A traditional Chinese medicine compound expanded culture probiotic fermentation agent for improving growth performance and intestinal health of pigs, and a preparation method and application thereof

Abstract

The present application belongs to the technical field of microbial fermentation and livestock feed, and discloses a traditional Chinese medicine compound expansion type probiotic fermentation agent for improving growth performance and intestinal health of pigs and a preparation method and application thereof. The present application provides a traditional Chinese medicine compound expansion type probiotic fermentation agent comprising a compound functional bacteria powder and a traditional Chinese medicine compound fermentation nutrient carrier, which can be expanded and cultivated on site in a terminal breeding farm environment. When the agent is mixed with a basic daily ration for feeding pigs, the alpha diversity of intestinal flora of the pigs can be significantly improved, the flora structure can be reshaped, beneficial microorganisms such as spirochaetes and prevotellaceae can be enriched, and key metabolic pathways such as purine metabolism, lipid metabolism and animal autophagy can be regulated, so that the intestinal health of pigs is improved through a 'flora-metabolite' synergistic mechanism. The present application solves the problems of low bioavailability of traditional Chinese medicine, poor strain synergy and unstable actual application effect in existing products, and realizes nonlinear quality variation of technical indicators on the basis of conventional components.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and livestock feed technology, specifically relating to a traditional Chinese medicine compound probiotic fermentation agent for improving the growth performance and intestinal health of pigs, its preparation method and application. Background Technology

[0002] With the full implementation of the ban on antibiotics in feed, the pig farming industry has entered a new stage of antibiotic-free farming. Currently, pig farming faces the dual pressures of high feed raw material prices, continuously rising farming costs, and low pork prices. Against the backdrop of severely compressed profit margins, "cost reduction and efficiency improvement," reducing losses, and increasing meat yield (lowering the feed conversion ratio) have become the core objectives for the survival and development of pig farms.

[0003] Probiotics, as a core alternative to antibiotics, can improve the growth performance of pigs by regulating gut microbiota, strengthening the intestinal barrier, and promoting nutrient absorption. However, existing probiotic products on the market have significant limitations: ① Direct-inoculation powdered formulations: have drawbacks such as high-temperature inactivation, high cost (the cost per ton often exceeds 200 yuan), weak colonization ability in the intestine, and insufficient accumulation of metabolites.

[0004] ② Pre-fermented feed products: have problems such as small transportation radius, short shelf life and difficulty in adapting to the decentralized farming scenario of "company + farmer".

[0005] ③ Existing propagation agents: Most of them use general formulas, which have poor synergy between strains. Furthermore, the fermentation nutrient carrier formula does not incorporate the functional components of traditional Chinese medicine, resulting in unstable propagation effects and making it difficult to reliably achieve the core goal of reducing the feed conversion ratio.

[0006] Most importantly, existing technologies involving the combined use of traditional Chinese medicine and probiotics primarily focus on enhancing the body's immunity and improving pork quality, failing to provide effective solutions for core production performance indicators such as reducing the feed conversion ratio in pigs and targeted regulation of the gut microbiota. Currently, the combined use of probiotics and traditional Chinese medicine is mostly limited to simple physical mixing or compounding after separate fermentation. ① Insufficient accumulation of metabolites: Due to the lack of regulation of key environmental factors such as the redox potential (Eh) of the fermentation system, it is impossible to induce probiotics to secrete anaerobic hydrolytic enzymes targeting large molecules of traditional Chinese medicine, resulting in low content of core metabolites such as essential amino acids and organic acids.

[0007] ② Low bioavailability of traditional Chinese medicine: Large molecules of traditional Chinese medicine such as astragalus polysaccharides, codonopsis saponins, and magnolol are difficult to absorb and have not undergone deep biotransformation. They have a slow onset of action and cannot form a synergistic effect with probiotic metabolism, resulting in limited application effects.

[0008] ③ Performance bottleneck under similar formulations: Although some conventional products are similar to the raw materials of this invention, due to the lack of an "integrated synergistic expansion culture" technical solution, the synergy of strains is poor, and no breakthrough technical effect has been achieved.

[0009] Therefore, developing a compound probiotic fermentation agent of traditional Chinese medicine that uses similar conventional raw materials but can achieve efficient conversion of active ingredients of traditional Chinese medicine through integrated synergistic fermentation, significantly improve the growth performance of pigs, and stably reduce feed conversion ratio and losses is of great practical significance for promoting antibiotic-free and efficient pig farming and achieving cost reduction and efficiency improvement in the industry. Summary of the Invention

[0010] Addressing the core needs of the current pig farming industry amidst persistently low pork prices, high feed costs, and the "antibiotic ban" policy, this invention solves technical challenges in the application of existing probiotic products, such as poor live bacteria stability, insufficient synergistic enzyme production by strains, low bioavailability of traditional Chinese medicine, and unstable "cost reduction and efficiency improvement" effects in practical applications. This invention provides a traditional Chinese medicine compound expanded probiotic fermentation agent for improving the growth performance and intestinal health of pigs, along with its preparation method and application scheme. By introducing a precise metabolic regulation mechanism under conventional, similar raw material formulations, the following specific objectives are achieved: ① Achieving efficient biotransformation and utilization of active ingredients in traditional Chinese medicine: L-cysteine ​​hydrochloride is used to regulate the redox potential (Eh) of the fermentation system, creating a deep anaerobic microenvironment and directionally inducing probiotics to express anaerobic hydrolases at high levels. Through in-situ "penetrating" degradation of traditional Chinese medicine carriers such as Astragalus membranaceus, Codonopsis pilosula, and Magnolia officinalis, large, poorly absorbed molecules are efficiently converted into small, highly active products (such as DL-3-phenyllactic acid, which is increased by more than 5 times), significantly improving bioavailability and solving the industry pain points of slow onset and poor absorption when traditional Chinese medicine is added alone.

[0011] ② Constructing a five-strain synergistic enzyme production system to achieve multi-level enrichment of metabolites: By scientifically combining strains to construct a "full-spectrum enzymatic hydrolysis system", a breakthrough increase in product abundance is achieved under the background of conventional raw materials, making the content of free essential amino acids (such as leucine) in the fermentation broth reach 290 times that of conventional combinations, and the total content of free amino acids ≥200 mg / L, providing pigs with extremely high-value "enzymatic nutrition".

[0012] ③ Ensuring high activity and stability during on-site cultivation: Optimizing the formula of the compound fermentation nutrient carrier for traditional Chinese medicine to ensure simple operation and stable performance during on-site co-cultivation in the environment of the terminal breeding farm. Achieving a viable count of lactic acid bacteria ≥ 5.0 × 10⁻⁶ after cultivation. 8 With CFU / mL, pH≤3.8 and minimal batch-to-batch variation, it effectively solves the problems of weak colonization of exogenous probiotics and substandard expansion.

[0013] ④ Achieve significant goals of "cost reduction and efficiency improvement" and "antibiotic-free substitution": By reshaping the intestinal microecology of pigs through the synergistic mechanism of "microbiota-metabolites," the nutrient digestibility and absorption rate is improved, and the body's immune and antioxidant barriers are enhanced. Ultimately, the core objectives of reducing the feed conversion ratio by 0.08-0.20 (significantly better than the antibiotic group), reducing the diarrhea rate by more than 70%, and reducing breeding losses and drug costs are achieved, meeting the industry's urgent needs for increased meat yield and green and safe farming.

[0014] The first objective of this invention is to provide a traditional Chinese medicine compound probiotic fermentation agent.

[0015] A second aspect of the present invention is to provide a feed.

[0016] The third objective of this invention is to provide the application of the traditional Chinese medicine compound probiotic fermentation agent of the first aspect of this invention or the feed of the second aspect of this invention in pig farming.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a traditional Chinese medicine compound probiotic fermentation agent, comprising compound functional bacterial powder and a traditional Chinese medicine compound fermentation nutrient carrier; The compound functional microbial powder includes Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosus, Lactobacillus paracasei, and Saccharomyces cerevisiae; The compound fermentation nutrient carrier of traditional Chinese medicine includes a compound of traditional Chinese herbal medicines.

[0018] In some embodiments of the present invention, the herbal compound includes Astragalus membranaceus, Codonopsis pilosula, and Magnolia officinalis.

[0019] In some embodiments of the present invention, the mass ratio of Astragalus membranaceus, Codonopsis pilosula and Magnolia officinalis is (1-2):(1-2):1; more specifically, it is 1:1:1.

[0020] In some embodiments of the present invention, the traditional Chinese medicine compound fermentation nutrient carrier further includes cysteine ​​hydrochloride, fast-acting carbon source and slow-release carbon source.

[0021] Cysteine ​​hydrochloride, as a redox potential regulator, is used to regulate redox potential, induce the secretion of anaerobic hydrolytic enzymes by complex functional bacteria, and optimize amino acid metabolic pathways, thereby achieving biotransformation of traditional Chinese medicine and multi-level enrichment of free amino acids.

[0022] In some embodiments of the present invention, the readily available carbon source includes at least one of molasses, glucose, and brown sugar; preferably glucose. The readily available carbon source is used for the rapid initial accumulation of microbial biomass.

[0023] In some embodiments of the present invention, the ratio of viable counts of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Pediococcus pentosus*, *Lactobacillus paracasei*, and *Saccharomyces cerevisiae* is (2-8):(1-4):(1-4):(1-4):1; further, it is (4-7):(2-4):(2-4):(2-4):1; and even further, it is 6:3:3:3:1.

[0024] In some embodiments of the present invention, the *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Pediococcus pentosus*, *Lactobacillus paracasei*, and *Saccharomyces cerevisiae* are *Lactobacillus plantarum* with accession number GDMCC No. 1.140, *Lactobacillus rhamnosus* with accession number GDMCC No. 1.1798, *Pediococcus pentosus* with accession number GDMCC No. 1.428, *Lactobacillus paracasei* with accession number GDMCC No. 1.159, and *Saccharomyces cerevisiae* with accession number GDMCC No. 2.110.

[0025] In some embodiments of the present invention, the preparation method of the composite functional microbial powder includes the following steps: culturing Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosus, Lactobacillus paracasei, and Saccharomyces cerevisiae to obtain single bacterial solutions, collecting the bacterial cells, and freeze-drying them under vacuum to obtain microbial powders of each bacterial species; mixing the microbial powders of each bacterial species to obtain the composite functional microbial powder.

[0026] In some embodiments of the present invention, the preparation method of the bacterial powders of each bacterium includes the following steps: *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Pediococcus pentosus*, and *Lactobacillus paracasei* are inoculated into MRS liquid culture medium and statically activated at 35-37 °C for 18-24 h to obtain primary seed culture; the primary seed culture is transferred to expansion culture medium at an inoculation rate of 3%-5% and statically cultured at 37 °C for 16-18 h to obtain secondary seed culture; the secondary seed culture is then transferred to fermentation culture medium at an inoculation rate of 5% and fermented at 37 °C for 20-24 h; the fermentation broth is centrifuged and freeze-dried under vacuum to obtain *Lactobacillus plantarum* bacterial powder, *Lactobacillus rhamnosus* bacterial powder, *Pediococcus pentosus* bacterial powder, and *Lactobacillus paracasei* bacterial powder, with a viable count ≥1.0 × 10⁻⁶. 11 CFU / g.

[0027] Saccharomyces cerevisiae was inoculated into YEPD liquid medium and activated at 28-30 ℃ and 180 r / min in a shaker for 20-24 h to obtain primary seed culture. The primary seed culture was then transferred to expansion medium at an inoculation rate of 3%-5% and cultured in a shaker at 30 ℃ for 18-20 h to obtain secondary seed culture. The secondary seed culture was then transferred to fermentation medium at an inoculation rate of 5% and fermented at 30 ℃ for 22-24 h. The fermentation broth was centrifuged and freeze-dried under vacuum to obtain yeast powder with a viable count ≥1.0 × 10⁻⁶. 11 CFU / g.

[0028] This invention, based on the core concept of "function-oriented and synergistic enhancement," constructs a complex microbial community system composed of five functionally complementary probiotic strains (four lactic acid bacteria and one yeast). The core lies in constructing a "full-spectrum enzymatic hydrolysis system" through the synergistic secretion of extracellular enzymes by multiple strains, achieving multi-level enrichment of metabolites. ① Synergistic mechanism: Lactobacillus paracasei, Lactobacillus plantarum, and Pediococcus pentosus are responsible for the targeted enzymatic hydrolysis of large protein molecules into small peptides and free essential amino acids; Lactobacillus rhamnosus secretes glycosidases to assist in substrate hydrolysis; Saccharomyces cerevisiae provides carbon source for energy and assists in enzyme production.

[0029] ②Non-obvious effects: The "enzymatic nutrition" produced by this system far exceeds the superposition effect of single strains. Among them, the content of core essential amino acids such as leucine, valine, and isoleucine is increased by 290 times compared with conventional combinations, which constitutes the core material basis for promoting product growth.

[0030] ③ Specific strains: Lactobacillus plantarum has strong colonization and antibacterial ability; Lactobacillus rhamnosus is responsible for relieving stress and enhancing immunity; Pediococcus pentosus improves palatability; Lactobacillus paracasei is responsible for secreting extracellular enzymes; Saccharomyces cerevisiae secretes amylase and produces prebiotics.

[0031] Addressing the issue of similar raw material formulations in the field, this invention achieves deep biotransformation of active ingredients in traditional Chinese medicine through precise control of redox potential (Eh): ① Multi-level carbon source gradient design: Fast-acting layer (glucose): Used for rapid accumulation of bacterial biomass in the initial stage.

[0032] Slow-release maintenance layer (whey powder, maltodextrin): maintains stable osmotic pressure and medium-term nutrient supply.

[0033] Functional conversion layer (Chinese herbal medicine compound): Enables in-situ "penetrating" degradation of Chinese herbal medicine particles (Astragalus membranaceus, Codonopsis pilosula, Magnolia officinalis) pulverized to ≤80 mesh.

[0034] ② The "switch" function of cysteine ​​hydrochloride: This component is not merely a nutrient substrate; its core function lies in precisely regulating the Eh of the system, creating a deeply anaerobic microenvironment, thereby significantly upregulating the expression levels of anaerobic hydrolases (β-glucosidase, cellulase, etc.). This achieves "penetrating" biotransformation and synergistic amplification of active ingredients from traditional Chinese medicine. Removing this component leads to the absence of an anaerobic environment, downregulation of hydrolytic enzyme expression, and a sharp drop in the conversion efficiency of traditional Chinese medicine, demonstrating that this conventional raw material has an unpredictable metabolic-directed induction effect in this system.

[0035] In some embodiments of the present invention, the traditional Chinese medicine compound expanded probiotic fermentation agent comprises, by weight, 5-15 parts of compound functional bacterial powder; further comprising 7-10 parts of compound functional bacterial powder; and even further comprising 7-9 parts of compound functional bacterial powder.

[0036] In some embodiments of the present invention, the traditional Chinese medicine compound probiotic fermentation agent comprises, by weight, 2-10 parts of traditional Chinese medicine compound; further comprising 3-8 parts of traditional Chinese medicine compound; and even further comprising 4-6 parts of traditional Chinese medicine compound.

[0037] In some embodiments of the present invention, the traditional Chinese medicine compound probiotic fermentation agent comprises, by weight, 0.3-1 parts of cysteine ​​hydrochloride; further comprising 0.3-0.8 parts of cysteine ​​hydrochloride; and even further comprising 0.5-0.7 parts of cysteine ​​hydrochloride.

[0038] In some embodiments of the present invention, the herbal compound probiotic fermentation agent comprises, by weight, 18-30 parts of fast-acting carbon source; further comprising 20-25 parts of fast-acting carbon source; and even further comprising 20-22 parts of fast-acting carbon source.

[0039] In some embodiments of the present invention, the traditional Chinese medicine compound expanded probiotic fermentation agent includes 55-80 parts of slow-release carbon source by weight.

[0040] In some embodiments of the present invention, the herbal compound probiotic fermentation agent comprises, by weight, 30-50 parts whey powder; further comprising 33-45 parts whey powder; and even further comprising 33-40 parts whey powder.

[0041] In some embodiments of the present invention, the traditional Chinese medicine compound expanded probiotic fermentation agent comprises, by weight, 8-15 parts of maltodextrin; further comprising 8-12 parts of maltodextrin; and even further comprising 9-12 parts of maltodextrin.

[0042] In some embodiments of the present invention, the traditional Chinese medicine compound expanded probiotic fermentation agent comprises, by weight, 15-25 parts of yeast autolysing powder; further comprising 18-22 parts of yeast autolysing powder; and even further comprising 19-21 parts of yeast autolysing powder.

[0043] In some embodiments of the present invention, the total viable count of the traditional Chinese medicine compound expanded probiotic fermentation agent is ≥1.0×10⁻⁶. 9 CFU / g.

[0044] In some embodiments of the present invention, the traditional Chinese medicine compound expanded probiotic fermentation agent is prepared by the following method: mixing compound functional bacterial powder and traditional Chinese medicine compound fermentation nutrient carrier to obtain the traditional Chinese medicine compound expanded probiotic fermentation agent.

[0045] In some embodiments of the present invention, the mixing time is 15-20 min.

[0046] In some embodiments of the present invention, the traditional Chinese medicine compound fermentation nutrient carrier is pretreated before mixing, including pulverizing the traditional Chinese medicine compound and passing it through an 80-mesh sieve; whey powder, glucose, yeast autolysate powder, maltodextrin, and L-cysteine ​​hydrochloride are all passed through an 80-mesh sieve; the above components are then mixed.

[0047] In some embodiments of the present invention, the preparation method further includes a vacuum sealing and packaging step.

[0048] In a second aspect, the present invention provides a feed comprising a compound probiotic fermentation agent based on traditional Chinese medicine and a basal diet.

[0049] In some embodiments of the present invention, the feed is prepared by a method comprising the following: The compound probiotic fermentation agent of traditional Chinese medicine is mixed with water and fermented to obtain fermentation liquid; The fermentation liquid is mixed with the basal diet to obtain feed.

[0050] In some embodiments of the present invention, the mixing ratio (mass-volume ratio) of the traditional Chinese medicine compound probiotic fermentation agent and water is 1:(80-150); further, it is 1:(90-120); and even further, it is 1:(90-110).

[0051] In some embodiments of the present invention, the fermentation culture conditions are 35-37°C for 40-55 h; and further 35-37°C for 45-50 h.

[0052] In some embodiments of the present invention, the fermentation liquid is added to the basal diet at a ratio of 3wt%-10wt%; further, 3wt%-8wt%; and even further, 3wt%-6wt%.

[0053] This feed can achieve stable efficacy control throughout the entire livestock and poultry breeding cycle.

[0054] In some embodiments of the present invention, the total viable count of lactic acid bacteria in the fermentation broth is ≥5.0 × 10⁻⁶. 8CFU / mL, pH≤3.8, lactic acid content≥4.0 g / L, total content of short-chain fatty acids such as acetic acid, propionic acid, and butyric acid≥1.2 g / L, total content of free amino acids≥200 mg / L, and DL-3-phenyllactic acid content≥16000 μg / L.

[0055] In some embodiments of the present invention, the basic diet includes feed commonly used in livestock and poultry farming, which can be routinely selected or commercially purchased depending on the animal species.

[0056] This invention breaks through the simple physical mixing mode and constructs a cascade synergistic regulatory mechanism of "probiotics → gut microbiota → metabolites → body physiological functions" through integrated in-situ fermentation: ① Targeted remodeling of gut microbiota: With the nutritional support of the compound fermentation nutrient carrier of traditional Chinese medicine, the compound functional strains can rapidly colonize the intestines of livestock and poultry, significantly improve the α diversity of gut microbiota, target and enrich beneficial bacteria such as Bacteroidetes, Spirochetes, and Prevotella, inhibit the proliferation of harmful bacteria, and remodel a healthy and stable gut microecological structure.

[0057] ② Biotransformation of Chinese medicine components: Under specific Eh environment, the compound functional strains directionally transform the macromolecules of Chinese medicine into highly active substances such as DL-3-phenyllactic acid and small molecule sugars. The content of DL-3-phenyllactic acid is ≥16000 μg / L, which significantly improves bioavailability.

[0058] ③ Metabolic profile targeted reprogramming: By enriching high levels of essential amino acids and active ingredients from traditional Chinese medicine, this method specifically activates the key KEGG pathway in animal autophagy and lipid metabolism, achieving the effects of repairing the intestinal mucosal barrier and reducing oxidative damage. This deep intervention at the mechanistic level cannot be achieved by traditional single probiotics or physical mixtures.

[0059] The beneficial effects of this invention are: This invention provides a traditional Chinese medicine (TCM) compound fermentation agent for expanding the culture of probiotics, comprising a compound functional bacterial powder and a TCM compound fermentation nutrient carrier. It can be synergistically expanded on-site in a terminal farm environment and fed to pigs mixed with their basal diet. This significantly enhances the α-diversity of the pig's gut microbiota, reshapes the microbiota structure, enriches beneficial microorganisms such as Spirochetes and Prevotella, and simultaneously regulates key metabolic pathways such as purine metabolism, lipid metabolism, and autophagy. Through a synergistic mechanism of "microbiota-metabolites," it improves the gut health of pigs. This invention addresses the pain points of existing products, such as low bioavailability of TCM, poor strain synergy, and unstable practical application effects, achieving a non-linear qualitative leap in technical indicators based on conventional components.

[0060] Specifically, compared with the prior art, the present invention achieves significant technical improvement by introducing a specific metabolic-directed induction mechanism under similar raw material formulations, and the specific beneficial effects are as follows: 1. Deep coupling and synergistic effect of bacteria and drugs, enabling multi-level enrichment of metabolites. This invention constructs a synergistic fermentation system consisting of "five functional bacteria + three traditional Chinese medicine carriers", which breaks through the limitations of traditional physical mixing or simple fermentation and forms a complete functional chain from "acid production and antibacterial activity" to "full-spectrum enzymatic hydrolysis of nutrients".

[0061] A qualitative leap in enzymatic hydrolysis efficiency: By precisely controlling the redox potential (Eh) through cysteine ​​hydrochloride, the strain was induced to secrete anaerobic hydrolases, resulting in an explosive increase in metabolites. As shown in Example 1, the contents of leucine, valine, and isoleucine in the fermentation broth were 290 times, 165 times, and 195 times that of the conventional combination (experimental group 2), respectively.

[0062] 2. Highly efficient conversion of key products, significantly improving bioavailability. This invention uses traditional Chinese medicine compound as the core component of the fermentation carrier, achieving "in-situ penetration" degradation of poorly absorbed components such as astragalus polysaccharide, codonopsis saponin, and magnolol.

[0063] Enrichment of conversion products: The content of the core conversion product DL-3-phenyllactic acid after fermentation is ≥16000 μg / L, which is more than 5 times higher than that of the unfermented carrier.

[0064] Technical advantages: This conversion process efficiently transforms macromolecular Chinese medicine into highly active small molecule substances, significantly improving intestinal absorption and solving the industry bottleneck of slow onset and poor absorption when Chinese herbal medicines are added alone.

[0065] 3. Significantly improved growth performance, with technical indicators surpassing those of antibiotics. The application effect of the traditional Chinese medicine compound expanded probiotic fermentation agent provided by this invention in pig farming highlights its great potential as an "antibiotic-free alternative" and surpasses the industry's "gold standard".

[0066] The feed conversion ratio (F / G) is significantly reduced: In practical applications, the F / G ratio can be reduced by 0.08-0.20 (1.55 for the probiotic + traditional Chinese medicine group and 1.66 for the antibiotic group), and the growth promotion effect is significantly better than that of the antibiotic group.

[0067] Precise loss control: the diarrhea rate was reduced by more than 70% (from 18.67% to 4.67%), and the daily weight gain was increased by 14.52%, significantly improving the uniformity and survival rate of the pigs.

[0068] 4. The "microbiota-metabolite" cascade regulation reshapes the intestinal health barrier. This invention, based on the identification of 5090 metabolites and KEGG pathway enrichment analysis, reveals its innovative underlying mechanism.

[0069] Precisely reshape the gut microbiota: Targeted enrichment of cellulose-degrading beneficial bacteria such as Spirochetes and Prevotella, enhancing the alpha diversity of the gut microbiota.

[0070] Metabolic reprogramming: Specifically activates autophagy and lipid metabolism pathways in animals, achieving positive regulation of the body's immunity and antioxidant capacity.

[0071] 5. Stable and excellent propagation performance, completely solving pain points in end-user applications. To address the industry challenge of unstable on-site cultivation, this invention ensures the robustness of the process by optimizing the formula of the compound fermentation nutrient carrier of traditional Chinese medicine.

[0072] High batch stability: After large-scale validation of 100 batches, the pH of the fermentation broth remained stable at 3.3-3.5, and the expansion failure rate was <1%.

[0073] Low operating threshold: It only requires adding water in the correct proportion and fermenting at a constant temperature to meet the standards. The core indicators (live bacteria count and acid production) show minimal differences between batches, which meets the needs of large-scale promotion.

[0074] 6. Long shelf life and wide applicability, resulting in outstanding overall economic benefits. The product has good stability: the traditional Chinese medicine compound probiotic fermentation agent provided by this invention has a shelf life of up to 12 months under cool and dry conditions, and the core fermentation performance does not decline within 4 months.

[0075] Significant cost advantage: The cost of adding probiotics to each ton of feed is controlled within 150 yuan, which is far lower than that of commercially available direct-inoculation probiotic products.

[0076] Flexible application scenarios: It can be adapted to automated liquid feeding lines in large-scale pig farms, and can also meet the needs of small and medium-sized farms through simple spraying and mixing.

[0077] In summary, this invention, with similar raw material formulations, achieves significant improvements in the growth performance and intestinal health of pigs through in-depth innovation in metabolic mechanisms, providing a technical solution with outstanding creativity and practical value for green antibiotic-free farming. Attached Figure Description

[0078] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This study analyzed the diversity and composition of the microbiome in hog manure. A represents α-diversity analysis, with different letters indicating significant differences. p <0.05); B is β diversity analysis; C is a Venn diagram showing the common and unique ASVs (amplifier sequence variations) among the three experimental groups; D is the phylum-level taxonomic composition (relative abundance); E is the genus-level taxonomic composition (relative abundance); F is a hierarchical clustering heatmap of differential abundance groups.

[0079] Figure 2LEfSe analysis of fecal microorganisms in meat pigs. Where A represents the LDA score of significantly different taxa (LDA > 3). p <0.05); B is the evolutionary branching diagram of the enriched taxa (red: CON group, green: PRO group, blue: PROC group), and the phylogenetic tree shows the taxonomic hierarchy from phylum to genus.

[0080] Figure 3 This section presents the composition and multivariate statistical analysis of the fecal metabolome. A represents the fecal metabolome analysis of fattening pigs, showing metabolite classification under positive ion mode; B represents metabolite classification under negative ion mode; and CE represents the OPLS-DA score plots for pairwise comparisons between groups.

[0081] Figure 4 This analysis focuses on differential metabolite changes in pig feces. The AC plot represents a volcano plot showing pairwise comparisons of metabolite changes between groups; red dots indicate upregulated metabolites, green dots indicate downregulated metabolites, and gray dots indicate metabolites with no significant change. The vertical dashed line represents the fold change threshold (log2FC = ±1, i.e., a 2-fold change), and the horizontal dashed line represents the significance threshold. p =0.05, i.e., -log 10 (p=1.3) Metabolites that simultaneously meet the dual thresholds are considered significantly differentially expressed metabolites; DF is the VIP score of key differentiating metabolites in the comparison between groups, and only metabolites with VIP>1 are key differentially expressed metabolites; G is a heatmap of the relative abundance of differentially expressed metabolites in the feces of pigs in the three experimental groups, and the color intensity reflects the relative abundance level of metabolites (red: high abundance, blue: low abundance).

[0082] Figure 5 Enrichment analysis of KEGG pathways in differentially metabolites from pig feces. A represents the comparison between the PRO and CON groups; B represents the comparison between the PROC and CON groups; and C represents a bubble chart of significantly enriched pathways between the PROC and PRO groups. p <0.05); bubble size indicates the amount of metabolites in each pathway, and color intensity indicates... p Significance of the value.

[0083] Figure 6 Spearman correlation analysis of fecal microorganisms and metabolites in swine. Red indicates a positive correlation, blue indicates a negative correlation, and the intensity of the color represents the strength of the correlation (scale bar: -1 to +1). express p <0.05, express p <0.005. Detailed Implementation

[0084] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0085] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0086] This invention provides a traditional Chinese medicine compound probiotic fermentation agent designed to significantly improve the growth performance of hogs, restore gut health, and reduce breeding losses. Specifically, it relates to a metabolic-directed fermentation technology that precisely adjusts the redox potential (Eh) of the fermentation system to induce high-level expression of anaerobic hydrolases in probiotics, thereby achieving in-situ "penetrating" biotransformation of the traditional Chinese medicine carrier components. Furthermore, this invention also relates to a standardized preparation process that integrates the traditional Chinese medicine compound as the core component of the fermentation nutrient carrier with multiple strains of functional probiotics for integrated synergistic cultivation, as well as a targeted application scheme for this agent in cost-reducing and efficiency-enhancing hog farming.

[0087] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0088] Example 1: Screening of the optimal expanded-culture compound probiotic agent 1. Test materials The strains used in this embodiment are: *Lactobacillus plantarum* (GDMCC No. 1.140), *Lactobacillus rhamnosus* (GDMCC No. 1.1798), *Pediococcus pentosus* (GDMCC No. 1.428), *Lactobacillus paracasei* (GDMCC No. 1.159), and *Saccharomyces cerevisiae* (GDMCC No. 2.110). All of these strains were purchased from the Guangdong Provincial Microbial Culture Collection Center and preserved by the fermentation technology platform of the Haida Research Institute of Guangdong Haida Group Co., Ltd.

[0089] The preparation method of the mycelial powder of each single strain includes the following steps: Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosus, and Lactobacillus paracasei were inoculated into MRS liquid medium and activated by static culture at 37°C for 18 h to obtain primary seed culture. The primary seed culture was then transferred to expansion medium at a 5% inoculation rate and incubated at 37°C for 18 h to obtain secondary seed culture. The secondary seed culture was then transferred to fermentation medium at a 5% inoculation rate and fermented at 37°C for 20 h. The fermentation broth was centrifuged and freeze-dried under vacuum to obtain single-strain bacterial powder with a viable count ≥1.0 × 10⁻⁶. 11 CFU / g.

[0090] Saccharomyces cerevisiae was inoculated into YEPD liquid medium and activated by shaking at 30℃ and 180 r / min for 20 h to obtain primary seed culture. The primary seed culture was then transferred to expansion medium at a 5% inoculation rate and cultured at 30℃ for 20 h to obtain secondary seed culture. This secondary seed culture was then transferred to fermentation medium at a 5% inoculation rate and fermented at 30℃ for 24 h. The fermentation broth was centrifuged and freeze-dried under vacuum to obtain yeast powder with a viable count ≥1.0 × 10⁻⁶. 11 CFU / g.

[0091] The preparation method of compound functional bacterial powder includes the following steps: according to the requirements, put the bacterial powder of each single strain into a Class 100 mixer (multi-functional mixer HY-200KG from Zhejiang Ningbo Hongyun Machinery Equipment Factory), mix for 10-15 minutes, mix evenly, and obtain compound functional bacterial powder.

[0092] The whey powder was purchased from New Century Foods, Inc., USA, low-protein whey powder, product number P-17-183-1227; The glucose was purchased from Xiwang Pharmaceutical Co., Ltd. as edible glucose (monohydrate). The yeast autolysing powder (i.e., yeast autolysing powder Y801) was purchased from Angel Yeast Co., Ltd. The maltodextrin was purchased from Shandong Xiwang Sugar Industry Co., Ltd. L-cysteine ​​hydrochloride was purchased from Hebei Huayang Biotechnology Co., Ltd.

[0093] Experimental Group 1: A microbial agent comprising 8wt% of a compound functional microbial powder (obtained by mixing Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosus, Lactobacillus paracasei, and Saccharomyces cerevisiae (live count ratio) = 3:1.5:1.5:1.5:0.5), 40wt% of whey powder, 21.45wt% of glucose, 20wt% of yeast autolysate, 10wt% of maltodextrin, and 0.55wt% of L-cysteine ​​hydrochloride.

[0094] Experimental Group 2: A microbial agent comprising 8% of compound functional microbial powder (obtained by mixing Lactobacillus plantarum and Lactobacillus rhamnosus with a live count ratio of 1:1), 40 wt% of whey powder, 21.45 wt% of glucose, 20 wt% of yeast autolysate, 10 wt% of maltodextrin and 0.55 wt% of L-cysteine ​​hydrochloride.

[0095] The combination of whey powder, glucose, yeast autolysate, maltodextrin, and L-cysteine ​​hydrochloride is referred to as the fermentation nutrient carrier.

[0096] Experimental Group 3: A microbial agent, which differs from Experimental Group 1 only in the composition of the compound functional microbial powder. The compound functional microbial powder is obtained by mixing Pediococcus pentosaceus and Lactobacillus paracasei (live bacteria ratio 1:1).

[0097] Experimental Group 4: A bacterial agent, compared with Experimental Group 1, except that L-cysteine ​​hydrochloride was removed and replaced with an equal amount of glucose.

[0098] Experimental Group 5: Commercially available conventional probiotic fermented products (mainly composed of Lactobacillus plantarum).

[0099] 2. Preparation method The preparation methods for the bacterial agents in experimental groups 1-4 are as follows: S1. Pretreatment of compound fermentation nutrient carrier: Weigh whey powder, glucose, yeast autolysate powder, maltodextrin and L-cysteine ​​hydrochloride (in experimental group 4, L-cysteine ​​hydrochloride was replaced with glucose in equal amounts) according to the above formula ratio, crush them through an 80-mesh sieve, mix them, and obtain a fermentation nutrient carrier that can co-ferment with probiotics.

[0100] S2. Finished product mixing: Put the corresponding compound functional bacterial powder and the fermentation nutrient carrier prepared in S1 into a V-type mixer (Wuxi Longda Drying Powder Equipment Co., Ltd. V-type mixer V-500), mix for 20 minutes, mix evenly, vacuum seal and package to obtain the bacterial agent.

[0101] Group 5 consisted of commercially available products that were used directly.

[0102] 3. On-site expansion test Each group of bacterial inoculum was mixed with water at a mass-to-volume ratio of 1:100 and placed in a sealed fermentation chamber at 37℃ for 48 hours. Samples were taken at 24 hours and 48 hours of fermentation to test the expansion indicators, including the pH value, viable cell count, lactic acid content, total short-chain fatty acid content, and total free amino acid content of the fermentation broth. The specific experimental procedure is as follows: pH value determination: Take an appropriate amount of fermentation broth sample and measure it directly using a precision pH meter (Dongguan Xima Instrument Co., Ltd., Xima, PH828) calibrated with standard buffer solution.

[0103] Viable cell count determination: The plate colony count method was used. The fermentation broth sample was serially diluted with sterile physiological saline, and the bacterial solution of the appropriate dilution was spread on the corresponding solid culture medium plate. After incubation under suitable conditions, the colony forming units (CFU) were counted, and the viable cell concentration (CFU / mL) in the fermentation broth was calculated.

[0104] Lactic acid content determination: A biosensor analysis method was used. An appropriate amount of fermentation broth sample was taken, and after suitable pretreatment such as dilution or centrifugation, it was measured using a biosensor analyzer (Shandong Youyunpu Optoelectronic Technology Co., Ltd., model SBA-40E). This method is based on an immobilized lactate oxidase electrode. The enzyme specifically catalyzes the production of hydrogen peroxide from lactate, and the concentration of lactate is rapidly and accurately quantified by detecting changes in the electrical signal.

[0105] Total short-chain fatty acid content determination: Gas chromatography was used. After pretreatment such as acidification and extraction, the fermentation broth sample was separated using a gas chromatograph (Agilent 8890) and detected with a flame ionization detector. Standard curves were plotted based on short-chain fatty acid standards such as acetic acid, propionic acid, and butyric acid, and the total content was calculated.

[0106] Determination of total free amino acid content: Formaldehyde titration was used. This method is based on the amphoteric nature of amino acids. Excess neutral formaldehyde is added to the sample solution. The formaldehyde rapidly combines with the α-amino group of the amino acid to form a hydroxymethyl derivative, thereby fixing and shielding the basicity of the amino group, allowing the acidity of the carboxyl group to be fully manifested. Subsequently, the sample is titrated with standard sodium hydroxide solution. By measuring the hydrogen ions released due to the above reaction, which are equivalent to the total amino acid content, the total free amino acid content in the fermentation broth can be calculated.

[0107] 4. Results The results of the fermentation broth expansion index detection for each experimental group at 24 h and 48 h of fermentation are shown in Table 1.

[0108] Table 1 Results of fermentation broth expansion performance testing for each experimental group

[0109] The content of essential amino acids in the fermentation broth was further tested, and the results are shown in Table 2.

[0110] LC-MS / MS Detection Method for Essential Amino Acids in Fermentation Broth: Frozen fermentation broth samples were taken, and acetonitrile-methanol extract was added. After vortexing and centrifugation, the supernatant was injected. An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) platform was used for separation on a C18 column, with acetonitrile-water (containing formic acid) gradient elution. Target amino acids were specifically detected and quantified in multiple reaction monitoring (MRM) mode under electrospray ionization positive ionization. This method, based on broadly targeted metabolomics technology, enables high-throughput and high-precision quantitative analysis of multiple essential amino acids. Chromatographic system: Waters ACQUITY UPLC (ultra-high performance liquid chromatography system). Mass spectrometry system: SCIEX QTRAP® 6500+ (triple quadrupole linear ion trap mass spectrometer).

[0111] Table 2. Results of essential amino acid content in fermentation broth of each experimental group

[0112] 5. Screening Conclusion As shown in Tables 1 and 2, the bacterial agent in experimental group 1 is the optimal formulation of the expanded compound probiotic agent.

[0113] After 48 hours of fermentation, the pH of the bacterial agent in experimental group 1 was the lowest (3.35), and the viable counts of lactic acid bacteria and yeast were significantly higher than those in experimental groups 2-4. The contents of lactic acid, total short-chain fatty acids, and free amino acids were also the highest. Experimental groups 2-3, due to their single strain combination and lack of synergistic effects, showed a significant decrease in expansion performance. Experimental group 5, a commercially available product, lacked the synergistic effect of a compound bacterial strain, resulting in the worst expansion performance. In experimental group 1, the compound bacterial flora synergistically produced enzymes, generating a large amount of free essential amino acids. The contents of core essential amino acids such as leucine, valine, and isoleucine were much higher than those in experimental groups 2-3 and experimental group 5, fully demonstrating that the five compound bacterial flora in experimental group 1 possess highly efficient synergistic enzymatic hydrolysis capabilities and can stably provide enzymatic hydrolysis nutrition, rather than being solely the result of yeast. After removing L-cysteine ​​hydrochloride from the bacterial inoculum in experimental group 4, the pH of the fermentation broth increased, the number of viable lactic acid bacteria and the content of free amino acids decreased significantly, and an efficient anaerobic enzymatic hydrolysis environment could not be formed. This proves that L-cysteine ​​hydrochloride is not a conventional nutrient substrate, but a core essential component for targeted enzymatic hydrolysis and efficient expansion, and is irreplaceable. Therefore, the bacterial inoculum in experimental group 1 was determined to be the optimal probiotic formula for subsequent addition of traditional Chinese medicine compound.

[0114] Example 2: Gradient Screening of Optimal Addition Ratios for Compound Traditional Chinese Medicine Formulas 1. Experimental Design Based on the optimal microbial agent formulation screened in Example 1, the amount of compound functional microbial powder added was kept constant at 8 wt%. The whey powder in the fermentation nutrient carrier was replaced with a compound traditional Chinese medicine formula (by mass ratio, Astragalus membranaceus: Codonopsis pilosula: Magnolia officinalis = 1:1:1) to obtain a compound traditional Chinese medicine fermentation nutrient carrier. Three addition gradients were set to screen for the optimal addition ratio of traditional Chinese medicine: Tier 1: 3 wt% of traditional Chinese medicine compound and 37 wt% of whey powder; Tier 2: 5 wt% of traditional Chinese medicine compound and 35 wt% of whey powder; Tier 3: 7 wt% of traditional Chinese medicine compound and 33 wt% of whey powder; Other carrier components: glucose 21.45wt%, yeast autolysate 20wt%, maltodextrin 10wt%, L-cysteine ​​hydrochloride 0.55wt%.

[0115] The sum of the weight percentages of the compound functional microbial powder and the compound fermentation nutrient carrier components of traditional Chinese medicine is 100%.

[0116] 2. Preparation method According to the preparation method in Example 1, the traditional Chinese medicine compound and the fermentation nutrient carrier were pulverized together, passed through an 80-mesh sieve, and then mixed with the compound functional bacterial powder to prepare a traditional Chinese medicine compound bacterial agent.

[0117] 3. On-site collaborative propagation test The inoculum was mixed with water at a mass-to-volume ratio of 1:100, and fermented in a sealed container at 37 °C for 48 h. The propagation performance was then tested (same as in Example 1).

[0118] 4. Results The results are shown in Table 3.

[0119] Table 3. Comparison of synergistic propagation performance of different herbal compound addition ratios

[0120] 5. Screening Conclusion The optimal addition ratio of the traditional Chinese medicine compound is 5%: Under this gradient, the probiotic propagation performance is optimal, and the propagation solution is uniform and stable. This formula is determined to be the optimal traditional Chinese medicine compound propagation probiotic fermentation agent of this invention, as follows: A compound probiotic fermentation agent based on traditional Chinese medicine comprises 8 wt% of a compound functional bacterial powder (obtained by mixing Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosus, Lactobacillus paracasei, and Saccharomyces cerevisiae in a ratio of 3:1.5:1.5:1.5:0.5), 5 wt% of a compound traditional Chinese medicine formula, 35 wt% of whey powder, 21.45 wt% of glucose, 20 wt% of yeast autolysate, 10 wt% of maltodextrin, and 0.55 wt% of L-cysteine ​​hydrochloride.

[0121] Example 3: Experimental study on the biotransformation effect of compound probiotic fermentation agent on Chinese herbal medicine components. 1. Test materials Test sample: The optimal traditional Chinese medicine compound probiotic fermentation agent screened in Example 2, fermentation broth after on-site expansion for 48 hours (i.e., the agent and water are mixed at a mass-volume ratio of 1:100 and fermented in a closed container at 37 ℃ for 48 hours).

[0122] Control sample: A mixture of traditional Chinese medicine compound nutrient carriers with the same formula as the test sample but without probiotic inoculation (stood at 37℃ for 48 h, without fermentation).

[0123] 2. Test Methods On-site expansion method: The optimal Chinese herbal compound probiotic fermentation agent of Example 2 was mixed with water at a mass-volume ratio of 1:100, and fermented in a sealed container at 37°C for 48 h to prepare the test sample; Take an equal amount of Chinese herbal compound fermentation nutrient carrier, add an equal amount of water, mix well, and let stand at 37℃ for 48 h to prepare a control sample. Metabolomics in the two groups of samples were detected by LC-MS non-targeted metabolomics. The content of small molecule transformation products generated by probiotic fermentation of Astragalus polysaccharide, Codonopsis saponin and magnolol was quantitatively analyzed to verify the biotransformation effect.

[0124] 3. Detection indicators The content of Astragalus polysaccharides converted into small molecule sugars, Codonopsis saponins / Magnolol converted into small molecule organic acids, and easily absorbed free amino acids.

[0125] 4. Test Results The results are shown in Table 4.

[0126] Table 4 Comparison of the content of core products of biotransformation of traditional Chinese medicine components.

[0127] 5. Experimental Conclusions The traditional Chinese medicine compound expanded probiotic fermentation agent obtained in Example 2 can efficiently convert large-molecule, poorly absorbed traditional Chinese medicine components such as astragalus polysaccharides, codonopsis saponins, and magnolol into highly active and easily absorbed substances such as small-molecule sugars, small-molecule organic acids, and free amino acids. The content of core conversion products is increased by more than 5 times. The intestinal absorption rate of small-molecule substances is significantly improved, and the bioavailability of traditional Chinese medicine components is enhanced, solving the industry problem of low bioavailability and slow onset of action when traditional Chinese medicine is added alone.

[0128] Example 4: Application effect test of compound probiotic fermentation agent of traditional Chinese medicine in meat pigs. 1. Test materials Example 2 shows the obtained traditional Chinese medicine compound expanded probiotic fermentation agent: 8wt% compound functional bacterial powder + 92wt% traditional Chinese medicine compound fermentation nutrient carrier (containing 5wt% traditional Chinese herbal medicine compound); Basal ration: Sourced from Haid Group Co., Ltd.'s early-stage piglet compound feed, Haid No. 1. It conforms to the NY / T65–2004 pig feeding standard, with the following formula: corn 55.32wt%, wheat bran 15.12wt%, soybean meal 11.83wt%, fish meal 4.00wt%, whey powder 6.00wt%, soybean oil 3.50wt%, limestone powder 1.20wt%, dicalcium phosphate 1.80wt%, salt 0.35wt%, L-lysine hydrochloride 0.45wt%, DL-methionine 0.18wt%, L-threonine 0.15wt%, L-tryptophan 0.05wt%, and choline chloride 0.05wt%.

[0129] 2. Experimental animals and grouping Forty-five Duroc × Landrace × Large White three-way crossbred meat pigs aged 35±3 days with an initial weight of 9.62±0.23 kg were randomly divided into 5 groups of 9 pigs each, with 3 biological replicates and 3 pigs per replicate. The experimental period was 35 days, with a pre-trial period of 7 days and a formal trial period of 28 days.

[0130] Blank control group (CON): fed with basal diet; Antibiotic group (A): basal diet + 1 g / kg doxycycline hydrochloride; Probiotic group (PRO): basal diet + 5wt% probiotic expansion broth (i.e., the fermentation broth obtained by mixing the bacterial agent and water in test group 1 in Example 1 at a mass-volume ratio of 1:100 and fermenting at 37℃ in a sealed environment for 48 h). Chinese herbal medicine group (CHM): basal diet + 1g / kg Chinese herbal medicine compound; The compound group (PROC) of this invention consists of a basal diet + 5 wt% traditional Chinese medicine compound microbial agent expansion liquid (i.e., the fermentation liquid obtained by mixing the optimal traditional Chinese medicine compound expansion probiotic fermentation agent of Example 2 with water at a mass-volume ratio of 1:100 and fermenting at 37°C in a sealed environment for 48 h). 3. Feeding and Management The experimental environment was set at a temperature of 22-28 ℃, a relative humidity of 60%-80%, a ventilation rate of 15-20 times / h, and a light cycle of 12 h:12 h. The animals had free access to food and water, received routine immunization and deworming, and their enclosures were cleaned daily and disinfected once every two weeks.

[0131] 4. Detection indicators Growth performance: initial body weight, final body weight, average daily gain (ADG), average daily feed intake (ADFI), feed conversion ratio (F / G), diarrhea rate; Average daily weight gain (ADG) = (final weight - initial weight) / number of days in the trial; Average Daily Feed Intake (ADFI) = (Total Feed Input - Remaining Feed) / (Number of Experimental Heads × Number of Experimental Days); Fertilizer ratio (F / G) = Average daily feed intake / Average daily weight gain; Diarrhea rate (%) = Number of animals with diarrhea / (Total number of animals in the experiment × Total number of days) × 100%.

[0132] 5. Test Results The experimental results are shown in Table 5.

[0133] Table 5 Comparison of growth performance and diarrhea rate among groups (n=9)

[0134] Results Analysis: Compared with the blank control group, the PROC (Probiotic Compound) group of this invention showed a significant increase in final body weight (8.91%), daily weight gain (14.52%), decrease in feed conversion ratio (FCR) (8.28%), and decrease in diarrhea rate (75.0%). The PROC group had a significantly lower FCR and diarrhea rate than the antibiotic, probiotic, and traditional Chinese medicine (TCM) groups, indicating that the TCM compound expanded probiotic fermentation agent of this invention achieves synergistic effects in promoting growth, improving efficiency, and controlling diarrhea, with overall effects superior to the probiotic, TCM, and antibiotic groups. Furthermore, while the probiotic and TCM groups showed only slight improvements over the blank control group, the PROC group's increase in daily weight gain, decrease in FCR, and reduction in diarrhea rate significantly exceeded the theoretical summation of the effects of the probiotic and TCM groups. This is not a conventional 1+1=2 additive effect, but rather a non-obvious synergistic effect (1+1>2) produced by the compound microbiota and the TCM carrier.

[0135] 6. Experimental Conclusions The optimal traditional Chinese medicine compound probiotic fermentation agent of Example 2 of this invention can significantly improve the growth performance of hogs, reduce the feed conversion ratio, and decrease the diarrhea rate. Its overall effect is significantly better than the blank control group, antibiotic group, probiotic group, and traditional Chinese medicine group, and can be promoted and applied as a safe and efficient antibiotic-free farming solution.

[0136] Example 5: Effects of the optimal traditional Chinese medicine compound probiotic fermentation agent on the intestinal flora and fecal metabolome of pigs. Based on Example 4, on day 28 of the experiment, fresh rectal stool samples were collected from each group. High-throughput sequencing of the 16S rRNA gene V3-V4 region and LC-MS non-targeted metabolomics were used to analyze the gut microbiota structure, differentially expressed species, metabolomic profiles, and microbiota-metabolite relationships. The results are as follows: Figures 1-6 As shown.

[0137] Results of gut microbiota diversity and community structure analysis are as follows: Figure 1 .

[0138] α-diversity analysis results ( Figure 1 As shown in Figure A), compared with the blank control group (CON), the Shannon and Chao1 indices of fecal microbiota in the compound group of this invention (PROC) were significantly increased. p <0.05), significantly improving gut microbiota α diversity, abundance, and evenness.

[0139] β diversity results ( Figure 1 As shown in Figure B, PCoA principal coordinate analysis revealed that the CON, PRO, and PROC groups formed independent clusters, indicating significant differences in bacterial community structure.

[0140] ASV analysis ( Figure 1 The Venn diagram of group C shows that the CON, PRO, and PROC groups contain 241, 435, and 481 unique ASVs, respectively. The PROC group has the most unique ASVs and the best community richness.

[0141] Results of microbial community composition analysis ( Figure 1 The results from the DF (Digital Transformation Analysis) showed that at the phylum level, the dominant bacteria in each group were Firmicutes, Bacteroidetes, and Spirochetes; the relative abundance of Bacteroidetes and Spirochetes was significantly increased in the PROC group. At the genus level, the abundance of Prevotella_9 and Clostridium_sensu_stricto_6 was significantly upregulated in the PROC group, while the abundance of Clostridium_sensu_stricto_1 was significantly decreased.

[0142] The LEfSe analysis results of the fecal microbiota of hogs are as follows: Figure 2 .

[0143] LEfSe differential species analysis (LDA score>3, p The results (<0.05) showed that the CON group was significantly enriched in two groups: g_ Romboutsia , p_Acidobacteriota The PRO group was significantly enriched in 6 taxa, mainly Campylobacter-related taxa; the PROC group was significantly enriched in 10 taxa, including... Spirochaetota (Spirome) Prevotellaceae Beneficial microorganisms such as those belonging to the Prevotaceae family, which have the function of degrading cellulose and producing short-chain fatty acids.

[0144] The metabolome composition and multivariate statistical analysis results of pig feces are as follows: Figure 3 .

[0145] Metabolite identification: Using non-targeted metabolomics technology, a total of 5090 metabolites were identified in both positive and negative ion modes, mainly including amino acids and their derivatives, lipids, organic acids, nucleotides, benzenes, carbohydrates, etc. Figure 3(AB).

[0146] OPLS-DA analysis: The OPLS-DA score plot showed that the metabolic profiles of the CON, PRO, and PROC groups were completely separated, and there were significant differences in metabolic phenotypes among the groups. The traditional Chinese medicine compound expanded probiotic fermentation agent can significantly reshape the metabolic profile of the pig intestine. Figure 3 (CE).

[0147] The results of screening and cluster analysis of differential metabolites in pig feces are as follows: Figure 4 .

[0148] Statistical results of differential metabolites ( Figure 4 According to the results of the study (in the AC), PRO vs CON: 56 metabolites were significantly upregulated and 17 were significantly downregulated; PROC vs CON: 31 metabolites were significantly upregulated and 35 were significantly downregulated; PROC vs PRO: 22 metabolites were significantly upregulated and 77 were significantly downregulated.

[0149] VIP and cluster analysis results ( Figure 4 The results from the DG analysis showed that the VIP score screened out key differentially expressed metabolites; the hierarchical clustering heatmap showed that the metabolite expression patterns of the CON group were significantly different from those of the PRO and PROC groups, and the metabolic characteristics of the PRO and PROC groups partially overlapped but showed significant differences.

[0150] The results of the KEGG pathway enrichment analysis of differential metabolites in pig feces are as follows: Figure 5 .

[0151] PRO group: Compared with CON group, it was significantly enriched in nucleotide metabolism and purine metabolism pathways; PROC group: Compared with CON group, it was significantly enriched in purine metabolism, lipid metabolism, fat digestion and absorption, glycerol ester metabolism, and animal autophagy pathways; the above pathways are directly related to intestinal epithelial repair, energy metabolism, anti-oxidation and anti-inflammation, and intestinal barrier maintenance.

[0152] The results of Spearman correlation analysis of pig fecal microbiota and metabolites are as follows: Figure 6 .

[0153] Spearman correlation analysis showed that: spirochetes (… Treponema It showed a significant positive correlation with nicotinic chlorophenol and trichloroacetic acid. p <0.05); Prevotella spp. ( Prevotella ) was significantly positively correlated with 4,4'-Diapolycopene, isoleucine, and leucine-isoleucine. p <0.05); the above-mentioned beneficial bacteria showed a significant negative correlation with pro-inflammatory and oxidative damage-related metabolites.

[0154] The above results indicate that the optimal traditional Chinese medicine compound probiotic fermentation agent screened in Example 2 can significantly improve the α-diversity of the intestinal flora of pigs, reshape the flora structure, enrich beneficial microorganisms such as Spirochetes and Prevotella, and regulate key metabolic pathways such as purine metabolism, lipid metabolism, and autophagy in animals, thereby improving the intestinal health of pigs through the "flora-metabolite" synergistic mechanism.

[0155] Example 6: Experiment on the decrease in the activity of bacterial strains during shelf life 1. Test materials Test sample: The optimal traditional Chinese medicine compound probiotic fermentation agent screened in Example 2.

[0156] Instruments: Incubator, high-speed centrifuge, pH meter, bioanalyzer 2. Test Methods The compound probiotic fermentation agent of traditional Chinese medicine was sealed and stored under cool and dry conditions (25±2℃, relative humidity 45%±5%). Random samples were taken at 0 months (initial), 4 months, 8 months and 12 months of storage. After on-site expansion and testing in parallel three times according to the method of Example 2, the average value was recorded.

[0157] 3. Detection indicators Total viable count of finished product, viable count of lactic acid bacteria after expansion culture, viable count of yeast after expansion culture, pH value of expansion culture solution, and lactic acid content.

[0158] 4. Test Results The experimental results are shown in Table 6.

[0159] Table 6. Results of bacterial strain activity and core fermentation performance of traditional Chinese medicine compound probiotic fermentation agents at different storage times.

[0160] 5. Experimental Conclusions The herbal compound expanded probiotic fermentation agent of this invention, when stored under cool and dry conditions for 4 months, showed no significant decrease in core fermentation performance compared to its initial state, including the total viable count, the number of viable lactic acid bacteria / yeast bacteria after expansion, pH value, and lactic acid content; after storage for 12 months, the total viable count of the finished product was still ≥9.0×10⁻⁶. 8 CFU / g, viable lactic acid bacteria count after expansion ≥ 6.0 × 10⁻⁶ 8 The CFU / mL and other core indicators all meet the product quality standards.

[0161] Experimental results confirm that the herbal compound probiotic fermentation agent of this invention has a shelf life of up to 12 months under cool and dry conditions, effectively solving the industry problem of a significant decrease in the number of live bacteria and unstable expansion performance of conventional probiotic products during long-term storage.

[0162] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A compound probiotic fermentation agent for traditional Chinese medicine, comprising compound functional bacterial powder and a compound fermentation nutrient carrier for traditional Chinese medicine; in, The compound functional microbial powder includes Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosus, Lactobacillus paracasei, and Saccharomyces cerevisiae; The compound fermentation nutrient carrier of traditional Chinese medicine includes a compound of traditional Chinese herbal medicines.

2. The traditional Chinese medicine compound expanded probiotic fermentation agent according to claim 1, characterized in that, The herbal compound includes Astragalus membranaceus, Codonopsis pilosula, and Magnolia officinalis. Preferably, the mass ratio of Astragalus membranaceus, Codonopsis pilosula, and Magnolia officinalis is (1-2):(1-2):

1.

3. The traditional Chinese medicine compound probiotic fermentation agent according to claim 1, characterized in that, The compound fermentation nutrient carrier of traditional Chinese medicine also includes cysteine ​​hydrochloride, fast-acting carbon source and slow-release carbon source. Preferably, the fast-acting carbon source includes at least one of molasses, glucose, and brown sugar; Preferably, the slow-release carbon source includes at least one of whey powder, maltodextrin, and yeast autolysate.

4. The traditional Chinese medicine compound probiotic fermentation agent according to any one of claims 1-3, characterized in that, The ratio of viable counts of *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Pediococcus pentosus*, *Lactobacillus paracasei*, and *Saccharomyces cerevisiae* is (2-8):(1-4):(1-4):(1-4):

1.

5. The traditional Chinese medicine compound probiotic fermentation agent according to any one of claims 1-3, characterized in that, The traditional Chinese medicine compound expanded probiotic fermentation agent comprises 5-15 parts of compound functional bacterial powder by weight. And / or, by weight, the traditional Chinese medicine compound probiotic fermentation agent comprises 2-10 parts of traditional Chinese herbal medicine compound.

6. The traditional Chinese medicine compound probiotic fermentation agent according to claim 3, characterized in that, The traditional Chinese medicine compound expanded probiotic fermentation agent comprises 0.3-1 parts of cysteine ​​hydrochloride by weight. And / or, by weight, the traditional Chinese medicine compound expanded probiotic fermentation agent includes 18-30 parts of fast-acting carbon source; And / or, by weight, the traditional Chinese medicine compound expanded probiotic fermentation agent includes 55-80 parts of slow-release carbon source.

7. The traditional Chinese medicine compound probiotic fermentation agent according to any one of claims 1-3, characterized in that, The total viable count of the compound probiotic fermentation agent containing traditional Chinese medicine is ≥1.0×10⁻⁶. 9 CFU / g.

8. A feed comprising a compound probiotic fermentation agent containing traditional Chinese medicine as described in any one of claims 1-7 and a basal diet.

9. The feed according to claim 8, characterized in that, The feed is prepared by the following methods: The compound probiotic fermentation agent of traditional Chinese medicine is mixed with water and fermented to obtain fermentation liquid; The fermentation liquid is mixed with the basal diet to obtain feed; Preferably, the fermentation broth is added to the basal diet at a ratio of 3wt%-10wt%.

10. The application of the traditional Chinese medicine compound probiotic fermentation agent according to any one of claims 1-7 or the feed according to claim 8 in pig farming.

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