Microbial compound microbial inoculant, preparation method and application thereof in preparation of product for relieving physiological disorder of chicken caused by aflatoxin B1

Abstract

The application discloses a microbial compound microbial inoculant, a preparation method and application of the microbial compound microbial inoculant in preparation of a product for relieving physiological function disorder of chickens caused by aflatoxin B1, and belongs to the field of microbial technology and livestock health breeding. Active ingredients of the microbial compound microbial inoculant are composed of plant lactobacillus and lactobacillus acidophilus; the plant lactobacillus has a preservation number of CGMCC No.35862; and the lactobacillus acidophilus has a preservation number of CGMCC No.35861. The microbial compound microbial inoculant shows a significant synergistic protection effect on AFB1-poisoned three yellow chickens through water feeding, can systematically relieve multidimensional physiological function disorder induced by AFB1, and shows excellent improvement effects in growth performance, intestinal health, liver metabolism and immune regulation and the like. The water feeding mode is simple in operation, small in stress, fast in absorption, avoids heat loss in feed processing, is high in utilization rate of live bacteria, and is especially suitable for intensive breeding.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and healthy animal husbandry, specifically relating to a microbial compound agent, its preparation method, and its application in the preparation of products that alleviate physiological dysfunction in chickens caused by aflatoxin B1. Background Technology

[0002] Aflatoxin B1 (AFB1) is a mycotoxin contaminant that seriously threatens the poultry farming industry. When Sanhuang chickens ingest feed contaminated with AFB1, it triggers a series of complex physiological disorders, ranging from growth inhibition and organ damage to immune dysfunction, resulting in significant economic losses. Currently, physical and chemical detoxification methods are limited in application due to drawbacks such as damage to feed nutrients, residual risks, or high costs. Developing green and efficient bio-mitigation strategies has become an urgent need for the industry. Probiotics, especially lactic acid bacteria, are considered a promising alternative due to their high safety profile and potential for intestinal regulation and toxin adsorption.

[0003] In current technologies, probiotic studies targeting AFB1 primarily focus on verifying the efficacy of single strains or simple combinations of different strains. While these studies have confirmed the potential value of some strains, they generally lack in-depth exploration of the interaction mechanisms between different bacterial species, and in particular, fail to effectively address the core challenge of how to systematically and multi-targetly reverse the multidimensional physiological disorders caused by AFB1. Single strains or their simple combinations often have limited effects and are difficult to simultaneously and efficiently improve the growth performance, metabolic health, digestive function, and immune status of affected poultry. Current applications lack dedicated compound formulations capable of systematically repairing the multi-system and multidimensional physiological dysfunctions caused by AFB1 in Sanhuang chickens. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a microbial compound agent, a preparation method, and its application in the preparation of products that alleviate physiological dysfunction in chickens caused by aflatoxin B1.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a microbial compound inoculant, the active ingredients of which are composed of Lactobacillus plantarum and Lactobacillus acidophilus; The *Lactobacillus plantarum* was deposited on September 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35862. The Lactobacillus acidophilus was deposited on September 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35861.

[0006] A second aspect of the present invention provides a method for preparing the microbial compound inoculant described in the first aspect, comprising: Lactobacillus plantarum and Lactobacillus acidophilus were inoculated into the culture medium, cultured, and mixed to obtain a microbial compound agent.

[0007] A third aspect of the present invention provides the application of the microbial compound agent described in the first aspect in the preparation of a product that alleviates the physiological dysfunction of Sanhuang chicken caused by aflatoxin B1.

[0008] A fourth aspect of the present invention provides a method for alleviating aflatoxin B1 poisoning in Sanhuang chickens, comprising adding the microbial compound agent described in the first aspect to the drinking water of Sanhuang chickens for free drinking, for continuous use for 10-15 days.

[0009] The beneficial effects of this invention are as follows: This invention provides a microbial compound agent composed of specific *Lactobacillus plantarum* and *Lactobacillus acidophilus* as active ingredients. When administered via drinking water, it exhibits a significant synergistic protective effect against AFB1-poisoned Sanhuang chickens, systematically alleviating AFB1-induced multidimensional physiological dysfunctions and demonstrating excellent improvements in growth performance, intestinal health, liver metabolism, and immune regulation. The drinking water administration method is simple to operate, causes minimal stress, and promotes rapid absorption, avoiding heat loss during feed processing. It also boasts high utilization of live bacteria, making it particularly suitable for intensive farming.

[0010] Microbial Preservation Information: The plant lactobacillus involved in this invention is plant lactobacillus ( Lactobacillus plantarum SCA31 was deposited on September 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35862.

[0011] The Lactobacillus acidophilus involved in this invention is Lactobacillus acidophilus ( Lactobacillus acidophilus SCA20 was deposited on September 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35861. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Figure 1The purpose of this study is to identify and perform phylogenetic analysis on *Lactobacillus plantarum* SCA31 and *Lactobacillus acidophilus* SCA20. (A) shows the plate culture morphology and microscopic images of *Lactobacillus plantarum* SCA31 and *Lactobacillus acidophilus* SCA20; (B) shows the phylogenetic analysis.

[0014] Figure 2 To evaluate the antibacterial activity of *Lactobacillus plantarum* SCA31 and *Lactobacillus acidophilus* SCA20; (A) shows the antibacterial phenotype of the strains against specific pathogens; (B) shows the diameter of the inhibition zone. Different letters on the column indicate significant differences between groups (P < 0.05).

[0015] Figure 3 The study investigated the effects of a microbial compound inoculant on the growth performance of AFB1-poisoned Sanhuang chickens. (A) represents the weight gain rate; (B) represents the specific growth rate (feedback ratio); and (C) represents the survival rate. Different letters on the columns indicate significant differences between groups (P < 0.05).

[0016] Figure 4 The study investigated the effects of a microbial compound inoculant on the intestinal morphology of AFB1-poisoned Sanhuang chickens. (A) represents an intestinal slice; (B) represents villus height; (C) represents crypt depth; (D) represents the villus-to-crypt ratio; (E) represents villus width; (F) represents the number of villus segments; and (G) represents the number of crypts. Different letters on the columns indicate significant differences between groups (P < 0.05).

[0017] Figure 5 The study investigated the effects of a microbial compound inoculant on the activity of intestinal digestive enzymes in AFB1-poisoned Sanhuang chickens. (A) represents lipase activity; (B) represents trypsin activity; and (C) represents α-amylase activity. Different letters on the column indicate significant differences between groups (P < 0.05).

[0018] Figure 6 This study investigated the effects of a microbial compound inoculant on the antioxidant function and liver damage indicators in the liver of AFB1-poisoned Sanhuang chickens. (A) represents SOD activity; (B) represents CAT activity; (C) represents GSH-Px activity; (D) represents serum MDA content; (E) represents serum AST content; and (F) represents serum ALT content. Different letters on the columns indicate significant differences between groups (P < 0.05).

[0019] Figure 7 This study investigated the effects of a microbial compound inoculant on serum immune markers in AFB1-poisoned Sanhuang chickens. (A) represents immunoglobulin A (IgA) concentration; (B) represents immunoglobulin G (IgG) concentration; (C) represents immunoglobulin M (IgM) concentration; (D) represents tumor necrosis factor-α (TNF-α) level; (E) represents interleukin-6 (IL-6) level; and (F) represents interleukin-4 (IL-4) level. Different letters on the columns indicate significant differences between groups (P < 0.05).

[0020] Figure 8 The effect of a microbial compound inoculant on the abundance of intestinal microbial species in AFB1-poisoned Sanhuang chickens was investigated. (A) is a bar chart of species abundance at the phylum level; (B) is a bar chart of species distribution at the genus level.

[0021] Figure 9 The study investigated the effects of a microbial compound inoculant on the species diversity and abundance of the gut microbiota in AFB1-poisoned Sanhuang chickens. (A) shows the inter-group difference analysis of the Alpha diversity index; (B) shows the species abundance clustering heatmap. Detailed Implementation

[0022] Given the lack of a systematic approach to repairing the physiological dysfunction in Sanhuang chickens caused by AFB1 in existing technologies, this invention proposes a microbial compound agent, its preparation method, and its application in the preparation of products that alleviate the physiological dysfunction in chickens caused by aflatoxin B1. This microbial compound agent can significantly improve the multi-system functional disorders caused by AFB1 through water-based feeding, providing a safe and efficient microbial solution for the biocontrol of aflatoxin in poultry farming practices.

[0023] A first typical embodiment of the present invention provides a microbial compound inoculant, the active ingredients of which are composed of Lactobacillus plantarum and Lactobacillus acidophilus; The *Lactobacillus plantarum* was deposited on September 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35862. The Lactobacillus acidophilus was deposited on September 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35861.

[0024] This invention, through extensive screening, unexpectedly obtained two specific bacterial strains with excellent host adaptability to Sanhuang chicken: *Lactobacillus plantarum* SCA31 and *Lactobacillus acidophilus* SCA20, from traditional kimchi. It was discovered that only when these two strains are combined in a specific ratio of viable bacteria can they produce a synergistic effect against AFB1 poisoning in Sanhuang chickens, achieving systemic repair of multi-system physiological dysfunction. Furthermore, this microbial compound agent can effectively reshape the intestinal flora structure of AFB1-poisoned Sanhuang chickens, increasing the abundance of beneficial bacteria and inhibiting pathogenic bacteria, systematically alleviating multi-system physiological dysfunction from a microecological perspective. This invention provides a dedicated microbial control product with strong host adaptability, safety, and high efficiency for the healthy breeding of Sanhuang chickens under the background of aflatoxin contamination, and has good prospects for industrial application.

[0025] Understandably, the beneficial bacteria include *Faecalibacterium* (…). Faecalibacterium ) and Megamonas spp. MegamonasBeneficial bacteria that produce butyric acid and other short-chain fatty acids; the pathogenic bacteria include, but are not limited to, Alisteria spp. Alistipes Bacteroides ( Bacteroides ) and Desulfurization Vibrio spp. Desulfovibri Bacteria associated with inflammation or metabolic disorders, such as Escherichia coli, Staphylococcus aureus, Salmonella enteritidis, and Riemerella anatipestifer.

[0026] In this invention, the live count ratio of *Lactobacillus plantarum* and *Lactobacillus acidophilus* is (1-15):1; the live bacteria concentration in the microbial compound agent is ≥1×10⁻⁶. 7 CFU / mL. Compared with single strains or other ratios, the (1-15):1 ratio showed superior effects in improving growth performance, gut health, antioxidant capacity and immune regulation, demonstrating the synergistic effect between specific strains and between strains and the host.

[0027] In this invention, the ratio of viable Lactobacillus plantarum to Lactobacillus acidophilus is (3-15):1.

[0028] In this invention, the ratio of viable Lactobacillus plantarum to Lactobacillus acidophilus is 3:1, 5:1, or 15:1.

[0029] In this invention, the live count ratio of *Lactobacillus plantarum* and *Lactobacillus acidophilus* is 5:1. Compared with single strains or other ratios, the 5:1 ratio shows the best effects in improving growth performance, gut health, antioxidant capacity, and immune regulation, demonstrating the synergistic effect between specific strains and between the strain and the host.

[0030] In this invention, the microbial compound agent also includes fermentation broth.

[0031] Understandably, the fermentation broth is essentially a liquid product of *Lactobacillus plantarum* and *Lactobacillus acidophilus* fermented in a specific culture medium, mainly comprising... (1) Active bacteria, namely Lactobacillus plantarum and Lactobacillus acidophilus, can directly adsorb AFB1, regulate the intestinal flora, and competitively exclude pathogens.

[0032] (2) Organic acids, such as lactic acid, butyric acid, acetic acid, etc., can lower the pH of the intestine, inhibit fungal growth, and improve the intestinal barrier function.

[0033] (3) Antibacterial substances, such as bacteriocins (i.e. lactobacilli), hydrogen peroxide, etc., can inhibit harmful bacteria such as Escherichia coli and reduce intestinal inflammation caused by AFB1.

[0034] (4) Enzymes, including proteases, amylases, lipases, etc., can aid digestion and improve the nutritional absorption disorder of chickens poisoned by AFB1.

[0035] (5) Other metabolites, such as vitamins (B group), extracellular polysaccharides, small molecule peptides, etc., can provide nutrition, enhance immune regulation, and assist in antioxidation.

[0036] (6) Culture medium residues, namely carbon sources (e.g., glucose) and nitrogen sources (e.g., peptone), protect the cells and provide energy for the cells, but are not used as active ingredients in microbial compound agents.

[0037] A second typical embodiment of the present invention provides a method for preparing the above-mentioned microbial compound inoculant, comprising: Lactobacillus plantarum and Lactobacillus acidophilus were inoculated into the culture medium, cultured, and mixed to obtain a microbial compound agent.

[0038] Since the two strains have significantly different growth characteristics, adopting a separate culture strategy can effectively avoid growth competition, ensure the stability of the activity and ratio of the two strains in the microbial compound agent, and thus ensure the consistency of their synergistic effect in animals.

[0039] In this invention, the culture medium is a liquid culture medium suitable for the growth of *Lactobacillus plantarum* and *Lactobacillus acidophilus*. For example, the culture medium can be MRS liquid medium. MRS liquid medium is a commonly used culture medium in the industry, which can meet the growth requirements of the bacteria during the culture process. This culture medium is only a preferred example and is not the only limitation; it can also be replaced with other conventional liquid culture media suitable for the growth of lactic acid bacteria. The synergistic effect between strains is determined by their own biological characteristics, and changing the culture medium will not change their inherent synergistic effect.

[0040] In this invention, the culture conditions are 35-39℃ and 150-200 rpm for 20-30 h.

[0041] Preferably, the culture conditions are 37°C and 180 rpm for 24 h.

[0042] The third typical embodiment of the present invention provides the application of the above-mentioned microbial compound agent in the preparation of a product that alleviates the physiological dysfunction of Sanhuang chicken caused by aflatoxin B1.

[0043] In this invention, the product is a drinking water agent. It is administered via drinking water, making it easy to operate, avoiding heat loss during feed processing, and offering high utilization of live bacteria with minimal stress on animals, making it particularly suitable for large-scale farms.

[0044] In this invention, the chicken includes the Sanhuang chicken.

[0045] In this invention, the physiological dysfunction includes any one or more of the following: growth and development dysfunction, digestive and absorptive dysfunction, metabolic and antioxidant dysfunction, immune and inflammatory dysfunction, and gut microbiota disorder.

[0046] In this invention, the growth and development disorder includes any one or more of the following: slow weight gain, decreased weight gain rate, and decreased survival rate. The digestive and absorptive dysfunction includes any one or more of the following: decreased intestinal digestive enzyme activity, decreased jejunal villus height, increased crypt depth, and decreased villus-crypt ratio. The metabolic and antioxidant dysfunction includes any one or more of the following: elevated liver damage indicators, decreased antioxidant enzyme activity, and elevated lipid peroxidation product content. The immune and inflammatory dysfunction includes any one or more of the following: elevated levels of pro-inflammatory factors, decreased levels of anti-inflammatory factors, and decreased levels of immunoglobulins. The gut microbiota dysbiosis includes any one or more of the following: decreased microbial diversity, decreased Firmicutes / Bacteroidetes ratio, reduced abundance of beneficial bacteria, and increased abundance of pathogenic bacteria.

[0047] In this invention, the liver damage indicators include any one or both of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The antioxidant enzymes include any one or more of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). The lipid peroxidation products include malondialdehyde (MDA). The intestinal digestive enzymes include any one or more of lipase, trypsin, and α-amylase; The serum pro-inflammatory factors include any one or two of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6); The anti-inflammatory factors include interleukin-4 (IL-4). The immunoglobulins include any one or more of IgA, IgG, and IgM.

[0048] A fourth typical embodiment of the present invention provides a method for alleviating aflatoxin B1 poisoning in Sanhuang chickens, comprising adding the aforementioned microbial compound agent to the drinking water of the Sanhuang chickens for free consumption, for continuous use for 10-15 days. This method of feeding via drinking water can significantly improve multi-system dysfunction caused by AFB1, providing a safe and efficient microbial solution for the biocontrol of aflatoxin in poultry farming practices.

[0049] In this invention, the dosage of the microbial compound agent is determined by the number of viable bacteria. When the microbial compound agent is added to drinking water, the total number of viable bacteria per liter of drinking water must be maintained at 1 × 10⁻⁶. 6 CFU to 1×10 10 CFU; the preferred range is 1×10⁻⁶ CFU / liter of drinking water. 7 CFU to 1×10 9 CFU; further preferred to be 5 × 10⁻⁶ CFU / liter of drinking water. 7 CFU to 1×10 8 CFU.

[0050] This invention discloses for the first time that a specific ratio of *Lactobacillus acidophilus* SCA20 and *Lactobacillus plantarum* SCA31, administered via drinking water, provides multi-system synergistic protection against AFB1 poisoning in Sanhuang chickens. Compared to single strains or other ratios, this specific compound ratio exhibits optimal effects in improving growth performance, intestinal health, antioxidant capacity, and immune regulation, demonstrating the synergistic effect between specific strains and between the strain and the host.

[0051] The microbial compound agent of the present invention can systematically alleviate the physiological dysfunction induced by AFB1 from five dimensions: growth performance, intestinal structure and function, liver antioxidant capacity, immune regulation and intestinal microecology. Among them, the improvement effect is most comprehensive and significant when Lactobacillus acidophilus SCA20 and Lactobacillus plantarum SCA31 are compounded at a live bacteria ratio of 1:5.

[0052] This invention uses a drinking water feeding method, which is simple to operate, avoids heat loss in feed processing, has a high utilization rate of live bacteria, and causes little stress to animals, making it especially suitable for large-scale farms.

[0053] This invention reveals for the first time the mechanism of action of the compound probiotics at the gut microbiota level. Experiments have confirmed that the compound probiotics can significantly improve the diversity of gut microbiota in AFB1-poisoned Sanhuang chickens, reshape the microbiota structure, increase the F / B ratio, promote the enrichment of butyrate-producing beneficial bacteria such as Lactobacillus and Faecalibacterium, while inhibiting pathogenic bacteria such as Desulfovibrio. This microecological regulatory effect is a crucial mechanism for the systemic alleviation of growth, digestion, immunity, and antioxidant dysfunction.

[0054] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0055] MRS agar and liquid medium, LB agar and liquid medium, and NB agar and liquid medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0056] Aflatoxin B1 was purchased from Shanghai Lianshuo Biotechnology Co., Ltd.

[0057] The bacterial genomic DNA extraction kit was purchased from Beijing Tiangen Biotech Co., Ltd.

[0058] The intestinal digestive enzyme activity assay kits (lipase, trypsin and α-amylase assay kits), liver antioxidant index assay kits (superoxide dismutase SOD, catalase CAT, glutathione peroxidase GSH-Px, malondialdehyde (MDA), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assay kits), and serum immune index assay kits (immunoglobulin A, immunoglobulin G, immunoglobulin M, tumor necrosis factor-α, interleukin-6 and interleukin-4) were all purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0059] All other conventional chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0060] Example 1: Isolation, identification, and antibacterial activity evaluation of strains SCA20 and SCA31 1.1 Isolation and Molecular Biological Identification of Strains Using traditional kimchi as the inoculum source, 1 mL of sample was inoculated into 100 mL of MRS liquid medium and cultured at 37℃ and 180 rpm for 24 h for enrichment. The enriched bacterial solution was then serially diluted 10-fold with 0.9% (w / v) sterile physiological saline, and 10 samples were selected. -6 10 -7 10 -8 Three dilutions, each 100 µL, were plated onto MRS agar plates and incubated at 37°C for 24 h. Morphologically typical single colonies were picked and purified on MRS plates using a streak plating method until pure cultures were obtained. The purified strains were inoculated into MRS liquid medium and cultured. Gram staining was performed on the bacterial culture, and cell morphology was observed under a light microscope. Genomic DNA was then extracted from the pure cultures and amplified by PCR using universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO:1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:2) for bacterial 16S rDNA. The PCR products were purified and sequenced. The obtained sequences were compared for homology using BLAST in the NCBI database, and a phylogenetic tree was constructed using neighbor-joining with MEGA 11 software to complete molecular biological identification.

[0061] like Figure 1 As shown, two strains, *Lactobacillus acidophilus* SCA20 and *Lactobacillus plantarum* SCA31, were obtained through screening. Figure 1As shown in Figure A, *Lactobacillus acidophilus* SCA20 forms white, rough-surfaced, frosted, translucent colonies with irregular edges on MRS plates; microscopic examination reveals them to be long rod-shaped. Figure 1 As shown in Figure B, its 16S rDNA sequence is similar to that of Lactobacillus acidophilus. Lactobacillus acidophilus The 1969 strain showed 99.72% homology, and phylogenetic analysis confirmed it to be... Lactobacillus acidophilus This strain was deposited on September 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35861. Figure 1 As shown in Figure A, *Lactobacillus plantarum* SCA31 forms white, smooth, moist, round colonies on MRS plates; microscopic examination reveals it to be a Gram-positive short rod-shaped bacterium. Figure 1 As shown in Figure B, its 16S rDNA sequence is similar to that of *Lactobacillus plantarum*. Lactobacillus plantarum The strain showed 99.93% homology, and phylogenetic analysis confirmed it to be... Lactobacillus plantarum This strain was deposited at the same collection center on September 10, 2025, with accession number CGMCC No. 35862.

[0062] 1.2 Fermentation culture of the strain The *Lactobacillus acidophilus* SCA20 and *Lactobacillus plantarum* SCA31 identified and preserved above were inoculated into MRS liquid medium for activation and expansion. Specifically, the strains were inoculated into fresh MRS liquid medium at a 2% (v / v) inoculum and cultured at 37°C and 180 rpm for 24 h to obtain a high-concentration bacterial fermentation broth. This fermentation broth can be used for the subsequent preparation of compound bacterial agents.

[0063] 1.3 Evaluation of antibacterial ability Four common avian pathogens were selected, including Escherichia coli (E. coli). Escherichia coli ATCC 25922, Staphylococcus aureus ( Staphylococcus aureus ATCC 12600, and Salmonella enteritidis preserved in the laboratory ( Salmonella Enteritidis ) and Riemerella anatipestifer ( Riemerella anatipestifer Each strain was cultured to the logarithmic growth phase, and the OD of the bacterial culture was adjusted. 595 The value is 0.22 (approximately 8.4 × 10⁻⁶). 8CFU / mL). Evaluation was performed using the agar well diffusion method: Four Oxford cups were evenly placed on an LB agar plate containing 1% agar. The above bacterial suspension was added to LB medium containing 1% agar at a 0.6% (v / v) inoculation rate at 55℃, mixed thoroughly, poured, and removed from the Oxford cups after solidification. 100 μL of the test bacterial suspension (single suspension of *Lactobacillus plantarum* SCA31, *Lactobacillus acidophilus* SCA20, or a 1:1 composite suspension thereof, all at a concentration of 1×10⁻⁶) was added to each well. 7 The concentration of CFU / mL was measured, with an equal volume of uninoculated MRS medium supernatant used as a negative control. Each treatment was performed in triplicate. Plates were incubated at 37°C for 24 hours, and the diameter of the inhibition zone was measured.

[0064] The results are as follows Figure 2 As shown, *Lactobacillus plantarum* SCA31 and *Lactobacillus acidophilus* SCA20 both exhibited varying degrees of inhibitory effects against the selected pathogens. The inhibition zone diameters of the compound bacterial suspension (SCA20:SCA31 = 1:1) against *Rimerella anatipestifer*, *Escherichia coli*, *Salmonella enteritidis*, and *Staphylococcus aureus* were 2.81 cm, 1.80 cm, 2.13 cm, and 1.65 cm, respectively, and its inhibitory effect was generally superior to that of the single-strain treatment groups. The results indicate that *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and their complex have in vitro inhibitory activity against common avian pathogens, and their combined use shows a synergistic effect.

[0065] Example 2: Experimental Design of a Three-Yellow Chicken Model Using Compound Probiotics to Alleviate AFB1 Poisoning 2.1 Laboratory Animals and Materials Seven-day-old healthy Sanhuang chickens were purchased from Greenland Agricultural Professional Cooperative (Suqian, China). Basic feed was provided by Guangyuan Feed Co., Ltd. (Jinan, China). Aflatoxin B1 (AFB1) was used as a standard in the formulation of the contaminated feed. The drinking water agent was a compound of *Lactobacillus plantarum* SCA31 and *Lactobacillus acidophilus* SCA20 fermentation broth prepared in Example 1. Before use, the concentration of both bacterial suspensions was adjusted to 1×10⁻⁶ with sterile physiological saline. 7 CFU / mL, and compounded according to the volume ratios shown in Table 1 to form intervention formulations with different ratios.

[0066] 2.2 Experimental Design Sixty Sanhuang chickens, after acclimatization, were randomly divided into four groups. Specific groupings and treatment protocols are shown in Table 1. The model group was fed a basal diet containing 50 μg / kg AFB1 for three consecutive days to establish a poisoning model. Afterward, they were fed the basal diet with free access to clean water. The low, medium, and high-dose treatment groups were established using the same method as the model group, and after modeling, intervention was achieved by adding corresponding proportions of compound probiotics to their drinking water. The experiment was conducted in a standardized greenhouse at the Shandong Academy of Sciences Institute of Biology, with controlled environmental conditions throughout. Regular cleaning and disinfection were performed, and all chickens had free access to food and water. The experiment lasted for 30 days. Chickens were fasted for 12 hours before the end of the experiment, followed by sample collection.

[0067] Table 1. Animal Experiment Grouping and Treatment Protocol

[0068] Example 3: Effects of compound probiotics on the growth performance of AFB1-poisoned Sanhuang chickens This embodiment evaluates the effect of compound probiotics on improving the growth performance of AFB1-poisoned Sanhuang chickens. The experimental animals were grouped and treated as in Example 2. At the beginning and end of the experiment, the weight of each chicken was measured, and the number of surviving chickens in each group was recorded. The weight gain rate and survival rate of the Sanhuang chickens were calculated using the following formulas.

[0069] Weight gain rate (%) = (Final weight – Initial weight) / Initial weight × 100% Feed conversion ratio = (final body weight – initial body weight) / food intake Survival rate (%) = (Number of surviving individuals at the end of the experiment / Total number of individuals at the beginning of the experiment) × 100% The results are as follows Figure 3 As shown, compared with the model group, the weight gain rate of the Sanhuang chickens in each probiotic treatment group was significantly increased. P <0.05, with the most significant improvement observed in the medium-dose group (Mid). Feed conversion ratio analysis showed that the feed utilization efficiency of each treatment group was significantly better than that of the model group ( P <0.05%, and the feed conversion ratio was lowest in the medium-dose group, indicating the best feed conversion effect. Regarding survival rate, all treatment groups were significantly higher than the model group ( P <0.05), with the medium-dose group showing the highest survival rate. These results indicate that the compound probiotic can effectively alleviate AFB1-induced growth inhibition in Sanhuang chickens, improve feed utilization efficiency and survival rate, with the medium-dose group (a mixture of Lactobacillus acidophilus SCA20 and Lactobacillus plantarum SCA31 at a live bacteria ratio of 1:5) showing the best effect on improving overall growth performance.

[0070] Example 4: Effects of compound probiotics on intestinal structure and digestive function of AFB1-poisoned Sanhuang chickens 4.1 Intestinal morphology analysis At the end of the experiment, five chickens were randomly selected from each group, and the intestines were dissected and separated. Approximately 3 cm of the middle section was taken, gently rinsed with pre-cooled physiological saline, and immediately fixed in 4% paraformaldehyde solution for 24 hours. After complete fixation, the tissue was trimmed, dehydrated, embedded in paraffin, sectioned, and stained with H&E according to standardized pathological examination procedures. After the sections passed microscopic examination, the entire slide was scanned using a PANNORAMIC panoramic slide scanner to obtain digital pathological images. Clear areas were observed and selected using CaseViewer 2.4 software, and images were taken at 10x and 50x magnification to ensure consistent background lighting. Subsequently, Image-ProPlus 6.0 software was used to randomly measure the height (VH), crypt depth (CD), and villus width of each intact intestinal slide in millimeters, calculate the VH / CD value, and count the number of villi and crypts.

[0071] The results are as follows Figure 4 As shown in the figure, observation of jejunal tissue sections revealed that, compared with the AFB1 model group, the morphology of intestinal villi in each probiotic treatment group was significantly improved. The model group showed sparse villi, reduced height, and disordered arrangement; while the treatment groups showed significantly restored villi structure, exhibiting increased height, denser arrangement, and intact morphology. The improvement was most significant in the medium-dose group, followed by the high-dose group, with the low-dose group also showing significant improvement. Further measurements showed that the medium-dose group (Mid) performed best in key morphological indicators such as villi height, villi-to-cryptotropy ratio, and villi number, and was significantly higher than the model group (…). P <0.05). The high-dose group (High) showed significantly better results than the model group in all indicators, but its overall effect was lower than the medium-dose group. While the low-dose group (Low) showed improvement, its villus height, villus-to-crypto ratio, and villus number were all lower than those in the medium- and high-dose groups. These results indicate that compound probiotics can effectively alleviate AFB1-induced intestinal morphological damage in a dose-dependent manner, and the medium-dose intervention had the most significant effect on intestinal structure repair.

[0072] 4.2 Determination of intestinal digestive enzyme activity Take another segment of jejunal contents or mucosal tissue homogenate and use a commercially available kit to determine the activities of lipase, trypsin, and α-amylase, respectively. Strictly follow the kit instructions for specific procedures. Enzyme activity is expressed as the amount of substrate catalyzed per gram of tissue protein per minute.

[0073] The results are as follows Figure 5 As shown, compared with the model group, the activities of lipase, trypsin, and α-amylase in each probiotic treatment group were significantly increased. P <0.05. Among them, the medium-dose group (Mid) performed best in all indicators, significantly higher than the model group and other dose groups ( P<0.05). The high-dose group was significantly better than the model group, but less effective than the medium-dose group; although the low-dose group showed improvement, its enzyme activity was still lower than that of the medium and high-dose groups, showing a dose-response trend of "medium > high > low". The above results indicate that the compound probiotic drinking water can effectively improve the intestinal digestive function of AFB1-poisoned Sanhuang chickens in a dose-dependent manner. Combined with the intestinal morphology results, it can be seen that the compound probiotic can not only significantly repair the intestinal morphological and structural damage caused by AFB1, but also effectively restore its digestive enzyme activity, thereby synergistically improving intestinal health at both structural and functional levels. Among them, the combination of Lactobacillus acidophilus SCA20 and Lactobacillus plantarum SCA31 at a live bacteria ratio of 1:5 showed the best effect.

[0074] Example 5: Effects of compound probiotics on antioxidant function and liver damage markers in the liver of AFB1-poisoned Sanhuang chickens At the end of the experiment, liver tissue and blood samples were collected from Sanhuang chickens for relevant index determination. Approximately 1.0 g of liver tissue was accurately weighed and added to 9 mL of pre-cooled 0.9% physiological saline. The mixture was mechanically homogenized in an ice-water bath to prepare a 10% (w / v) tissue homogenate. After centrifugation at 2500 r / min for 10 min, the supernatant was collected and appropriately diluted with physiological saline according to the linear range of each index. Following strict adherence to the kit instructions, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) content in the supernatant were measured. Simultaneously, blood samples were collected, and serum was separated by centrifugation after standing. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were measured according to the corresponding kit instructions.

[0075] The results are as follows Figure 6 As shown, the AFB1 model group of Sanhuang chickens exhibited significant hepatic oxidative stress and tissue damage. The activities of hepatic antioxidant enzymes (SOD, CAT, GSH-Px) were all significantly reduced. P <0.05%, the content of lipid peroxidation product MDA was significantly increased ( P <0.05, while the serum liver injury markers ALT and AST activities were significantly increased ( P<0.05). All probiotic treatment groups effectively improved the above-mentioned liver oxidative stress and liver injury indicators, with the medium-dose group showing the most comprehensive recovery effect. Compared with the model group, the medium-dose group significantly increased the activity of liver antioxidant enzymes (SOD, CAT, GSH-Px) and significantly reduced the content of lipid peroxidation product MDA. At the same time, the serum liver injury markers ALT and AST activities in this group also decreased significantly, and the reduction was the most significant among all treatment groups. The results indicate that this compound probiotic can effectively alleviate AFB1-induced liver oxidative damage and reduce hepatocyte damage, with the medium-dose (SCA20:SCA31 = 1:5) showing the most comprehensive and significant improvement effect.

[0076] Example 6: Effects of compound probiotics on serum immune indicators in AFB1-poisoned Sanhuang chickens At the end of the experiment, after a 12-hour fast, five chickens from each group were randomly selected for wing vein blood collection. Blood samples were collected in sterile centrifuge tubes and centrifuged at 4°C and 2500 rpm for 20 min to obtain serum, which was then stored at -80°C for later use. Before testing, the serum samples were appropriately diluted with 0.9% physiological saline according to the optimal linear range for each indicator. Subsequently, the levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM), as well as the contents of pro-inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and anti-inflammatory factor interleukin-4 (IL-4) in the serum were measured strictly according to the instructions of the corresponding commercially available kits.

[0077] The results are as follows Figure 7 As shown, the AFB1 model group of Sanhuang chickens exhibited significant immune dysfunction. Serum levels of pro-inflammatory factors TNF-α and IL-6 were significantly elevated (…). P <0.05), while the levels of anti-inflammatory factor IL-4 and immunoglobulins IgA, IgG, and IgM were significantly reduced ( P <0.05%. All probiotic treatment groups effectively regulated the aforementioned immune imbalance: compared with the model group, the treatment groups showed significantly lower serum pro-inflammatory factor levels, significantly higher anti-inflammatory factor IL-4 levels, and significantly restored levels of immunoglobulins IgA, IgG, and IgM. P <0.05). Among them, the medium-dose treatment group showed the most comprehensive and significant improvement in all indicators, with its immunoglobulin and anti-inflammatory factor levels closest to the healthy range and pro-inflammatory factors reduced to the lowest level; the high-dose and low-dose groups also showed significant regulatory effects, but the effects were inferior to those of the medium-dose group.

[0078] Example 7: Effects of compound probiotics on the intestinal microbiota of AFB1-poisoned Sanhuang chickens 7.1 Species abundance analysis at the phylum and genus levels To investigate the regulatory effect of compound probiotics on the intestinal microecology of AFB1-poisoned Sanhuang chickens, 16S rRNA gene sequencing analysis was performed on the cecal contents. At the phylum level ( Figure 8 In Group A, the model group (Mod) was dominated by Bacteroides (67.75%), with a relatively low abundance of Firmicutes (24.81%), and an F / B ratio of only 0.37. After intervention with compound probiotics, the intestinal flora structure of each treatment group changed significantly. Compared with the model group, the abundance of Firmicutes in the medium-dose treatment group (Mid) significantly increased to 39.91%, the abundance of Bacteroides decreased to 48.99%, and the F / B ratio increased to 0.81. The trend of this ratio change is highly consistent with the trend of the weight gain rate of Sanhuang chickens in each treatment group in Example 3, further confirming the close relationship between the optimization of intestinal flora structure and the improvement of host growth performance. It is worth noting that the F / B ratios of the low-dose and high-dose groups were 0.54 and 0.72, respectively, showing a dose-dependent recovery characteristic.

[0079] At the genus level ( Figure 8 (B) The relative abundance of conditionally pathogenic bacteria, such as *Desulfovibrio*, was significantly increased in the model group. After intervention with the compound probiotics, *Ligilactobacillus* was significantly enriched in the medium-dose treatment group, indicating that the exogenous probiotics successfully colonized and played a regulatory role in the gut microbiota. Simultaneously, the relative abundance of *Desulfovibrio* was significantly reduced in the treatment groups, especially in the medium-dose group. Furthermore, compared with the model group, the relative abundance of other potentially beneficial bacteria genera (such as *Faecalibacterium*) was also increased in the medium-dose group. These results indicate that the compound probiotics can effectively antagonize AFB1-induced gut microbiota dysbiosis, promote the proliferation of beneficial bacteria, and inhibit the growth of pathogenic bacteria.

[0080] 7.2 Alpha diversity and genus-level cluster analysis Alpha diversity analysis results ( Figure 9 A) showed that, compared with the model group, the Chao1 index in the medium-dose treatment group was significantly increased ( P The value <0.05 indicates that the intervention of compound probiotics can effectively improve the species richness of the gut microbiota and help restore the decline in microbiota diversity caused by AFB1.

[0081] Genus-level species abundance clustering heatmap ( Figure 9 B) further revealed differences in gut microbiota composition among different treatment groups. Cluster analysis showed that the gut microbiota structure of the model group and each treatment group was clearly distinguishable. Compared with the model group, the *Alisteria* genus (…) was significantly higher in each treatment group. Alistipes Bacteroides ( Bacteroides) and Desulfurization Vibrio spp. Desulfovibri The relative abundance of bacteria genera associated with inflammation or metabolic disorders, such as *O*, was significantly reduced; while the abundance of *Femobacterium* (*O*) was significantly lower. Faecalibacterium ) and Megamonas spp. Megamonas Beneficial bacteria genera that produce short-chain fatty acids such as butyric acid were significantly enriched in the treatment group. Among them, *Faecalibacterium* showed the most significant dominance in the medium-dose group, while *Megalomonas* showed a higher enrichment level in the high-dose group, demonstrating a dose-dependent characteristic of gut microbiota regulation.

[0082] Based on the results in 7.1 and 7.2, the compound probiotic of this invention can systematically alleviate AFB1-induced physiological dysfunction at the microecological level by reshaping the intestinal flora structure of AFB1-poisoned Sanhuang chickens, improving flora diversity, restoring the F / B ratio, promoting the colonization of probiotics (such as Lactobacillus and Faecalibacterium), and inhibiting the overgrowth of pathogenic bacteria (such as Desulfovibrio and Bacteroides). This finding provides an important mechanistic explanation for the multi-system synergistic protective effect of the compound probiotic of this invention from the perspective of intestinal flora.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microbial compound inoculant, characterized in that, Its active ingredients consist of Lactobacillus plantarum and Lactobacillus acidophilus; The *Lactobacillus plantarum* was deposited at the China General Microbiological Culture Collection Center on September 10, 2025, with accession number CGMCC No. 35862. The Lactobacillus acidophilus was deposited on September 10, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35861.

2. The microbial compound inoculant as described in claim 1, characterized in that, The viable count ratio of *Lactobacillus plantarum* and *Lactobacillus acidophilus* is (1-15):1; the viable count concentration in the microbial compound agent is ≥1×10⁻⁶. 7 CFU / mL; Preferably, the live count ratio of *Lactobacillus plantarum* to *Lactobacillus acidophilus* is (3-15):

1. Preferably, the ratio of viable Lactobacillus plantarum to Lactobacillus acidophilus is 3:1, 5:1 or 15:1; Preferably, the ratio of viable Lactobacillus plantarum to Lactobacillus acidophilus is 5:

1.

3. The microbial compound inoculant as described in claim 1, characterized in that, The microbial compound agent also includes fermentation broth.

4. A method for preparing a microbial compound inoculant according to any one of claims 1-3, characterized in that, include: Lactobacillus plantarum and Lactobacillus acidophilus were inoculated into the culture medium, cultured, and mixed to obtain a microbial compound agent.

5. The preparation method according to claim 4, characterized in that, The culture medium is a liquid culture medium suitable for the growth of *Lactobacillus plantarum* and *Lactobacillus acidophilus*. Preferably, the culture medium is MRS liquid culture medium; Preferably, the culture conditions are 35-39℃ and 150-200 rpm for 20-30 h.

6. The use of the microbial compound agent according to any one of claims 1-3 in the preparation of a product that alleviates the physiological dysfunction in chickens caused by aflatoxin B1.

7. The application as described in claim 6, characterized in that, The product is a drinking water preparation; Preferably, the chicken includes the Sanhuang chicken.

8. The application as described in claim 6, characterized in that, The physiological dysfunctions include any one or more of the following: growth and development dysfunction, digestive and absorptive dysfunction, metabolic and antioxidant dysfunction, immune and inflammatory dysfunction, and gut microbiota dysbiosis.

9. The application as described in claim 8, characterized in that, The aforementioned growth and developmental dysfunction includes any one or more of the following: slow weight gain, decreased weight gain rate, and decreased survival rate; The digestive and absorptive dysfunction includes any one or more of the following: decreased intestinal digestive enzyme activity, decreased jejunal villus height, increased crypt depth, and decreased villus-crypt ratio. The metabolic and antioxidant dysfunction includes any one or more of the following: elevated liver damage indicators, decreased antioxidant enzyme activity, and elevated lipid peroxidation product content. The immune and inflammatory dysfunction includes any one or more of the following: elevated levels of pro-inflammatory factors, decreased levels of anti-inflammatory factors, and decreased levels of immunoglobulins. The gut microbiota dysbiosis includes any one or more of the following: decreased microbial diversity, decreased Firmicutes / Bacteroidetes ratio, reduced abundance of beneficial bacteria, and increased abundance of pathogenic bacteria.

10. The application as described in claim 9, characterized in that, The liver injury indicators include any one or both of ALT and AST. The antioxidant enzymes include any one or more of SOD, CAT, and GSH-Px; The lipid peroxidation products include MDA; The intestinal digestive enzymes include any one or more of lipase, trypsin, and α-amylase; The serum pro-inflammatory factors include any one or both of TNF-α and IL-6; The anti-inflammatory factor includes IL-4; The immunoglobulins include any one or more of IgA, IgG, and IgM.

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