Fermented traditional Chinese medicine, feed additive and application thereof

Abstract

The application provides a fermented traditional Chinese medicine, a feed additive and application thereof, and relates to the technical field of microbial fermentation. Bacillus subtilis The application uses Bacillus subtilis (Bacillus subtilis) strain XNW-KCYB1 to ferment a traditional Chinese medicine composition, so that the content of active ingredients in the fermented traditional Chinese medicine extract can be significantly improved. In-vitro antibacterial tests show that the fermented traditional Chinese medicine has significant inhibitory effects on Escherichia coli, Streptococcus suis, Haemophilus parasuis, Aspergillus flavus, Fusarium graminearum and porcine reproductive and respiratory syndrome virus. When the fermented traditional Chinese medicine is used for animal breeding, the growth performance of animals can be significantly improved; meanwhile, the immune organ index, humoral immune capacity and cellular immune capacity of animals can be significantly improved, so that the immune performance of animals is improved, and the fermented traditional Chinese medicine has application value and industrialization prospect for healthy production of animals.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a fermented traditional Chinese medicine, a feed additive, and their applications. Background Technology

[0002] With the overuse of antibiotics in animal husbandry, the increasing drug resistance of pathogens, environmental pollution, and meat safety issues have become increasingly prominent. Finding efficient, low-toxicity, and residue-free antibiotic alternatives has become a hot research topic in the industry. Traditional Chinese medicine, with its rich natural active ingredients, multi-target pharmacological effects, and lower risk of drug resistance, has shown significant advantages in regulating animal immunity and preventing viral diseases.

[0003] However, traditional Chinese medicine has significant limitations in its application. First, the active ingredients are mostly encased in tough plant cell walls, making it difficult for unprocessed powders to be fully absorbed by animals, resulting in resource waste. Second, some medicinal materials have a bitter taste, and direct addition can easily lead to a decrease in animal feed intake. Third, the bioconversion rate of crude Chinese medicine powder in the gastrointestinal tract is low, making it difficult to quickly reach an effective concentration to address acute infections. Currently, many Bacillus strains have been reported for fermentation of Chinese medicine, but their cellulase activity and conversion efficiency for specific Chinese medicine compositions vary, and research on practical problems in livestock and poultry farming, such as viral diseases and mycotoxin contamination, is still insufficient. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a fermented traditional Chinese medicine that, after fermenting the traditional Chinese medicine composition using Bacillus subtilis strain XNW-KCYB1, can improve animal growth performance and immune performance, and has the ability to inhibit pathogenic microorganisms, and can be used as a safe alternative to antibiotics in animal feed.

[0005] This invention provides a fermented traditional Chinese medicine, which utilizes Bacillus subtilis (B. subtilis) Bacillus subtilis The traditional Chinese medicine composition was obtained by fermenting strain XNW-KCYB1; the traditional Chinese medicine composition includes the following raw materials in parts by weight: Astragalus membranaceus 30-50 parts, Atractylodes macrocephala 30-45 parts, Isatis indigotica 10-25 parts, Anemarrhena asphodeloides 5-20 parts, Morus alba root bark 1-15 parts, Magnolia officinalis 1-15 parts, Ginkgo biloba leaf 1-14 parts and Glycyrrhiza uralensis 8-22 parts; the Bacillus subtilis strain XNW-KCYB1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20241806.

[0006] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 40-45 parts of Astragalus membranaceus, 35-40 parts of Atractylodes macrocephala, 15-20 parts of Isatis indigotica, 10-15 parts of Anemarrhena asphodeloides, 5-10 parts of Morus alba root bark, 5-10 parts of Magnolia officinalis, 5-10 parts of Ginkgo biloba leaf, and 12-18 parts of Glycyrrhiza uralensis.

[0007] Preferably, the mass-to-volume ratio of the traditional Chinese medicine composition and the bacterial solution of Bacillus subtilis strain XNW-KCYB1 is (1~5) g : (0.025~0.5) ml; The viable cell concentration of the Bacillus subtilis strain XNW-KCYB1 in the bacterial culture is ≥1×10⁻⁶. 9 CFU / mL.

[0008] This invention provides a feed additive, comprising the fermented traditional Chinese medicine.

[0009] The present invention provides an animal feed comprising the fermented traditional Chinese medicine or the feed additive and a basal diet; wherein the fermented traditional Chinese medicine or feed additive accounts for 4% to 6% of the mass of the basal diet.

[0010] This invention provides the application of Bacillus subtilis strain XNW-KCYB1, the fermented traditional Chinese medicine, and the feed additive in the preparation of animal feed or animal husbandry that is resistant to pathogenic microorganisms; the Bacillus subtilis strain XNW-KCYB1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20241806.

[0011] Preferably, the pathogenic bacteria include at least one of fungi, bacteria, and viruses.

[0012] Preferably, the bacteria include at least one of the following: Escherichia coli (Escherichia coli) Escherichia coli Streptococcus suis ( Streptococcus suis ) and Haemophilus parasuis ( Haemophilus parasuis The fungi include Aspergillus flavus ( ); Aspergillus flavus ) and / or Fusarium graminearum ( Fusarium graminearum ).

[0013] Preferably, the virus includes porcine reproductive and respiratory syndrome virus (PRRSV).

[0014] Preferably, the animal husbandry includes improving the growth performance and / or improving the immune performance of the animals.

[0015] This invention provides a fermented traditional Chinese medicine, obtained by fermenting a traditional Chinese medicine composition using Bacillus subtilis strain XNW-KCYB1. The fermentation composition using the aforementioned Bacillus subtilis strain XNW-KCYB1 can effectively degrade plant cell walls, significantly increasing the content of small molecule active ingredients such as astragaloside, flavonoid aglycones, smilax saponins, glycyrrhetinic acid, magnolol, total free polyphenols, soluble functional oligosaccharides, and free amino acids. p<0.05), thereby improving the bioavailability of traditional Chinese medicine. In vitro antibacterial tests confirmed that the fermented traditional Chinese medicine had significant inhibitory effects on pathogenic microorganisms such as Escherichia coli, Streptococcus suis, Haemophilus parasuis, Aspergillus flavus, and Fusarium graminearum. The effect was significantly higher than that of the probiotic group and the unfermented traditional Chinese medicine group. It can be seen that the traditional Chinese medicine composition, after fermentation by Bacillus subtilis strain XNW-KCYB1, significantly improved the ability to resist pathogenic microorganisms and can be used as an alternative to antibiotics in animal feed. In addition, the fermented traditional Chinese medicine can inhibit the replication of porcine reproductive and respiratory syndrome virus, which has a significant effect on the safe growth of poultry and livestock. Animal experiments showed that animal feed with the addition of the fermented traditional Chinese medicine can significantly increase the average daily weight gain of animals, reduce the feed conversion ratio and diarrhea rate; at the same time, it can increase the spleen index, thymus index and serum IgG, IgA, IL-10, TNF-α and IFN-γ levels, and enhance the body's immunity. The fermented traditional Chinese medicine provided by this invention has multiple functions such as promoting growth, preventing diarrhea, regulating immunity, antibacterial and antiviral properties, providing an efficient, safe and green technical solution for antibiotic-free aquaculture. Attached Figure Description

[0016] Figure 1 This is a gel electrophoresis image of the PCR product of Bacillus subtilis XNW-KCYB1 in this invention. Lane M represents DNA Marker, and lanes 1 and 2 both represent two repeat wells for the identification of Bacillus subtilis XNW-KCYB1. Figure 2 The following are performance comparison charts of Bacillus subtilis XNW-KCYB1 and strains screened in the same batch: (a) is a comparison chart of viable cell counts; (b) is a qualitative comparison chart of cellulase production plates; (c) is a statistical chart of the ratio of cellulase clear zone diameter to colony diameter (D / d); and (d) is a statistical chart of the ratio of cellulase clear zone diameter to colony diameter (D / d) for strain 21 and four commercially available product strains. Figure 3 The following are monitoring indicators for the fermentation process of traditional Chinese medicine: (a) is a dynamic change graph of viable cell count within 4 days of fermentation; (b) is a curve of pH value change during fermentation of the fermented traditional Chinese medicine group (Treat group) and the unfermented control group (CK group); and (c) is a qualitative graph of cellulase production on a plate during fermentation. Figure 4 Figure 1 shows the efficacy and mechanism of fermented traditional Chinese medicine supernatant against porcine reproductive and respiratory syndrome virus (PRRSV). (a) shows the GFP fluorescence effect of the supernatant against PRRSV after 4 days of fermentation in the fermented traditional Chinese medicine group (Treat group) and the unfermented control group (CK group); (b) shows the TCID of PRRSV in the cell supernatant after treatment with different concentrations of fermented traditional Chinese medicine supernatant. 50(c) Results of titer; (d) Results of PRRSV N protein expression in cells after treatment with supernatant of fermented Chinese medicine at different concentrations (Western Blot); (d) Results of relative expression of PRRSV NSP9 gene in cells after treatment with supernatant of fermented Chinese medicine at different concentrations (qPCR).

[0017] Figure 5 This is a comparative graph showing the effects of each group on the growth performance of piglets; where (a) is the average daily weight gain (ADG); (b) is the average daily feed intake (ADFI); (c) is the feed conversion ratio (F / G); and (d) is the diarrhea rate; the graph shows the differences between the control group (CON), the traditional Chinese medicine group (TCM), the probiotic group (PRO), the physical mixing group (MIX), and the fermented traditional Chinese medicine group (FTCM); Figure 6 The graph shows the comparison of the effects of each group on the immune performance indicators of piglets; where (a) is the spleen index; (b) is the thymus index; (c) is the serum IgG level; (d) is the serum IgA level; (e) is the serum IL-10 level; (f) is the serum TNF-α level; and (g) is the serum IFN-γ level.

[0018] Biological Preservation Instructions Bacillus subtilis ( Bacillus subtilis XNW-KCYB1 was deposited on August 16, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20241806. Detailed Implementation

[0019] This invention provides a fermented traditional Chinese medicine, which utilizes Bacillus subtilis (B. subtilis) Bacillus subtilis The traditional Chinese medicine composition was obtained by fermenting strain XNW-KCYB1; the traditional Chinese medicine composition includes the following raw materials in parts by weight: Astragalus membranaceus 30-50 parts, Atractylodes macrocephala 30-45 parts, Isatis indigotica 10-25 parts, Anemarrhena asphodeloides 5-20 parts, Morus alba root bark 1-15 parts, Magnolia officinalis 1-15 parts, Ginkgo biloba leaf 1-14 parts and Glycyrrhiza uralensis 8-22 parts; the Bacillus subtilis strain XNW-KCYB1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20241806.

[0020] In this invention, the Bacillus subtilis strain XNW-KCYB1 was isolated from fresh feces of healthy piglets in a pig farm in Guangxi Zhuang Autonomous Region. The Bacillus subtilis strain XNW-KCYB1 exhibits high cellulase production, with cellulase activity significantly higher than other Bacillus strains isolated in the same batch and several commercially available Bacillus subtilis products. Furthermore, the Bacillus subtilis strain XNW-KCYB1 exhibits rapid growth, reaching a viable count of 15.57 × 10⁻⁶ cells after 48 hours of culture. 8 CFU / mL. The cellulase described can decompose cellulose and hemicellulose in the cell walls of plants in traditional Chinese medicine, allowing the active ingredients such as astragalus polysaccharides and saponins encapsulated within the cell walls to be fully released. At the same time, the enzyme system secreted by the strain can further biotransform macromolecules into small molecule active ingredients, significantly improving the bioavailability of traditional Chinese medicine.

[0021] In this invention, the preferred weight percentage of Astragalus membranaceus is 40-45 parts, and most preferably 42 parts. Astragalus membranaceus is the dried root of Astragalus mongholicus or Astragalus membranaceus, both belonging to the Fabaceae family. It is slightly warm in nature, sweet in taste, and enters the spleen and lung meridians. It has the effects of tonifying qi and strengthening the exterior, enhancing the body's vitality, improving resistance, and aiding in tissue repair.

[0022] In this invention, the preferred weight percentage of Atractylodes macrocephala is 35-40 parts, and most preferably 38 parts. Atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala, a plant of the Asteraceae family. It is warm in nature, bitter and sweet in taste, and enters the spleen and stomach meridians. It has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis. It has a good effect on improving digestive function and eliminating dampness in the body. In the formula, it assists Astragalus membranaceus to enhance the effect of replenishing qi and strengthening the spleen, and promotes the digestion and absorption of nutrients.

[0023] In this invention, the preferred weight percentage of Isatis root is 15-20 parts, and most preferably 18 parts. Isatis root is the dried root of Isatis indigotica, a plant of the Brassicaceae family. It is cold in nature, bitter in taste, and enters the heart and stomach meridians. It has the effects of clearing heat and detoxifying, cooling blood and relieving sore throat, and is often used for symptoms such as fever, sore throat and influenza.

[0024] In this invention, the preferred weight percentage of Anemarrhena asphodeloides is 10-15 parts, and most preferably 12 parts. Anemarrhena asphodeloides is the dried rhizome of the Liliaceae plant Anemarrhena asphodeloides. It is cold in nature, bitter and sweet in taste, and enters the lung, stomach, and kidney meridians. It has the effects of clearing heat and purging fire, nourishing yin and moistening dryness, and is suitable for states such as thirst due to excess heat or internal disturbance due to deficiency heat. In this invention, the preferred weight percentage of the mulberry bark is 5-10 parts, and most preferably 8 parts. Mulberry bark is the dried root bark of the mulberry tree (Morus alba), a plant of the Moraceae family. It is cold in nature, sweet in taste, and enters the lung meridian. It can clear lung heat, relieve asthma, promote diuresis, and reduce swelling, and is particularly effective for coughs caused by lung heat.

[0025] In this invention, the preferred weight percentage of Magnolia officinalis is 5-10 parts, and most preferably 8 parts. Magnolia officinalis is the dried bark, root bark, and branch bark of Magnolia officinalis or Magnolia officinalis var. ...

[0026] In this invention, the preferred weight percentage of the ginkgo leaves is 5-10 parts, and most preferably 7 parts. Ginkgo leaves are the dried leaves of the ginkgo plant (Ginkgo biloba). They are neutral in nature, sweet, bitter, and astringent in taste, and enter the heart and lung meridians. They can promote blood circulation, remove blood stasis, clear the meridians, and relieve pain. They also have a certain relieving effect on cough and asthma.

[0027] In this invention, the preferred weight percentage of licorice is 12-18 parts, and most preferably 15 parts. Licorice is the dried root and rhizome of Glycyrrhiza uralensis, Glycyrrhiza inflata, or Glycyrrhiza glabra, all belonging to the legume family. It is neutral in nature, sweet in taste, and enters the heart, lung, spleen, and stomach meridians. It has multiple functions, including tonifying the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, and harmonizing other medicines. It is a commonly used auxiliary medicine in prescriptions.

[0028] In this invention, the herbal composition is formulated according to the traditional Chinese medicine theory of "principal, assistant, adjuvant, and guide." Astragalus membranaceus is the principal herb, tonifying qi and raising yang, strengthening the defensive qi and consolidating the exterior; Atractylodes macrocephala is the assistant herb, strengthening the spleen and tonifying qi, assisting the qi-tonifying effect of Astragalus membranaceus; Isatis indigotica, Anemarrhena asphodeloides, Morus alba root bark, Magnolia officinalis, and Ginkgo biloba leaf are the adjuvant herbs, among which Isatis indigotica and Anemarrhena asphodeloides clear heat and detoxify, nourish yin and moisten dryness to counteract the warming and drying effects of the principal and assistant herbs, while Morus alba root bark, Magnolia officinalis, and Ginkgo biloba leaf purge the lungs, relieve asthma, promote qi circulation and remove blood stasis to treat concurrent symptoms; Glycyrrhiza uralensis is the guiding herb, harmonizing the effects of all the herbs. The entire formula works synergistically to tonify qi and strengthen the body, clear heat and detoxify, resolve phlegm and relieve asthma. This invention does not restrict the source of the above-mentioned herbal ingredients; any commercially available herbal ingredients can be used.

[0029] In this invention, fermenting the traditional Chinese medicine composition with the Bacillus subtilis strain XNW-KCYB1 can significantly improve the inhibitory ability of the traditional Chinese medicine composition against pathogenic microorganisms, and enhance the effect of the traditional Chinese medicine composition on improving animal growth performance and immune function.

[0030] In this invention, the preferred mass-to-volume ratio of the traditional Chinese medicine composition and the bacterial suspension of Bacillus subtilis strain XNW-KCYB1 is (1~5) g:(0.025~0.5) ml; it can be 2 g:0.03 ml, 3 g:0.04 ml, 2 g:0.3 ml, or 4 g:0.08 ml. The preferred viable cell concentration of the Bacillus subtilis strain XNW-KCYB1 bacterial suspension is ≥1×10⁻⁶. 9 CFU / mL, which can be 2×10 9 CFU / mL, 3×10 9 CFU / mL, 5×10 9 CFU / mL or 8×109 CFU / mL. The preferred method for culturing the bacterial culture includes streaking the *Bacillus subtilis* XNW-KCYB1 onto LB solid medium plates to activate and obtain single colonies; inoculating the single colonies into LB liquid medium for seed culture to obtain a seed culture; and inoculating the seed culture into LB liquid medium for fermentation culture to obtain the bacterial culture. The activation culture time is 22-26 h, and can also be 23-25 ​​h. In a specific embodiment of the invention, the activation culture time is 24 h. The seed culture time is 10-15 h; in a specific embodiment of the invention, the seed culture time is 12 h. The inoculation amount of the seed culture is 1%-3%, and in a specific embodiment of the invention, the inoculation amount of the seed culture is 2%. The fermentation culture time is 24-48 h, and can also be 36-42 h, specifically 37 h, 38 h, 39 h, or 42 h. The culture temperature is 33-40℃, which can be 33℃, 34℃, 35℃, 36℃, or 39℃; in a specific embodiment of the present invention, the culture temperature is 37℃. In a specific embodiment of the present invention, a 1:4 mass-to-volume ratio is used for fermentation. Using the aforementioned mass-to-volume ratio and the aforementioned viable cell concentration for fermentation, on the one hand, provides sufficient nutrients for the Bacillus subtilis strain XNW-KCYB1, promoting rapid proliferation of the strain and continuous secretion of highly active cellulase; on the other hand, the appropriate inoculum size avoids slow fermentation start-up and incomplete enzymatic hydrolysis due to insufficient inoculation, and also prevents nutrient competition or accumulation of metabolic waste due to excessive inoculation.

[0031] In this invention, the preparation method of the fermented traditional Chinese medicine preferably includes the following steps: Fermented traditional Chinese medicine is obtained by inoculating the traditional Chinese medicine composition with Bacillus subtilis strain XNW-KCYB1 and then carrying out aerobic fermentation.

[0032] In this invention, before the aerobic fermentation, it is preferable to separately or in combination pulverize each raw material in the traditional Chinese medicine composition. The pulverization process preferably includes passing the pulverized material through a 40-mesh sieve and collecting the sieved powder to obtain the traditional Chinese medicine composition powder for fermentation. After the pulverization process, it is preferable to sterilize the traditional Chinese medicine composition powder. The sterilization method is preferably autoclaving. The autoclaving method preferably involves mixing the traditional Chinese medicine composition powder and sterile water and then autoclaving at 121°C for 30 minutes. The mass ratio of the traditional Chinese medicine composition powder to sterile water is (1~5):(5~10). In a specific embodiment of this invention, the sterilization process involves adding 200 g of traditional Chinese medicine powder to 800 mL of sterile water and autoclaving at 121°C for 30 minutes to obtain a sterilized fermentation substrate system.

[0033] In this invention, Bacillus subtilis strain XNW-KCYB1 is added to the sterilized fermentation substrate system for aerobic fermentation. The inoculum amount of Bacillus subtilis strain XNW-KCYB1 is 0.5% to 5% of the fermentation substrate system volume, which can be 1%, 2%, 3%, or 4%. In a specific embodiment of this invention, a 2% inoculum amount is used for fermentation. The preferred temperature for aerobic fermentation is 30-40℃, which can be 31℃, 33℃, 35℃, or 39℃. The preferred time for aerobic fermentation is 48-120 h, which can be 60 h, 72 h, 84 h, or 108 h, with 96 h being the most preferred. The aerobic fermentation is preferably shake fermentation. The 30-40℃ temperature range is the suitable growth temperature range for Bacillus subtilis strain XNW-KCYB1. Within this temperature range, the strain has active metabolism and efficient enzyme secretion system, enabling rapid proliferation and continuous production of highly active cellulase. The shaking fermentation can be carried out using a constant-temperature shaker, or by mechanical stirring or oscillation in a fermentation tank, to achieve thorough mixing of the material and the microorganisms and ensure adequate oxygen supply. This shaking fermentation allows for full contact between the microorganisms, enzyme solution, and herbal powder, preventing sedimentation of the herbal particles, improving the uniformity and efficiency of enzymatic hydrolysis, and thus accelerating the breaking down of plant cell walls and the release of active substances. In this embodiment of the invention, fermentation is specifically carried out at 37°C in a constant-temperature shaker at a rotation speed of 180 r / min for 96 h.

[0034] In this invention, after fermentation, the fermentation product is preferably post-treated. The post-treatment preferably includes centrifuging, filtering, or drying the fermentation product to separate solid and liquid phases, obtaining a solid fermentation product, and collecting the supernatant to obtain the fermentation broth. The centrifugation speed is preferably 6000~10000 r / min, and can be 7000 r / min, 8000 r / min, or 9000 r / min. The centrifugation time is preferably 5~15 min, and can be 7 min, 8 min, or 12 min. The filtration preferably includes plate and frame filtration, microfiltration, or membrane filtration to remove solid residues and bacterial cells, obtaining a clear fermentation supernatant. The drying treatment is preferably freeze-drying, low-temperature drying, and / or vacuum drying. The freeze-drying preferably includes pre-freezing the fermentation broth at -40℃ to -60℃, followed by removing excess moisture through sublimation. The low-temperature drying preferably includes placing the fermentation broth in a low-temperature drying oven and drying it at a temperature of 40℃ to 60℃ until the moisture content of the material drops below 10%. The vacuum drying preferably involves placing the fermentation broth in a vacuum drying oven for drying until the moisture content of the material drops below 10%. The solid-state fermentation product is then pulverized and sieved to obtain fermentation powder. This invention provides a feed additive comprising the fermented traditional Chinese medicine. The feed additive preferably comprises the fermentation broth and / or the fermentation powder.

[0035] This invention provides an animal feed comprising the fermented traditional Chinese medicine or the feed additive and a basal diet; the fermented traditional Chinese medicine or feed additive accounts for 4% to 6% of the basal diet mass, and can be up to 5%. When the addition amount is less than 4%, the absolute content of the active ingredients in the fermented traditional Chinese medicine in the basal diet is low, insufficient to fully exert the effects of growth performance enhancement, immune performance enhancement, and antiviral efficacy. Within the addition range of 4% to 6%, the fermented traditional Chinese medicine and the basal diet can achieve good compatibility, ensuring that animals ingest sufficient small molecule active substances to exert the effects of promoting growth, preventing diarrhea, enhancing immunity, and inhibiting pathogenic microorganisms, without negatively affecting feed palatability and animal feeding behavior. This animal feed can be widely used in the breeding of livestock such as pigs, cattle, and sheep, especially suitable for animals in the weaning period or under stress, and can significantly improve animal health and production performance without relying on antibiotics, with good economic benefits and application prospects.

[0036] This invention provides the application of Bacillus subtilis strain XNW-KCYB1, the fermented traditional Chinese medicine, and the feed additive in the preparation of animal feed or animal husbandry that is resistant to pathogenic microorganisms; the Bacillus subtilis strain XNW-KCYB1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20241806.

[0037] In this invention, the animal husbandry preferably includes improving animal growth performance and / or improving animal immune performance. Improving animal production performance preferably includes at least one of increasing average daily weight gain, reducing feed conversion ratio, and reducing diarrhea rate.

[0038] In this invention, the average daily gain (ADG) directly reflects the animal's growth rate and nutrient assimilation efficiency per unit time. In animal husbandry, an increased average daily gain means that animals can more efficiently convert nutrients in feed into body tissues, thereby shortening the fattening cycle and reducing feeding costs. In the embodiments of this invention, the average daily gain of piglets in the fermented traditional Chinese medicine group was significantly higher than that in the control group, directly demonstrating that the fermented traditional Chinese medicine of this invention can effectively promote the anabolic metabolism of animals and improve growth performance.

[0039] In this invention, the feed conversion ratio (F / G) is a core indicator for measuring the economic benefits of animal husbandry. A lower F / G indicates that the animal gains more weight per unit weight of feed consumed, and that feed utilization efficiency is higher. In the embodiments of this invention, the F / G ratio of the fermented traditional Chinese medicine group was significantly lower than that of the control group and the probiotic group, indicating that fermented traditional Chinese medicine improved the animal's feed utilization rate and reduced nutrient waste.

[0040] In this invention, the diarrhea rate directly reflects the intestinal health and disease resistance of animals. Diarrhea is often caused by intestinal flora imbalance, pathogen infection, or digestive dysfunction; severe diarrhea can lead to dehydration, malnutrition, and even death. In the embodiments of this invention, the diarrhea rate in the fermented traditional Chinese medicine group was significantly lower than that in the other groups. This indicates that the active ingredients in the fermented traditional Chinese medicine can protect the intestinal mucosal barrier and enhance local immune function. Therefore, this invention demonstrates a significant effect in maintaining animal intestinal health.

[0041] In this invention, the improvement of animal immune performance preferably includes at least one of improving the animal's spleen index, improving the animal's thymus index, improving the animal's humoral immunity, and improving the animal's cellular immunity.

[0042] In this invention, the spleen index (the ratio of spleen weight to animal body weight) and thymus index (the ratio of thymus weight to animal body weight) are important morphological indicators for evaluating the body's non-specific immune function and the development of immune organs. The spleen is the largest peripheral immune organ, rich in B lymphocytes and macrophages, responsible for filtering blood, producing antibodies, and clearing pathogens. The thymus is a central immune organ for the differentiation, development, and maturation of T lymphocytes, and its weight directly reflects the body's cellular immune potential. When animals are malnourished or immunosuppressed, the spleen and thymus atrophy, and their indices decrease; conversely, an increase in immune organ indices indicates that immune function has been effectively activated or enhanced. In the embodiments of this invention, the spleen index and thymus index of piglets in the fermented traditional Chinese medicine group were significantly higher than those in the control group, directly demonstrating that the fermented traditional Chinese medicine of this invention can promote the growth and development of immune organs, laying the foundation for establishing a solid immune barrier for the body.

[0043] In this invention, enhancing the humoral immunity of animals preferably includes increasing the levels of immunoglobulin G and / or immunoglobulin A in the animal's serum. Immunoglobulins are the main effector molecules of humoral immunity: IgG is the most abundant antibody in serum, with functions such as neutralizing viruses, opsonizing phagocytosis, and activating complement, and is the core of systemic immune protection; IgA is the main antibody for mucosal immunity, acting as the first line of defense against pathogens on the mucosal surfaces of the digestive and respiratory tracts. In the embodiments of this invention, the levels of IgG and IgA in the serum of animals in the fermented traditional Chinese medicine group were significantly higher than those in the control group and other experimental groups, indicating that fermented traditional Chinese medicine can promote B lymphocyte activation and antibody secretion, thereby enhancing the body's humoral immune defense against pathogens such as bacteria and viruses.

[0044] In this invention, enhancing the cellular immunity of animals preferably includes increasing the level of any one of interleukin-10, interferon-γ, and tumor necrosis factor-α in serum. Cellular immunity is mainly mediated by T lymphocytes, exerting its effects through the secretion of cytokines. Interleukin-10 (IL-10) is an important anti-inflammatory cytokine that can prevent tissue damage caused by excessive inflammatory responses. Interleukin-γ (IFN-γ) is a Th1-type cytokine with antiviral, macrophage activation, and antigen presentation promotion functions. Tumor necrosis factor-α (TNF-α) participates in inflammatory responses and immune regulation. These three factors together constitute the balance of the cellular immune network. In the embodiments of this invention, the levels of IFN-γ, TNF-α, and IL-10 in the serum of animals in the fermented traditional Chinese medicine group were significantly increased, indicating that the fermented traditional Chinese medicine can simultaneously activate both pro-inflammatory and anti-inflammatory immune pathways, enhancing the body's ability to clear pathogens while preventing damage caused by excessive immunity, thus achieving benign regulation of cellular immunity.

[0045] In this invention, the animals preferably include livestock, such as pigs, cattle, and sheep, which are commercially raised animals. Livestock in large-scale farming face problems such as weaning stress, pathogen infection, and the prohibition of antibiotics, making the demand for safe and effective antibiotic alternatives particularly urgent. The livestock can be weaned young animals, including piglets, calves, and lambs. For example, piglets, whose digestive systems are not fully developed and whose immune functions are not yet mature during the weaning stage, are highly susceptible to diarrhea, growth retardation, and even death, making them the most sensitive and representative animal model for testing the effectiveness of feed additives.

[0046] In this invention, the pathogenic microorganisms preferably include fungi, bacteria, and viruses. The bacteria include at least one of the following: *Escherichia coli* (…). Escherichia coli Streptococcus suis ( Streptococcus suis ) and Haemophilus parasuis ( Haemophilus parasuis ).

[0047] In this invention, *Escherichia coli* is the most common opportunistic pathogen in livestock and poultry farming, causing yellow-white scours, edema, and septicemia in animals, leading to mass mortality in severe cases. As a representative of Gram-negative bacteria, its drug resistance is becoming increasingly prominent. In this embodiment of the invention, to further investigate the effect of fermentation on the antibacterial activity of traditional Chinese medicine (TCM), fermented TCM, a single TCM composition, a single *Bacillus subtilis* strain (probiotic group), and a physical mixture of TCM composition and *Bacillus subtilis* strain were used as experimental subjects. Antibacterial tests were conducted using the Oxford cup method. The results showed that the diameter of the inhibition zone against *E. coli* in the fermented TCM group reached 22.50±1.00 mm, significantly better than the probiotic group (16.00±0.00 mm) and the physical mixture group (15.83±0.58 mm). The single TCM group (TCM) showed no antibacterial effect, indicating that TCM can only acquire potent anti-*E. coli* activity after fermentation with *Bacillus subtilis* strain.

[0048] In this invention, *Streptococcus suis* is an important bacterial pathogen in pigs, causing meningitis, arthritis, endocarditis, and septicemia, resulting in significant economic losses. Some serotypes can also infect humans. In this embodiment, to further investigate the effect of fermentation on the antibacterial activity of traditional Chinese medicine (TCM), fermented TCM, a single TCM composition, a single *Bacillus subtilis* strain (probiotic group), and a physical mixture of TCM composition and *Bacillus subtilis* strain were used as experimental subjects. The Oxford cup method was used to conduct antibacterial tests. The inhibition zone diameter of the fermented TCM group against *Streptococcus suis* was 27.00±1.50 mm, significantly higher than that of the probiotic group (19.33±0.58 mm), the physical mixture group (19.00±0.50 mm), and the TCM group (14.00±1.00 mm), indicating that fermentation of TCM with *Bacillus subtilis* strain can enhance its anti-*Streptococcus suis* activity.

[0049] In this invention, *Haemophilus parasuis* is an opportunistic pathogen of the swine upper respiratory tract that can cause Glaser's disease, manifested as fibrinous polyserositis, arthritis, and meningitis. Weaned piglets are particularly susceptible, and drug-resistant strains are prevalent. In the embodiments of this invention, the fermented traditional Chinese medicine group exhibited the largest inhibition zone diameter against *Haemophilus parasuis*, reaching 29.50±2.18 mm, significantly higher than the probiotic group (20.67±1.15 mm), the physical mixture group (20.50±1.00 mm), and the single traditional Chinese medicine group (15.00±1.00 mm), which showed no inhibitory effect. This indicates that fermentation of traditional Chinese medicine with *Bacillus subtilis* strains can enhance its potent anti-*Haemophilus parasuis* activity.

[0050] In this invention, the fungus preferably includes Aspergillus flavus (… Aspergillus flavus ) and / or Fusarium graminearum ( Fusarium graminearum ).

[0051] In this invention, *Aspergillus flavus* and *Fusarium graminearum* are common toxin-producing fungi in feed ingredients, primarily found in corn, wheat, soybean meal, and other feed ingredients. *Aspergillus flavus* can produce aflatoxin B1, which is highly hepatotoxic and carcinogenic; *Fusarium graminearum* mainly produces deoxynivalenol and zearalenone, leading to anorexia, vomiting, reproductive disorders, and immunosuppression in animals, seriously endangering animal health and food safety. In the embodiments of this invention, fermented traditional Chinese medicine, a single traditional Chinese medicine composition, a single *Bacillus subtilis* strain (probiotic group), and a physical mixture of traditional Chinese medicine composition and *Bacillus subtilis* strain were used as experimental subjects. The plate confrontation method was used for the experiment. The results showed that the fermented traditional Chinese medicine group achieved an inhibition rate of 65.02±0.14% against *Aspergillus flavus* and 46.84±0.15% against *Fusarium graminearum*, both significantly better than the probiotic group and the physical mixture group, while the single traditional Chinese medicine group showed no inhibitory effect. This indicates that the fermented traditional Chinese medicine obtained after fermentation by Bacillus subtilis XNW-KCYB1 produces metabolites with antifungal activity. Furthermore, the fermentation treatment of Bacillus subtilis and traditional Chinese medicine enhances the antibacterial properties of Bacillus subtilis itself, which can effectively inhibit the growth of toxin-producing fungi, thereby reducing the risk of mycotoxin contamination in feed and achieving the dual technical effect of ensuring feed safety and protecting animal health.

[0052] In this invention, the virus preferably includes porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is a single-stranded positive-sense RNA virus belonging to the Arteriviridae family and is the main pathogen causing porcine reproductive and respiratory syndrome (PRRS). This virus primarily infects porcine alveolar macrophages, leading to immunosuppression and secondary bacterial and viral infections. Clinically, it manifests as reproductive disorders in pregnant sows, such as abortion, stillbirth, and mummified fetuses, as well as respiratory diseases in pigs of all ages, especially piglets, with high mortality rates, causing significant economic losses to the pig industry. Due to the high variability and antibody-dependent enhancement of PRRSV, the protective effect of traditional vaccines is limited, and there are currently no specific antiviral drugs. Therefore, developing safe and effective anti-PRRSV alternatives is of significant clinical importance. In this embodiment of the invention, embryonic epithelial cells are infected with a PRRSV-GFP strain labeled with green fluorescent protein, and fluorescence observation and TCID monitoring are performed. 50The antiviral effects of fermented traditional Chinese medicine (TCM) were systematically evaluated using methods such as virus titer determination, Western blotting, and qPCR. Results showed that the GFP fluorescence intensity in the fermented TCM group was significantly lower than that in the unfermented control group; the virus titer in the cell supernatant decreased significantly with increasing TCM concentration; the expression abundance of the viral N protein was significantly inhibited; and the relative expression level of the viral NSP9 gene mRNA was also significantly reduced. These results consistently indicate that the fermented TCM obtained by fermentation with Bacillus subtilis XNW-KCYB1 can effectively inhibit the adsorption, entry, or replication of PRRSV, blocking viral proliferation at both the protein and gene levels. Therefore, the fermented TCM of this invention demonstrates outstanding potential in anti-PRRSV activity and can provide a green feed additive solution for the prevention and control of porcine reproductive and respiratory syndrome (PRRS).

[0053] This invention provides a fermented traditional Chinese medicine, obtained by fermenting a traditional Chinese medicine composition using Bacillus subtilis strain XNW-KCYB1. The strain XNW-KCYB1 exhibits a significant characteristic of high cellulase production, with cellulase activity significantly higher than other Bacillus strains isolated in the same batch and several commercially available products; the viable cell count after 48 hours of cultivation reaches (15.57±0.39)×10⁻⁶. 8 The strain XNW-KCYB1, with a concentration of CFU / mL, exhibits rapid proliferation and the ability to stably secrete cellulase over a long period. When used for fermentation of traditional Chinese medicine, this strain efficiently degrades the cellulose and lignin skeleton in plant cell walls, fully releasing active ingredients such as astragalus polysaccharides and saponins encapsulated within the cell walls. Simultaneously, the enzyme system secreted by the strain further biotransforms macromolecules (such as glycosides, proteins, and polysaccharides) into smaller active ingredients (astragalin, flavonoids, smilax saponins, glycyrrhetinic acid, magnolol, total free polyphenols, soluble oligosaccharides, free amino acids, etc.), significantly improving the bioavailability of traditional Chinese medicine. Compared with unfermented traditional Chinese medicine, single-agent probiotics, or physical mixtures, this fermented traditional Chinese medicine has the following advantages: In animal husbandry, it can significantly improve the average daily weight gain and average daily feed intake, reduce the feed conversion ratio and diarrhea rate, and comprehensively improve growth performance; in terms of immune enhancement, it can significantly increase spleen index, thymus index, and serum IgG, IgA, IL-10, TNF-α, and IFN-γ levels, enhancing animal immunity from multiple levels of immune organs, humoral immunity, and cellular immunity; in terms of antibacterial activity, it has significant inhibitory effects on pathogenic bacteria such as Escherichia coli, Streptococcus suis, and Haemophilus parasuis, as well as toxin-producing fungi such as Aspergillus flavus and Fusarium graminearum, reducing the risk of feed mycotoxin contamination; in terms of antiviral activity, it can effectively inhibit the reproduction of porcine reproductive and respiratory syndrome virus (PRRSV), reducing viral titers and the expression of N protein and NSP9 genes. In summary, this fermented traditional Chinese medicine has multiple functions including growth promotion, antidiarrheal, immune regulation, antibacterial, and antiviral effects, providing an efficient, safe, and green technical solution for antibiotic-free farming.

[0054] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0055] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1 Isolation, identification and preservation of Bacillus subtilis strain XNW-KCYB1 1. Sample source and isolation culture medium The samples in this embodiment were sourced from fresh feces of healthy piglets from farms in different regions across China, including Hubei Province, Guangxi Zhuang Autonomous Region, Hunan Province, and Shandong Province. Among them, the Bacillus subtilis strain (strain number 21) obtained through screening originated from piglet feces from a farm in Guangxi Zhuang Autonomous Region.

[0057] The culture medium used was LB liquid medium, composed of the following components: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1 L distilled water, pH 7.0–7.2. LA solid medium was prepared by adding 15 g / L agar powder to the LB liquid medium.

[0058] 2. Isolation and purification of bacterial strains (1) Weigh 5 g of fecal sample, add it to 45 mL of sterile PBS buffer, and vortex in a constant temperature shaker at 37 ℃ and 180 r / min for 30 min, then let it stand for 30 min to obtain the sample suspension.

[0059] (2) Take 2 mL of the supernatant of the sample suspension obtained in step (1) and transfer it to a 2 mL sterile cryovial. Treat it in a water bath at 80°C for 10 min.

[0060] (3) Take 100 μL of the sample suspension after water bath treatment in step (2), and dilute it in a 10-fold gradient to obtain 10 μL of the sample suspension. -1 10 -2 10 -3 Sample dilutions of different gradients.

[0061] (4) Take 100 μL of each gradient dilution and spread it evenly on LA solid culture medium plates. Invert the plates and incubate them in a 37 ℃ constant temperature incubator for 24 h.

[0062] (5) Pick a single colony grown on the plate in step (4) and streak it onto a fresh LA solid medium plate for purification. Place the streaked plate in a 37 ℃ constant temperature incubator for 24 h. Observe that the colony morphology on the plate is consistent, that is, pure colonies are obtained, indicating that the isolation and purification are completed.

[0063] 3. Strain identification and preservation The single colonies obtained after purification in step 2 (5) were cultured in liquid medium, and the bacterial cells were collected. Genomic DNA was extracted as a PCR template. 16S rDNA fragment amplification was performed using upstream primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 1) and downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 2). The primers were synthesized by Wuhan Qingke Biotechnology Co., Ltd. The total volume of the PCR reaction system was 25 μL, including: 12.5 μL of 2× Rapid Tap Master Mix, 1 μL of upstream primer, 1 μL of downstream primer, 2 μL of DNA template, and ddH2O added to a final volume of 25 μL. The amplification program was: 95℃ pre-denaturation for 5 min; then 95℃ denaturation for 15 s, 55℃ annealing for 15 s, and 72℃ extension for 35 s, for a total of 30 cycles; finally, a final extension at 72℃ for 5 min, and storage at 4℃. Five μL of PCR product was subjected to 1% agarose gel electrophoresis. After confirming the amplification of a specific band of approximately 1500 bp, the remaining PCR product was sent to Wuhan Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The obtained 16S rDNA sequence was compared with known sequences in the NCBI database using a BLAST tool.

[0064] After PCR molecular identification, more than 60 strains of Bacillus were obtained, and the strains were preserved in ultra-low temperature glycerol tubes in the laboratory at -80℃.

[0065] Example 2 Comparative experiment on the viable count and cellulase production capacity of Bacillus subtilis More than 60 strains of Bacillus obtained in Example 1 were streaked onto LB solid plates for activation and cultured at 37°C for 24 h. Single colonies were picked and inoculated into LB liquid medium and cultured at 37°C and 180 r / min for 12 h to prepare seed culture. The seed culture was transferred to shake flasks containing LB liquid medium at a 2% inoculum and cultured at 37°C and 180 r / min for 48 h. After culturing, the bacterial culture was evenly spread onto LB solid plates using the dilution plating method and cultured at 37°C for 24 h. The viable cell count and cellulase activity were then measured.

[0066] Cellulase activity was detected using a plate assay with sodium carboxymethyl cellulose as the substrate. 1 μL of sample was spotted onto the plate and incubated at 37 ℃ for 48 h. Congo red was then used for color development. A comprehensive screening was conducted by comparing the viable cell count and cellulase activity of various strains. A Bacillus strain with high viable cell count and strong cellulase production capacity was selected, designated as strain 21 (subsequently named XNW-KCYB1). This strain achieved a viable cell count of (15.57±0.39)×10⁻¹⁰ after 48 h of incubation. 8 The CFU / mL cellulase activity of this strain was significantly higher than that of other strains. The comparison results between this strain and the other four strains are shown in Tables 1 and 2 below. Figure 2 (a) Figure 2 (b) and Figure 2 As shown in (c). To verify the cellulase production advantage of the strain of the present invention compared with existing commercially available products, four commercially available Bacillus subtilis powders were selected as controls, numbered 1#, 2#, 3#, and 4#, respectively. 0.1 g of each powder was accurately weighed, dissolved thoroughly in 10 mL of PBS, and its cellulase activity was determined using the same conditions and methods as in Example 2. The results were compared with those of strain 21. The experimental results are as follows. Figure 2 As shown in (d), the cellulase activity of Bacillus subtilis strain 21 of the present invention is significantly higher than that of most commercially available Bacillus subtilis powder products (2#, 3#, 4#), and is comparable to or slightly better than one of the products (1#), showing a good advantage in enzyme production.

[0067] Table 1. Statistical table of viable bacteria count of Bacillus subtilis XNW-KCYB1 and strains from the same batch.

[0068] Note: The letters a, b, c, and d following the data in the same row in the table represent the significance levels between the experimental groups. Groups marked with completely different letters in the same row indicate significant differences. p <0.05); groups labeled with the same letter (including combinations of letters) indicate no significant difference.

[0069] Table 2. Statistical table of the ratio of clear zone diameter to colony diameter (D / d) for cellulase production by different strains.

[0070] Note: The letters a, b, c, and d following the data in the same row in the table represent the significance levels between the experimental groups. Groups marked with completely different letters in the same row indicate significant differences. p <0.05); groups labeled with the same letter (including combinations of letters) indicate no significant difference.

[0071] Example 3 Activation of fermentation strains and preparation of seed culture 1. Source of strain Bacillus subtilis ( Bacillus subtilis XNW-KCYB1 was deposited on August 16, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20241806.

[0072] 2. Preparation of Bacillus subtilis seed culture Take Bacillus subtilis XNW-KCYB1 cryovials, streak them onto LB solid medium, and incubate aerobically at 37°C for 24 h. Pick a single colony and inoculate it into 5 mL of LB liquid medium, incubate at 37°C with shaking at 180 rpm for 12 h. Transfer to 100 mL of LB liquid medium at a 2% inoculum rate, and incubate at 37°C with shaking at 180 rpm for 24–48 h to obtain viable counts ≥1 × 10⁻⁶. 9 Seed culture of CFU / mL.

[0073] Example 4 Preparation of fermented traditional Chinese medicine 1. Experimental Traditional Chinese Medicine Composition In this embodiment, the traditional Chinese medicine composition consists of the following raw materials in parts by weight: Astragalus membranaceus 42 parts, Atractylodes macrocephala 38 parts, Isatis indigotica 18 parts, Anemarrhena asphodeloides 12 parts, Morus alba root bark 8 parts, Magnolia officinalis 8 parts, Ginkgo biloba leaf 7 parts, and Glycyrrhiza uralensis 15 parts. The above-mentioned traditional Chinese medicines are pulverized separately and passed through a 40-mesh sieve, then mixed evenly in proportion to obtain the powdered traditional Chinese medicine composition for fermentation.

[0074] 2. Fermentation of Traditional Chinese Medicine Compositions Weigh 200 g of the above-mentioned traditional Chinese medicine composition, add 800 mL of sterile water, mix thoroughly, and autoclave at 121 ℃ for 30 minutes. After cooling to room temperature, inoculate with Bacillus subtilis seed culture (viable count ≥ 1 × 10⁻⁶) at an inoculation rate of 2% (v / v). 9 The control group (CK) consisted of samples treated in the same manner but inoculated with an equal volume of LB liquid medium (CFU / mL). Both groups were subjected to aerobic fermentation at 37 °C and 180 r / min for 96 h in a constant-temperature shaker. The resulting fermentation product was then placed in a freeze dryer, with pre-freezing at -40 °C to -60 °C for 6–10 h. Sublimation drying was then performed under a vacuum below 20 Pa until the moisture content of the sample dropped below 5%, yielding a dried powder.

[0075] Fermentation results as follows Figure 3 As shown in the figure. Experimental results indicate that the viable cell count on day 0 of fermentation is approximately 1.5 × 10⁻⁶.7 CFU / mL, viable cell count increased to 1.4 × 10⁻⁶ after 1 day of fermentation. 9 CFU / mL, viable bacterial count reached 1.5 × 10⁻⁶ after 2 days. 9 The concentration of CFU / mL remained above 6.3-6.4 until the end of fermentation. The control group maintained a stable pH of 6.3-6.4; the experimental group started at pH 6.37, rising to 6.78 after 1 day, and then to 7.18 after 2 days, maintaining a pH of around 7.2, consistent with the increasing trend of viable cell count. Clear zones were detected in the experimental group on days 2 and 4, indicating that Bacillus subtilis XNW-KCYB1 continuously produced cellulase; the control group showed no activity.

[0076] 3. Determination of the content of free aglycones, polyphenols, amino acids and oligosaccharides in the traditional Chinese medicine composition before and after fermentation Weigh 1.0 g of the sample powder before and after fermentation, and perform the following determinations respectively: (1) Detection of free aglycones and polyphenols: After ultrasonic extraction with 80% methanol for 30 min, centrifugation and filtration, the contents of astragaloside, flavonoid aglycones (such as kaempferol and quercetin), smilax saponin, glycyrrhetinic acid and magnolol were determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) in multiple reaction monitoring (MRM) mode. At the same time, the total soluble polyphenols were determined at 765 nm by the Folin-Ciocalteu method.

[0077] (2) Detection of water-soluble components: The supernatant was extracted with hot water at 80 °C for 30 min, centrifuged, and the total amount of free amino acids was determined at 570 nm using the ninhydrin method. The remaining supernatant was precipitated with 80% ethanol to remove macromolecular polysaccharides, and the content of soluble oligosaccharides (degree of polymerization 2~10) was determined by high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD).

[0078] All the above determinations were performed in triplicate. The experimental results are shown in Table 3. After fermentation with Bacillus subtilis XNW-KCYB1, the contents of astragalin, flavonoid aglycone, smilax saponin, glycyrrhetinic acid, magnolol, total free polyphenols, soluble functional oligosaccharides, and free amino acids in the traditional Chinese medicine composition were significantly increased. p <0.05). This indicates that the strain of the present invention can effectively convert glycosides, polyphenols and macromolecular nutrients in traditional Chinese medicine into small molecule active ingredients with higher bioavailability, significantly improving the absorption and utilization effect of the traditional Chinese medicine composition.

[0079] Table 3 Comparison of the content of major active ingredients in samples before and after fermentation

[0080] Note: Different lowercase letters (a, b) on the superscript of data in the same row indicate significant differences between groups. p <0.05).

[0081] Example 5 In vitro antibacterial experiment of fermented Chinese medicine 1. Fermented traditional Chinese medicine antagonizes pathogenic bacteria The Oxford cup method was used to detect the antibacterial activity of different treatment groups against *Escherichia coli*, *Streptococcus suis*, and *Haemophilus parasuis*. Five treatment groups were set up in the experiment, and the sample preparation methods were as follows: Control group (CON) was prepared with PBS (blank control); Traditional Chinese medicine group (TCM) consisted of 1 g of unfermented TCM powder, which was added to 9 mL of PBS to prepare a suspension; Probiotic group (PRO) consisted of 1 g of *Bacillus subtilis* XNW-KCYB1 bacterial agent powder, which was added to 9 mL of PBS to prepare a suspension; Mixed group (MIX) consisted of unfermented TCM powder and bacterial agent powder mixed at a mass ratio of 49:1, with 1 g of the mixed powder added to 9 mL of PBS to prepare a suspension; Fermented TCM group (FTCM) consisted of 1 g of fermented TCM powder prepared in Example 4, which was added to 9 mL of PBS to prepare a suspension. Each of the above suspensions was vortexed for 5 min, sonicated for 10 min, and then centrifuged at 10000 r / min for 10 min. The supernatant was then filtered through a 0.22 μm microporous membrane for sterilization to obtain the sample to be tested. Inhibition zone determination: Each pathogen suspension was added to the corresponding solid culture medium at a volume ratio of 1% (final concentration approximately 10). 6 After mixing (CFU / mL), pour the mixture into a petri dish containing an Oxford cup. Once the agar has solidified, remove the Oxford cup and add 100 μL of the sample to each well. Incubate at 37°C for 12 h and measure the diameter of the inhibition zone. Each sample has three replicates.

[0082] Table 4 Comparison of antibacterial activities against three pathogens in different treatment groups

[0083] Note: Different lowercase letters (a, b, c, d) in the superscript of the data in the same row indicate significant differences between groups. p <0.05).

[0084] The diameter of the inhibition zone against the three pathogens in different treatment groups was determined using the Oxford cup method, and the results are shown in Table 4. The fermented herbal medicine group (FTCM) showed significantly better antibacterial effects against the three pathogens than the probiotic group, the herbal medicine group, and the mixed group, indicating that the Bacillus subtilis XNW-KCYB1 fermented herbal medicine composition has synergistically enhanced broad-spectrum antibacterial activity.

[0085] 2. Fermented traditional Chinese medicine antagonizes pathogenic fungi. The inhibition rates of the five treatment groups (CON, TCM, PRO, MIX, FTCM) against Aspergillus flavus and Fusarium graminearum were determined using the plate confrontation method. Under aseptic conditions, mycelial blocks were excised from the edge of Aspergillus flavus and Fusarium graminearum colonies using a sterile punch (5 mm diameter). The mycelial blocks were then inoculated onto the center of PDA plates with the mycelial side facing down. Samples from each treatment group were thoroughly mixed, centrifuged, and the supernatant was collected. 5 μL of the supernatant was then spotted onto the plate at a distance of 2 cm from the center of the mycelial block. Each treatment was repeated in triplicate. Plates without the test samples (containing an equal volume of sterile PBS) served as the blank control group. The inoculated PDA plates were incubated upside down at 28°C. After the mycelia in the blank control group had fully colonized the plates, the mycelial growth radius was observed and measured. The inhibition rate was calculated according to Formula III. .

[0086] The results of the antagonistic fungal experiments are shown in Table 5. The inhibition rates of the fermented Chinese medicine group against both pathogenic fungi were significantly better than those against the probiotic group and the mixed group, confirming that fermentation treatment can synergistically enhance the antifungal ability of the Chinese medicine composition.

[0087] Table 5. Inhibition rates of different treatment groups against the two pathogenic fungi

[0088] Note: Different lowercase letters (a, b, c, d) in the superscript of the data in the same row indicate significant differences between groups. p <0.05).

[0089] Example 6 In vitro antiviral experiments of fermented traditional Chinese medicine 1. Cells and Viruses The experiment used Marc-145 cells (growth medium: DMEM containing 10% fetal bovine serum) as the model. The viral strain used was PRRSV-GFP (Porcine Reproductive and Respiratory Syndrome Virus) labeled with green fluorescent protein. The PRRSV-GFP strain used in this example was constructed according to existing technology. Specific technical details of this preparation method can be found in existing literature (e.g., Wang et al., Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein). BioMed Research International (2014). Those skilled in the art can obtain the PRRSV-GFP strain used in this embodiment based on the above-described literature.

[0090] The fermentation products of the fermented Chinese medicine group and the CK group after 4 days of fermentation were centrifuged at 8000 r / min for 10 min, and the supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain the final product. The final product was then diluted to different concentrations according to experimental requirements for cell treatment.

[0091] 2. Preliminary screening of antiviral activity The antiviral activity of fermented traditional Chinese medicine was detected using fluorescence observation. Marc-145 cells were seeded in 96-well plates, and when the cell confluence reached 80%–90%, PRRSV-GFP was inoculated at MOI=1. Subsequently, different concentrations of fermented samples from 0 days and 4 days of fermentation were added. Forty-eight hours after infection, the fluorescence intensity of GFP in each group was observed using an inverted fluorescence microscope. The fluorescence quantity and intensity of the fermented group and the unfermented group at the same concentration were compared to preliminarily determine the effect of fermentation process on enhancing the antiviral efficacy of the drug.

[0092] 3. Determination of viral titer To further accurately evaluate the inhibitory effect of fermented traditional Chinese medicine on viral replication, cell culture supernatants from each group were collected and analyzed using TCID50. 50 Virus titers were determined using the Reed-Muench method. The virus titers of each group were calculated, and the inhibition rate of virus proliferation in the fermentation group was analyzed.

[0093] 4. Viral load detection (RT-qPCR) Total RNA was extracted from cells in each group, reverse transcribed, and the relative mRNA expression level of the PRRSV NSP9 gene was detected using RT-qPCR. β-actin was used as an internal reference gene, and the relative fold change was calculated using the 2^(-ΔΔCt) method to verify the antiviral activity of the fermentation product at the gene transcription level. The primer sequences used are as follows: NSP9-F (SEQ ID NO: 3): 5'-CTAAGAGAGGTGGCCTGTCG-3'; NSP9-R (SEQ ID NO: 4): 5'-GAGACTCGGCATACAGCACA-3'; β-actin-F (SEQ ID NO: 5): 5'-CTCCATCATGAAGTGCGACGT-3'; β-actin-R (SEQ ID NO: 6): 5'-GTGATCTCCTTCTGCATCCTGTC-3'.

[0094] 5. Viral protein expression analysis (Western Blot) To verify drug efficacy at the translational level, cell lysates were collected, subjected to SDS-PAGE electrophoresis, and transferred to a PVDF membrane. An N protein-specific antibody was added to detect viral protein expression, which was normalized using the internal control protein GAPDH.

[0095] 6. Experimental Results Experimental results showed that fermented traditional Chinese medicine could significantly reduce the GFP fluorescence intensity of PRRSV ( Figure 4 (a), while significantly reducing viral titer ( Figure 4 (b) Inhibit viral N protein expression ( Figure 4 c) and NSP9 gene expression ( Figure 4 (d).

[0096] Example 7 The effect of fermented feed on piglet production performance 1. Experimental Design Seventy-five healthy weaned piglets of similar age and weight (initial weight approximately 7.00 ± 0.50 kg) were randomly divided into five groups, with three replicates per group and five piglets per replicate. The experimental period was 28 days. All piglets had free access to feed and water, and their immunization program was performed according to standard farm practices. At the beginning and end of the experiment, the piglets were weighed on an empty stomach, and their daily feed intake and diarrhea were recorded.

[0097] 2. Experimental Grouping Control group (CON): Basal diet. The basal diet used in this example was purchased from Guangxi Yangxiang Group Co., Ltd., whose products are compound feed for pigs with stable nutritional components that meet the nutritional requirements of the experimental pigs.

[0098] Traditional Chinese Medicine Group (TCM): 5% (w / w) of the unfermented traditional Chinese medicine composition powder described in Example 4 was added to the basal diet.

[0099] Probiotic group (PRO): Add 5% (w / w) Bacillus subtilis inoculum (live count ≥1×10⁻⁶) to the basal diet. 9 CFU / kg feed).

[0100] Mixed group (MIX): basal diet + 4.9% of the unfermented Chinese herbal medicine composition described in Example 4 + 0.1% Bacillus subtilis inoculum.

[0101] Fermented Chinese Medicine Group (FTCM): Freeze-dried sample of fermented Chinese medicine composition prepared by adding 5% (w / w) of Example 4 to the basal diet.

[0102] 3. Indicator Measurement On days 1 and 28 of the experiment, piglets were weighed on an empty stomach, and their feed intake was recorded. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F / G) were calculated. Diarrhea was observed and recorded daily for each piglet, and the diarrhea rate was calculated using Formula I. .

[0103] 4. Experimental Results After a 28-day feeding trial, the growth performance results of each group of piglets were as follows: Figure 5 As shown. Compared with the other groups, the fermented herbal medicine group (FTCM) significantly increased the average daily weight gain of piglets ( ). Figure 5 (a), while significantly reducing the material weight ratio ( Figure 5 (c) and diarrhea rate ( Figure 5 (d). The above results indicate that the traditional Chinese medicine composition fermented by Bacillus subtilis can effectively improve the growth performance of piglets and reduce the incidence of diarrhea.

[0104] Example 8 The effect of fermented feed on the immune performance of piglets 1. Calculation of Immune Organ Index The experimental groups were the same as in Example 5. On day 28 of the experiment, blood was collected from the anterior vena cava of two piglets randomly selected from each replicate, and the serum was separated. After blood collection, all piglets were slaughtered, and the spleen and thymus were completely removed and weighed for calculation of the immune organ index. The calculation of the immune organ index is given by Formula II: .

[0105] 2. Measurement of immune factor indicators Serum immune markers were measured using an ELISA kit to determine the levels of immunoglobulin G (IgG) and immunoglobulin A (IgA) in serum.

[0106] Cytokine assay: Serum levels of interleukin-10 (IL-10), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were measured using an ELISA kit.

[0107] 3. Experimental Results After a 28-day feeding trial, the non-specific immunity level of piglets in the fermented traditional Chinese medicine group was significantly improved. The results of immune performance in each group are as follows: Figure 6 As shown. Compared with the control group, the spleen index of piglets in the fermented traditional Chinese medicine group ( Figure 6 a) and thymus index ( Figure 6 The significantly increased levels of IgG (b) in the serum of this group of piglets indicate that the development of their immune organs has been effectively promoted. Regarding humoral immunity, the serum levels of IgG (b) in this group of piglets were significantly higher. Figure 6 c) and IgA ( Figure 6The levels of IL-10 in the serum of piglets in this group were significantly higher than those in the control group, indicating a stronger humoral immune response. Regarding cellular immunity, the serum levels of IL-10 in this group of piglets were significantly higher than those in the control group. Figure 6 (e), TNF-α ( Figure 6 f) and IFN-γ ( Figure 6 The levels of g) in the middle range increased significantly.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fermented traditional Chinese medicine, characterized in that, To utilize Bacillus subtilis ( Bacillus subtilis The traditional Chinese medicine composition was obtained by fermentation of strain XNW-KCYB1; The traditional Chinese medicine composition comprises the following raw materials in parts by weight: Astragalus membranaceus 30-50 parts, Atractylodes macrocephala 30-45 parts, Isatis indigotica 10-25 parts, Anemarrhena asphodeloides 5-20 parts, Morus alba root bark 1-15 parts, Magnolia officinalis 1-15 parts, Ginkgo biloba leaf 1-14 parts, and Glycyrrhiza uralensis 8-22 parts. The Bacillus subtilis strain XNW-KCYB1 is deposited at the China Center for Type Culture Collection (CCTCCNO), with accession number CCTCCNO: M 20241806.

2. The fermented traditional Chinese medicine according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following raw materials in parts by weight: Astragalus membranaceus 40-45 parts, Atractylodes macrocephala 35-40 parts, Isatis indigotica 15-20 parts, Anemarrhena asphodeloides 10-15 parts, Morus alba root bark 5-10 parts, Magnolia officinalis 5-10 parts, Ginkgo biloba leaf 5-10 parts, and Glycyrrhiza uralensis 12-18 parts.

3. The fermented traditional Chinese medicine according to claim 1 or 2, characterized in that, The mass-to-volume ratio of the traditional Chinese medicine composition and the bacterial solution of Bacillus subtilis strain XNW-KCYB1 is (1~5) g : (0.025~0.5) ml; The viable cell concentration of the Bacillus subtilis strain XNW-KCYB1 in the bacterial culture is ≥1×10⁻⁶. 9 CFU / mL.

4. A feed additive, characterized in that, Includes the fermented traditional Chinese medicine as described in any one of claims 1 to 3.

5. An animal feed, characterized in that, Includes the fermented traditional Chinese medicine as described in any one of claims 1 to 3 or the feed additive and basal diet as described in claim 4; The fermented Chinese medicine or feed additives account for 4% to 6% of the basal diet weight.

6. The application of Bacillus subtilis strain XNW-KCYB1, the fermented traditional Chinese medicine according to any one of claims 1 to 3, and the feed additive according to claim 4 in the preparation of animal feed or animal breeding that is resistant to pathogenic microorganisms; The Bacillus subtilis strain XNW-KCYB1 is deposited at the China Center for Type Culture Collection (CCTCCNO), with accession number CCTCCNO: M 20241806.

7. The application according to claim 6, characterized in that, The pathogenic microorganisms include at least one of fungi, bacteria, and viruses.

8. The application according to claim 7, characterized in that, The bacteria include at least one of the following: Escherichia coli (Escherichia coli) Escherichia coli Streptococcus suis ( Streptococcus suis ) and Haemophilus parasuis ( Haemophilus parasuis ); The fungi include Aspergillus flavus ( Aspergillus flavus ) and / or Fusarium graminearum ( Fusarium graminearum ).

9. The application according to claim 7, characterized in that, The viruses mentioned include porcine reproductive and respiratory syndrome virus (PRRSV).

10. The application according to claim 6, characterized in that, The animal husbandry mentioned includes improving animal growth performance and / or improving animal immune performance.

Images