Compound bacteria fermented feed for improving intestinal health of animals and preparation method thereof

Abstract

This invention discloses a compound bacterial fermented feed for improving animal intestinal health and its preparation method. The feed consists of soybean meal, compound bacterial liquid, buffer regulator, and other components; wherein the compound bacterial liquid is a mixture of Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae, and the buffer regulator is a mixture of citric acid, disodium hydrogen phosphate, and light calcium carbonate. The preparation method includes steps such as substrate pretreatment, constructing a buffer system, two-stage fermentation, and post-ripening drying. This application achieves a metabolic relay of enzymatic hydrolysis, acidification, and flavor modification through staged inoculation and dynamic pH control of the buffer system, effectively solving the competitive inhibition of multiple strains. The final feed product has a crude protein content ≥40% (dry basis), a free amino acid percentage of ≥11.8% of total protein, a phytic acid phosphorus degradation rate ≥88.5%, a pH of 4.0-4.5, and a pure, sour and fragrant flavor, which can significantly improve animal intestinal health, increase nutrient utilization, and reduce diarrhea rate and fecal phosphorus emissions.

Description

Technical Field

[0001] This invention relates to the field of microbial fermented feed technology, specifically to a compound bacterial fermented feed for improving animal intestinal health and its preparation method. Background Technology

[0002] Microbial fermentation technology is an important way to improve the nutritional value of feed and improve the intestinal health of animals. Among them, multi-strain solid-state fermentation has become a research hotspot in this field due to its advantages such as simulating the natural digestive environment of animals and low production costs. However, existing solid-state fermentation technologies generally face a common technical challenge: when multiple functional strains such as lactic acid bacteria, Bacillus subtilis, and yeast are simultaneously inoculated or simply sequentially in the same system, different strains are prone to fierce metabolic competition and microecological imbalance due to differences in growth conditions (such as pH and oxygen requirements) and metabolic pathways (acid production, enzyme production, and flavor substance production). For example, the rapid reproduction of lactic acid bacteria will cause a sharp drop in the pH of the system, which not only inhibits the secretion activity of extracellular proteases and amylases in Bacillus subtilis, but also interferes with the metabolism of ethanol and flavor substances in yeast. Ultimately, this leads to incomplete degradation of anti-nutritional factors (such as phytic acid and macromolecular proteins), unstable content of functional metabolites (such as small peptides and organic acids) in the product, and the finished product often has an unpleasant sour and rancid taste, which seriously restricts the improvement of fermented feed quality and its large-scale application.

[0003] To address these challenges, existing technologies have attempted to mitigate interference between bacterial strains through stepwise fermentation strategies. For example, Chinese patent application CN104664169A discloses a bio-enhancer for ruminant feed and its preparation method. This method employs a three-stage solid-state fermentation process, inoculating the entire volume of *Candida utilis*, *Lactobacillus plantarum*, and *Bacillus subtilis* in sequence. By staggering the peak proliferation periods of the strains through sequential inoculation, the antagonistic problem of simultaneous fermentation is alleviated to some extent. However, the phased logic of this technical solution is based on a simple time sequence. After the yeast produces acid and changes the initial pH, the lactic acid bacteria proliferate rapidly, causing the environmental acidity to rise sharply. Subsequently, Bacillus subtilis faces high acid stress, and the number of viable bacteria is significantly limited. The essence of the process is still the successive proliferation of each strain in different time periods. The metabolic processes of each stage are relatively fragmented, and the deep synergy and organic relay of the metabolic activities of different strains in terms of function are not achieved. There is still much room for improvement in the degradation efficiency of macromolecular proteins and anti-nutritional factors such as phytate phosphorus in feed. Secondly, the fermentation process lacks active pH control methods and relies entirely on the adaptability of the strains themselves. It is difficult to maintain a neutral environment in the early stage of fermentation to ensure the enzyme production efficiency of Bacillus subtilis, and it is also impossible to accurately control the pH drop in the middle and late stages of fermentation. After the lactic acid bacteria produce acid, the pH drops sharply, which can easily inhibit the activity of Bacillus subtilis and yeast. This can also be seen from the fact that the OD600 needs to be frequently detected in the embodiments of the specification to passively adapt to the growth state of the strains.

[0004] Chinese patent application CN112335788A discloses a bio-enhancer for ruminant feed and its preparation method, employing a microbial-enzyme co-fermentation technology. While this method achieves surface coupling of enzymatic hydrolysis and fermentation by simultaneously adding exogenous compound enzyme preparations (such as protease, phytase, and cellulase) and compound microbial agents (various lactic acid bacteria, Bacillus subtilis, and yeast), effectively shortening fermentation time and reducing anti-nutritional factor content, the simultaneous inoculation of all microbial strains does not fundamentally solve the problem of metabolic competition between strains. Furthermore, it lacks a segmented aerobic-anaerobic relay process and a dynamic pH buffer system. Although the added enzyme preparations can initially compensate for some endogenous enzyme deficiencies, their activity rapidly declines due to pH fluctuations and protease degradation as fermentation progresses, resulting in an inability to achieve optimal temporal matching between enzymatic hydrolysis and fermentation.

[0005] In summary, how to achieve orderly temporal and spatial regulation of the metabolic activities of multiple bacterial strains in a solid-state fermentation system, enabling efficient metabolic relay among different functional stages such as enzymatic pretreatment, acidification and antibacterial treatment, and flavor modification, while synergistically maintaining the dynamic stability of the fermentation microenvironment (especially pH), and ultimately achieving fermented feed with deep degradation of antinutritional factors, high abundance of functional metabolites, and excellent product flavor, remains a pressing technical challenge in this field. This invention addresses the shortcomings of the existing technologies by providing a novel solution. Summary of the Invention

[0006] To address the aforementioned challenges, one objective of this invention is to provide a compound microbial fermented feed for improving animal gut health. This fermented feed comprises the following components by weight: 40-60 parts soybean meal, 20-35 parts corn flour, 10-20 parts wheat bran, 5-15 parts compound microbial solution, and 0.5-2.0 parts buffer regulator. Soybean meal serves as a protein source and phytic acid carrier, corn flour provides fermentable sugars, and wheat bran provides dietary fiber and loosens the matrix structure to facilitate gas exchange. The compound bacterial solution is composed of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae in a live cell ratio of (1-3):(1-2):(0.5-1.5); the buffer regulator is composed of citric acid, disodium hydrogen phosphate and light calcium carbonate in a mass ratio of (1.5-2.5):(0.8-1.5):1.

[0007] Furthermore, the compound bacterial solution is composed of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae in a live cell ratio of 3:2:1.5, and the buffer regulator is composed of citric acid, disodium hydrogen phosphate and light calcium carbonate in a mass ratio of 1.5:0.8:1.

[0008] Furthermore, the compound bacterial solution is composed of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae in a live cell ratio of 1:2:0.5, and the buffer regulator is composed of citric acid, disodium hydrogen phosphate and light calcium carbonate in a mass ratio of 2.5:1.5:1.

[0009] Furthermore, the final feed product should have a moisture content of <12%, a free amino acid content of ≥11.8% of total protein, a phytic acid phosphorus degradation rate of ≥88.5%, and a pH of 4.0. 4.5.

[0010] A method for preparing a compound microbial fermented feed for improving animal gut health, characterized by the following steps: (1) Base material pretreatment: Mix soybean meal, corn flour and wheat bran evenly, add water to adjust the moisture content of the base material to 38%-42%, sterilize by moist heat at 110-121℃ for 20-30 minutes, and cool to 35-40℃. (2) Buffer system construction: Dissolve the buffer regulator in sterile water and spray it into the base material after step (1). Stir and mix for 10-20 minutes to stabilize the pH value of the system at 6.0-6.5. The buffer regulator is composed of citric acid, disodium hydrogen phosphate and light calcium carbonate in a mass ratio of (1.5-2.5):(0.8-1.5):1. When preparing the buffer regulator, citric acid and disodium hydrogen phosphate should be premixed first and then mixed with calcium carbonate.

[0011] (3) Fermentation: The first stage is aerobic fermentation: 50%-60% of the total amount of Bacillus subtilis bacterial solution is added to the substrate treated in step (2), and aerobic fermentation is carried out for 12-24 hours at a temperature of 28-37℃ and a relative humidity of 60%-70%. Bacillus subtilis is the dominant strain in the first stage. Its optimal growth pH is 6.5-7.5 and its optimal temperature is 28-37℃. It is an aerobic or facultative anaerobic bacterium and can secrete alkaline protease, neutral protease, amylase and phytase. In the early stage of fermentation, this strain is responsible for breaking down the cell wall barrier of soybean meal, degrading macromolecular proteins into small peptides and free amino acids, converting phytic acid phosphorus into inositol and inorganic phosphorus, and consuming free oxygen in the system to create a low redox potential environment for subsequent anaerobic bacteria.

[0012] The second stage involves anaerobic or microaerobic fermentation: The remaining Bacillus subtilis culture, all Lactobacillus plantarum culture, and all Saccharomyces cerevisiae culture are added, and the substrate moisture content is adjusted to 40%-50%. Fermentation continues for 36-60 hours under anaerobic or microaerobic conditions at 30-35℃. This second stage primarily inhibits the excessive proliferation of Bacillus subtilis and promotes the metabolism of lactic acid bacteria and yeast. The endpoint is determined by a stable pH of 4.0-4.5 with a change of less than 0.1 over four consecutive hours. Lactobacillus plantarum, as the core functional bacterium in the second stage, has an optimal pH of 6.0-6.5 and an optimal temperature of 30-37℃. As a facultative anaerobic bacterium, it produces lactic acid through fermentation, causing the environmental pH to steadily decrease to the 4.0-4.5 range in the later stages of fermentation, thereby effectively inhibiting the growth of Escherichia coli, Salmonella, and other miscellaneous bacteria. Saccharomyces cerevisiae is also introduced in the second stage. Its optimal pH is 4.5-5.5 and its optimal temperature is 28-32℃. Under facultative anaerobic conditions, it utilizes amino acids and reducing sugars to produce flavor substances such as esters and alcohols, while also synthesizing B vitamins, which significantly improves the palatability of the feed.

[0013] (4) Post-fermentation and drying: After the fermentation is completed, the base material is naturally post-fermented for 4-6 hours to further convert the residual reducing sugars. Then, it is dried at 45-55℃ until the moisture content is less than 12%. The finished product is obtained by crushing and sieving.

[0014] Furthermore, in step (1), the heating rate of moist heat sterilization is less than or equal to 3°C / min to prevent local overheating that could lead to loss of amino acid Maillard reaction.

[0015] Furthermore, the stirring rate in step (2) is 150-250 rpm.

[0016] Furthermore, the aeration ratio for aerobic fermentation in step (3) is 0.5-1.0 vvm.

[0017] Furthermore, in step (3), the ventilation ratio needs to be adjusted to 0.05-0.1vvm when using the micro-aerobic mode, and intermittent ventilation should be used for 5 minutes every 2 hours to maintain dissolved oxygen below 0.5mg / L.

[0018] Furthermore, the sieve used in step (4) has a mesh size of 40-80.

[0019] The beneficial effects are as follows: The core idea of ​​this application is to adopt a metabolic time-series decoupling strategy. Instead of pursuing the synchronous proliferation of all strains in the same environment, it uses a phased inoculation and buffer system design to make the metabolic peaks of different strains staggered in time and complementary in space, forming a metabolic relay of enzymatic pretreatment-acidification inhibition-flavor modification.

[0020] Specifically, in the first stage of fermentation, Bacillus subtilis dominates the metabolism, secreting proteases to convert large protein molecules into small peptides and free amino acids, secreting phytase to convert phytate phosphorus into inositol and inorganic phosphorus, and secreting amylase to convert starch into reducing sugars. Simultaneously, it consumes oxygen to lower the system's redox potential. In the second stage of fermentation, Lactobacillus plantarum and Saccharomyces cerevisiae work synergistically under anaerobic or microaerobic conditions. Lactobacillus plantarum fermentation produces lactic acid, lowering the pH and inhibiting the growth of other microorganisms. Saccharomyces cerevisiae utilizes amino acids to produce isoamyl alcohol, 2-phenylethanol, esters, and other flavor compounds, giving the feed its unique sour and aromatic flavor. Furthermore, the remaining Bacillus subtilis culture added in the second stage forms spores and enters a dormant state under high acidity and low oxygen conditions. After the feed enters the animal's digestive tract, these spores re-germinate under suitable intestinal conditions, continuing to exert their beneficial effects. Meanwhile, the buffer system consists of a buffer pair (citric acid and disodium hydrogen phosphate) and a backup alkali source (light calcium carbonate), which can dynamically regulate the pH value of the system. Citric acid has multi-stage dissociation characteristics, allowing the citric acid and disodium hydrogen phosphate buffer pair to provide continuous buffering capacity within the pH range of 5.8-6.5. In the early stage of fermentation, when the substrate pH is maintained at 6.0-6.5, this buffer pair can effectively resist the slight alkaline or acidic fluctuations produced by Bacillus subtilis metabolism, maintaining pH stability. As lactic acid bacteria produce a large amount of acid in the second stage, when the local pH drops below 5.0, light calcium carbonate begins to slowly dissolve, releasing calcium ions and carbon dioxide through an acid-base neutralization reaction, preventing the pH from dropping excessively below 3.5 and inhibiting yeast activity. This design can ensure the near-neutral environment required by Bacillus subtilis in the early stage of fermentation, while creating an acidic metabolic window for lactic acid bacteria and yeast, achieving a smooth transition of pH from 6.5 to 4.0.

[0021] Experimental data show that, compared with the comparative examples, the crude protein content (dry basis) of the finished product of Example 1 reached 42.6%, the proportion of free amino acids in the total protein reached 12.7%, the phytic acid phosphorus degradation rate reached 91.2%, the small peptide content reached 85.4 mg / g, the total amount of volatile flavor substances reached 186.5 μg / g, the final product pH was stable at 4.2, and the sensory score reached 9.2 points; the spore rate of Bacillus subtilis was 62.3%, and the simulated intestinal germination rate reached 89.6%, which were significantly higher than those of the comparative examples. Animal experiments (Table 5) show that the average daily weight gain of pigs in the Example 1 group was 820 g, the feed efficiency was 0.443, the diarrhea rate was only 1.2%, and the fecal phosphorus emission was 2.85 g / kg, all of which were significantly better than those of the comparative examples. The above results demonstrate that the present invention, through the synergy of staged inoculation and a dynamic buffer system, can achieve a metabolic relay of enzymatic pretreatment, acidification and antibacterial action, and flavor modification, deeply degrading anti-nutritional factors, enriching functional metabolites, improving feed palatability, and effectively improving animal growth performance, reducing diarrhea rate, and reducing phosphorus emissions in actual breeding, thus possessing outstanding value for promotion and application. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0023] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention. The technical solutions of the various embodiments can be combined with each other, but only on the basis that those skilled in the art can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope claimed by the invention.

[0024] The dry basis mentioned in this application refers to the calculation method based on the absolute dry matter weight after moisture removal. For example, if a fresh sample of fermented feed weighs 100g and has a moisture content of 12%, then the dry basis weight is 88g. The ventilation ratio (vvm) mentioned in this application refers to the volume of air introduced per kilogram of dry material per minute. The anaerobic mode mentioned in this application is achieved by stopping ventilation, while the micro-aerobic mode is achieved by maintaining dissolved oxygen below 0.5mg / L. In the composition of the base material of this application, the ratio of 40-60 parts soybean meal, 20-35 parts corn flour, and 10-20 parts wheat bran is determined according to the nutritional requirements of conventional livestock and poultry feed. Those skilled in the art can expect that adjustments within this range can achieve the purpose of the invention. A base material moisture content of 38%-42% is the conventional operating range for solid-state fermentation. Below 38%, the fermentation rate is limited, and above 42%, the material viscosity increases, which is not conducive to ventilation. Those skilled in the art can adjust within this range according to the actual material characteristics.

[0025] In the embodiments of this invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of this invention, unless specifically indicated, the technical means used are all conventional means well-known to those skilled in the art. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all conventional reagent products that can be obtained commercially.

[0026] Raw material source: Lactobacillus plantarum ( Lactobacillus plantarum The strain is identified by its accession number CGMCC No. 12554. Bacillus subtilis (Bacillus subtilis The strain with accession number ACCC No. 10619 is: Saccharomyces cerevisiae ( Saccharomyces cerevisiae The strain is identified by its accession number ACCC No. 20065. Light calcium carbonate, supplied by Shandong Aokai Chemical Co., Ltd., model Aokai AK-011; All other raw and auxiliary materials are commercially available.

[0027] Example 1 This embodiment provides a compound microbial fermented feed for improving animal gut health and its preparation method, the preparation method including the following steps: (1) Pretreatment of base material: Mix 50 parts soybean meal, 28 parts corn flour and 15 parts wheat bran evenly according to the mass ratio (total base material 93 parts), add water to adjust the moisture content of the base material to 40%, put the mixed base material into a solid fermentation tank, and pass saturated steam for moist heat sterilization, raising the temperature to 121°C at 3°C / min and holding for 20min. After sterilization, cool the base material to 40°C.

[0028] (2) Buffer system construction: Take 1.5 parts of buffer regulator and dissolve it in 10% of the mass of the base material in sterile water (i.e., 9.3 parts of sterile water) to prepare a buffer solution; spray the buffer solution evenly into the base material after step (1) and mix continuously at 200 rpm for 15 min. Take multiple samples to measure the pH and confirm that the pH value of the system is stable at 6.2; the buffer regulator is composed of citric acid, disodium hydrogen phosphate and light calcium carbonate in a mass ratio of 2:1:1; when preparing the buffer regulator, citric acid and disodium hydrogen phosphate should be premixed first and then mixed with calcium carbonate.

[0029] (3) Fermentation: The compound bacterial solution was prepared by mixing *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* in a live cell ratio of 1.5:1.8:1.2, with a total dosage of 10 parts. The live *Lactobacillus plantarum* count in the compound bacterial solution was 5 × 10⁻⁶. 8 CFU / mL, Bacillus subtilis viable count was 6 × 10⁻⁶ 8 CFU / mL, viable count of Saccharomyces cerevisiae: 4 × 10⁻� 8 CFU / mL. Fermentation is carried out in two stages: First stage (aerobic fermentation): Introduce 55% of the total amount of Bacillus subtilis culture solution and ferment aerobicly for 18 hours under the conditions of ventilation ratio of 0.8vvm, temperature of 35℃ and relative humidity of 65%. The second stage (micro-aerobic fermentation): Add the remaining 45% Bacillus subtilis culture, all Lactobacillus plantarum culture, and all Saccharomyces cerevisiae culture, adjust the substrate moisture content to 45%, and continue fermentation for 48 hours at a temperature of 32℃ under micro-aerobic conditions. Adjust the ventilation ratio to 0.05vvm and adopt intermittent ventilation (ventilate for 5 minutes every 2 hours) to maintain dissolved oxygen below 0.5mg / L.

[0030] (4) Post-fermentation and drying: After the base material fermentation is completed, it is naturally post-fermented for 5 hours, and then dried at 50℃ until the moisture content is less than 12%. The finished product is obtained by crushing it through a 60-mesh sieve.

[0031] Example 2 The difference between this embodiment and Example 1 is that the amount of the compound bacterial solution is 5 parts, the amount of the buffer regulator is 0.5 parts, and the ratio of viable Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae in the compound bacterial solution is 3:2:1.5 (high proportion of Lactobacillus plantarum); the buffer regulator is a mixture of citric acid, disodium hydrogen phosphate, and light calcium carbonate in a mass ratio of 1.5:0.8:1 (low buffer capacity ratio). All other components, process steps, and process parameters are the same as in Example 1.

[0032] Example 3 The difference between this embodiment and Example 1 is that the amount of the compound bacterial solution is 15 parts, the amount of the buffer regulator is 2.0 parts, and the ratio of viable bacteria of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* in the compound bacterial solution is 1:2:0.5 (high *Bacillus subtilis* ratio). The buffer regulator is a mixture of citric acid, disodium hydrogen phosphate, and light calcium carbonate in a mass ratio of 2.5:1.5:1 (high buffer capacity ratio). All other components, process steps, and process parameters are the same as in Example 1.

[0033] Example 4 The difference between this embodiment and Embodiment 1 is that, in step (1), the wet heat sterilization temperature is 110℃ and the time is 30 min; in step (3), the aerobic fermentation in the first stage has an ventilation ratio of 0.5 vvm, a temperature of 37℃, and a time of 24 h; in the second stage, the moisture content of the substrate is adjusted to 40%, and the conditions are changed to anaerobic (aeration is stopped) and fermented at 30℃ for 48 h; in step (4), the drying temperature is 55℃ and the sieve mesh is 80 mesh. The remaining components, process steps, and process parameters are the same as in Embodiment 1.

[0034] Example 5 The difference between this embodiment and Embodiment 1 is that in step (1), the heating rate of moist heat sterilization is controlled at 2℃ / min, the sterilization temperature is 121℃, and the sterilization time is 20min; in step (2), the stirring rate is 150rpm; in step (3), the aerobic fermentation ventilation rate in the first stage is 1.0vvm, the temperature is 28℃, and the time is 12h; in the second stage, the moisture content of the substrate is adjusted to 50%, and fermentation continues for 48h under micro-aerobic conditions at a temperature of 35℃, with the ventilation ratio adjusted to 0.1vvm; in step (4), the natural ripening time is 6h, the drying temperature is 45℃, and the sieve mesh is 40 mesh. The remaining components, process steps, and process parameters are the same as in Embodiment 1.

[0035] Comparative Example 1 The difference between this comparative example and Example 1 is that step (3) fermentation adopts a simultaneous inoculation method: the three bacterial liquids of Lactobacillus plantarum, Bacillus subtilis, and Saccharomyces cerevisiae are all simultaneously inoculated into the substrate treated in step (2), without staged fermentation, and are continuously fermented for 66 hours under the conditions of a ventilation ratio of 0.8 vvm and a temperature of 35°C, without switching between aerobic and microaerobic conditions. The remaining components, process steps, and process parameters are the same as in Example 1.

[0036] Comparative Example 2 The difference between this comparative example and Example 1 is that no buffer conditioner is added in step (2), and only an equal amount of sterile water is sprayed onto the base material. The remaining components, process steps, and process parameters are the same as in Example 1.

[0037] Comparative Example 3 The difference between this comparative example and Example 1 is that the buffer regulator in step (2) is composed only of citric acid and disodium hydrogen phosphate in a mass ratio of 2:1 (excluding light calcium carbonate), and the total amount used is the same as in Example 1. The remaining components, process steps, and process parameters are the same as in Example 1.

[0038] Comparative Example 4 The difference between this comparative example and Example 1 is that in step (3), the first stage does not introduce any microorganisms and is an empty fermentation stage (only the substrate is placed under the same temperature and humidity conditions for 18 hours); in the second stage, all Bacillus subtilis, all Lactobacillus plantarum, and all Saccharomyces cerevisiae are introduced. The remaining components, process steps, and process parameters are the same as in Example 1.

[0039] Comparative Example 5 The difference between this comparative example and Example 1 is that in step (3), the first stage involves inoculating the entire Bacillus subtilis culture and fermenting aerobically for 24 hours; the second stage involves inoculating the entire Lactobacillus plantarum culture and fermenting under microaerobic conditions for 24 hours; and the third stage involves inoculating the entire Saccharomyces cerevisiae culture and fermenting under microaerobic conditions for 24 hours. The remaining components, process steps, and process parameters, including microaerobic conditions, are the same as in Example 1.

[0040] Test Example 1: The test subjects were the final feed products provided in each embodiment and comparative example. Test method: Crude protein content (on a dry basis): Performed according to GB / T6432-2018 "Determination of Crude Protein in Feed - Kjeldahl Method". Test procedure: Crush the final fermented feed product and pass it through a 0.42mm standard sieve. Accurately weigh approximately 0.5g of the sample (accurate to 0.0001g) and place it in a digestion tube. Add 0.2g of copper sulfate, 6g of potassium sulfate, and 12mL of concentrated sulfuric acid to the digestion tube, mix well, and place it on a digestion furnace. Digest at 420℃ until the solution turns into a blue-green transparent liquid, and continue digestion for at least 1 hour. After cooling the digestate, place it in an automatic Kjeldahl nitrogen analyzer, add 40% sodium hydroxide solution to excess, and perform steam distillation. Collect the distillate with 2% boric acid absorbent solution (containing mixed indicator). Titrate the distillate with 0.1mol / L hydrochloric acid standard solution until the solution changes from green to light purple-red as the endpoint. Calculation: Crude protein content (%) = [(V1-V0)×c×0.014×6.25] / m×100, where: V1 is the volume of hydrochloric acid consumed in the sample titration (mL), V0 is the volume of hydrochloric acid consumed in the blank titration (mL), c is the concentration of the hydrochloric acid standard solution (mol / L), m is the sample mass (g), and 6.25 is the nitrogen-to-protein conversion factor. Dry basis content = Wet basis content / (1-Moisture content)×100% Moisture content: Perform the test according to GB / T6435-2014 "Determination of Moisture in Feed". Test steps: Place a clean weighing dish in an oven at (105±2)℃ and dry for 1 hour. Remove it and cool it to room temperature in a desiccator. Weigh it (accurate to 0.0001g), repeating until constant weight (the difference between two weighings ≤ 0.001g). Accurately weigh approximately 2g of the sample (accurate to 0.0001g) and spread it evenly in the weighed dish that has reached constant weight. Place the weighing dish in an oven at (105±2)℃ and dry it with the lid off for 4 hours. During this time, remove it, cover it, and cool it in a desiccator for 30 minutes before weighing. Place it back in the oven and dry for 1 hour, cool it, and weigh it. Repeat until constant weight (the difference between two weighings ≤ 0.001g). Calculate: Moisture content (%) = (m1-m2) / (m1-m0)×100, where: m0 is the mass of the weighing dish (g), m1 is the total mass of the sample and weighing dish before drying (g), and m2 is the total mass of the sample and weighing dish after drying (g).

[0041] Final product pH value: Follow the procedure outlined in T / SDFA002.3-2023 "Fermented Feed Raw Materials Part 3: pH Determination". Test steps: Accurately weigh 10.0g (accurate to 0.1g) of the final fermented feed product and place it in a 250mL Erlenmeyer flask. Add 90mL of distilled water (solid-liquid ratio 1:9). Place the flask on a magnetic stirrer and stir for 30min at room temperature to ensure thorough sample dispersion. After extraction, allow to stand for 5min to allow solid phase precipitation. Measure the pH value of the supernatant using a calibrated pH meter (calibrated at three points using standard buffer solutions of pH 4.00, 6.86, and 9.18). Immerse the electrode in the supernatant and record the data after the reading stabilizes (change ≤0.01 / min). Perform three parallel measurements on the same sample and take the arithmetic mean as the final result. Precision requires a range of ≤0.10 between the three measurements.

[0042] Free amino acid content (as a percentage of total protein): The test was performed according to GB / T18246-2019 "Determination of Amino Acids in Feed" – acid extraction method. The procedure was as follows: Accurately weigh approximately 2g of the final fermented feed product (accurate to 0.0001g), place it in a 50mL centrifuge tube, add 20mL of 5% sulfosalicylic acid solution (or 0.1mol / L hydrochloric acid solution), and vortex to mix. Place the centrifuge tube in an ultrasonic cleaner for ultrasonic extraction for 30min, or extract by shaking at room temperature for 60min to ensure complete dissolution of free amino acids. Centrifuge at 4000rpm for 15min and collect the supernatant. Take an appropriate amount of the supernatant and derivatize it using a pre-column derivatization method (such as OPA / FMOC derivatization) compatible with an automated amino acid analyzer. Separate and quantify the free amino acids using an automated amino acid analyzer (equipped with a cation exchange column and a UV / fluorescence detector), and calculate the content of each free amino acid using the external standard method. Calculation: Total free amino acids (%) = Sum of the contents of each free amino acid as a percentage of total protein (%) = Total free amino acids / Crude protein content (dry basis) × 100%.

[0043] Phytate phosphorus degradation rate: Following the test procedure in T / CBFIA02002-2020 "Determination of Phytase Phosphorus Release Rate - Spectrophotometric Method": Accurately weigh 2g each of the pre-fermentation substrate and the post-fermentation product (accurate to 0.0001g), and place them separately in 250mL Erlenmeyer flasks. Add 50mL of 0.2mol / L hydrochloric acid solution to the Erlenmeyer flasks, and extract by shaking at room temperature for 2 hours to ensure complete dissolution of phytate phosphorus. Filter the extract using qualitative filter paper, discard the initial filtrate, and collect the subsequent filtrate for testing.

[0044] Phytic acid phosphorus determination (ammonium vanadylmolybdate colorimetric method): Take an appropriate amount of filtrate into a 50 mL volumetric flask, add 10 mL of ammonium vanadylmolybdate colorimetric solution, and dilute to the mark with distilled water. Shake well. After standing at room temperature for 15 min for color development, measure the absorbance at a wavelength of 415 nm using a spectrophotometer. Simultaneously, plot a phosphorus standard curve (prepare a series of standard solutions with potassium dihydrogen phosphate, and determine the color using the same method). Calculation: Phytic acid phosphorus content (mg / g) = Phosphorus content obtained from the standard curve / Sample mass; Phytic acid phosphorus degradation rate (%) = (C0-C1) / C0×100%, where: C0 is the phytic acid phosphorus content (mg / g) in the substrate before fermentation, and C1 is the phytic acid phosphorus content (mg / g) in the finished product after fermentation.

[0045] Bacillus subtilis viable count: The test was conducted according to GB / T26428-2010 "Detection of Bacillus subtilis in Feed Microbial Preparations" and the general principles of the plate count method. Test procedure: Accurately weigh 10g (accurate to 0.1g) of the final fermented feed product and place it in an Erlenmeyer flask containing 90mL of sterile physiological saline (0.85wt% NaCl). Shake for 20min to prepare a plate count of 10... -1 Diluent. It was then serially diluted 10-fold with sterile physiological saline to a final concentration of 10. -6 ~10 -8 Selective culture medium for Bacillus subtilis (such as mannitol-polymyxin agar, MYP) was sterilized and cooled to approximately 50°C before pouring into plates. 0.1 mL of an appropriate dilution was evenly spread onto the surface of the selective plate, with three replicates for each dilution. The plates were inverted and incubated at (30±1)°C for 24–48 h. Plates with colony counts between 30 and 300 were selected for counting, and the number of typical colonies was recorded (Bacillus subtilis typically appears as pink colonies with a precipitate ring on MYP). The viable count (CFU / g) was calculated as: viable count (CFU / g) = colony count × dilution factor / spread volume (mL), expressed as logarithmic (logCFU / g).

[0046] Viable count of *Lactobacillus plantarum*: The test was conducted according to GB4789.35-2023 "National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria Examination" and the plate count method. The test procedure was the same as for *Bacillus subtilis*. MRS agar medium was used, sterilized and cooled to approximately 50°C. 0.1 mL of the appropriate dilution was evenly spread on the surface of an MRS plate, with three replicates for each dilution. The plates were inverted and incubated in a (37±1)°C anaerobic incubator (or a sealed container with an anaerobic gas-generating bag) for 48 hours.

[0047] Counting: Select plates with colony counts between 30 and 300 for counting (Lactobacillus plantarum appears as milky white, round, raised colonies on MRS medium). Calculation: Same as the calculation method for Bacillus subtilis.

[0048] Saccharomyces cerevisiae viable count: The test was conducted according to GB4789.15-2016 "National Food Safety Standard - Microbiological Examination of Food: Counting of Molds and Yeasts" and the plate count method. The test procedure was the same as for Bacillus subtilis. Bengal red agar (containing chloramphenicol) was used, sterilized and cooled to approximately 50°C. 0.1 mL of the appropriate dilution was evenly spread on the surface of a Bengal red agar plate, with three replicates for each dilution. The plates were inverted and incubated at (28±1)°C for 48-72 hours. Plates with colony counts between 30-300 were selected for counting (Saccharomyces cerevisiae appears as pink, round, moist colonies on Bengal red agar). The counting method was the same as for Bacillus subtilis.

[0049] Total volatile flavor compounds: These were determined using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The procedure was as follows: Accurately weigh 5.0 g of the final fermented feed product and place it in a 20 mL headspace sample vial. Add an internal standard (e.g., 2-octanol, final concentration 1 μg / mL) and immediately seal the vial. Place the vial in a water bath at (60±1) °C for 30 min to equilibrate the volatile components in the headspace. Insert the SPME extraction head (50 / 30 μm DVB / CAR / PDMS fiber head) into the headspace vial and perform headspace adsorption extraction at 60 °C for 30 min. Insert the extraction head into the GC-MS inlet and desorb at 250 °C for 5 min.

[0050] The chromatographic column used for GC-MS analysis was a DB-Wax capillary column (30m × 0.25mm × 0.25μm). The carrier gas was high-purity helium (≥99.999%), with a flow rate of 1.0 mL / min. The specific temperature program was: 40℃ for 3 min, increased to 200℃ at 5℃ / min, then increased to 230℃ at 10℃ / min, and held for 5 min. The mass spectrometry conditions were: EI ion source, electron energy 70 eV, scan range m / z 35-450. Qualitative analysis was performed by comparison with the NIST mass spectrometry library, and semi-quantitative calculation (μg / g) was performed by the ratio of the peak area of ​​each volatile component to the peak area of ​​the internal standard. Total amount calculation: Total volatile flavor substances = sum of the contents of each detected volatile component.

[0051] Small peptide content: Trichloroacetic acid (TCA) precipitation-Folin-Ciocalteu method (Lowry method) was used for testing. Test procedure: Accurately weigh 2 g (accurate to 0.0001 g) of the final fermented feed product, add 20 mL of 15% TCA solution, and vortex to mix. Let stand at room temperature for 30 min to precipitate large protein molecules. Centrifuge at 4000 r / min for 15 min, and collect the supernatant (containing small peptides and free amino acids). Take an appropriate amount of the supernatant and determine the soluble protein / peptide content using the Folin-Ciocalteu method (Lowry method); separately, take another portion of the supernatant and determine the free amino acid content according to the free amino acid determination method. Calculation: Small peptide content (mg / g, dry basis) = (Total soluble protein / peptide in supernatant - Total free amino acids) / Sample dry basis mass.

[0052] Lactic acid content: High-performance liquid chromatography (HPLC) was used for testing. The procedure was as follows: Accurately weigh 5g of the final fermented feed product (accurate to 0.01g), add 50mL of distilled water, and extract by shaking for 30min. Centrifuge at 4000rpm for 15min, and filter the supernatant through a 0.22μm microporous membrane. HPLC analysis: The chromatographic column was a C18 reversed-phase column (250mm × 4.6mm, 5μm); the mobile phase was 0.01mol / L potassium dihydrogen phosphate solution (pH 2.7), the flow rate was 0.8mL / min; the detector was a UV detector with a detection wavelength of 210nm; the column temperature was 30℃; a standard curve was plotted using lactic acid standards, and quantification was performed using the external standard method. The lactic acid concentration in the sample was determined from the standard curve and converted to mg / g of sample.

[0053] Procedure for determining the spore count rate of Bacillus subtilis: Take the final product of fermented feed, dilute and spread it according to the viable cell count determination method, and divide it into two groups: The first group is directly spread and cultured on MYP plates (total viable cell count); the second group is heated in an 80℃ water bath for 10 min before being spread and cultured (spore germination count only); Calculation: Spore count rate (%) = Number of colonies after heating / Number of colonies without heating × 100% Simulated intestinal germination rate determination procedure: Take the final product of fermented feed and prepare 10 according to the viable count determination method. -2 Diluent. Take 5 mL of the diluent and add it to 45 mL of simulated intestinal fluid (containing 0.05 mol / L phosphate buffer, pH 6.8, and 0.1% trypsin). Incubate anaerobically at 37℃ for 4 h. After incubation, take samples and perform spread counting according to the Bacillus subtilis viable count method. Calculation: Simulated intestinal germination rate (%) = (Number of viable bacteria after simulated intestinal fluid treatment / Original total number of viable bacteria) × 100% Sensory evaluation: A standard sensory evaluation method commonly used in the feed industry was employed. The testing procedure involved selecting 10 trained evaluators (with experience in sensory evaluation of feed or food). The sample numbers for each example and comparative example were randomly assigned. 50g of each sample was placed in a uniform opaque container and allowed to equilibrate at room temperature for 30 minutes.

[0054] Evaluation dimensions: Aroma (weight 40%): Assess the intensity of the sour and aromatic flavor, presence of any sour or off-odors, etc. Color (weight 20%): Assess color uniformity, presence of mold, etc. Texture (weight 20%): Assess looseness, presence of lumps, etc. Overall Acceptability (weight 20%): Overall level of acceptability Scoring criteria: A 10-point scale (1-10 points) is used, with 1 point being the worst and 10 points being the best. Each sample is scored independently by 10 evaluators, and the average score is taken.

[0055] Data statistics: Calculate the weighted total score of each sample across the four dimensions and then calculate the average.

[0056] The test results are shown in Table 1-4.

[0057] Table 1. Test results of performance indicators for each embodiment and comparative example. Table 2. pH changes during the fermentation process of Examples 1-5 and Comparative Examples 2-3 Table 3. Results of flavor compound tests in Example 1 and comparative examples. Table 4. Results of Bacillus subtilis test in Example 1 and each comparative example. As shown in Tables 1 to 4, the examples significantly outperformed the comparative examples in key indicators such as crude protein content, free amino acid ratio, phytic acid phosphorus degradation rate, small peptide content, and total volatile flavor compounds. Furthermore, the final product pH value remained consistently within the target range of 4.0-4.5, and the sensory scores were significantly higher. This trend may be attributed to the synergistic effect of the metabolic time-series decoupling strategy and the dynamic buffer system employed in this invention.

[0058] Specifically, Example 1, as the optimal formulation, exhibits the most balanced performance across all indicators. This is presumably because in the first stage, Bacillus subtilis fully secretes proteases and phytases under a near-neutral environment, providing ample small peptides, free amino acids, and fermentable sugar substrates for subsequent lactic acid bacteria and yeast metabolism. In the second stage, the inoculation of Lactobacillus plantarum and Saccharomyces cerevisiae occurs in a low redox potential environment following aerobic depletion, resulting in moderate lactic acid production and active yeast flavor metabolism. The final product contains high levels of esters and alcohols (especially isoamyl alcohol and 2...). It is rich in phenylethanol, with a pure sour and fragrant flavor and no sour or rancid taste. In Example 2, due to the increased proportion of Lactobacillus plantarum and the reduced buffer capacity, the pH dropped faster. Although the lactic acid content was higher, the phytic acid phosphorus degradation rate and the total amount of flavor substances decreased. It is speculated that the excessive acidification in the early stage partially inhibited the enzyme activity of Bacillus subtilis and yeast metabolism. In Example 3, the proportion of Bacillus subtilis was increased and the buffer capacity was enhanced. Although the protein degradation and phytic acid degradation effects were outstanding, the lactic acid accumulation was insufficient, the pH of the final product was higher, the flavor modification was slightly weaker, and the sensory score was slightly lower than that of Example 1. Examples 4 and 5 were adjusted in terms of aeration strategy or sterilization and drying parameters, but the core inoculation sequence and buffer system remained unchanged. Therefore, the performance was close to that of Example 1, with only slight fluctuations in viable cell count and flavor substances due to differences in aerobic intensity or heat treatment.

[0059] Comparative Example 1 used a simultaneous inoculation method, where all strains competed for nutrients and space simultaneously. The enzyme production window of Bacillus subtilis was compressed by the rapid acid production of lactic acid, and the activity of yeast was inhibited at low pH. Therefore, the protein degradation rate, flavor substances, and viable Bacillus subtilis count all decreased significantly, and the final product had an excessively low pH, resulting in a severely sour and rancid taste. Comparative Example 2 (without buffer regulator) experienced a sudden drop in pH during fermentation, almost completely inhibiting the metabolism of Bacillus subtilis and yeast. This may be because under acute acid stress, most Bacillus subtilis did not initiate the energy-consuming spore differentiation program, but instead underwent irreversible acidolysis and death. It is speculated that the low spore rate in Comparative Example 2 was not due to the failure of acid stress to induce spores, but rather to the excessively severe acid shock causing premature apoptosis of spore-producing mother cells. Comparative Example 3 (without calcium carbonate) had some buffer pair, but lacked a backup alkali source. The pH still dropped too quickly during fermentation, resulting in significantly lower phytic acid degradation and flavor generation compared to the examples containing calcium carbonate. This indicates that the slow-release neutralization effect of light calcium carbonate is crucial when the pH drops below 5.0. Comparative Example 4 (first-stage empty fermentation) lacked the pre-enzymatic hydrolysis and aerobic process of Bacillus subtilis. In the second stage, the simultaneous inoculation of all three bacteria resulted in metabolic disorder. Although the final product had a suitable pH, the efficiency of protein and phytic acid degradation was significantly insufficient, and the flavor profile was lower than in the Example. Comparative Example 5 (three-stage sequential inoculation) exhibited some temporal sequence, but the bacterial strains at each stage were completely separated, failing to form a metabolic relay (Bacillus subtilis was not added again in the second stage, and the late-stage inoculation of yeast lacked the synergistic effect of the acidic environment created by lactic acid bacteria). This resulted in all its indicators being lower than in the Example. Notably, although its Bacillus subtilis spore rate and simulated intestinal germination rate were higher, the content of free amino acids and small peptides was still significantly lower than in Example 1, indicating insufficient functional synergy.

[0060] As shown in Table 2, all examples exhibited a smooth transition from an initial pH of 6.2 to a final product pH of 4.0-4.5, verifying the dynamic pH regulation capability of the buffer system. Comparative Examples 2 and 3, however, showed a sharp drop, further confirming the crucial role of light calcium carbonate and the buffer in preventing a rapid decrease in acidity. In Table 4, the spore rate (62.3%) and simulated intestinal germination rate (89.6%) of Bacillus subtilis in Example 1 were significantly higher than those in the comparative examples. This is presumably because the two-stage inoculation strategy allowed the bacteria proliferating in the first stage to fully form spores under the high-acid, low-oxygen environment of the second stage. In contrast, in the groups with simultaneous inoculation or lack of buffer, Bacillus subtilis was under prolonged acid stress, resulting in insufficient spore formation and consequently affecting its probiotic effect after reaching the intestines. Overall, the data in Tables 1 to 4 indicate that this application, through the organic combination of staged inoculation and the buffer system, can achieve a metabolic relay of enzymatic hydrolysis, acidification, and flavor modification, solving the technical challenge of competitive inhibition by multiple strains.

[0061] Test Example 2 Test method: 500 pigs of similar age and parity, weighing 40±2kg, with half males and half females, were randomly divided into 10 groups and fed with the microbial feeds formulated in the examples and comparative proportions. The experiment lasted for 30 days. The pigs were weighed on an empty stomach at fixed times at the beginning and end of each week, and the starting weight and ending weight of each stage were recorded to calculate the average daily weight gain of each pig at each growth stage and throughout the entire period. At the same time, the number of diarrhea episodes was recorded to calculate the diarrhea rate, and the phosphorus emissions were measured by collecting feces. The experimental results are shown in Table 5.

[0062] Table 5. Effects of microbial feeds in various examples and comparative examples on pigs. As shown in Table 5, the average daily weight gain, feed efficiency, and diarrhea rate of pigs in each embodiment group were significantly better than those in the control group, and the phosphorus emissions in the feces were significantly reduced. This indicates that the fermented feed of the present invention has practical application advantages in improving animal intestinal health, increasing nutrient utilization, and reducing phosphorus emissions.

[0063] In Example 1, with similar daily feed intake, the highest daily weight gain was observed. This may be related to the high content of small peptides, free amino acids, and rich flavor compounds in the feed, which effectively promote feed intake and digestion. Simultaneously, the diarrhea rate was low, possibly attributed to the suitable lactic acid content and pH value in the final product, which effectively inhibited intestinal pathogens without excessive acidification that could damage the intestinal mucosa. From the perspectives of fecal phosphorus emission and phytic acid degradation rate, Example 1 showed a substantial improvement in phosphorus utilization. Although the daily weight gain and conversion rate of Examples 2 and 3 were slightly lower than those of Example 1, they were still far superior to the comparative example. Example 2, due to its higher lactic acid content, also had a lower diarrhea rate, but its slightly inferior flavor might have affected feed intake. Example 3, due to more thorough phytic acid degradation, had lower phosphorus emission, but insufficient lactic acid led to a slightly higher diarrhea rate, reflecting that different formulations have different focuses, with Example 1 showing the best overall balance.

[0064] The diarrhea rates of Comparative Example 1 (simultaneous inoculation) and Comparative Example 2 (no buffer) were as high as 6.8% and 7.2%, respectively, with daily weight gain of only 630 g and 580 g. This is presumably due to the high levels of anti-nutritional factors in the feed, low protein digestibility, and unpleasant sour taste leading to decreased feed intake. Additionally, the excessively low pH of 3.4-3.6 may have directly irritated the intestines, causing diarrhea. While the breeding effects of Comparative Example 3 (without calcium carbonate) and Comparative Example 4 (empty fermentation) were slightly better than the former two, they were still significantly lower than the example. This indicates that even with some buffering or later inoculation of microorganisms, without prior enzymatic hydrolysis and dynamic pH control, the ideal intestinal health improvement effect cannot be achieved. Comparative Example 5 (three-stage sequential inoculation) had a daily weight gain of 670 g and a conversion rate of 0.432, close to the level of the example, but the diarrhea rate was still as high as 7.0%. This is presumably because although its flavor compounds were relatively rich, the content of small peptides and free amino acids was insufficient, and the Bacillus subtilis spore rate was low, resulting in insufficient probiotic effects after reaching the intestines and failure to effectively competitively exclude pathogens.

[0065] The above are merely embodiments of the present invention and are 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 principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A compound microbial fermented feed for improving animal gut health, characterized in that, The product is composed of the following components by weight: 40-60 parts soybean meal, 20-35 parts corn flour, 10-20 parts wheat bran, 5-15 parts compound bacterial solution, and 0.5-2.0 parts buffer regulator; wherein the compound bacterial solution is prepared by compounding Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae in a live cell ratio of 1-3:1-2:0.5-1.5; and the buffer regulator is prepared by compounding citric acid, disodium hydrogen phosphate and light calcium carbonate in a mass ratio of 1.5-2.5:0.8-1.5:

1.

2. The compound microbial fermented feed for improving animal intestinal health according to claim 1, characterized in that, The compound bacterial solution is composed of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae in a live cell ratio of 3:2:1.5, and the buffer regulator is composed of citric acid, disodium hydrogen phosphate and light calcium carbonate in a mass ratio of 1.5:0.8:

1.

3. The compound microbial fermented feed for improving animal intestinal health according to claim 1, characterized in that, The compound bacterial solution is composed of Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae in a live cell ratio of 1:2:0.5, and the buffer regulator is composed of citric acid, disodium hydrogen phosphate and light calcium carbonate in a mass ratio of 2.5:1.5:

1.

4. The compound microbial fermented feed for improving animal intestinal health according to claim 1, characterized in that, The final feed product has a moisture content of <12%, a free amino acid content of ≥11.8% of total protein, a phytic acid phosphorus degradation rate of ≥88.5%, and a pH of 4.

0. 4.

5.

5. A method for preparing a compound microbial fermented feed for improving animal intestinal health as described in any one of claims 1 to 4, characterized in that, The following steps were followed to prepare the following: (1) Base material pretreatment: Mix soybean meal, corn flour and wheat bran evenly, add water to adjust the moisture content of the base material to 38%-42%, sterilize by moist heat at 110-121℃ for 20-30 minutes, and cool to 35-40℃. (2) Buffer system construction: Dissolve the buffer regulator in sterile water and spray it into the base material after step (1). Stir and mix for 10-20 minutes to stabilize the pH value of the system at 6.0-6.

5. (3) Fermentation: First stage fermentation: First, add 50%-60% of the total amount of Bacillus subtilis bacterial solution to the substrate treated in step (2), and then perform aerobic fermentation for 12-24 hours at a temperature of 28-37℃ and a relative humidity of 60%-70%. Second stage fermentation: Add the remaining Bacillus subtilis culture, all Lactobacillus plantarum culture, and all Saccharomyces cerevisiae culture, adjust the moisture content of the substrate to 40%-50%, and continue fermentation for 36-60 hours under anaerobic or micro-aerobic conditions at a temperature of 30-35℃. (4) Post-fermentation and drying: After the fermentation is completed, the base material is naturally post-fermented for 4-6 hours, dried at 45-55℃ until the moisture content is less than 12%, and then crushed and sieved to obtain the finished product.

6. The preparation method according to claim 5, characterized in that, The heating rate of the moist heat sterilization in step (1) is less than or equal to 3℃ / min.

7. The preparation method according to claim 5, characterized in that, The stirring speed in step (2) is 150-250 rpm.

8. The preparation method according to claim 5, characterized in that, The aeration ratio for aerobic fermentation in step (3) is 0.5-1.0vvm.

9. The preparation method according to claim 5, characterized in that, In step (3), the ventilation ratio needs to be adjusted to 0.05-0.1vvm when using the micro-aerobic mode, and intermittent ventilation should be used for 5 minutes every 2 hours to maintain dissolved oxygen below 0.5mg / L.

10. The preparation method according to claim 5, characterized in that, The sieve used in step (4) has a mesh size of 40-80.

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