Fermentation process of biological fermentation material

Abstract

The invention discloses a fermentation process of a biological fermentation material, and belongs to the technical field of fermented feeds, and the process sequentially comprises nine procedures of micro powder carrier preparation, substrate pretreatment, strain propagation primary fermentation, first stirring, dry and wet fermentation, second stirring, finished product bagging, finished product third fermentation, third stirring and cooling. A plant micro powder carrier with the particle size smaller than or equal to 300 micrometers is adopted, a substrate treatment mode that free water is not added in the whole process is adopted, high-concentration viable bacteria fermentation liquor obtained through compound strain propagation is combined with compound enzyme for dry-wet synergistic fermentation, and temperature-control and humidity-control after-ripening fermentation control of three-time stirring and breathable film bagging is adopted for assistance. And high-activity planting of lactic acid bacteria and saccharomycetes in the fermented material is realized. The safe and stable fermented product obtained by the method has good free-running property, palatability and nutrition release effect, and is suitable for feeding various animals such as ruminant livestock and poultry.

Description

Technical Field

[0001] This invention relates to the technical field of fermentation materials, and in particular to a fermentation process for biological fermentation materials. Background Technology

[0002] With the increasing scale of livestock and poultry farming, traditional feed systems face a series of challenges, including the restriction and prohibition of antibiotic use, uneven growth performance, and high levels of manure emissions. Against this backdrop, bio-fermented feed, with its comprehensive advantages such as improving animal gut health, increasing feed utilization, reducing the growth of harmful microorganisms, and reducing pollution during the farming process, is gradually becoming an important direction for the development of the feed industry.

[0003] The market currently offers a wide variety of so-called "fermented feeds," but they generally suffer from problems such as crude fermentation processes, insufficient microbial activity, poor process stability, and low nutrient conversion efficiency. For example, some products use dormant dry powder microbial cultures, simply mixing them into raw materials and free water before sealing and fermentation. This neglects the systematic control of the microecological environment, microbial propagation mechanisms, and fermentation kinetics, ultimately resulting in a limited number of probiotics in the product, incomplete fermentation, or even excessive levels of contaminating bacteria. Feed produced in this way not only fails to significantly improve feed intake and growth performance in animals but may also cause intestinal stress, hindering its widespread application in large-scale farming.

[0004] Furthermore, existing technologies have the following limitations: On the one hand, most fermentation processes fail to effectively address issues such as large carrier particle size, poor adsorption capacity, and imprecise moisture control, resulting in uneven colonization of microorganisms, uneven fermentation, or easy clumping. On the other hand, conventional processes lack systematic segmented control over different fermentation stages, such as microbial propagation, substrate conditioning, wet-heat fermentation, and post-fermentation, affecting the reproduction and metabolism of beneficial microorganisms and the activity indicators of the final product. Especially in a "free water" environment, it easily leads to mold growth and toxin accumulation, seriously affecting feed safety and storage stability.

[0005] Therefore, there is an urgent need to develop a bio-fermentation process with the core functions of compound strain propagation, precise humidity control, construction of non-free water environment, multi-stage fermentation synergy, and dynamic control of microbial community. The entire chain should be optimized from aspects such as process flow, equipment matching, and packaging form to improve fermentation stability and product functionality, and achieve a synergistic effect of high efficiency, high safety, high palatability, and low cost of fermented feed, so as to meet the urgent needs of modern aquaculture for green, safe, and efficient feed products. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a fermentation process for biological fermentation materials.

[0007] A fermentation process for a biological fermentation material includes the following nine steps:

[0008] S1 micronized carrier preparation: The plant-based carrier was pulverized to an average particle size ≤300μm and dried at below 55℃ to a moisture content ≤12%;

[0009] S2 substrate pretreatment: Soybean by-products or sweet potato by-products are dehydrated by pressure filtration to adjust the moisture content to 60%–85%, without adding free water throughout the process;

[0010] S3 strain propagation through primary fermentation: Using whey powder (3%–6% of the total culture medium mass) and brown sugar (1%–3% of the total culture medium mass) as nutrient sources, the compound strain was propagated at 32℃–38℃ and pH 5.5–6.2 for 12–24 hours to obtain a viable cell concentration ≥1×10⁻⁶. 9 Fermentation broth with CFU / g;

[0011] S4 First stirring: Mix the micro powder carrier from step S1 with the substrate pretreated in step S2 and stir until homogeneous;

[0012] S5 Dry and wet fermentation: Add the fermentation liquid and compound enzyme from step S3 to the well-stirred material. The total amount of fermentation liquid and compound enzyme added accounts for 2% to 6% of the total mass of the material. After mixing thoroughly, ferment under anaerobic conditions at 30℃ to 42℃ for 18 to 36 hours.

[0013] S6 Second mixing: After the material has undergone dry and wet fermentation, it is passed through a vibrating screen and mixed again;

[0014] S7 Finished Product Bagging: Pack the material into double-sided 20-mil thick microporous breathable membrane packaging bags, with a packing density of 0.5~0.7kg / L;

[0015] S8 finished product third fermentation: Place the bagged material in a 28℃~45℃ insulated room and continue fermentation for 24h~72h;

[0016] S9 Third stirring and cooling: Take out the bagged material, break the bag and stir for 3 minutes, then let it cool naturally to obtain the finished biological fermentation material with pH≤4.0 and water activity≤0.60.

[0017] By pulverizing the plant-based carrier to an average particle size ≤300μm, the specific surface area is effectively increased, facilitating the adsorption, fixation, and full contact of the inoculum with the fermentation substrate. Drying to ≤12% moisture content below 55℃ ensures good flowability and low water activity of the carrier, helping to control the overall humidity of the fermentation material and inhibit the growth of contaminating microorganisms. Timely cooling to <40℃ avoids heat damage to the inoculum activity, ensuring the success rate of subsequent inoculation. Pressure filtration dehydration of sweet potato or soybean by-products controls the moisture content to the range of 40%–55%, effectively creating a low-activity water environment without free water, providing a stable microecological foundation for anaerobic fermentation. Eliminating free water significantly reduces the risk of mold and spoilage bacteria growth, improving product safety and shelf life. Whey powder and brown sugar are used to provide protein and carbon sources, respectively. Under optimized conditions of 32℃–38℃ and pH 5.5–6.2, the complex inoculum is propagated, effectively increasing the number and activity of bacteria, achieving a viable cell concentration ≥1×10⁻⁶. 9 The high-efficiency fermentation broth with CFU / g significantly enhances subsequent fermentation efficiency and the product's probiotic function.

[0018] The first stirring process thoroughly mixes the micro-powder carrier and substrate in a specific ratio, ensuring even nutrient distribution and promoting subsequent inoculation and diffusion of the microorganisms. This creates a loosely structured, well-aerated mixed substrate, laying a homogeneous foundation for the fermentation microenvironment. Dry and wet fermentation involves adding propagation broth and a compound enzyme (2%–6% total amount) and fermenting under anaerobic conditions at 30℃–42℃ for 18–36 hours. This effectively promotes the proliferation of beneficial microorganisms, while the compound enzyme synergistically degrades anti-nutritional factors such as proteins and non-starch polysaccharides, improving feed utilization and nutrient release efficiency. After dry and wet fermentation, a vibrating screen removes clumps, followed by re-stirring. This effectively eliminates uneven fermentation in certain areas, improves product consistency, and ensures uniform redistribution of the microbial community in the material, enhancing the microecological stability of the final product. The material is packaged in double-sided 20-mil thick microporous breathing membrane bags (filling density 0.5–0.7 kg / L), ensuring both respiration and moisture control while preventing excessive oxygen entry and spoilage. This creates a suitable microenvironment for post-fermentation, facilitating flavor precipitation and the formation of a sour and aromatic flavor.

[0019] The final fermentation stage, conducted at 28℃~45℃ for 24h~72h, promotes further degradation of residual substrates, producing metabolites such as lactic acid and small peptides. This results in a final product with uniform acidity and stable flavor, while also enhancing probiotic activity and competitive advantage. After stirring the bag for 3 minutes, it is allowed to cool naturally to break up fermentation clumps, terminating the fermentation process and lowering the product temperature. This maintains bacterial activity and product flavor, providing stable quality control assurance for packaging and use.

[0020] Preferably, the compound microbial strain comprises Lactobacillus plantarum, Enterococcus faecalis, Pediococcus pentosaceus, and Saccharomyces cerevisiae, and the ratio of each microbial strain at the end of propagation is (5-8):(1-3):(1-3):(1-2).

[0021] Synergistic interactions among the microbial communities create a stable fermentation ecosystem. Lactic acid bacteria rapidly lower the pH and inhibit harmful bacteria; yeast enhances flavor and enzyme activity; and Enterococcus faecalis and Pediococcus pentosaceus strengthen stress resistance and intestinal barrier function. The ratio is controlled at (5–8):(1–3):(1–3):(1–2) to ensure a balance between symbiosis and metabolic efficiency.

[0022] Preferably, the complex enzyme is composed of acidic protease, neutral protease, xylanase and β-glucanase, and the total enzyme activity is controlled at 5000-10000 U / kg based on the acidic protease activity.

[0023] The synergistic action of acidic protease, neutral protease, xylanase, and β-glucanase can comprehensively degrade anti-nutritional factors, release small protein peptides and soluble sugars, improve feed digestibility, reduce feed conversion ratio, and enhance daily weight gain in animals; while controlling enzyme activity within a reasonable range prevents enzyme inactivation and waste.

[0024] Preferably, the fermentation in step S5 is considered complete when the temperature of the mixture increases by ≥5°C and the pH drops to 4.2–4.5.

[0025] This dual-indicator control can reflect the metabolic activity of the microbial community and effectively prevent over-fermentation or stagnation. It is a key control point for dynamically monitoring the maturity of fermentation and helps to ensure the stability of product batches.

[0026] Preferably, the air permeability of the microporous breathing membrane is 1000–1200 mL / (m²). 2 • 24h • 0.1MPa), water vapor transmission rate 3–5 g / (m 2 •24h).

[0027] This packaging structure effectively regulates the gas concentration and humidity inside the bag, preventing condensation and secondary mold growth, while maintaining a suitable CO2 concentration and moisture balance. It is one of the core elements for ensuring the safety, flavor stability, and survival of beneficial bacteria in the finished product.

[0028] Preferably, during the fermentation of the finished product in step S8, the CO2 concentration inside the bag is maintained at 3% to 8%.

[0029] A moderately anaerobic environment can activate the metabolism of lactic acid bacteria, increase the content of organic acids and the ability to inhibit bacteria, while avoiding excessive anaerobicness that could lead to putrefaction, gas production or microbial imbalance, thus improving the functionality and safety of fermented materials.

[0030] Preferably, the micronized carrier in step S1 is cooled to <40°C immediately after vacuum drying before proceeding to step S4.

[0031] The above operations can reduce the impact of residual heat on the activity of subsequent fermentation strains, reduce the risk of protein denaturation and oxidation, and improve process continuity and fermentation stability.

[0032] Preferably, in step S5, the relative humidity of the dry and wet fermentation is maintained at 65% to 80%, and intermittent venting is performed for 2 minutes at 6h, 12h and 24h of fermentation.

[0033] Maintaining a reasonable humidity range keeps the microbial metabolism active and prevents clumping; intermittently releasing metabolic heat and accumulated gases at different times helps prevent the formation of "dead zones" in the anaerobic system, improving fermentation uniformity and final product quality.

[0034] Preferably, the viable count of lactic acid bacteria in the obtained bio-fermentation material is ≥1×10⁻⁶. 8 CFU / g, yeast viable count ≥1×10⁻⁶ 6 CFU / g, Salmonella not detected, and aflatoxin B1 content ≤20μg / kg.

[0035] A high number of viable bacteria indicates that the product has strong biological functions, such as antibacterial and digestive-promoting effects; the absence of Salmonella and the compliance of aflatoxin with national standards are key safety indicators to ensure that the product can be safely used in livestock and poultry feed.

[0036] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following aspects:

[0037] This invention constructs a three-stage fermentation system consisting of "primary fermentation for strain propagation, dry and wet fermentation, and fermentation within the finished product bag," coupled with three stirring and vibrating sieving operations throughout the process. This ensures that the strain remains in an active reproductive state throughout the entire fermentation process, significantly improving the colonization ability and metabolic efficiency of beneficial bacteria, and overcoming common problems in existing technologies such as uneven fermentation and strain activity attenuation. Compared to the traditional method of directly fermenting with added dry powder strains, this invention first propagates the compound strain to a viable cell concentration ≥1×10⁻⁶. 9 CFU / g effectively improved the density of initiating bacteria and community stability during fermentation.

[0038] This invention employs a pressure filtration and water control process during substrate pretreatment, eliminating the addition of free water throughout the entire process. Combined with the adsorption characteristics of the micronized carrier, it constructs a low water activity, high aeration, and weakly free moisture environment, effectively inhibiting the growth of contaminating bacteria and the release of toxins, thus endowing the product with excellent microbial stability and shelf-life safety. The composite strain design integrates *Lactobacillus plantarum*, *Enterococcus faecalis*, *Pediococcus pentosaceus*, and *Saccharomyces cerevisiae*, proliferating in a synergistic ratio to achieve multiple functions including lactic acid production, flavor enhancement, and probiotic colonization. Simultaneously, it incorporates a complex of enzymes such as acidic protease, neutral protease, xylanase, and β-glucanase to synergistically degrade proteins and non-starch polysaccharides, improving nutrient release efficiency and digestibility.

[0039] Regarding fermentation environment control, this invention employs double-sided 20-micron thick microporous breathing membrane bags with a packing density of 0.5–0.7 kg / L, and conducts the final fermentation process in an insulated room within the range of 28℃–45℃. The CO2 concentration inside the bag is controlled at 3%–8%, achieving a combined effect of oxygen inhibition, humidity control, and heat dissipation in biological fermentation environment regulation. This avoids the problems of off-odors, gas production, and mold growth caused by excessive sealing in traditional fermentation materials. The final product has a pH ≤ 4.0 and a water activity ≤ 0.60, exhibiting good flowability, uniform particle size, and non-stickiness, making it suitable for transportation and storage.

[0040] The test results showed that the number of viable lactic acid bacteria in the bio-fermentation material obtained by this fermentation process was ≥1×10⁻⁶. 8 CFU / g, yeast viable count ≥1×10⁻⁶ 6 CFU / g, Salmonella not detected, aflatoxin B1 content ≤20μg / kg, fully compliant with feed safety standards, and possesses comprehensive effects such as regulating animal intestinal flora, increasing feed intake, enhancing stress resistance, and reducing the incidence of diarrhea. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the embodiments.

[0042] Example 1

[0043] This embodiment discloses a fermentation process for biological fermentation materials, including the following steps:

[0044] Preparation of S1 micronized carrier:

[0045] Select plant-based raw materials that meet the "GB 13078-2017 Feed Hygiene Standard", such as rice bran, corn, or wheat bran (in this example, rice bran and corn are mixed at a mass ratio of 70:30). Grind these materials using a hammer mill until the average particle size is no greater than 300 μm. Immediately after grinding, dry the materials with hot air circulation at 50°C until the moisture content is ≤12%. After drying, rapidly cool the materials to <40°C to prevent heat damage to the inoculum, and then set aside for later use.

[0046] S2 substrate pretreatment:

[0047] A mixture of sweet potato distiller's grains and soybean residue (mass ratio 1:1) was selected as the substrate and filtered at room temperature using a plate and frame filter press until the moisture content was controlled at 75%. No free water was added during the entire process, and all substrates used were food-grade by-products with good resistance to contaminants and dietary fiber structure.

[0048] S3 strain propagation and primary fermentation:

[0049] Dissolve whey powder (5% of the culture medium mass) and brown sugar (2% of the culture medium mass) in sterile water and adjust the pH to 5.8. Inoculate with a compound bacterial culture under aseptic conditions; its composition is as follows:

[0050] Lactobacillus plantarum: 8 portions

[0051] Enterococcus faecalis: 2 portions

[0052] Pediococcus pentosaceus: 2 portions

[0053] Brewing yeast: 1 part

[0054] The total vaccination dose was 1×10 7 The concentration of CFU / mL was increased using a shake flask or tank culture method, and the culture was carried out at 35℃ and pH 5.8 for 18 hours to obtain a compound bacterial fermentation broth. At the end of fermentation, the total viable bacterial concentration in the broth was ≥1×10⁻⁶. 9 CFU / g.

[0055] S4 First stirring:

[0056] Mix the micronized carrier obtained in step S1 with the substrate obtained in step S2 at a mass ratio of 1:1, and mix for 10 minutes using a horizontal ribbon mixer to ensure uniform mixing and no agglomeration.

[0057] S5 Dry and Wet Fermentation

[0058] Add the fermentation broth and compound enzyme preparation obtained in step S3 to the mixture obtained in step S4 at a ratio of 5% of the total mass. The compound enzyme is added with a total enzyme activity of 8000 U / kg based on the acidic protease activity meter. The composition is as follows:

[0059] Acidic protease: 4000 U / kg

[0060] Neutral protease: 2000 U / kg

[0061] Xylanase: 1000 U / kg

[0062] β-glucanase: 1000 U / kg

[0063] After thorough mixing, the mixture was placed in a fermentation tank and subjected to solid-state fermentation for 30 hours under anaerobic conditions at 35℃ and 70% relative humidity. Intermittent venting was performed for 2 minutes at 6h, 12h, and 24h of fermentation. Fermentation was considered complete when the material temperature increased by ≥5℃ and the pH dropped to 4.3.

[0064] S6 Second Stirring:

[0065] After fermentation, the material is sieved through a 2mm aperture vibrating screen to remove large particles and clumps, and then stirred again for 5 minutes using a horizontal mixer to ensure uniform distribution of the inoculum.

[0066] S7 finished product packaging:

[0067] Using an air permeability of 1100 mL / (m 2 •24h•0.1MPa), water vapor transmission rate is 4g / (m 2 Fermentation materials were filled into double-sided 20-mil thick microporous breathing membrane bags (24h), with the filling density controlled at 0.6kg / L, and then sealed and placed in an insulated environment.

[0068] S8 finished product third fermentation:

[0069] Place the packaging bags in an insulated room at 38°C for 48 hours, maintaining a CO2 concentration of 5% inside the bags. Do not disturb the bags during fermentation.

[0070] S9 Third stirring and cooling:

[0071] After fermentation, break the bag and stir for 3 minutes, then let it cool naturally to room temperature to obtain the finished fermented material with pH ≤ 4.0 and water activity ≤ 0.60.

[0072] Example 2

[0073] Preparation of S1 micronized carrier:

[0074] Select plant-based raw materials that meet the "GB 13078-2017 Feed Hygiene Standard", such as rice bran, corn, or wheat bran (in this example, rice bran and corn are mixed at a mass ratio of 70:30). Grind these materials using a hammer mill until the average particle size is no greater than 300 μm. Immediately after grinding, dry the materials with hot air circulation at 50°C until the moisture content is ≤12%. After drying, rapidly cool the materials to <40°C to prevent heat damage to the inoculum, and then set aside for later use.

[0075] S2 substrate pretreatment:

[0076] A mixture of sweet potato distiller's grains and soybean residue (mass ratio 1:1) was selected as the substrate and filtered at room temperature using a plate and frame filter press until the moisture content was controlled at 75%. No free water was added during the entire process, and all substrates used were food-grade by-products with good resistance to contaminants and dietary fiber structure.

[0077] S3 strain propagation and primary fermentation:

[0078] Dissolve whey powder (6% of the culture medium mass) and brown sugar (3% of the culture medium mass) in sterile water and adjust the pH to 5.8. Inoculate with a compound bacterial culture under aseptic conditions; its composition is as follows:

[0079] Lactobacillus plantarum: 8 portions

[0080] Enterococcus faecalis: 2 portions

[0081] Pediococcus pentosaceus: 2 portions

[0082] Brewing yeast: 1 part

[0083] The total vaccination dose was 1×10 7 The concentration of CFU / mL was determined using a tank culture method, and the culture was carried out at 38℃ and pH 5.8 for 24 hours to obtain a compound bacterial fermentation broth. At the end of fermentation, the total viable bacterial concentration in the broth was ≥1×10⁻⁶. 9 CFU / g.

[0084] S4 First stirring:

[0085] The micronized carrier obtained in step S1 and the substrate obtained in step S2 are mixed at a mass ratio of 1:1 and mixed for 15 minutes using a horizontal ribbon mixer to ensure uniform mixing and no agglomeration.

[0086] S5 dry and wet fermentation:

[0087] To the mixture obtained in step S4, add the fermentation broth and compound enzyme preparation obtained in step S3 at a ratio of 6% of the total mass. The compound enzyme is added with a total enzyme activity of 10,000 U / kg based on an acidic protease activity meter. The composition is as follows:

[0088] Acidic protease: 5000 U / kg

[0089] Neutral protease: 2500 U / kg

[0090] Xylanase: 1500 U / kg

[0091] β-glucanase: 1000 U / kg

[0092] After thorough mixing, the mixture was placed in a fermentation tank and subjected to solid-state fermentation for 36 hours under anaerobic conditions at 42℃ and 80% relative humidity. Intermittent venting was performed for 2 minutes at 6h, 12h, and 24h of fermentation. Fermentation was considered complete when the material temperature increased by ≥5℃ and the pH dropped to 4.3.

[0093] S6 Second Stirring:

[0094] After fermentation, the material is sieved through a 2mm aperture vibrating screen to remove large particles and clumps, and then stirred again for 10 minutes using a horizontal mixer to ensure uniform distribution of the inoculum.

[0095] S7 finished product packaging:

[0096] Uses an air permeability of 1200 mL / (m 2 •24h•0.1MPa), water vapor transmission rate is 5g / (m 2 Fermentation materials were filled into double-sided 20-mil thick microporous breathing membrane bags (24h), with the filling density controlled at 0.7kg / L, and then sealed and placed in an insulated environment.

[0097] S8 finished product third fermentation:

[0098] Place the packaging bags in an insulated room at 45℃ for 72 hours, maintaining a CO2 concentration of 8% inside the bags. Do not disturb the bags during fermentation.

[0099] S9 Third stirring and cooling:

[0100] After fermentation, break the bag and stir for 5 minutes, then let it cool naturally to room temperature to obtain the finished fermented material with pH ≤ 4.0 and water activity ≤ 0.60.

[0101] Example 3

[0102] This embodiment discloses a fermentation process for biological fermentation materials, including the following steps:

[0103] Preparation of S1 micronized carrier:

[0104] Select plant-based raw materials that meet the "GB 13078-2017 Feed Hygiene Standard", such as rice bran, corn, or wheat bran (in this example, rice bran and corn are mixed at a mass ratio of 70:30). Grind these materials using a hammer mill until the average particle size is no greater than 300 μm. Immediately after grinding, dry the materials with hot air circulation at 50°C until the moisture content is ≤12%. After drying, rapidly cool the materials to <40°C to prevent heat damage to the inoculum, and then set aside for later use.

[0105] S2 substrate pretreatment:

[0106] A mixture of sweet potato distiller's grains and soybean residue (mass ratio 1:1) was selected as the substrate and filtered at room temperature using a plate and frame filter press until the moisture content was controlled at 75%. No free water was added during the entire process, and all substrates used were food-grade by-products.

[0107] S3 strain propagation and primary fermentation:

[0108] Dissolve whey powder (3% of the culture medium mass) and brown sugar (1% of the culture medium mass) in sterile water and adjust the pH to 5.8. Inoculate with a compound bacterial culture under aseptic conditions; its composition is as follows:

[0109] Lactobacillus plantarum: 8 portions

[0110] Enterococcus faecalis: 2 portions

[0111] Pediococcus pentosaceus: 2 portions

[0112] Brewing yeast: 1 part

[0113] The total vaccination dose was 1×10 7 The concentration of CFU / mL was determined by shaking flask incubation at 32℃ and pH 5.8 for 12 hours to obtain the complex bacterial fermentation broth. At the end of fermentation, the total viable bacterial concentration was ≥1×10⁻⁶ CFU / mL. 9 CFU / g.

[0114] S4 First stirring:

[0115] Mix the micronized carrier obtained in step S1 with the substrate obtained in step S2 at a mass ratio of 1:1, and mix for 5 minutes using a horizontal ribbon mixer to ensure uniform mixing.

[0116] S5 dry and wet fermentation:

[0117] To the mixture obtained in step S4, add the fermentation broth and compound enzyme preparation obtained in step S3 at a ratio of 2% of the total mass. The compound enzyme is added with a total enzyme activity of 5000 U / kg based on an acidic protease activity meter. The composition is as follows:

[0118] Acidic protease: 2500 U / kg

[0119] Neutral protease: 1500 U / kg

[0120] Xylanase: 500 U / kg

[0121] β-glucanase: 500 U / kg

[0122] After thorough mixing, the mixture was placed in a fermentation tank and subjected to solid-state fermentation for 18 hours under anaerobic conditions at 30℃ and 65% relative humidity. Intermittent venting was performed for 2 minutes at 6h, 12h, and 24h of fermentation. Fermentation was considered complete when the material temperature increased by ≥5℃ and the pH dropped to 4.3.

[0123] S6 Second Stirring:

[0124] After fermentation, the material is sieved through a 2mm aperture vibrating screen to remove large particles and clumps, and then stirred again for 3 minutes using a horizontal mixer to ensure uniform distribution of the inoculum.

[0125] S7 finished product packaging:

[0126] Use a breathability of 1000 mL / (m 2 •24h•0.1MPa), water vapor transmission rate is 3g / (m 2 Fermentation materials were filled into double-sided 20-mil thick microporous breathing membrane bags (24h), with the filling density controlled at 0.5kg / L, and then sealed and placed in an insulated environment.

[0127] S8 finished product third fermentation:

[0128] Place the packaging bag in an insulated room at 28°C for 24 hours to ferment, maintaining a CO2 concentration of 3% inside the bag. Do not disturb the bag during fermentation.

[0129] S9 Third stirring and cooling:

[0130] After fermentation, break the bag and stir for 2 minutes, then let it cool naturally to room temperature to obtain the finished fermented material with pH ≤ 4.0 and water activity ≤ 0.60.

[0131] Comparative Example 1

[0132] This comparative example uses a simplified fermentation method commonly found in conventional feed companies, omitting strain propagation, three-stage stirring, and microenvironment control. The process flow is as follows:

[0133] S1 carrier and substrate mixing:

[0134] Unprocessed wheat bran and sweet potato residue were mixed at a mass ratio of 1:1 without particle size control or drying. The raw materials were used directly in a wet state on site, with a moisture content of approximately 65%.

[0135] S2 bacterial strain addition:

[0136] Purchase commercially available dry powder dormant single-strain bacteria (Lactobacillus plantarum, yeast, etc.) and add them at 1% of the total mass of the fermentation material. No propagation step is performed; they are directly mixed with the material.

[0137] S3 mixed with water for fermentation:

[0138] To ensure the fluidity of the mixture, add free water until the total moisture content of the material reaches 70%. No compound enzyme preparation is added. The material is sealed in a woven bag and allowed to undergo natural anaerobic fermentation at room temperature (25℃) for 48 hours without intermittent degassing or temperature and humidity control.

[0139] S4 Finished Product Processing:

[0140] After fermentation is complete, open the packaging, stir, and use directly as "fermented feed".

[0141] Testing items

[0142] To comprehensively evaluate the performance of the prepared bio-fermentation material and ensure its nutritional quality, safety, and microecological activity, the following tests were conducted:

[0143] First, the basic nutritional components of the fermented feed were tested, specifically including moisture, crude protein, crude fat, and crude ash. Moisture content was tested according to GB / T 6435 "Determination of Moisture in Feed" to assess the product's dryness and storage stability; crude protein content was determined according to GB / T 6432 "Determination of Crude Protein in Feed" to reflect the nutrient conversion efficiency of the fermentation substrate; crude fat was determined according to GB / T 6433 "Determination of Crude Fat in Feed"; and crude ash was determined according to GB / T 6438 "Determination of Crude Ash in Feed" to assess the inorganic mineral levels.

[0144] Secondly, regarding the microecological activity of the bio-fermented material, the focus was on testing the viable counts of lactic acid bacteria and yeast. The viable count of lactic acid bacteria was tested according to GB / T 4789.35 "Food Hygiene Microbiology Examination: Method for Counting Lactic Acid Bacteria," and the viable count of yeast was tested according to GB / T 4789.15 "Food Hygiene Microbiology Examination: Method for Counting Yeast and Mold." These indicators reflect the colonization activity of probiotics and the effectiveness of bio-fermentation.

[0145] To ensure feed safety, it is necessary to test for Salmonella and aflatoxin B1 levels. Salmonella testing should be conducted according to the "Microbial Limits in Feed Hygiene Standard GB / T13091," and it should be undetectable. Aflatoxin B1 levels should be determined according to "Determination of Aflatoxin B1 in Feed NY / T2071," and the content should not exceed 20 μg / kg, meeting the safety standards for livestock and poultry feed.

[0146] In addition, the pH value and water activity of the finished product were tested. The pH value was measured by the electrode method to assess the metabolic activity of lactic acid bacteria and the degree of fermentation completion; the ideal value should be less than or equal to 4.0. The water activity was determined according to the standard GB / T 22266 "Determination of Water Activity in Food" and controlled within ≤0.60 to ensure the stability of the finished product against mold and for storage.

[0147] Finally, sensory evaluation is conducted, including appearance, color, clumping, odor, and particle uniformity. The consistency and palatability of the product are judged by manual observation and smell.

[0148] The test results are shown in Table 1 below.

[0149] Table 1

[0150]

[0151]

[0152] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A fermentation process for a biological fermentation material, characterized in that, The process consists of the following nine steps: S1 micronized carrier preparation: The plant-based carrier was pulverized to an average particle size ≤300μm and dried at below 55℃ to a moisture content ≤12%; S2 substrate pretreatment: Soybean by-products or sweet potato by-products are dehydrated by pressure filtration to adjust the moisture content to 60%–85%, without adding free water throughout the process; S3 strain propagation through primary fermentation: Using whey powder (3%–6% of the total culture medium mass) and brown sugar (1%–3% of the total culture medium mass) as nutrient sources, the compound strain was propagated at 32℃–38℃ and pH 5.5–6.2 for 12–24 hours to obtain a viable cell concentration ≥1×10⁻⁶. 9 Fermentation broth with CFU / g; S4 First stirring: Mix the micro powder carrier from step S1 with the substrate pretreated in step S2 and stir until homogeneous; S5 Dry and wet fermentation: Add the fermentation liquid and compound enzyme from step S3 to the well-stirred material. The total amount of fermentation liquid and compound enzyme added accounts for 2% to 6% of the total mass of the material. After mixing thoroughly, ferment under anaerobic conditions at 30℃ to 42℃ for 18 to 36 hours. S6 Second mixing: After the material has undergone dry and wet fermentation, it is passed through a vibrating screen and mixed again; S7 Finished Product Bagging: Pack the material into double-sided 20-mil thick microporous breathable membrane packaging bags, with a packing density of 0.5~0.7kg / L; S8 finished product third fermentation: Place the bagged material in a 28℃~45℃ insulated room and continue fermentation for 24h~72h; S9 Third stirring and cooling: Take out the bagged material, break the bag and stir for 3 minutes, then let it cool naturally to obtain the finished biological fermentation material with pH≤4.0 and water activity≤0.

60.

2. The fermentation process for a biological fermentation material according to claim 1, characterized in that, The compound microbial strain includes Lactobacillus plantarum, Enterococcus faecalis, Pediococcus pentosus, and Saccharomyces cerevisiae. At the end of the propagation, the ratio of each microbial strain is (5-8):(1-3):(1-3):(1-2).

3. The fermentation process for a biological fermentation material according to claim 1, characterized in that, The complex enzyme consists of acidic protease, neutral protease, xylanase and β-glucanase, and the total enzyme activity is controlled at 5000-10000 U / kg based on the acidic protease activity.

4. The fermentation process for a biological fermentation material according to claim 1, characterized in that, Step S5 is considered complete when the temperature of the mixture increases by ≥5℃ and the pH drops to 4.2-4.

5.

5. The fermentation process for a biological fermentation material according to claim 1, characterized in that, The air permeability of the microporous breathing membrane is 1000–1200 mL / (m²). 2 • 24h • 0.1MPa), water vapor transmission rate 3–5 g / (m 2 •24h).

6. The fermentation process for a biological fermentation material according to claim 1, characterized in that, During the fermentation of the finished product in step S8, the CO2 concentration inside the bag is maintained at 3% to 8%.

7. The fermentation process for a biological fermentation material according to claim 1, characterized in that, After vacuum drying, the micronized carrier in step S1 is immediately cooled to <40°C before proceeding to step S4.

8. The fermentation process for a biological fermentation material according to claim 1, characterized in that, In step S5, the relative humidity for dry and wet fermentation is maintained at 65%–80%, and intermittent venting is performed for 2 minutes at 6h, 12h, and 24h of fermentation.

9. The fermentation process for a biological fermentation material according to claim 1, characterized in that, The viable count of lactic acid bacteria in the resulting bio-fermented material is ≥1×10⁻⁶. 8 CFU / g, yeast viable count ≥1×10⁻⁶ 6 CFU / g, Salmonella not detected, and aflatoxin B1 content ≤20μg / kg.

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