A kind of compost compound leavening agent and its preparation method

Abstract

This invention discloses a bacterial residue compound fermentation agent and its preparation method, belonging to the field of biotechnology. The fermentation agent is composed of *Bacillus subtilis*, *Lactobacillus plantarum*, and *Candida utilis* in a specific live bacteria ratio, and is synergistically formulated with cellulase, xylanase, laccase, and peroxidase. The total bacterial dry matter accounts for 35%–45%, and the compound enzyme system accounts for 55%–65%. The preparation method includes: step S1, bacterial culture and powder preparation; step S2, bacterial powder mixing and compounding; step S3, enzyme powder preparation; and step S4, mixing and sieving. This invention achieves efficient degradation of lignocellulose, enrichment of crude protein, and efficient chemical degradation of aflatoxin B1, zearalenone, and vomitoxin through dynamic pH control and metabolic flux distribution. When used for corn cob treatment, this fermentation agent significantly improves the production performance, nutrient digestibility, and antioxidant and immune functions of beef cattle.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a microbial residue composite fermentation agent and its preparation method. Background Technology

[0002] With the increasing scale and intensification of my country's livestock industry, the contradiction between the supply and demand of feed resources has become increasingly prominent, and the prices of traditional raw materials remain high, hindering the sustainable development of the livestock industry. Developing and utilizing unconventional feed resources that are widely available and low in cost has become an important direction for alleviating feed shortages and reducing breeding costs. Corn cobs, as a major by-product of corn processing, have a huge annual output, but due to their high lignocellulose content, low digestibility, poor palatability, and susceptibility to mold and fungal toxin production during storage, they have long been unused and mostly discarded or incinerated, resulting in resource waste and environmental pressure. Therefore, how to efficiently convert corn cobs through biotechnology to improve their nutritional value, eliminate toxin risks, and verify their safety and effectiveness in ruminant diets is a key issue that urgently needs to be addressed.

[0003] To address these needs, existing technologies primarily employ microbial fermentation or enzyme preparations to treat corn cobs. Single-strain fermentation has limited ability to break down stubborn structures such as lignin, resulting in insignificant nutritional improvements; single enzyme preparations lack sustained biotransformation capabilities and struggle to simultaneously achieve toxin degradation and nutrient remodeling. In recent years, researchers have attempted to construct a "microbial-enzyme synergistic" system, leveraging the complementarity of microbial metabolism and enzyme catalysis to achieve deep structural deconstruction and functional remodeling of corn cobs. This strategy has shown some success in improving in vitro digestibility and reducing some anti-nutritional factors.

[0004] However, as beef cattle farming demands increasingly stringent requirements for feed safety, functionality, and economic efficiency, existing microbial-enzyme compound technologies have revealed deep-seated contradictions. The interactions between different microorganisms and enzyme preparations are complex; improper selection or formulation of strains can easily lead to metabolic conflicts, nutrient competition, or decreased fermentation efficiency. For moldy corn cobs, the endogenous mycotoxins are structurally stable and persistently toxic; conventional fermentation often only achieves limited adsorption, failing to substantially degrade them. Existing solutions primarily focus on nutritional enhancement, neglecting the potential threats of toxin residues to animal health and product safety. Furthermore, even with good in vitro results, the true impact on beef cattle production performance, nutrient digestibility, and metabolism in vivo lacks systematic evaluation. Inappropriate fermentation products may even induce oxidative stress or immunosuppression, offsetting nutritional gains. The root cause lies in the lack of precise design of existing microbial-enzyme compound systems targeting the specific structure of corn cobs, toxin types, and the physiological characteristics of ruminants. They have failed to establish a four-in-one functional integration mechanism of "degradation—conversion—efficiency enhancement—detoxification," resulting in a technical bottleneck between improving feed value and ensuring animal health. Summary of the Invention

[0005] The purpose of this invention is to provide a microbial residue compound fermentation agent and its preparation method, so as to solve the problems of low nutrient utilization rate of corn cob, serious mycotoxin contamination, and functional fragmentation of existing microbial enzyme systems in the prior art.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: On one hand, a compound fermentation agent for bacterial residue contains the following component: Bacillus subtilis CGMCC No. 1.529, with a viable cell concentration of not less than 1.0 × 10⁻⁶. 9 CFU / g; Lactobacillus plantarum CGMCC No. 1.3867, with a viable bacterial concentration of not less than 8.0 × 10⁻⁶. 8 CFU / g; Candida utilis CGMCC No. 2.1180, with a viable cell concentration of not less than 5.0 × 10⁻⁶. 8 CFU / g; cellulase activity not less than 15000 U / g; xylanase activity not less than 10000 U / g; laccase activity not less than 2000 U / g; peroxidase activity not less than 1500 U / g. The above components are mixed according to the following mass ratio, wherein the total dry matter of Bacillus subtilis, Lactobacillus plantarum, and Candida utilis accounts for 35% to 45% of the total mass of the starter culture, and the complex enzyme system accounts for 55% to 65% of the total mass of the starter culture. The starter culture is in solid powder form, with a moisture content controlled below 8%, and is stored sealed at 4°C with a shelf life of not less than 12 months.

[0007] The aforementioned microbial residue composite fermentation agent is a precise blend of specific functional microbial communities and directed enzyme systems, used for deep biotransformation of corn cobs. This process simultaneously optimizes nutrient structure, efficiently degrades multiple mycotoxins, and significantly improves production performance, nutrient digestibility, and antioxidant and immune functions in beef cattle feed. To achieve the above objectives, this invention constructs a functional strain combination centered on Bacillus subtilis, Lactobacillus plantarum, and Candida utilis, synergistically combining them with a multi-enzyme system composed of cellulase, xylanase, laccase, and peroxidase. This forms an integrated fermentation system with substrate-specific recognition, targeted toxin cleavage, and directed synthesis of metabolites. During fermentation, this system establishes a dynamic pH regulation mechanism and metabolic flux distribution strategy to ensure efficient depolymerization of lignocellulose, effective enrichment of crude protein, and breakage of mycotoxin chemical bonds. Simultaneously, it generates immunomodulatory secondary metabolites, thereby achieving a triple synergistic effect of nutrient supply, toxin clearance, and maintenance of physiological homeostasis in ruminants.

[0008] The specific application method of the bacterial residue compound fermentation agent of this invention for treating corn cobs is as follows: Take dried corn cobs, crush them to a particle size of 2-3 mm, adjust the moisture content to 55%-60%, and mix them evenly with the bacterial residue compound fermentation agent at a ratio of 8-12 grams per kilogram of dry matter. Pack the mixture into an anaerobic fermentation bag, vacuum seal it, and ferment it at a constant temperature of 28-32℃ for 72-96 hours. After fermentation, the material is light brown, has a sour aroma, a stable pH value between 4.2 and 4.8, and no musty or off-odors. Testing shows that after fermentation, the neutral detergent fiber content of the corn cobs decreases by more than 22%, the acid detergent lignin content decreases by more than 18%, the crude protein content increases by more than 35%, and the reducing sugar content increases by more than 40%. The degradation rate of aflatoxin B1 is not less than 92%, the degradation rate of zearalenone is not less than 88%, and the degradation rate of vomitoxin is not less than 85%. This fermentation product can be directly used as a component of beef cattle diets, replacing part of the conventional energy and protein feed, with an addition ratio of 15%-25% of the dry matter of the diet.

[0009] The core technology of this invention lies in constructing a four-in-one functional integration mechanism of "degradation-conversion-enhancement-detoxification". *Bacillus subtilis* secretes alkaline protease and amylase to promote the decomposition of non-structural carbohydrates, while simultaneously producing antimicrobial peptides to inhibit the growth of other microorganisms. *Lactobacillus plantarum* metabolizes and produces lactic acid, rapidly lowering the environmental pH, inhibiting mold regeneration, and activating the catalytic activity of laccase and peroxidase. *Candida utilis* utilizes released monosaccharides to synthesize cell proteins and secretes antioxidants such as glutathione. Cellulase and xylanase work synergistically to hydrolyze the cellulose and hemicellulose backbone, disrupting the cell wall structure. Laccase and peroxidase specifically recognize the phenolic hydroxyl groups and lactone ring structures in fungal toxin molecules, achieving chemical degradation through oxidative coupling and ring-opening reactions. These four components form a metabolic relay in the spatiotemporal dimension: the enzyme system first deconstructs the substrate, releasing soluble sugars and phenolic precursors; the microbial community then utilizes the carbon source to proliferate and secrete metabolites, while maintaining a suitable pH to ensure enzyme activity; the final product is rich in digestible nutrients, free of toxic residues, and contains immunomodulatory factors.

[0010] Furthermore, the fermented corn cob compound fermentation agent described in this invention exhibits significant physiological regulatory effects in beef cattle feed. Experiments show that adding 20% ​​fermented corn cob to the diet can increase the average daily weight gain of beef cattle by 12.3%, reduce the feed conversion ratio by 9.7%, increase the apparent digestibility of dry matter by 11.5%, and increase the apparent digestibility of crude protein by 13.2%. Serum biochemical indicators show significantly increased levels of total protein, albumin, and globulin, while alanine aminotransferase and aspartate aminotransferase activities remained within the normal range, indicating no liver function damage. Regarding antioxidant indicators, serum total antioxidant capacity increased by 18.6%, superoxide dismutase and glutathione peroxidase activities increased by 15.4% and 14.8%, respectively, and malondialdehyde content decreased by 21.3%, confirming effective alleviation of oxidative stress. Regarding immune indicators, IgA, IgG, and IgM concentrations increased by 16.2%, 13.7%, and 12.9%, respectively, indicating enhanced mucosal and humoral immunity. The aforementioned effects stem from the synergistic effect of bioactive substances such as short-chain fatty acids, nucleotides, and β-glucan in the fermentation products, rather than from the supplementation of a single nutrient.

[0011] In a preferred embodiment of the present invention, the live bacteria ratio of Bacillus subtilis, Lactobacillus plantarum, and Candida utilis in the bacterial residue compound fermentation agent is strictly controlled at 1.2:1.0:0.8. This ratio has been verified as the optimal ratio through orthogonal experiments, maximizing the metabolic complementarity among the bacterial communities. If the ratio is unbalanced, such as an excess of Lactobacillus, the pH will drop too quickly, leading to inhibited yeast activity; if an excess of Bacillus subtilis is present, excessive hydrolysis by proteases may produce bitter peptides, affecting palatability. Similarly, the activity ratio of laccase to peroxidase in the compound enzyme system must be maintained at 4:3. This ratio ensures broad-spectrum degradation of fungal toxins with different chemical structures. Laccase mainly targets zearalenone containing phenolic hydroxyl groups, while peroxidase is more specific to vomitoxin containing lactone rings; the two work synergistically to cover the main toxin types.

[0012] On the other hand, a method for preparing a microbial residue composite fermentation agent, applied to the microbial residue composite fermentation agent, includes the following steps: Step S1: High-density liquid fermentation culture was carried out on Bacillus subtilis, Lactobacillus plantarum, and Candida utilis, respectively, with strict limits on culture medium composition and process parameters. Bacillus subtilis was cultured in LB liquid medium with an initial pH of 7.2 at 37°C with shaking for 18 hours at 200 rpm. After obtaining the logarithmic growth phase, the bacterial cells were collected by centrifugation and freeze-dried to prepare bacterial powder. Lactobacillus plantarum was cultured in MRS liquid medium with an initial pH of 6.2 at 37°C with static culture for 24 hours. After obtaining the stationary phase, the bacterial cells were collected by centrifugation and freeze-dried to prepare bacterial powder. Candida utilis was cultured in YPD liquid medium with an initial pH of 5.8 at 30°C with shaking for 36 hours at 180 rpm. After obtaining the late logarithmic growth phase, the bacterial cells were collected by centrifugation and freeze-dried to prepare bacterial powder. Step S2: Mix the above three types of bacterial powder in a mass ratio of 1.2:1.0:0.8 to form a composite bacterial residue matrix; Step S3: Weigh cellulase, xylanase, laccase, and peroxidase in an activity ratio of 15:10:2:1.5, add sterile maltodextrin as a carrier, mix thoroughly, and then spray dry to form enzyme powder. Step S4: Mix the composite microbial residue substrate and enzyme powder in a sterile mixer at a mass ratio of 4:6 for 30 minutes, and then pass the mixture through an 80-mesh sieve to obtain the microbial residue composite fermentation agent.

[0013] In the preparation method described above, the bacterial cells must be washed three times with 0.85% sterile physiological saline before freeze-drying to remove culture medium residues and avoid interfering with the subsequent fermentation process. When preparing the enzyme powder, the maltodextrin carrier must be pre-sterilized at 121℃ for 20 minutes and cooled to room temperature before use to prevent microbial contamination. The mixing process must be carried out in a clean room with a relative humidity of less than 40%, and the mixing time must not be less than 30 minutes to ensure uniform distribution of the bacterial enzymes. The finished product is packaged in aluminum foil composite bags with a built-in desiccant; each bag weighs 500 grams net and is sealed and vacuum-sealed to ensure product stability.

[0014] The technical solution described in this invention addresses three major technical bottlenecks in existing microbial-enzyme composite systems: first, the functions of microbial strains and enzyme systems are disconnected, lacking metabolic coupling; second, toxin degradation relies on physical adsorption, failing to achieve chemical elimination; and third, the impact of fermentation products on metabolic homeostasis in animals is ignored. Through precise design of microbial-enzyme combinations, strict control of the preparation process, and clear application parameters, this invention, for the first time, realizes the transformation of corn cobs from low-value waste into a highly safe and functional feed ingredient, providing an industrially scalable technical path for the efficient utilization of unconventional feed resources.

[0015] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention constructs a four-in-one functional integration mechanism of "degradation-conversion-enhancement-detoxification" through a specific combination and precise ratio of Bacillus subtilis, Lactobacillus plantarum, Candida utilis, cellulase, xylanase, laccase, and peroxidase. This significantly improves the nutritional value of corn cobs and efficiently degrades various fungal toxins, fundamentally solving the industry problems of low utilization rate and high toxin residues in corn cobs.

[0016] When the fermentation product of this invention is added to the diet of beef cattle, it can increase the average daily weight gain by 12.3%, reduce the feed conversion ratio by 9.7%, and increase the digestibility of dry matter and crude protein by more than 11.5%. At the same time, it enhances the body's antioxidant capacity and immune function, achieving a synergistic effect of nutrient supply, toxin removal and maintenance of physiological homeostasis.

[0017] This invention achieves spatiotemporal synergy between enzymatic hydrolysis and microbial fermentation through metabolic relay and dynamic pH regulation mechanisms, avoiding the problems of functional fragmentation and metabolic conflict in traditional bacterial-enzyme combinations; and utilizes the targeted action of laccase and peroxidase to achieve chemical degradation of toxins, rather than physical adsorption, fundamentally eliminating toxic residues.

[0018] The preparation method of this invention has clear parameters and standardized process. The finished product has a moisture content of ≤8% and a shelf life of ≥12 months. The fermentation conditions are mild and the addition amount is low, making it easy to scale up production and application. It provides a reliable and replicable technical path for the high-value utilization of unconventional feed resources such as corn cobs. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the process flow for the preparation method of the bacterial residue composite fermentation agent according to the present invention; Figure 2 This is a schematic diagram illustrating the functional mechanism of the bacterial residue compound fermentation agent described in this invention in corn cob treatment and beef cattle feeding applications. Detailed Implementation

[0020] The features and exemplary embodiments of various aspects of the present invention will now be described in detail. To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely intended to explain the present invention and not to limit the present invention. For those skilled in the art, the present invention can be practiced without some of these specific details. The following description of the embodiments is merely to provide a better understanding of the present invention by illustrating examples of the invention.

[0021] This invention provides a microbial residue composite fermentation agent and its preparation method. The microbial residue composite fermentation agent is composed of a specific functional microbial community and a directed enzyme system compounded in a precise ratio. It enables deep biotransformation of corn cobs, simultaneously optimizing nutrient structure and efficiently degrading multiple mycotoxins. In beef cattle feed, it significantly improves production performance, nutrient digestibility, and the body's antioxidant and immune functions. The technical solution of this invention will be described in detail below with reference to specific embodiments.

[0022] The fermentation residue compound fermentation agent contains Bacillus subtilis CGMCC No. 1.529, Lactobacillus plantarum CGMCC No. 1.3867, Candida utilis CGMCC No. 2.1180, as well as cellulase, xylanase, laccase, and peroxidase. The viable concentration of Bacillus subtilis is not less than 1.0 × 10⁻⁶. 9 CFU / g, viable concentration of Lactobacillus plantarum not less than 8.0 × 10⁻⁶. 8 The CFU / g concentration of *Candida utilis* is not less than 5.0 × 10⁻⁶. 8 CFU / g; cellulase activity not less than 15000 U / g, xylanase activity not less than 10000 U / g, laccase activity not less than 2000 U / g, and peroxidase activity not less than 1500 U / g. The above components are mixed according to the following mass ratios, wherein the total dry matter of Bacillus subtilis, Lactobacillus plantarum, and Candida utilis accounts for 35% to 45% of the total mass of the starter culture, and the complex enzyme system accounts for 55% to 65% of the total mass of the starter culture. The starter culture is in solid powder form, with a moisture content controlled below 8%, and should be stored sealed at 4℃ with a shelf life of not less than 12 months.

[0023] In a preferred embodiment of the present invention, the live bacterial ratio of Bacillus subtilis, Lactobacillus plantarum, and Candida utilis is strictly controlled at 1.2:1.0:0.8. This ratio has been verified as the optimal ratio through orthogonal experiments, maximizing the metabolic complementarity among the bacterial communities. If the ratio is unbalanced, such as with an excess of Lactobacillus, the pH will drop too quickly, leading to inhibited yeast activity; if Bacillus subtilis is excessive, excessive hydrolysis by proteases may produce bitter peptides, affecting palatability. Similarly, the activity ratio of laccase to peroxidase in the complex enzyme system must be maintained at 4:3. This ratio ensures broad-spectrum degradation of fungal toxins with different chemical structures. Laccase mainly targets zearalenone containing phenolic hydroxyl groups, while peroxidase is more specific to vomitoxin containing lactone rings; the two work synergistically to cover the main toxin types.

[0024] The preparation method of the bacterial residue compound fermentation agent, applied to the above-mentioned bacterial residue compound fermentation agent, includes the following steps: Step S1 involves high-density liquid fermentation cultures of Bacillus subtilis, Lactobacillus plantarum, and Candida utilis, with strictly defined culture medium components and process parameters. Bacillus subtilis was cultured in LB liquid medium at an initial pH of 7.2 at 37°C with shaking for 18 hours at 200 rpm. After obtaining the logarithmic growth phase culture, the cells were collected by centrifugation and freeze-dried to produce a bacterial powder. Lactobacillus plantarum was cultured in MRS liquid medium at an initial pH of 6.2 at 37°C with static incubation for 24 hours. After obtaining the stationary phase culture, the cells were collected by centrifugation and freeze-dried to produce a bacterial powder. Candida utilis was cultured in YPD liquid medium at an initial pH of 5.8 at 30°C with shaking for 36 hours at 180 rpm. After obtaining the late logarithmic growth phase culture, the cells were collected by centrifugation and freeze-dried to produce a bacterial powder. Before freeze-drying, the bacterial cells were washed three times with 0.85% sterile physiological saline to remove culture medium residues and avoid interfering with subsequent fermentation processes.

[0025] Step S2: Mix the three types of bacterial powders in a mass ratio of 1.2:1.0:0.8 to form a composite bacterial residue matrix.

[0026] In step S3, cellulase, xylanase, laccase, and peroxidase are weighed out at an activity ratio of 15:10:2:1.5, and sterile maltodextrin is added as a carrier. After thorough mixing, the mixture is spray-dried into enzyme powder. When preparing the enzyme powder, the maltodextrin carrier must be pre-sterilized at 121℃ for 20 minutes and cooled to room temperature before use to prevent microbial contamination.

[0027] Step S4: Mix the composite microbial substrate and enzyme powder at a mass ratio of 4:6 in a sterile mixer for 30 minutes, then pass through an 80-mesh sieve to obtain the microbial substrate composite fermentation agent. The mixing process must be carried out in a clean room with a relative humidity below 40%, and the mixing time must not be less than 30 minutes to ensure uniform distribution of the microorganisms and enzymes. The finished product is packaged in aluminum foil composite bags with a built-in desiccant. Each bag weighs 500 grams net and is sealed and vacuum-sealed to ensure product stability.

[0028] The specific application method of using a microbial residue compound fermentation agent to treat corn cobs is as follows: Take dried corn cobs, crush them to a particle size of 2-3 mm, adjust the moisture content to 55%-60%, and mix them evenly with 8-12 grams of microbial residue compound fermentation agent per kilogram of dry matter. Pack the mixture into anaerobic fermentation bags, vacuum seal them, and ferment at a constant temperature of 28-32℃ for 72-96 hours. After fermentation, the material is light brown, has a sour aroma, a stable pH value between 4.2 and 4.8, and no musty or off-odors. Testing shows that after fermentation, the neutral detergent fiber content of the corn cobs decreases by more than 22%, the acid detergent lignin content decreases by more than 18%, the crude protein content increases by more than 35%, and the reducing sugar content increases by more than 40%. The degradation rate of aflatoxin B1 is no less than 92%, the degradation rate of zearalenone is no less than 88%, and the degradation rate of vomitoxin is no less than 85%. This fermentation product can be directly used as a component of beef cattle diets, replacing part of the conventional energy and protein feed, with an addition ratio of 15%-25% of the dry matter in the diet.

[0029] In Example 1, dried corn cobs were crushed to a particle size of 2.5 mm, and the moisture content was adjusted to 58%. The mixture was added at a ratio of 10 g of bacterial residue compound fermentation agent per kilogram of dry matter. The ratio of live Bacillus subtilis, Lactobacillus plantarum, and Candida utilis in the bacterial residue compound fermentation agent was 1.2:1.0:0.8, and the activity ratio of cellulase, xylanase, laccase, and peroxidase in the compound enzyme system was 15:10:2:1.5. The material was placed in an anaerobic fermentation bag, vacuum-sealed, and fermented at a constant temperature of 30°C for 84 hours. After fermentation, the pH of the material was 4.5, the neutral detergent fiber content decreased from 48.6% to 37.2%, the acid detergent lignin content decreased from 22.1% to 18.3%, the crude protein content increased from 5.8% to 7.9%, and the reducing sugar content increased from 1.2% to 1.7%. Aflatoxin B1 decreased from 85.3 μg / kg to 6.1 μg / kg, zearalenone from 120.5 μg / kg to 13.8 μg / kg, and vomitoxin from 98.7 μg / kg to 14.2 μg / kg. The fermentation products were added to beef cattle diets at a ratio of 20%. After 90 days of feeding, the average daily weight gain reached 1.42 kg / d, the feed conversion ratio was 6.8:1, the dry matter digestibility was 68.3%, and the serum IgG concentration was 28.6 mg / mL.

[0030] In Example 2, the same corn cob raw material and processing method as in the previous examples were used, but the fermentation temperature was adjusted to 28°C and the fermentation time was extended to 96 hours. After fermentation, the pH was 4.3, neutral detergent fiber decreased to 36.8%, crude protein increased to 8.1%, and aflatoxin B1 decreased to 5.8 μg / kg. Feeding trials in beef cattle showed an average daily weight gain of 1.39 kg / d, a feed conversion ratio of 6.9:1, a serum total antioxidant capacity of 12.4 U / mL, and a malondialdehyde content of 4.1 nmol / mL. This indicates that although low-temperature, long-term fermentation slightly reduces weight gain efficiency, it provides superior antioxidant effects and is suitable for beef cattle feeding during periods of high heat stress.

[0031] In Example 3, the moisture content of corn cobs was adjusted to 60%, the amount of starter culture added was 12 g / kg dry matter, the fermentation temperature was 32°C, and the fermentation time was 72 hours. After fermentation, the pH was 4.7, the neutral detergent fiber content decreased to 38.1%, crude protein was 7.7%, and reducing sugar was 1.8%. The toxin degradation rate was slightly lower than in the previous examples, but the feed intake of beef cattle increased by 3.2%, indicating that higher moisture and temperature can improve palatability and are suitable for fattening cattle with poor appetite in the later stages.

[0032] In Example 4, the fermentation product from the first example was used, but the proportion added to the diet was adjusted to 25%. After 90 days of feeding, the average daily weight gain reached 1.45 kg / d, the feed conversion ratio was 6.7:1, the crude protein digestibility was 72.1%, the IgA concentration was 1.98 mg / mL, the neutral detergent fiber content was 37.2%, the acid detergent lignin content was 18.3%, the crude protein content was 7.9%, the reducing sugar content was 1.7%, the aflatoxin B1 content was 6.1 μg / kg, the zearalenone content was 13.8 μg / kg, and the vomitoxin content was 14.2 μg / kg. The results indicate that increasing the proportion added within a reasonable range can further improve production performance, but fecal morphology needs to be monitored to prevent mild diarrhea, and it is recommended to provide adequate drinking water and buffer salts.

[0033] To verify the effectiveness of the technical solution of the present invention, multiple comparative examples were set up for comparative analysis.

[0034] In Comparative Example 1, corn cobs were treated with only Bacillus subtilis, at a concentration of 1.0 × 10⁻⁶ per gram. 9 CFU, other conditions were the same as in Example 1. After fermentation, neutral detergent fiber only decreased to 44.2%, crude protein increased to 6.5%, aflatoxin B1 only decreased to 72.4 μg / kg, the average daily weight gain of beef cattle was 1.18 kg / d, and the feed conversion ratio was 8.2:1, indicating that a single strain of bacteria cannot achieve deep degradation and detoxification.

[0035] In Comparative Example 2, corn cobs were treated with only cellulase and xylanase, without any microbial strains. The activity ratio of cellulase to xylanase was 15:10. After fermentation, the reducing sugar increased to 1.9%, but the crude protein was only 5.9%. The pH remained above 6.0, mold proliferated, and the contents of neutral detergent fiber and acid detergent lignin did not decrease effectively. The toxin content remained unchanged, with aflatoxin B1 still at 85.3 μg / kg, zearalenone still at 120.5 μg / kg, and vomitoxin still at 98.7 μg / kg. Beef cattle showed mild liver damage and increased ALT activity. The beef cattle's production performance, dry matter digestibility, and serum immune indicators were not effectively improved, proving that the aseptic system could not inhibit contaminating bacteria and had no detoxification function.

[0036] In Comparative Example 3, the ratio of live Bacillus subtilis, Lactobacillus plantarum, and Candida utilis was 2:1:1, and the enzyme system was the same as in Example 1. The fermentation pH dropped to 3.9, yeast activity was inhibited, crude protein was only 7.2%, and beef cattle feed intake decreased by 8%, indicating that the imbalance of the bacterial ratio disrupted metabolic balance.

[0037] In Comparative Example 4, laccase was removed from the enzyme system, retaining only cellulase, xylanase, and peroxidase. The degradation rate of zearalenone in zearalenone was only 45%, with a residual amount of 66.3 μg / kg. This resulted in abnormally elevated serum estrogen levels in beef cattle, leading to false estrus, demonstrating that laccase is indispensable for the treatment of certain toxins.

[0038] In Comparative Example 5, the fermentation time was shortened to 48 hours. The material pH was 5.2, indicating incomplete toxin degradation. Aflatoxin B1 residue was 32.6 μg / kg, and the serum MDA content of beef cattle increased while the antioxidant capacity decreased, suggesting that insufficient fermentation time was insufficient to complete toxin lysis.

[0039] In Comparative Example 6, corn cobs were treated with commercially available common compound probiotics and universal cellulase. The commercially available common compound probiotics contained lactic acid bacteria and yeast, but the strains were not specified. Nutritional improvement after fermentation was limited; no significant increase was observed in nutritional indicators such as neutral detergent fiber, acid detergent lignin, crude protein, and reducing sugar. The toxin degradation rate was less than 50%, with aflatoxin B1 residue >40 μg / kg, zearalenone residue >60 μg / kg, and vomitoxin residue >50 μg / kg. Beef cattle production performance showed no significant difference compared to the control group, with an average daily weight gain of approximately 1.15 kg / d, a feed conversion ratio of approximately 8.3:1, a dry matter digestibility of approximately 60%, and a serum IgG concentration of approximately 22 mg / mL, demonstrating the ineffectiveness of the non-specific bacterial-enzyme combination.

[0040] In Comparative Example 7, the peroxidase activity in the fermentation agent was insufficient, only 500 U / g. The degradation rate of vomitoxin was only 60%, with a residual amount of 39.5 μg / kg. The degradation rates of aflatoxin B1 and zearalenone may also have been affected. Beef cattle exhibited decreased appetite and mild neurological symptoms. The beef cattle's production performance, feed efficiency, and health indicators failed to achieve the effects described in this invention, confirming that the specific degradation effect of peroxidase on vomitoxin is irreplaceable.

[0041] In Comparative Example 8, the corn cobs were not crushed and were fermented whole. Uneven fermentation occurred within the material, with increased mold growth in the central area. Indicators such as neutral detergent fiber, acid detergent lignin, crude protein, and reducing sugar could not be effectively improved, resulting in a moldy state. Toxin content even increased, with elevated levels of aflatoxin B1, zearalenone, and vomitoxin. Cattle refused to eat the feed, and no production performance or health benefits were obtained, demonstrating that physical pretreatment is a necessary prerequisite.

[0042] This invention constructs a functional strain combination centered on Bacillus subtilis, Lactobacillus plantarum, and Candida utilis, synergistically combining them with a multi-enzyme system consisting of cellulase, xylanase, laccase, and peroxidase. This results in an integrated fermentation system capable of substrate-specific recognition, targeted toxin cleavage, and directed synthesis of metabolites. During fermentation, this system establishes a dynamic pH regulation mechanism and metabolic flux distribution strategy, ensuring efficient depolymerization of lignocellulose, effective enrichment of crude protein, and breakage of fungal toxin chemical bonds. Simultaneously, it generates secondary metabolites with immunomodulatory activity, thereby achieving a triple synergistic effect of nutrient supply, toxin clearance, and maintenance of physiological homeostasis in ruminants.

[0043] Specifically, *Bacillus subtilis* secretes alkaline protease and amylase to promote the breakdown of non-structural carbohydrates, while simultaneously producing antimicrobial peptides to inhibit the growth of other microorganisms. *Lactobacillus plantarum* metabolizes lactic acid, rapidly lowering the environmental pH, inhibiting mold regeneration, and activating the catalytic activity of laccase and peroxidase. *Candida utilis* utilizes released monosaccharides to synthesize cell proteins and secretes antioxidants such as glutathione. Cellulase and xylanase work synergistically to hydrolyze the cellulose and hemicellulose backbone, disrupting the cell wall structure. Laccase and peroxidase specifically recognize the phenolic hydroxyl groups and lactone ring structures in fungal toxin molecules, achieving chemical degradation through oxidative coupling and ring-opening reactions. These four processes form a metabolic relay across time and space: the enzyme system first deconstructs the substrate, releasing soluble sugars and phenolic precursors; the microbial community then utilizes the carbon source to proliferate and secrete metabolites, while maintaining a suitable pH to ensure enzyme activity; the final product is rich in digestible nutrients, free of toxic residues, and contains immunomodulatory factors.

[0044] The fermented corn cob compound fermentation agent of this invention exhibits significant physiological regulatory effects in beef cattle feed. Experiments show that adding 20% ​​fermented corn cob to the diet can increase the average daily weight gain of beef cattle by 12.3%, reduce the feed conversion ratio by 9.7%, increase the apparent digestibility of dry matter by 11.5%, and increase the apparent digestibility of crude protein by 13.2%. Serum biochemical indicators show significantly increased levels of total protein, albumin, and globulin, while alanine aminotransferase and aspartate aminotransferase activities remained within the normal range, indicating no liver function damage. Regarding antioxidant indicators, serum total antioxidant capacity increased by 18.6%, superoxide dismutase and glutathione peroxidase activities increased by 15.4% and 14.8%, respectively, and malondialdehyde content decreased by 21.3%, confirming effective alleviation of oxidative stress. Regarding immune indicators, IgA, IgG, and IgM concentrations increased by 16.2%, 13.7%, and 12.9%, respectively, indicating enhanced mucosal and humoral immunity. The aforementioned effects stem from the synergistic effect of bioactive substances such as short-chain fatty acids, nucleotides, and β-glucan in the fermentation products, rather than from the supplementation of a single nutrient.

[0045] The table below summarizes the key performance indicators of Examples 1 to 4 and Comparative Examples 1 to 8:

[0046] Note: NDF is neutral detergent fiber, ADL is acid detergent lignin, CP is crude protein, AFB1 is aflatoxin B1, ZEN is zearalenone, DON is vomitoxin, ADG is average daily weight gain, FCR is feed conversion ratio, and DMdigest is dry matter digestibility.

[0047] In summary, this invention, through precise design of the bacterial-enzyme combination, strict control of the preparation process, and clear application parameters, has for the first time achieved the transformation of corn cobs from low-value waste into a high-safety functional feed ingredient. It overcomes the technical bottlenecks in existing bacterial-enzyme composite systems, such as the functional disconnect between bacterial strains and enzyme systems, reliance on physical adsorption for toxin degradation, and neglect of the impact of fermentation products on metabolic homeostasis in animals. This provides an industrially scalable technical path for the efficient utilization of unconventional feed resources. Those skilled in the art can reproduce all the technical effects of this invention based on the content disclosed in this specification without any inventive effort.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A compound fermentation agent for bacterial residue, characterized in that: The bacterial residue compound fermentation agent is a solid powder containing Bacillus subtilis CGMCC No. 1.529, Lactobacillus plantarum CGMCC No. 1.3867, Candida utilis CGMCC No. 2.1180, as well as cellulase, xylanase, laccase and peroxidase; The viable concentration of Bacillus subtilis is not less than 1.0 × 10⁻⁶. 9 CFU / g, viable concentration of Lactobacillus plantarum not less than 8.0 × 10⁻⁶. 8 The CFU / g concentration of *Candida utilis* is not less than 5.0 × 10⁻⁶. 8 CFU / g; cellulase activity not less than 15000 U / g, xylanase activity not less than 10000 U / g, laccase activity not less than 2000 U / g, peroxidase activity not less than 1500 U / g; the total dry matter of the Bacillus subtilis, Lactobacillus plantarum and Candida utilis accounts for 35% to 45% of the total mass of the starter culture, and the complex enzyme system accounts for 55% to 65% of the total mass of the starter culture; the moisture content is not higher than 8%.

2. The bacterial residue compound fermentation agent according to claim 1, characterized in that, The ratio of viable Bacillus subtilis, Lactobacillus plantarum, and Candida utilis is 1.2:1.0:0.

8.

3. The bacterial residue compound fermentation agent according to claim 1, characterized in that, The activity unit ratio of cellulase, xylanase, laccase and peroxidase is 15:10:2:1.

5.

4. The bacterial residue compound fermentation agent according to claim 1, characterized in that, The activity ratio of laccase to peroxidase is 4:

3.

5. The bacterial residue compound fermentation agent according to claim 1, characterized in that, The method for applying the microbial residue compound fermentation agent in the bioconversion of corn cobs includes: crushing corn cobs to a particle size of 2 mm to 3 mm, adjusting the moisture content to 55% to 60%, adding 8 to 12 grams of the microbial residue compound fermentation agent per kilogram of dry matter, mixing well, loading into an anaerobic container and sealing it under vacuum, and fermenting at 28°C to 32°C for 72 to 96 hours.

6. The bacterial residue compound fermentation agent according to claim 1, characterized in that, After fermentation, the pH value of the resulting material is 4.2 to 4.8, the neutral detergent fiber content is reduced by more than 22%, the acid detergent lignin content is reduced by more than 18%, the crude protein content is increased by more than 35%, the aflatoxin B1 degradation rate is not less than 92%, the zearalenone degradation rate is not less than 88%, and the vomitoxin degradation rate is not less than 85%.

7. A method for preparing a microbial residue composite fermentation agent, applied to the microbial residue composite fermentation agent according to any one of claims 1 to 6, characterized in that, Includes the following steps: Step S1: Bacillus subtilis, Lactobacillus plantarum and Candida utilis were cultured in liquid fermentation, and the cells were collected by centrifugation and freeze-dried to produce bacterial powder. Step S2: Mix the three types of bacterial powder in a mass ratio of 1.2:1.0:0.8 to form a composite bacterial residue matrix; Step S3: Cellulase, xylanase, laccase and peroxidase are mixed with sterile maltodextrin carrier at an activity ratio of 15:10:2:1.5 and then spray-dried to prepare enzyme powder; Step S4: Mix the composite microbial residue substrate and enzyme powder at a mass ratio of 4:6 under sterile conditions for more than 30 minutes, and pass through an 80-mesh sieve to obtain the microbial residue composite fermentation agent.

8. The method for preparing the bacterial residue compound fermentation agent according to claim 7, characterized in that, The *Bacillus subtilis* was cultured in LB liquid medium with an initial pH of 7.2 at 37°C and 200 rpm for 18 hours with shaking. The *Lactobacillus plantarum* was cultured in MRS liquid medium with an initial pH of 6.2 at 37°C for 24 hours with static incubation. The *Candida utilis* was cultured in YPD liquid medium with an initial pH of 5.8 at 30°C and 180 rpm for 36 hours with shaking.

9. The method for preparing the bacterial residue compound fermentation agent according to claim 7, characterized in that, Each bacterial cell was washed three times with 0.85% sterile physiological saline before freeze-drying; the maltodextrin carrier was sterilized at 121℃ for 20 minutes before use.

10. The method for preparing the bacterial residue compound fermentation agent according to claim 7, characterized in that, Step S2 is performed in a clean environment with a relative humidity of less than 40%.

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