of harmful substances; regulating the immune balance, interacting with intestinal immune cells to precisely regulate the immune response, and enhancing the body's defense against pathogens; and potentially improving diseases related to metabolic disorders. As a typical beneficial bacterium, the increase in the abundance of Lactobacillus contributes to the diversity and stability of the intestinal flora, promotes nutrient absorption and metabolism, and enhances immune function.

[0114] Example 4

[0115] This example is an optimization experiment of an improved prescription. In our previous animal experiments, it was found that the basic prescription fermented by Bacillus cereus PE-Z showed significant effects in promoting the development of the immune organ spleen and promoting the proliferation and improvement of beneficial bacteria Akkermansia and Lactobacillus in the intestine compared with the simple basic prescription and the simple PE-Z bacterial solution. However, to further optimize its clinical application value, the research group carried out addition of flavors, subtraction of flavors, and substitution treatments on this prescription, and selected the following 4 prescriptions from numerous prescriptions and conducted relevant animal experiments, which are as follows:

[0116] 1. Test animals: 54 SPF-grade KM male mice with a body weight of 18 ± 2 g, purchased from the Experimental Animal Center of Jiangxi University of Traditional Chinese Medicine (License No.: SYXK (Gan) 2020-0005, No36003020251100017035).

[0117] 2. Test methods

[0118] (1) Preparation of Bacillus cereus seed solution: The same as in Example 3

[0119] (2) Preparation of the medicinal liquids of each fermentation prescription:

[0120] Prescription 1 (basic prescription): 20 parts of red dates, 15 parts of astragalus root, 10 parts of atractylodes macrocephala, 10 parts of dried tangerine peel, 10 parts of poria cocos, 5 parts of smoked plum, 10 parts of hawthorn, and 2 parts of licorice.

[0121] Prescription 2 (Modified Prescription: Hawthorn and Dried Plum omitted): Astragalus membranaceus 15 parts, Atractylodes macrocephala 10 parts, Jujube 20 parts, Poria cocos 10 parts, Tangerine peel 10 parts, and Licorice 2 parts. The two herbs omitted from this prescription are hawthorn and dried plum. Hawthorn is sour and sweet, slightly warm in nature, and enters the spleen, stomach, and liver meridians, promoting digestion and strengthening the stomach (effectively relieving greasy and meaty food stagnation). Dried plum is sour and astringent, neutral in nature, and enters the liver, spleen, lung, and large intestine meridians. Its main functions are to astringe yin and generate fluids, and to astringe the intestines. In this prescription, hawthorn primarily promotes digestion, while dried plum has an astringent effect. To verify the core functions of hawthorn and dried plum in "promoting digestion and astringing yin" and their necessity in combination with other herbs, this prescription was modified by removing or replacing these herbs to accurately determine whether they are essential to the formula, providing a basis for determining the optimal prescription.

[0122] Prescription 3 (Replace hawthorn and dried plum in the basic prescription with polygonatum and malt respectively): Astragalus membranaceus 15 parts, Atractylodes macrocephala 10 parts, jujube 20 parts, Poria cocos 10 parts, tangerine peel 10 parts, polygonatum 10 parts, malt 10 parts, and licorice 2 parts. The two replaced herbs in this prescription are: Polygonatum sibiricum, which is sweet and neutral in nature. It enters the spleen, lung, and kidney meridians, and has the effects of nourishing yin and moistening the lungs, tonifying the spleen and replenishing qi, and nourishing the kidneys and replenishing essence. Combined with astragalus membranaceus, it serves as the principal herb, possessing the characteristic of "dual tonification of qi and yin, and simultaneous regulation of the lungs, spleen, and kidneys"; and malt, which is sweet and neutral in nature, enters the spleen and stomach meridians, and has the effects of "sweet and neutral digestion, and soothing the liver and regulating qi." Malt has a strong and comprehensive digestive effect (effectively digesting various types of food stagnation, such as rice, noodles, meat, and eggs). This prescription focuses on tonifying qi and nourishing yin to promote digestion rather than astringing.

[0123] Prescription 4 (Modified Prescription 2: Adding Polygonatum and Malt to the basic prescription): Astragalus membranaceus 15 parts, Polygonatum 10 parts, Atractylodes macrocephala 10 parts, Jujube 20 parts, Poria cocos 10 parts, Tangerine peel 10 parts, Hawthorn 10 parts, Malt 10 parts, Mume 5 parts, and Licorice 2 parts. The biggest difference between this modified prescription and the basic prescription is that this prescription uses "two principal herbs," that is, Polygonatum sibiricum and Astragalus membranaceus are both principal herbs, with Astragalus membranaceus tonifying Qi and Polygonatum sibiricum nourishing Yin, forming a "dual tonification of Qi and Yin." Another major difference is that the digestive aid in Prescription 1 (the basic prescription) is Hawthorn, while Prescription 4 (Modified Prescription 2) combines Hawthorn with Malt. The added Malt contains protease and amylase, which are good at eliminating various food stagnations such as rice, noodles, meat, and eggs, enhancing the digestive effect of Hawthorn on greasy meat, and also expanding its digestive effect on rice, noodles, and starchy foods. The difference between this modified formula and the substitute formula is that the substitute formula uses malt as a digestive aid, while the modified formula uses a combination of malt and hawthorn. By comparing the experimental results of formula 4 (modified formula 2) with other formulas, the necessity of adding Polygonatum and malt was clarified. The combined effects of the herbs in formula 4 both tonify Qi and nourish Yin, and prevent stagnation of the tonifying herbs through digestive aids, achieving a balance of medicinal properties of "tonification with unblocking, tonification without stagnation." Overall, formula 4, through the combined approach of "strengthening the spleen + tonifying Qi and nourishing Yin + promoting digestion and eliminating stagnation," constructs a system of efficacy that "supports the body's resistance and consolidates its foundation, while simultaneously tonifying and unblocking."

[0124] The preparation method of fermented traditional Chinese medicine liquid is as follows: All medicinal materials in the traditional Chinese medicine composition are pulverized into powder and poured into a decoction pot. 8-10 times the volume of distilled water is added and soaked for 30 minutes, then decocted for 1 hour. The decoction is filtered out, and the residue is then decocted again with 8-10 times the volume of distilled water for 1 hour, and the decoction is filtered out again. The above decoction is rotary evaporated to a concentration of 0.5 g / mL, and then sterilized after being prepared at a ratio of 6:4 (rotary evaporated traditional Chinese medicine liquid: ordinary broth). The prepared Bacillus cereus PE-Z seed solution is diluted to 5 × 10⁻⁶. 7 The culture medium was inoculated with 4% of the traditional Chinese medicine broth fermentation medium and cultured on a shaker at 160 rpm / min for 48 hours.

[0125] (3) Animal grouping and treatment: After 3 days of acclimatization, mice were randomly divided into 6 groups according to body weight: blank control group, Astragalus polysaccharide group (traditional immune enhancer control drug), Formula 1 (basic formula) fermentation group, Formula 2 (reduced flavor formula) fermentation group, Formula 3 (replaced flavor formula) fermentation group, and Formula 4 (added flavor formula) fermentation group, with 9 mice in each group. The administration period was from 6 pm to 8 am the next day, and the drugs were administered according to Table 9. At 8 am the next day, the water was replaced and the mice were allowed free access to drink. The administration was carried out for 5 days, stopped for 2 days, and then continued for another 5 days.

[0126] Based on the preliminary experimental results and referring to the dosage of the basic formula in Example 2, the dosage of each fermentation group in this example is 1.5%, which is lower than the dosage of 2% in the basic formula in Example 2. The purpose of the adjustment is that if the dosage of the improved formula is too high, the advantages of the improved formula may be masked by the saturation of the effect or the data fluctuation.

[0127] After the drug administration cycle ended, mice were fasted for 12 hours but allowed free access to water. Blood was collected from the eyeballs, and the serum was collected by centrifugation for subsequent experiments. Mice were euthanized by cervical dislocation, and the spleens were removed, weighed, and the spleen index was calculated. The livers were placed in cryovials and flash-frozen in liquid nitrogen for temporary storage. Finally, all cryovials in liquid nitrogen were transferred to a -80°C freezer for unified storage for subsequent experiments.

[0128] Table 9. Grouping and Treatment of Experimental Animals

[0129]

[0130] (4) Test indicators and detection methods:

[0131] ① Antioxidant function assay: Antioxidant function indicators, total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), and catalase (CAT), were detected using ELISA kits (all manufactured by Nanjing Jiancheng Biotechnology Co., Ltd.). All procedures must be strictly followed according to the kit instructions.

[0132] ② Spleen Index in Mice: The spleen was collected, rinsed with physiological saline, blotted dry with filter paper, and weighed. The spleen index (mg / g) was calculated using the following formula: Spleen Index (mg / g) = Spleen weight (mg) / Body weight (g).

[0133] ③ Serum IgG antibody level: The level was detected using an immunoglobulin G (IgG) ELISA kit (manufactured by Shanghai Enzyme-Linked Biotechnology Co., Ltd.).

[0134] 3. Test Results

[0135] The effects of each prescription on spleen index and IgG are shown in Table 10:

[0136] Table 10. Effects of each prescription on spleen index and IgG.

[0137]

[0138] Note: In the table, the superscript ** indicates a highly significant difference compared to the blank control group (p < 0.01), and * indicates a significant difference (p < 0.05). ## indicates a highly significant difference compared to the Astragalus polysaccharide group (p < 0.01), # indicates a significant difference (p < 0.05), and no label indicates no significant difference (p > 0.05). The same applies to the following tables.

[0139] As shown in Table 10, regarding the spleen index, the fermentation groups of Formula 3 (Substitute Formula) and Formula 4 (Additional Formula 2) were significantly higher than the blank control group (p < 0.01). The spleen indices of the other treatment groups were higher than the blank group, but the difference was not significant (p > 0.05). Regarding the serum IgG level, only the fermentation group of Formula 4 (Additional Formula 2) was significantly higher than the blank group (p < 0.01), while the other treatment groups did not show significant differences from the blank group (p > 0.05). In summary, the fermentation group of Formula 4 (Additional Formula 2) can significantly improve the immunity of mice.

[0140] The effects of each formulation on the antioxidant indicators of mice are shown in Table 11:

[0141] Table 11 Effects of each formulation on antioxidant indices in mice

[0142]

[0143] Table 11 shows that there was no significant difference in liver SOD levels among the groups. The glutathione peroxidase (GSH-PX) level in the Astragalus polysaccharide group was significantly higher than that in the control group (p < 0.01). Compared with the control group, prescriptions 1, 3, and 4 all significantly increased CAT levels, with prescriptions 3 and 4 also significantly increasing GSH-GPX levels. This indicates that these three prescriptions have a positive effect on enhancing the body's antioxidant capacity (especially CAT-related indicators), and prescriptions 3 and 4 have a more comprehensive antioxidant effect.

[0144] Example 5

[0145] To further confirm the immune-enhancing effect of the fermentation prescription 4 (modified prescription 2) of Bacillus cereus PE-Z, in this example, Bacillus cereus (BC-SLP) isolated from the human medicine Bifidobacterium quadruple tablets was used as the control strain to compare the immune enhancement effects of Bacillus cereus PE-Z and BC-SLP fermenting prescription 4 on mice. The specific steps are as follows:

[0146] 1. Experimental strains

[0147] ① Bacillus cereus (Bacillus cereus-SLP, abbreviated as BC-SLP), which is the Bacillus cereus isolated from the human medicine Bifidobacterium quadruple tablets in this study. After identification, it was preserved and used as the control strain in this example.

[0148] ② The preserved strain Bacillus cereus (Bacillus cereus) strain PE-Z (abbreviated as PE-Z strain) in Example 1

[0149] 2. Experimental animals: 36 SPF-grade KM male mice with a body weight of 18 ± 2 g were purchased from the Experimental Animal Center of Jiangxi University of Traditional Chinese Medicine (License number: SYXK (Gan) 2020 - 0005)

[0150] 3. Methods:

[0151] Treatment 1: Preparation of the modified prescription 2 medicinal liquid: 15 parts of Astragalus membranaceus, 10 parts of Polygonatum sibiricum, 20 parts of red dates, 10 parts of Atractylodes macrocephala, 10 parts of Citrus reticulata, 10 parts of Poria cocos, 5 parts of Prunus mume, 10 parts of Crataegus pinnatifida, 10 parts of Hordeum vulgare, and 2 parts of Glycyrrhiza uralensis were crushed into powder and poured into a decocting pot. After adding 8 - 10 times the distilled water and soaking for 30 min, it was decocted for 1 h. After filtering out the decoction, 8 - 10 times the distilled water was added again and decocted for 1 h. After filtering, the decoctions were combined and stored in a -20°C refrigerator, and diluted before use.

[0152] Treatment 2: Ferment the traditional Chinese medicine liquid in Treatment 1 using the method in Example 4, and the strain used was: Bacillus cereus BC-SLP.

[0153] Treatment 3: Ferment the traditional Chinese medicine liquid in Treatment 1 using the method in Example 4, and the strain used was: Bacillus cereus PE-Z.

[0154] Animal experiment: After 3 days of adaptive feeding, the mice were randomly divided into 4 groups according to body weight stratification, namely the blank control group, Treatment 1 group (unfermented modified group 2), Treatment 2 group (BC-SLP fermentation group), and Treatment 3 group (PE-Z fermentation group), with 9 mice in each group. The dosing scheme was the same as in Example 4.

[0155] 4. Test indicators and detection methods

[0156] (1) The enzyme production capacity (protease, amylase, cellulase) of Bacillus cereus PE-Z and BC-SLP was compared using the clear zone method.

[0157] (2) Spleen index in mice: The spleen was taken, rinsed with physiological saline, dried with filter paper, and weighed. The organ index was calculated using the following formula: Spleen index (mg / g) = Spleen weight (mg) / Body weight (g).

[0158] (3) Serum IgG antibody levels were detected using an immunoglobulin G (IgG) ELISA kit (manufactured by Shanghai Enzyme-Link Biotechnology Co., Ltd.).

[0159] 5. Results: The comparison results of extracellular enzyme production by the two Bacillus cereus strains are shown in Table 12:

[0160] Table 12 Comparison of extracellular enzyme production by two Bacillus cereus strains (diameter of clear zone / colony diameter)

[0161]

[0162] As shown in Table 12, the protease secreted by Bacillus cereus PE-Z was significantly higher than that of BC-SLP (p < 0.05), the cellulase secreted was extremely significantly higher than that of BC-SLP (p < 0.01), and there was no significant difference in the amount of amylase secreted.

[0163] The spleen index and IgG results for each group are shown in Table 13.

[0164] Table 13 Spleen index and IgG results for each group

[0165]

[0166] As shown in Table 13, the spleen index of mice treated with treatment 3 was significantly higher than that of the blank control group (p < 0.05), and the serum IgG level of mice treated with treatment 3 was significantly higher than that of the blank group (p < 0.01). While the spleen index and IgG levels of treatments 1 and 2 were increased, they were not significantly different from those of the blank group. Therefore, the fermentation system of PE-Z strain and this traditional Chinese medicine composition is significantly more effective in enhancing the body's immune function than the fermentation system of BC-SLP strain and this traditional Chinese medicine composition.

[0167] The effects of each formulation on the antioxidant indices of mouse liver are shown in Table 14:

[0168] Table 14 Effects of each formulation on antioxidant indices in mice

[0169]

[0170]

[0171] As shown in Table 14, CAT levels in treatment group 3 were significantly higher than those in the blank control group, while those in treatment group 2 were significantly higher. GSH-PX levels in treatment group 3 were significantly higher than those in the blank control group, while there was no significant difference between treatment group 2 and the blank control group. This indicates that treatment group 3, fermented with Bacillus cereus PE-Z, exhibited superior antioxidant activity compared to treatment group 2, fermented with BC-SLP. T-SOD levels in treatment group 1 were significantly higher than those in the blank control group, and T-SOD levels in treatment group 3 were higher than those in treatment group 2, but neither was significantly different from the blank control group.

[0172] GSH-Px and CAT are key antioxidant enzymes in the body. After PE-Z fermented traditional Chinese medicine, the activities of these two enzymes were significantly / extremely significantly higher than those in the blank control group, indicating that they can more efficiently remove reactive oxygen species such as hydrogen peroxide and lipid peroxides from the body, reduce oxidative stress damage to cells (especially cells of metabolically active organs such as the liver), and protect the integrity of biological membrane structures and biological macromolecules (such as proteins and DNA).

[0173] In summary, treatment 3 (formula 4) obtained by fermenting the traditional Chinese medicine composition with Bacillus cereus PE-Z can significantly improve the immunity of animals and significantly enhance the antioxidant capacity of the animal body.

[0174] In this embodiment, the traditional Chinese medicine composition consisting of 15-20 parts Astragalus membranaceus, 10-15 parts Atractylodes macrocephala, 15-20 parts Jujube, 10-15 parts Poria cocos, 10-15 parts Citrus reticulata peel, 10-15 parts Crataegus pinnatifida, 5-8 parts Prunus mume, 2-5 parts Glycyrrhiza uralensis, 10-15 parts Polygonatum sibiricum, and 10-15 parts Hordeum vulgare malt can achieve the desired effect. Within this range, the spleen index of the fermented traditional Chinese medicine liquid is 3.01-4.13 mg / g, and the IgG reaches 10.34-14.32 mg / mL, which are significantly higher than those of the blank group.

[0175] Example 6

[0176] This example is a fermentation process optimization experiment, as detailed below:

[0177] 1. Herbal decoction (Prescription 4): Weigh out 15g of Astragalus membranaceus, 10g of Polygonatum sibiricum, 20g of jujube, 5g of dried plum, 10g of dried tangerine peel, 10g of Atractylodes macrocephala, 10g of hawthorn, 10g of malt, 10g of Poria cocos, and 2g of licorice. Crush them and place them in a decoction pot. Add 8-10 times the weight of the herbs in water and soak for 30 minutes. Boil for 1 hour and filter. Use the dregs as above and decoct a second time with 8-10 times the weight of the herbs for 1 hour. Combine the two decoctions and concentrate to 0.5g / mL.

[0178] 2. Activation and preparation of seed culture for the microbial strain: Same as in Example 3.

[0179] 3. Construction of the glucose standard curve: Prepare a 0.1 mg / mL glucose standard solution. Pipette 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, and 1.0 mL into test tubes respectively, add double-distilled water to a final volume of 1 mL, then add 4 mL of freshly prepared anthrone-concentrated sulfuric acid solution (weigh 0.1 g anthrone, dissolve in 100 mL of 80% concentrated sulfuric acid, and mix thoroughly). Heat the prepared solution in boiling water for 10 min, then cool to room temperature. Measure the absorbance at 625 nm using a UV spectrophotometer. Plot the standard curve with glucose concentration on the x-axis and absorbance on the y-axis. The results are shown below. Figure 5 As shown, the linear regression equation for glucose is Y = 6.0214x + 0.0101, r 2 =0.9931. It exhibits good linearity in the range of 0.04–0.1 mg / g.

[0180] 4. Screening of fermentation media for traditional Chinese medicine: Based on the prepared formula 4, liquid nutrient broth was added in proportion to design seven fermentation media, named media 1-7 respectively. These media were dispensed into 50mL Erlenmeyer flasks, with the components shown in Table 13, and autoclaved for later use. The cultured seed culture was inoculated into each of the seven fermentation media at a rate of 3%, and cultured at 37℃ and 160r / min for 48h with shaking (all culture conditions were obtained from preliminary experiments). The polysaccharide content was then determined.

[0181] Table 15 Screening of fermentation media for traditional Chinese medicine

[0182]

[0183] The results are shown in Table 15 and Figure 6 As shown, the polysaccharide content was highest in broth culture medium with a volume percentage of 40% and in rotary evaporated liquid of traditional Chinese medicine with a volume percentage of 60%.

[0184] 5. Single-factor experiments: The optimal culture medium obtained after screening (40% broth medium and 60% rotary evaporated Chinese medicine liquid) was used as the basal culture medium. The culture conditions were set as follows: culture temperature 37℃, liquid volume 20mL (50mL Erlenmeyer flask), constant temperature shaker speed 160r / min, inoculum size 3%, fermentation for 2 days. Based on these conditions, each experimental condition was optimized separately, as follows:

[0185] (1) Determination of optimal fermentation time: Using time as the variable, four groups were set up: 1d, 2d, 3d, and 4d, with two replicates for each group. After fermentation was completed, the polysaccharide content was measured to evaluate the effect of time on the fermentation results of Bacillus cereus PE-Z. Each group had three replicates. Figure 7 As shown: the polysaccharide content was highest after 2 days of fermentation.

[0186] (2) Determination of optimal inoculum size: The cultured liquid inoculum was inoculated into the traditional Chinese medicine broth medium at inoculum sizes of 1%, 2%, 3%, 4%, and 5%, respectively. After fermentation, the polysaccharide content was measured to evaluate the effect of inoculum size on the fermentation results of Bacillus cereus PE-Z. Each group was replicated three times. Figure 8 As shown, the polysaccharide content is highest when the inoculum concentration is 4%.

[0187] (3) Determination of optimal liquid volume: 10 mL, 20 mL, and 30 mL of culture medium were added to 50 mL Erlenmeyer flasks, representing liquid volumes of 20%, 40%, and 60%, respectively. Each experimental group was tested in triplicate. After fermentation was complete, the polysaccharide content was measured to determine the optimal liquid volume. Figure 9 As shown, the polysaccharide content was highest when the liquid volume was 20 mL (40%).

[0188] (4) Determination of the optimal shaking speed: The shaking speed was set to 100 rpm / min, 160 rpm / min, 180 rpm / min, and 200 rpm / min, with three replicates for each group. After fermentation was completed, the polysaccharide content was measured to determine the optimal shaking speed. Figure 10 As shown, the polysaccharide content is highest when the shaker speed is 160 rpm / min.

[0189] 6. Orthogonal Experiment: Fermentation conditions were optimized through orthogonal experiments. Based on the results of single-factor experiments, an L9(3)2 orthogonal experimental design was used. 4 An orthogonal array with three replicates was used to optimize the fermentation conditions of the compound traditional Chinese medicine and to evaluate the primary and secondary effects of each single factor on the fermentation effect. The levels of the orthogonal factors are shown in Table 16, and the optimal fermentation parameters were determined. Finally, the rationality of the results was verified under the optimal process conditions.

[0190] Table 16 Orthogonal Experimental Design

[0191]

[0192] The results are shown in Table 17:

[0193] Table 17 Orthogonal Experimental Design and Results Analysis

[0194]

[0195] In Table 17, the factor with the highest average polysaccharide value (k) is selected as the optimal level, and the order of importance is determined based on the magnitude of the average range (R). Therefore, the order of importance of the factors affecting fermentation is: fermentation time > rotation speed > inoculum quantity > liquid volume. The optimal combination of fermentation processes is A2 B3C2D2, i.e., fermentation time 2 days, liquid volume 20%, inoculum quantity 4%, and rotation speed 160 r / min.

[0196] (3) Results of the repeatability verification of the extraction process: Based on the optimal extraction process conditions obtained from the orthogonal experiment in Table 17, three parallel experiments were conducted to measure the polysaccharide content. The results are shown in Table 18.

[0197] As shown in Table 18, the average polysaccharide content of Formula 4 was 50.94% when fermented according to the optimal extraction process conditions obtained in Table 17. The RSD of polysaccharide content in the three batches was 0.73%, and the repeatability was good (see Table 18), indicating that the process has high feasibility and repeatability.

[0198] Table 18 Repeatability Validation Results

[0199]

[0200]

[0201] Therefore, based on the optimization results of the preparation process in Tables 17 and 18, the optimal process for fermenting traditional Chinese medicine is as follows: After pulverizing the traditional Chinese medicine, place it in a decoction pot, add 8-10 times the weight of the medicinal material in water and soak for 30 minutes, boil for 1 hour, filter, and decoct the residue with 8-10 times the weight of water as above. Combine the secondary extracts and concentrate to 0.5 g / mL. Inoculate the seed liquid of Bacillus cereus PE-Z into a sterilized traditional Chinese medicine broth culture medium for fermentation. The fermentation conditions are: fermentation time 2 days, liquid volume 20%, inoculum 4%, and rotation speed 160 rpm / min. The traditional Chinese medicine broth culture medium consists of 40% broth culture medium and 60% traditional Chinese medicine rotary evaporation liquid.

[0202] In summary, through extensive strain screening, this invention has identified *Bacillus cereus* strain PE-Z as a suitable fermentation microorganism for the traditional Chinese medicine composition of this application. The traditional Chinese medicine composition of this application is developed to enhance animal immunity and improve intestinal flora. The composition comprises *Astragalus membranaceus*, *Atractylodes macrocephala*, jujube, *Poria cocos*, tangerine peel, hawthorn, dried plum, licorice, *Polygonatum sibiricum*, and / or malt. After fermentation with strain PE-Z, this composition significantly increases the content of polysaccharides in the traditional Chinese medicine, significantly increases the abundance of *AKK* and *Lactobacillus* in the intestines, and significantly improves animal immunity. Furthermore, orthogonal optimization yielded the optimal culture medium and optimized the fermentation process.

[0203] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. Bacillus cereus strain PE-Z, with accession number GDMCC NO: 66471.

2. A fermented traditional Chinese medicine composition comprising the Bacillus cereus strain PE-Z as described in claim 1.

3. The fermented traditional Chinese medicine composition according to claim 2, characterized in that, The fermented traditional Chinese medicine composition consists of 15-20 parts of Astragalus membranaceus, 10-15 parts of Atractylodes macrocephala, 15-20 parts of jujube, 10-15 parts of Poria cocos, 10-15 parts of dried tangerine peel, 10-15 parts of hawthorn, 5-8 parts of dried plum, and 2-5 parts of licorice.

4. The application of the fermented traditional Chinese medicine composition as described in claim 3 in improving the intestinal flora Akkermansia and Lactobacillus.

5. The fermented traditional Chinese medicine composition according to claim 2, characterized in that, The fermented traditional Chinese medicine composition consists of 15-20 parts of Astragalus membranaceus, 10-15 parts of Atractylodes macrocephala, 15-20 parts of jujube, 10-15 parts of Poria cocos, 10-15 parts of dried tangerine peel, 10-15 parts of hawthorn, 5-8 parts of dried plum, 2-5 parts of licorice, 10-15 parts of Polygonatum sibiricum, and 10-15 parts of malt.

6. The application of the fermented traditional Chinese medicine composition as described in claim 5 in enhancing immunity.

7. The application of Bacillus cereus strain PE-Z as described in claim 1 in increasing the polysaccharide content of fermented traditional Chinese medicine compositions.

8. A method for preparing the fermented traditional Chinese medicine composition as described in any one of claims 2-5, characterized in that, The method is as follows: (1) After pulverizing the Chinese herbal composition, place it in a Chinese herbal decoction pot, add 8-10 times the weight of the herbal material in water, boil for 1 hour and take the extract. The residue is then extracted a second time with 8-10 times the weight of water as above. The two extracts are combined and concentrated by rotary evaporation to 0.5 g / mL. (2) Inoculate the seed culture of Bacillus cereus strain PE-Z into sterilized Chinese medicine broth culture medium, place it in a constant temperature shaker at 160 rpm / min, and ferment it at 37℃ for 48 h.

9. The method according to claim 8, characterized in that, The fermentation conditions for the herbal broth culture medium are as follows: fermentation time 2 days, liquid volume percentage 20%, inoculum volume percentage 4%, and rotation speed 160 rpm / min.

10. The method according to claim 8, characterized in that, The herbal broth culture medium consists of 40% broth culture medium and 60% rotary evaporation liquid of traditional Chinese medicine.

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