Forage leavening agent capable of increasing crude protein, reducing crude fiber and improving digestibility

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a pasture leavening agent for improving crude protein, reducing crude fiber and improving digestibility, which comprises three strains, namely aspergillus niger, candida utilis and lactobacillus pentosus. The invention relates to a self-induced peptide premix, which is composed of a covalent complex, a lignin-hemicellulose-L-arabinose, a phosphorylated urea-glycine chelate, a synthetic self-induced peptide premix and a carrier. According to the method, a specific initial carbon source is provided for aspergillus niger through LHLAC to start fiber degradation, synchronous slow release of a nitrogen source is achieved through PUGC, and protein anabolism of candida utilis is activated through sAIP. According to the scheme, a time sequence synergistic mechanism of fiber degradation, protein synthesis and acidification preservation is constructed, the problem of low efficiency caused by mismatching of all metabolic processes in the prior art is solved, and effective degradation of crude fibers of forage grass, great increase of crude protein content and remarkable improvement of final digestibility are realized.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility. Background Technology

[0002] Silage fermentation is a common technique for preserving forage and improving its feed value. By adding microbial fermentation agents such as lactic acid bacteria to forage, soluble sugars can be converted into lactic acid, rapidly lowering the pH of the system, thereby inhibiting the growth of putrefactive microorganisms and achieving long-term preservation of forage.

[0003] However, existing silage technologies still face challenges in further enhancing the nutritional value of forage. The complex structure of lignocellulose in forage not only makes it difficult for lactic acid bacteria to utilize directly but also limits the source of fermentation substrates, resulting in high fiber content in the fermentation products and affecting their digestibility and absorption. To address this issue, existing technologies attempt to combine strains capable of degrading fiber; however, these strains have a slow initial activity in the early stages of fermentation, and the process of degrading fiber and releasing fermentable sugars is difficult to match in time with the subsequent metabolic activities of other microorganisms.

[0004] Meanwhile, to increase the crude protein content of fermentation products, non-protein nitrogen sources such as urea are often added technically. However, ordinary urea hydrolyzes too quickly in the fermentation system, and the large amount of ammonia released in a short period of time not only raises the pH value and interferes with the normal acid production of lactic acid bacteria, but the released nitrogen source is also difficult to be efficiently synthesized into cell protein by strains such as gluten-producing yeast because it cannot be supplied synchronously with the carbon source produced by the slow degradation of fiber, resulting in low nitrogen source utilization.

[0005] Therefore, how to effectively regulate the sequence and reaction rate of key steps such as fiber degradation, protein synthesis and acidification preservation in the fermentation system, so as to achieve temporal synergy among carbon source release, nitrogen source supply and microbial metabolic activities, is a technical problem to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility. This solves the problem in existing forage fermentation technologies where metabolic competition and temporal mismatch among different functional microorganisms lead to mutual inhibition of fiber degradation, protein conversion, and acidification preservation processes, resulting in a lack of synergistic effects and ultimately affecting the improvement of the nutritional value of fermented feed.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention provides a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility.

[0008] In one specific embodiment, the forage fermentation agent comprises the following components in parts by weight: 0.5-1.5 parts of Aspergillus niger freeze-dried mycelium powder; 1.0-2.5 parts of Candida utilis freeze-dried mycelium powder; 0.5-1.5 parts of Lactobacillus pentosus freeze-dried mycelium powder; 8.0-12.0 parts of lignin-hemicellulose-L-arabinose covalent complex; 10.0-15.0 parts of phosphorylated urea-glycine chelate; 1.0-2.0 parts of synthetic self-inducible peptide premix; and 100 parts of corn starch carrier.

[0009] The lignin-hemicellulose-L-arabinose covalent complex is a chemically synthesized product that, in the early stages of fermentation, serves as a specific substrate and can only be effectively decomposed by the enzyme system secreted by Aspergillus niger. This provides a preferential carbon source for the growth of Aspergillus niger and the secretion of cellulase, thereby providing an initial reaction time window for the degradation of forage fiber.

[0010] The phosphorylated urea-glycine chelate is a chemically synthesized slow-release non-protein nitrogen source. Its decomposition rate is related to the metabolic activity of the Candida utilis during fermentation. It can release urea and glycine simultaneously during the middle stage of fermentation when the concentration of available carbon source produced by fiber degradation increases, providing nitrogen source and amino acid precursors for the synthesis of cell protein by the Candida utilis.

[0011] The premixed synthetic self-inducible peptide contains a synthetic self-inducible peptide whose amino acid sequence is related to the Candida utilis protein synthesis pathway. Its function is to activate the Candida utilis population as an exogenous signaling molecule during the mid-fermentation stage, enabling it to simultaneously enter a highly efficient protein synthesis metabolic state.

[0012] In one specific embodiment, the synthetic self-inducible peptide premix is ​​prepared by mixing synthetic self-inducible peptides and trehalose in a mass ratio of 1:(100-200).

[0013] In one specific embodiment, the *Aspergillus niger* strain is *Aspergillus niger* A-L1 obtained through screening using a lignin-hemicellulose-L-arabinose covalent complex as the sole carbon source. The *Candida utilis* strain is *Candida utilis* C-P2 obtained through screening using phosphorylated urea-glycine chelate as the main nitrogen source. The *Lactobacillus pentosaccharide* strain is *Lactobacillus pentosaccharide* L-X3 obtained through screening using xylose as the main carbon source. In the later stages of fermentation, the *Lactobacillus pentosaccharide* utilizes the pentose sugars produced from fiber degradation and the remaining hexose sugars for lactic acid fermentation, thereby lowering the pH of the fermentation system.

[0014] A second aspect of the present invention provides a method for preparing the aforementioned forage fermentation agent, the method comprising the following steps: S1. Preparation of lignin-hemicellulose-L-arabinose covalent complex; S2. Preparation of phosphorylated urea-glycine chelate; S3. Preparation of a premix for synthesizing self-induced peptides; S4. Aspergillus niger, Candida utilis, and Lactobacillus pentosus were cultured separately and prepared into freeze-dried bacterial powders. S5. Mix the products obtained in S1 to S4 with the corn starch carrier to obtain the forage fermentation agent.

[0015] In one specific embodiment, the specific method of S1 is as follows: hemicellulose and phosphorus oxychloride are activated by reacting in N,N-dimethylformamide at 25-30°C for 4-6 hours; then lignin is added and reacted at 40-50°C for 8-12 hours; then L-arabinose and triethylamine are added and reacted at 60-70°C for 10-15 hours. After the reaction is completed, the product is obtained by washing with alcohol, drying and pulverizing.

[0016] In one specific embodiment, the specific method of S2 is as follows: urea and phosphorus oxychloride are reacted in pyridine at 0-15°C to prepare phosphorylated urea; then the phosphorylated urea is reacted with glycine in an aqueous solution at pH 7.0-7.5 at 50-60°C for 4-6 hours, and after the reaction is completed, the product is obtained by alcohol precipitation, drying and pulverization.

[0017] In one specific embodiment, the specific method of S3 is as follows: the self-inducible peptide is synthesized using the Fmoc solid-phase synthesis method, purified by reversed-phase high-performance liquid chromatography, and then mixed with trehalose to prepare the self-inducible peptide premix.

[0018] A third aspect of the present invention provides a method for applying the aforementioned forage fermentation agent.

[0019] The method includes the steps of mixing the forage fermentation agent with the forage to be fermented, and then compacting, sealing, and anaerobic fermentation.

[0020] In one specific embodiment, the amount of the forage fermentation agent added is 300-500g per ton of forage to be fermented. The moisture content of the forage to be fermented is adjusted to 60%-68%. The anaerobic fermentation temperature is 25-35℃, and the fermentation time is 25-35 days.

[0021] This invention provides a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility. It has the following beneficial effects: 1. In this invention, the lignin-hemicellulose-L-arabinose covalent complex provides a carbon source for specific Aspergillus niger strains, promotes the secretion of cellulase and hemicellulose, and thereby degrades the fiber structure in the forage raw material. This process reduces the content of neutral detergent fiber and acid detergent fiber in the fermentation products and converts the indigestible fiber into sugars that can be utilized by subsequent microorganisms.

[0022] 2. This invention uses phosphorylated urea-glycine chelate as a slow-release nitrogen source, which coordinates the release of nitrogen with the supply of sugars produced by fiber degradation in time, avoiding the decrease in nitrogen source utilization and the increase in pH value caused by the rapid release of ammonia. At the same time, the synthesis of self-inducible peptides can activate the protein synthesis of Candida utilis, and promote the synthesis of cell protein when carbon and nitrogen sources are sufficient, thereby increasing the crude protein content of fermentation products.

[0023] 3. The degradation of fiber structure and the synthesis of bacterial protein in this invention together increase the total amount of digestible and absorbable substances in the product. In addition, the sugars produced by fiber degradation also provide a substrate for the growth and acid production of Lactobacillus pentosus, which can rapidly reduce the pH value of the fermentation system, inhibit the growth of other bacteria, and ensure the storage stability of the final product. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example: Example 1 This embodiment provides a method for preparing a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility.

[0026] Accurately weigh the following components: Aspergillus niger A-L1 freeze-dried mycelial powder: 1.0 kg; Lyophilized Candida utilis C-P2 bacterial powder: 1.8 kg; Lactobacillus pentosus L-X3 lyophilized bacterial powder: 1.0 kg; Lignin-hemicellulose-L-arabinose covalent complex: 10.0 kg; Phosphorylated urea-glycine chelate: 12.5 kg; Synthetic self-inducible peptide premix: 1.5 kg; Corn starch carrier: 72.2 kg.

[0027] Add all the weighed components together into the V-type mixer.

[0028] In an environment of 28℃ and relative humidity below 40%, start the mixer and mix at a low speed of 30 rpm for 35 minutes until the materials are evenly mixed. After mixing, immediately discharge the material from the outlet and aseptically package and seal it to obtain the finished forage fermentation agent.

[0029] Example 2 This embodiment provides a method for preparing a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility.

[0030] Accurately weigh the following components: Aspergillus niger A-L1 freeze-dried mycelial powder: 0.5 kg; Lyophilized Candida utilis C-P2 bacterial powder: 1.0 kg; Lactobacillus pentosus L-X3 lyophilized bacterial powder: 0.5 kg; Lignin-hemicellulose-L-arabinose covalent complex: 8.0 kg; Phosphorylated urea-glycine chelate: 10.0 kg; Synthetic self-inducible peptide premix: 1.0 kg; Corn starch carrier: 79.0 kg.

[0031] Add all the weighed components together into the horizontal ribbon mixer.

[0032] In an environment of 25℃ and relative humidity below 35%, start the mixer and mix at 40 rpm for 30 minutes until the materials are evenly mixed. After mixing, immediately discharge the material from the outlet, and vacuum package and seal it to obtain the finished forage fermentation agent.

[0033] Example 3 This embodiment provides a method for preparing a forage foster that increases crude protein, reduces crude fiber, and improves digestibility.

[0034] Accurately weigh the following components: Aspergillus niger A-L1 freeze-dried mycelial powder: 1.5kg; Lyophilized Candida utilis C-P2 bacterial powder: 2.5 kg; Lactobacillus pentosus L-X3 lyophilized bacterial powder: 1.5kg; Lignin-hemicellulose-L-arabinose covalent complex: 12.0 kg; Phosphorylated urea-glycine chelate: 15.0 kg; Synthetic self-inducible peptide premix: 2.0 kg; Corn starch carrier: 65.5 kg.

[0035] Add all the weighed components together into the three-dimensional motion mixer.

[0036] In an environment of 30℃ and relative humidity below 45%, start the mixer and mix at 20 rpm for 40 minutes until the materials are evenly mixed. After mixing, immediately discharge the material from the outlet, and then package and seal it with nitrogen to obtain the finished forage fermentation agent.

[0037] Comparative example: Comparative Example 1 Compared with Example 1, the difference is that the lignin-hemicellulose-L-arabinose covalent complex, phosphorylated urea-glycine chelate, and synthetic self-inducible peptide premix are not added, and their weights are replaced by corn starch carrier, while the rest are the same.

[0038] Comparative Example 2 The difference from Example 1 is that the lignin-hemicellulose-L-arabinose covalent complex was not added, but replaced with 10.0 kg of glucose; all other aspects were the same.

[0039] Comparative Example 3 Compared with Example 1, the difference is that phosphorylated urea-glycine chelate is not added, and instead 4.7 kg of ordinary urea with stoichiometric nitrogen content is added. The remaining weight of 12.5-4.7=7.8 kg is supplemented by corn starch carrier. Everything else is the same.

[0040] Comparative Example 4 Compared with Example 1, the difference is that no synthetic self-inducible peptide premix was added, and its weight was replaced by corn starch carrier, while the rest were the same.

[0041] Comparative Example 5 The difference compared to Example 1 is as follows: Instead of adding the lignin-hemicellulose-L-arabinose covalent complex, 10.0 kg of pure hemicellulose was used. Phosphorylated urea-glycine chelate was not added; instead, 12.5 kg of commercially available coated slow-release urea was used. Everything else remained the same.

[0042] Test example: Test Example 1: Fermentation Treatment of Forage and Analysis of Conventional Nutrient Components and Fermentation Quality Experimental steps: Raw material preparation: Take the same batch of air-dried corn stalks and chop them to a length of 2.0-3.5cm using a chaff cutter. Adjust the final moisture content to 65.0%±0.5% by spraying an appropriate amount of deionized water and mixing thoroughly. Take samples to determine their initial nutrient composition; Experimental Grouping and Inoculation: Eight experimental groups were prepared, including three example groups and five comparative groups. 20 kg of each of the moisture-containing straw prepared in step S1 was weighed and spread evenly on a clean plastic sheet. 8.0 g of each of the KOM fermentation agent prepared in Examples 1-3 and Comparative Examples 1-5 (addition ratio of 400 g / ton) was weighed and evenly sprinkled onto the corresponding straw, and repeatedly stirred with a shovel for 15 minutes to ensure the fermentation agent and straw were evenly mixed. Fermentation process: Each mixture is separately loaded into a 50L polyethylene fermentation tank, with manual compaction performed as filling until the material can no longer be compressed. After filling, the tank lid is tightened and a one-way exhaust valve is installed. All fermentation tanks are placed in a constant-temperature fermentation chamber at 28℃±1℃ for anaerobic fermentation in the dark for 30 days. Sample preparation: After fermentation, open each fermentation tank and randomly sample from the top, middle, and bottom of the tank. Mix the three samples thoroughly to obtain a representative sample for the group. A portion of the fresh sample is used for pH and organic acid analysis. Another portion of the sample is dried in a 65℃ oven to constant weight, then pulverized using a grinder and passed through a 1mm sieve to obtain a dry powder for subsequent nutrient composition and in vitro digestibility analysis. Indicator Measurement: Crude protein content: determined according to the Kjeldahl method in the national standard GB / T6432-2018 "Determination of crude protein in feed".

[0043] Neutral detergent fiber and acid detergent fiber content: determined according to the provisions of the national standard GB / T20806-2006 "Determination of Neutral Detergent Fiber (NDF) in Feed" and the acid detergent fiber section.

[0044] pH value: Weigh 10g of fresh sample, add 90mL of deionized water, shake and mix for 30 minutes, filter with gauze, and measure the pH value of the filtrate using a calibrated pH meter.

[0045] Organic acid content: The above filtrate was filtered through a 0.22 μm filter membrane, and the contents of lactic acid and acetic acid were determined by high performance liquid chromatography (HPLC).

[0046] In vitro dry matter digestibility (IVDMD): determined using a two-step enzymatic digestion method.

[0047] The measurement results are recorded in Table 1.

[0048] Table 1. Performance test data of fermentation products from the examples and comparative examples. Table 1 shows that, compared with the raw materials and the fermentation products of Comparative Examples 1-5, the fermentation products of Examples 1-3 have higher crude protein content, lower neutral detergent fiber and acid detergent fiber content, lower pH value, and higher in vitro dry matter digestibility. This result indicates that the fermentation agent formulation of the present invention, through the combination of a lignin-hemicellulose-L-arabinose covalent complex, a phosphorylated urea-glycine chelate, and a synthetic self-inducible peptide, can establish an orderly and synergistic microbial metabolic process under the combined action of three functional strains.

[0049] Compared to Example 1, Comparative Example 2, lacking the lignin-hemicellulose-L-arabinose covalent complex, exhibited significantly higher NDF and ADF contents than Example 1, indicating a lack of specific substrate support for fiber-degrading bacteria in the early stages of fermentation, leading to reduced fiber degradation efficiency. Comparative Example 4, lacking the ability to synthesize self-inducible peptides, had a lower crude protein content than Example 1, demonstrating that even with sufficient carbon and nitrogen sources, the efficiency of cell protein synthesis is limited without the synchronous activation of the *Candida utilis* community by exogenous signaling molecules.

[0050] Comparative Example 3 used ordinary urea instead of phosphorylated urea-glycine chelate, and its product had a higher pH and lower crude protein content than Example 1. This is related to the rapid hydrolysis of urea releasing ammonia, leading to a decrease in nitrogen source utilization efficiency. Comparative Example 5 used conventional hemicellulose and coated urea, and its various indicators were inferior to Examples 1-3, indicating that the specific chemical structure designed in this invention has a special role in regulating microbial metabolism. Comparative Example 1 served as a blank control, and its various indicators showed the least improvement, further confirming the necessity of the specific chemical regulation module combination in this invention to achieve the technical effects.

[0051] Test Example 2: Comparative Test of Conventional Nutrient Components of Fermentation Products Experimental description: This test case aims to determine the content of crude protein, neutral detergent fiber, and acid detergent fiber in fermentation products after treatment with different starter cultures.

[0052] Sample source: The samples used in this test example are all dry fermentation product powders prepared in Test Example 1, which have been dried, pulverized and sieved, including the sample groups corresponding to Examples 1-3 and Comparative Examples 1-5, as well as the unfermented raw material group.

[0053] Crude protein content determination: Weigh 0.5-1.0g of sample, accurate to 0.0001g, and place it in a dry Kjeldahl flask; Add the catalyst and 10-15 mL of concentrated sulfuric acid, and digest in a fume hood on an electric furnace until the liquid turns a clear blue-green color. After the digestion solution is cooled, it is transferred to a Kjeldahl nitrogen analyzer, and excess sodium hydroxide solution is added for distillation. Ammonia is absorbed by boric acid solution. Titrate the receiving solution with standard hydrochloric acid titrant and record the volume consumed. Calculate the crude protein content of the sample according to the calculation formula in the national standard GB / T6432-2018.

[0054] Determination of neutral detergent fiber and acid detergent fiber content: Weigh approximately 1.0g of the sample, accurate to 0.0001g, and place it in a special filter bag; For NDF determination, the filter bag is placed in a container with neutral detergent and heated to boiling for 60 minutes on a fiber analyzer. For ADF determination, the filter bag is placed in a container with acidic washing solution and heated to boiling for 60 minutes on a fiber analyzer. After boiling, wash the filter bag with hot water and acetone respectively until the washing solution is neutral and colorless; The washed filter bags were dried in an oven at 105℃ until constant weight, and then weighed. Calculate the NDF and ADF contents of the sample according to the calculation formula in the national standard GB / T20806-2006.

[0055] The measurement results are recorded in Table 2.

[0056] Table 2. Results of conventional nutrient composition analysis of fermentation products from the examples and comparative examples. Table 2 shows that, compared to Comparative Examples 1-5 and the unfermented raw material, the fermentation products of Examples 1-3 exhibited a significant increase in crude protein content, while the contents of neutral detergent fiber and acid detergent fiber significantly decreased. These changes reflect the synergistic effect of the various components in the starter culture during the fermentation process. By constructing an ordered microbial metabolic process, the starter culture facilitated the degradation of fiber components in forage and the conversion of non-protein nitrogen into microbial protein.

[0057] The reduced NDF and ADF content in the products of Examples 1-3 is attributed to the application of the lignin-hemicellulose-L-arabinose covalent complex. This substance provides a specific initial carbon source for Aspergillus niger A-L1, enabling it to gain a growth advantage in the early stages of fermentation and thus secrete sufficient amounts of cellulase and hemicellulase. These enzymes act on the cell wall structure of the forage grass, breaking it down into fermentable sugars that can be utilized by subsequent microorganisms. Comparative Example 2, due to the use of glucose instead of LHLAC, lacked specific initiation for Aspergillus niger, resulting in insufficient fiber degradation, and its NDF and ADF contents were higher than those of Examples 1-3.

[0058] The significant increase in crude protein content in the products of Examples 1-3 is a result of the effective coordination between the two stages of fiber degradation and protein synthesis. During the later stages of fermentation, when fiber degradation produces a large amount of fermentable sugars, the phosphorylated urea-glycine chelate simultaneously releases urea and glycine as nitrogen sources. Simultaneously, the synthesis of self-induced peptide molecules activates the protein synthesis metabolic pathway of *Candida utilis* C-P2. This temporal and concentrational matching of carbon source, nitrogen source, and metabolic activation signal enables the yeast to efficiently utilize the fermentation substrate to synthesize cell protein. Data from Comparative Examples 3 and 4 show that the absence of either PUGC or sAIP disrupts this synchronization mechanism, leading to reduced cell protein synthesis efficiency and ultimately a lower crude protein content compared to the examples.

[0059] Test Example 3: Comparative Test of Fermentation Quality and Stability Experimental description: This test case aims to evaluate the degree of acidification and fermentation mode of products treated with different starter cultures by measuring the pH value and organic acid content of the fermentation products.

[0060] Sample source: The samples used in this test example are all representative fresh samples prepared in Test Example 1 that have not been dried, including the sample groups corresponding to Examples 1-3 and Comparative Examples 1-5, as well as the unfermented raw material group.

[0061] Preparation of sample extract: Accurately weigh 10.0g of each group of fresh samples and place them in a 250mL Erlenmeyer flask; Place the conical flask on a shaker and extract by shaking at 200 rpm for 30 minutes; After extraction, the mixture was filtered through four layers of medical gauze, and the filtrate was collected.

[0062] pH value measurement: Take the filtrate obtained in step S2 and measure its pH value using a laboratory pH meter calibrated with the three-point method (pH 4.00, 6.86, 9.18). Repeat the measurement three times for each sample and take the average value.

[0063] Determination of organic acid content: Take the filtrate obtained in step S2, filter it through a 0.22 μm aqueous filter membrane, and collect the filtrate into a sample vial; The contents of lactic acid and acetic acid in the filtrate were analyzed using high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); mobile phase: 0.005 mol / L sulfuric acid solution; flow rate: 0.6 mL / min; column temperature: 35 °C; the content of lactic acid and acetic acid in the sample was quantitatively calculated using the standard curve method, and the results were expressed on a dry matter (DM) basis.

[0064] The measurement results are recorded in Table 3.

[0065] Table 3. Analysis results of pH value and organic acid content of fermentation products from the examples and comparative examples. Table 3 shows that the fermentation products of Examples 1-3 have lower pH values ​​and higher lactic acid content, while the acetic acid content remains at a low level. The product pH value rapidly decreases to around 4.0, and lactic acid is the main fermentation product, indicating that the fermentation process is dominated by highly efficient homolactic fermentation. This is attributed to the temporal synergy of microbial metabolism in the present invention. In the later stages of fermentation, the large amount of fermentable sugars, especially pentose sugars, produced by Aspergillus niger from the degradation of fiber provide sufficient substrate for the proliferation and acid production activity of Lactobacillus pentosus L-X3.

[0066] Compared to the examples, none of the comparative examples achieved the desired acidification effect. Comparative Example 1, lacking a chemical regulation module, resulted in insufficient fermentable substrate production, limiting lactic acid bacteria growth, low total acid production, and a high pH. Comparative Example 2, using glucose as a carbon source, also produced acid, but its lactic acid yield was lower than that of the examples, indicating that the mixed sugars continuously released from fiber degradation are more conducive to the sustained acid production of *Lactobacillus pentosus*. This confirms the correlation between substrate supply and the fiber degradation and acidification stages.

[0067] Comparative Example 3 exhibited the highest pH value and the lowest lactic acid yield. This was because the rapid hydrolysis of ordinary urea produced a large amount of ammonia, leading to an increase in the pH value of the fermentation system. This alkaline environment severely inhibited the growth and metabolic activities of lactic acid bacteria, causing the acidification process to fail. Comparative Example 5, using conventional hemicellulose and coated urea, also showed inferior acid production compared to the examples, indicating that the LHLAC and PUGC with specific structures in this invention are more effective than conventional substitutes in establishing an ordered substrate release chain. Ultimately, only when the three stages of fiber degradation, protein synthesis, and acidification preservation are effectively time-controlled can a fermentation product with low pH value and high lactic acid content be obtained.

[0068] Test Example 4: In Vitro Digestibility Comparison Test Experimental description: This test case aims to simulate the digestion process in animals using a two-step enzymatic hydrolysis method, and to determine and compare the in vitro dry matter digestibility (IVDMD) of products treated with different fermentation agents.

[0069] Sample source: The samples used in this test example are all dry fermentation product powders prepared in Test Example 1, which have been dried, pulverized and sieved, including the sample groups corresponding to Examples 1-3 and Comparative Examples 1-5, as well as the unfermented raw material group.

[0070] Pepsin digestion stage: Accurately weigh approximately 0.5g of each sample group, accurate to 0.0001g, and record it as W1. Place it in a special filter bag and seal it. Simultaneously, set up a blank filter bag containing no sample as a blank control. Place the filter bag into a beaker containing 1.0g of pepsin and 1000mL of 0.1mol / L hydrochloric acid solution, and adjust the pH to 1.9; Place the beaker in a 39°C constant temperature water bath and incubate with continuous shaking for 48 hours.

[0071] Cellulase digestion stage: After 48 hours, remove the filter bag from the beaker and rinse it with deionized water until neutral. Transfer the filter bag to a new beaker containing 0.1g cellulase and 1000mL phosphate buffer (pH 6.8); Place the new beaker in a 39°C constant temperature water bath and incubate with continuous shaking for 48 hours.

[0072] Result calculation: After cultivation, remove the filter bag, wash it with deionized water, and then dry it in a 65℃ oven until constant weight. Weigh the bag and record the final weight as W2. Treat the blank filter bag in the same way and weigh it, recording the weight as Wb. In vitro dry matter digestibility (IVDMD) is calculated using the following formula: IVDMD(%)=[(W1-(W2-Wb)) / W1]×100; The measurement results are recorded in Table 4.

[0073] Table 4. Results of in vitro dry matter digestibility (IVDMD) analysis of fermentation products from the examples and comparative examples. Table 4 shows that the in vitro dry matter digestibility (IVDMD) of the fermentation products in Examples 1-3 was significantly higher than that of all comparative examples and unfermented raw materials. As a comprehensive indicator, IVDMD directly reflects the potential for the utilization of nutrients in feed by organisms. The improved digestibility achieved in the Example groups is the ultimate manifestation of the synergistic effect of various functional components in the fermentation agent at specific time points, jointly and deeply modifying the chemical composition of the raw materials.

[0074] The increase in IVDMD primarily stems from the effective degradation of recalcitrant components such as cellulose and hemicellulose. In the fermentation system of the example, the lignin-hemicellulose-L-arabinose covalent complex provided a specific substrate for the early proliferation and enzyme production activity of Aspergillus niger A-L1, ensuring the effective breakdown of forage cell wall structures. This process converts previously indigestible polysaccharides into usable nutrients, directly contributing to the increase in IVDMD. Comparative Example 2, lacking LHLAC, had a lower IVDMD value than the example, directly demonstrating the decisive role of fiber degradation efficiency in final digestibility.

[0075] Another contribution to in vitro digestibility comes from the newly synthesized, easily digestible microbial proteins during fermentation. After fiber degradation provides a sufficient carbon source, phosphorylated urea-glycine chelates and self-induced peptides work synergistically to provide a slow-release nitrogen source matched to the carbon source release rate and activate the protein synthesis metabolism of *Candida utilis* C-P2. This process converts inorganic nitrogen into high-value biological proteins, replacing some of the recalcitrant plant proteins and increasing total digestible nutrients. The IVCMD values ​​of Comparative Examples 3 and 4 were lower than those of the Example, indicating that the nitrogen source release pattern and the metabolic state of the yeast community also have a significant impact on the digestibility of the final product. Therefore, the overall digestibility of the fermentation product can only be maximized when the two core processes of fiber degradation and protein synthesis are precisely aligned in terms of time and substrate.

[0076] The method described in this embodiment can be used to perform the above-described forage fermentation agent preparation embodiment, and its principle and technical effects are similar, so they will not be repeated here.

Claims

1. A forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility. Includes, characterized in that, The forage fermentation agent consists of the following components in parts by weight: Aspergillus niger freeze-dried mycelium powder: 0.5-1.5 parts; Freeze-dried Candida utilis powder: 1.0-2.5 parts; Lyophilized Lactobacillus pentosolicus powder: 0.5-1.5 parts; Lignin-hemicellulose-L-arabinose covalent complex: 8.0-12.0 parts; Phosphorylated urea-glycine chelate: 10.0-15.0 parts; Synthetic self-inducible peptide premix: 1.0-2.0 parts; Corn starch carrier: Add to 100 parts.

2. The forage fermentation agent according to claim 1, which increases crude protein, reduces crude fiber, and improves digestibility, is characterized in that, The lignin-hemicellulose-L-arabinose covalent complex and the phosphorylated urea-glycine chelate are both chemically synthesized products, and the self-inducible peptide premix contains a self-inducible peptide related to the protein synthesis metabolic pathway of the Candida utilis.

3. The forage fermentation agent according to claim 1, which increases crude protein, reduces crude fiber, and improves digestibility, is characterized in that... The Aspergillus niger strain is Aspergillus niger strain A-L1, obtained by screening with lignin-hemicellulose-L-arabinose covalent complex as the sole carbon source; the Candida utilis strain is Candida utilis strain C-P2, obtained by screening with phosphorylated urea-glycine chelate as the main nitrogen source; and the Lactobacillus pentosus strain is L-X3, obtained by screening with xylose as the main carbon source.

4. The forage fermentation agent according to claim 1, which increases crude protein, reduces crude fiber, and improves digestibility, is characterized in that... The synthetic self-inducible peptide premix is ​​prepared by mixing synthetic self-inducible peptides and trehalose in a mass ratio of 1:(100-200).

5. The method for preparing a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility according to claim 1, characterized in that, Includes the following steps: S1. Preparation of lignin-hemicellulose-L-arabinose covalent complex; S2. Preparation of phosphorylated urea-glycine chelate; S3. Preparation of a premix for synthesizing self-induced peptides; S4. The Aspergillus niger, the Candida utilis, and the Lactobacillus pentosus are cultured separately and then freeze-dried into bacterial powder. S5. The lignin-hemicellulose-L-arabinose covalent complex, the phosphorylated urea-glycine chelate, the synthetic self-inducible peptide premix, the three freeze-dried bacterial powders, and the corn starch carrier are mixed to obtain the forage fermentation agent.

6. The method for preparing a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility according to claim 5, characterized in that, The specific method of S1 is as follows: Hemicellulose was activated by reacting phosphorus oxychloride with N,N-dimethylformamide at 25-30°C for 4-6 hours; then lignin was added and reacted at 40-50°C for 8-12 hours. Then add L-arabinose and triethylamine and react at 60-70℃ for 10-15 hours.

7. The method for preparing a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility according to claim 5, characterized in that, The specific method of S2 is as follows: Phosphorylated urea was prepared by reacting urea with phosphorus oxychloride in pyridine at 0-15°C. The phosphorylated urea and glycine are then reacted in an aqueous solution at pH 7.0-7.5 at 50-60°C for 4-6 hours.

8. The method for preparing a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility according to claim 5, characterized in that, The specific method of S3 is as follows: The self-inducible peptide was synthesized using the Fmoc solid-phase synthesis method, purified by reversed-phase high-performance liquid chromatography, and then mixed with trehalose to prepare the self-inducible peptide premix.

9. The method for preparing a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility according to claim 1, characterized in that, The process includes mixing the forage fermentation agent with the forage to be fermented, and then compacting, sealing, and anaerobic fermentation.

10. The method for preparing a forage fermentation agent that increases crude protein, reduces crude fiber, and improves digestibility according to claim 9, characterized in that, The amount of the forage fermentation agent added is 300-500g per ton of forage to be fermented; The moisture content of the forage to be fermented is adjusted to 60%-68%; The anaerobic fermentation temperature is 25-35℃, and the fermentation time is 25-35 days.

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