Method for preparing live pig feed through mixed solid-state fermentation of feed rape and rapeseed meal

Abstract

The invention discloses a method for preparing live pig feed through mixed solid-state fermentation of feed rape and rapeseed meal. The method specifically comprises the following steps: harvesting feed rape from a full-bloom stage to a final bloom stage, adding rapeseed dregs, and uniformly mixing to obtain a fermentation substrate with the water content of 55-65%; spraying the compound enzyme bacterium preparation on the fermentation substrate, uniformly stirring, finally compacting by using a packer, sealing, packing and storing, and fermenting for 45 days; the compound enzyme bacterial preparation comprises a bacterial agent and an enzyme agent; the fungicide comprises lactobacillus plantarum, bacillus subtilis, saccharomyces cerevisiae and aspergillus niger; the enzyme agent is prepared from phytase, cellulase, hemicellulase, pectinase, tannase, xylanase and alpha-galactosidase. The feed rape and the rapeseed meal are subjected to mixed solid fermentation, so that the content of phytic acid, glucosinolate and crude fiber is greatly reduced, the content of crude protein is increased, and the nutritional quality of the feed is remarkably improved. The fermented feed is used for replacing part of corn, soybean meal and wheat bran, and the use amount of concentrated feed in live pig breeding is reduced.

Description

Technical Field

[0001] This invention relates to the field of feed, and more particularly to a method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal. Background Technology

[0002] Rapeseed is one of my country's most important oilseed crops, with a wide planting area. my country's annual rapeseed production exceeds 15 million tons, and rapeseed meal production exceeds 9 million tons. Rapeseed meal has a high crude protein content (35%–45%), and its true protein content is high, with a balanced amino acid composition, rich in lysine, methionine, and cysteine, which are key nutrients for promoting animal growth and development. However, due to the high content of anti-nutritional factors such as phytic acid, erucic acid, tannins, and glucosinolates (hereinafter referred to as glucosinolates) in rapeseed meal, its use as animal feed seriously affects palatability and reduces the digestibility of nutrients. It also adversely affects the healthy growth and production performance of livestock and poultry, posing risks to safe farming. Because of the high content of anti-nutritional factors, rapeseed meal often cannot be fully utilized, only being used as part of fish feed and soil fertilizer, resulting in a significant waste of high-protein sources. Rapeseed, on the other hand, has a high crude protein content (15%–30%), a rich variety of amino acids, and a relatively large biomass, making it an ideal green fodder. However, due to its high water content of 80% to 90%, feeding it as a single feed can cause bloating and diarrhea in livestock and poultry, affecting their feed intake.

[0003] As people's living standards continue to improve, their demands for pork production and quality are also increasing. Currently, domestic pig farming feed mainly consists of concentrated feeds such as corn, soybean meal, and wheat bran, the vast majority of which are imported. In 2024, my country imported over 2 million tons of soybean meal and over 20 million tons of corn from Argentina, Brazil, the United States, Australia, and other countries. The shortage and high cost of pig farming feed are pressing issues that need to be addressed.

[0004] Solid-state fermentation is one of the effective methods to reduce the content of anti-nutritional factors such as phytic acid, glucosinolates, and tannins in rapeseed meal and forage rapeseed plants. Solid-state fermentation technology can improve the palatability of feed and the digestibility of various nutrients, while reducing the risk of disease in livestock after consuming rapeseed meal with high levels of these anti-nutritional factors. Currently, there are many research reports on the reduction of anti-nutritional factors through solid-state fermentation of rapeseed meal both domestically and internationally, but most are limited to the fermentation of single substrates, with fewer reports on mixed fermentation of multiple substrates and optimization of the fermentation process. Achieving simultaneous degradation of anti-nutritional factors in multiple mixed substrates is relatively difficult because different fermentation substrates have different adaptability to added degrading enzymes and microbial agents. The temperature and pH required for enzymes and microbial agents to achieve optimal enzyme activity also differ among different substrates. Different substances have different spatial structures, resulting in different steric hindrance effects and varying enzyme adsorption. These factors make it difficult to achieve degradation effects in mixed fermentation of multiple substrates. Compared to mixed-substrate fermentation, single-substrate fermentation conditions are easier to control. However, in daily use as a feed additive for pigs, mixed substrates are the most common. Therefore, reducing anti-nutritional factors through solid-state fermentation of mixed substrates is an urgent problem to be solved.

[0005] To address the above problems, this invention aims to utilize solid-state fermentation of forage rapeseed and rapeseed meal, adding suitable microbial and enzyme preparations under specific moisture conditions. Through synergistic microbial-enzyme interaction, the content of anti-nutritional factors in the rapeseed meal is reduced, while the crude protein content of the fermentation substrate is increased, thus improving its nutritional quality. This fully leverages the advantages of forage rapeseed plants, such as high biomass yield and balanced nutrients, while further improving the effective utilization rate of rapeseed meal, expanding forage resources, and reducing livestock costs.

[0006] Chinese invention patent CN202510070450 discloses a method for mixed-culture co-fermentation of rapeseed meal. This method involves enzymatically hydrolyzing the rapeseed meal with cellulase, followed by fermentation with *Lactobacillus brevis*, *Lactobacillus casei*, *Lactobacillus paracasei*, *Lactobacillus rhamnosus*, and *Lactobacillus delbrueckii* to increase the soluble peptides and reduce the content of anti-nutritional factors in the rapeseed meal, thereby preparing rapeseed meal polypeptides. Chinese invention patent CN202310880502 discloses a method using *Bacillus subtilis* (5-7 × 10⁻⁶)... 7 cfu / g), brewer's yeast (6×10) 6 cfu / g), Aspergillus niger (2.5×10) 7A method for solid-state fermentation of rapeseed meal using CFU / g and cellulase (100 U / g) is described, involving fermentation for 48 hours at a moisture content of 50%–52% and a temperature of 35℃–37℃ to prepare the finished rapeseed meal product. Publication number CN202211565207 uses rapeseed meal as the fermentation substrate and utilizes a mixed inoculum, cellulase, pectinase, and protease for fermentation to prepare the finished rapeseed meal product. Publication number CN202211579443 uses rapeseed straw and pig feed as the fermentation substrate, and involves adding fermentation agents at a moisture content of 65% followed by sealed solid-state fermentation to prepare fermented pig feed. Publication number CN202411527615 also uses rapeseed meal as the fermentation substrate, and involves inoculating Bacillus subtilis H8 strain (concentration of 2×10⁻⁶) at 30℃. 7 The fermentation process involves fermenting rapeseed meal (cfu / ml) for 45-50 hours. Most of the methods described above involve solid-state fermentation of a single substrate, such as rapeseed meal or crop straw, to enhance its nutritional value. However, research on the simultaneous solid-state fermentation of rapeseed meal and forage rapeseed plants to develop fermented pig feed has not been reported. Directly using rapeseed meal prepared using the methods described above as pig feed can easily lead to poor nutrient absorption, decreased feed intake, reduced digestibility, and even weakened disease resistance in pigs. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal.

[0008] To achieve the above objectives, the present invention provides a method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal, characterized in that...

[0009] Harvest rapeseed from its peak flowering period to its end flowering period, add rapeseed meal and mix well to form a fermentation substrate, so that the moisture content of the fermentation substrate is 55% to 65%.

[0010] Spray the compound enzyme preparation evenly onto the fermentation substrate, mix thoroughly with a mixer, and finally compact and seal with a packing machine. Pack and store. It can be opened and used gradually after 45 days of fermentation.

[0011] The compound enzyme preparation includes microbial agents and enzymes; the microbial agents include four types of microbial agents: Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger; the enzymes include phytase, cellulase, hemicellulase, pectinase, tanninase, xylanase, and α-galactosidase.

[0012] Furthermore, the harvesting of forage rapeseed from its peak flowering period to its end flowering period refers to cutting the rapeseed at a height of 10-20cm from the ground during its peak flowering period to its end flowering period, harvesting fresh forage rapeseed plants, and then crushing them into 1-2mm coarse pieces using a straw crusher.

[0013] Furthermore, the water content of the fermentation substrate is 60%.

[0014] Furthermore, the viable counts of Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger in the bacterial agent are 1:1:2:1.

[0015] Furthermore, the dosage of the bacterial agent in the compound enzyme preparation, taking 1 kg of fermentation substrate as an example, is ≥1.0 × 10⁻⁶ live Lactobacillus plantarum bacteria. 10 CFU / g; Bacillus subtilis viable count ≥1.0 × 10⁻⁶ 10 CFU / g; viable count of Saccharomyces cerevisiae 2.0 × 10⁻⁶ 10 cfu / g; viable Aspergillus niger count ≥1.0×10⁻⁶ 10 cfu / g.

[0016] Furthermore, the weight ratio of phytase, cellulase, hemicellulase, pectinase, tanninase, xylanase, and α-galactosidase in the enzyme preparation is as follows: phytase 50 mg; cellulase 100 mg; hemicellulase 10 mg; pectinase 200 mg; tanninase 10 mg; xylanase 100 mg; α-galactosidase 50 mg.

[0017] Furthermore, the preparation method of the compound enzyme preparation is as follows, taking the amount required for 1 kg of fermentation substrate as an example:

[0018] Preparation of microbial agents: Add 50g of brown sugar to 50ml of sterile water, and then add the required Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger in sequence;

[0019] Enzyme preparation: Weigh 50 mg of phytase with an enzyme activity ≥ 5 U / mg, and bring the volume to 2 ml with sterile water at 50℃, then adjust the pH to 5.0.

[0020] Weigh 100 mg of cellulase with an enzyme activity ≥3 U / mg, and bring the volume to 2 ml with sterile water at 40°C, then adjust the pH to 3.0.

[0021] Weigh 10 mg of cellulase with an enzyme activity ≥1.5 U / mg, and bring the volume to 1 ml with sterile water at 40°C, adjusting the pH to 5.0.

[0022] Weigh 200 mg of pectinase with an enzyme activity ≥40 U / mg, and bring the volume to 2 ml with sterile water at 50°C, then adjust the pH to 4.0.

[0023] Weigh 10 mg of tanninase with an enzyme activity ≥0.3 U / mg, and bring the volume to 1 ml with sterile water at 30°C, adjusting the pH to 4.7.

[0024] Weigh 100 mg of xylanase with an enzyme activity ≥50 U / mg, and dilute to 1 ml with sterile water at 50°C, then adjust the pH to 5.0.

[0025] Weigh 50 mg of α-galactosidase with an enzyme activity ≥2 U / mg, and bring the volume to 1 ml with sterile water at 37°C, adjusting the pH to 5.5.

[0026] The enzyme solution is obtained by mixing the enzymes that have been brought to a constant volume.

[0027] Simply mix the prepared bacterial agent and enzyme agent thoroughly.

[0028] This invention also protects the prepared pig feed.

[0029] Furthermore, when using it, add 30% to 40% by weight and mix it with 60% to 70% concentrate.

[0030] This invention calculates the required weight of rapeseed and rapeseed meal to be added per 1 kg of fermentation substrate by measuring the average moisture content of the rapeseed and rapeseed meal. The calculation is based on the following formula: Fresh weight of rapeseed (kg): X, Fresh weight of rapeseed meal (kg): Y, Moisture content of rapeseed (%): W X (Measured value), Rapeseed meal moisture content (%): W Y (Measured value), X+Y=1.

[0031] Formula for calculating the amount of rapeseed added to a substrate with 55% moisture content: X = (0.55 - W) Y ) / (W X -W Y Formula for calculating the amount of rapeseed added to a substrate with 60% moisture content: X = (0.6 - W) Y ) / (W X -W Y Formula for calculating the amount of rapeseed added to a substrate with 65% moisture content: X = (0.65 - W) Y ) / (W X -W Y Taking the derivation of the formula for calculating the fresh weight of rapeseed in a substrate with 55% moisture content as an example: (X·W) X +Y·W Y ) / 1=0.55①, Y=1-X②, substituting ② into ①, we get: X·W X +(1-X)·W Y =0.55, X·W X +W Y -X·W Y =0.55, X·(W X -W Y ) = 0.55 - W YThen: X = (0.55 - W) Y ) / (W X -W Y The formulas for calculating the fresh weight of rapeseed in substrates with 60% and 65% moisture content are derived similarly.

[0032] The feed of this invention can be used in combination with concentrated feeds such as corn, soybean meal, and wheat bran as pig feed, with an addition ratio of 30% to 40% by weight. When mixed with 60% to 70% concentrated feed, it achieves good results. This comprehensively improves the nutritional quality of the feed while reducing the amount of concentrated feed used in pig farming.

[0033] This invention utilizes solid-state fermentation of feed rapeseed and rapeseed meal to significantly reduce the content of phytic acid, glucosinolates and crude fiber, while increasing the crude protein content, thus significantly improving the nutritional quality of the feed (Table 4).

[0034] The crude protein content increased by 10.85% after fermentation, reaching a highly significant level (p < 0.01). This is because the technical solution of this invention includes a large amount of bacterial enzyme preparations. Under the synergistic fermentation effect of bacteria and enzymes, large protein molecules can be broken down into small peptides and amino acids. Simultaneously, the proteases secreted by Bacillus subtilis and Lactobacillus can also promote the decomposition of large protein molecules, resulting in an overall increase in crude protein content. Furthermore, lactic acid bacteria and yeast continuously multiply during fermentation, significantly increasing the microbial protein content. This is one reason why lactic acid bacteria and yeast can be used as feed additives to improve the nutritional quality of feed. At the same time, with the degradation of anti-nutritional factors, some proteins originally bound by anti-nutritional factors such as glucosinolates and phytic acid gradually separate, also increasing the overall crude protein content. Since the technical solution of this invention not only adds an appropriate amount of phytase, but Bacillus subtilis and Aspergillus niger can also release some phytase during fermentation, resulting in a better degradation effect on phytic acid, the phytic acid degradation rate is as high as 98.82%, reaching a highly significant level (p < 0.01). The degradation rate of glucosinolates reached 53.31%, which was also highly significant (p < 0.01). The degradation rate of crude fiber reached 27.03%, which was also highly significant (p < 0.01). The degradation of crude fiber was relatively difficult. With the addition of pectinase and xylanase, the lignin and pectin network structures surrounding the cellulose were disrupted, and then cellulase and hemicellulase were used to maximize the adsorption and degradation of cellulose. However, due to the high degree of lignification in rapeseed straw from full bloom to the end of flowering, the specific spatial structure of lignin aromatic ring macromolecules, the steric hindrance formed by its three-dimensional network structure, the non-productive adsorption of cellulase, and the co-precipitation of phenolic substances with cellulase, which inhibits cellulase hydrolysis, this may be one of the key factors leading to the difficulty in optimizing the degradation of crude fiber. The technical solution of this invention has poor optimization and degradation effects on tannins, possibly because the optimal enzyme activity temperature for tanninase is 30–40°C, while the harvest time for silage fermentation of rapeseed is from full bloom to the end of flowering (mid-to-late March), with an average air temperature of 25°C. Therefore, it is difficult to achieve the optimal enzyme activity effect at room temperature. Simultaneously, the gallic acid produced by tanninase decomposition of tannins repolymerizes with other polyphenols, which is also one of the key factors leading to the poor optimization and degradation effect of tannins. Attached Figure Description

[0035] Figure 1 This is a screening diagram of microbial agents for the mixed fermentation of rapeseed meal and feed.

[0036] Figure 2 This is a graph showing the changes in crude protein and anti-nutritional factors at different water contents during enzyme screening experiments.

[0037] Figure 3This is a graph showing the changes of five nutritional indicators before and after fermentation in various treatments of the synergistic fermentation experiment of compound enzyme and bacterial preparations. Detailed Implementation

[0038] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0039] Example 1:

[0040] 1. Experimental Materials and Preliminary Preparations

[0041] 1.1 Forage rapeseed

[0042] The rapeseed variety used in this fermentation experiment was Shengsi No. 2, provided by the Guizhou Provincial Rapeseed Research Institute. Shengsi No. 2 rapeseed was sown by broadcasting in late September 2023 in Jinbi Town, Qianxi City, with a density controlled at 40,000–50,000 plants / 667m². 2 The rapeseed was managed in the field according to conventional rapeseed cultivation and management practices. It reached its peak flowering period in mid-March 2024, with an average temperature of around 25℃. The rapeseed was harvested during the period from peak flowering to the end of flowering to prepare materials for subsequent fermentation experiments.

[0043] 1.2 Rapeseed Meal

[0044] High-quality rapeseed meal was purchased from Guizhou Youyan Chunxiang Ecological Grain and Oil Technology Co., Ltd., and then crushed for later use.

[0045] 1.3 Enzyme and bacterial preparations

[0046] Phytase, cellulase, hemicellulase, pectinase, and other enzyme preparations were purchased from Beijing Solarbio Science & Technology Co., Ltd. *Lactobacillus plantarum* (live count ≥ 1.0 × 10⁻⁶) 10 CFU / g), Bacillus subtilis (live count ≥ 1.0 × 10⁻⁶) 10 CFU / g), Saccharomyces cerevisiae (live count ≥ 2.0 × 10⁻⁶) 10 CFU / g), Aspergillus niger (live count ≥1.0×10⁻⁶) 10 CFU / g was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. Brown sugar was purchased from a supermarket, and purified water was obtained from the molecular laboratory of the Guizhou Rapeseed Research Institute. All other experimental equipment and chemical reagents required for this study were provided by the Guizhou Rapeseed Research Institute.

[0047] 1.4 Determination of moisture content in fermentation substrate

[0048] Before harvesting forage rapeseed, the plant moisture content was measured. Two rapeseed plants were randomly selected from three different locations in the field during the full-blown to the end-blooming stage, and their weight was recorded. Fresh plant samples were blanched at 105℃ for 20 minutes and dried at 65℃ to constant weight. The average moisture content of the plants at the three locations was calculated. The same method was used to determine and calculate the average moisture content of rapeseed meal, and the data was recorded. In this experiment, the average moisture content of the forage rapeseed was 82.18%, and the average moisture content of the rapeseed meal was 3.20%.

[0049] 1.5 Preparation of Fermentation Substrate Raw Materials

[0050] After measuring the moisture content of the forage rapeseed, harvest the fresh rapeseed plants as soon as possible during the peak flowering to the end of the flowering period of the No. 2 forage rapeseed, cutting them 10-20cm above the ground. Use a straw crusher to crush the rapeseed into 1-2mm coarse pieces for later use. At the same time, use a crusher to crush the lumpy rapeseed meal for later use.

[0051] 1.6 Inoculation, Cultivation and Preparation of Compound Fermentation Enzymes and Microbial Preparations

[0052] Each enzyme preparation was diluted to volume with sterile water at a specific temperature according to its corresponding dosage, and the pH was adjusted to the optimal value. Specific dosages and optimal pH values ​​are shown in Table 1. The microbial agent preparation method was as follows: 5g of brown sugar was added to 50ml of sterile water, followed by the sequential addition of four microbial agents: *Lactobacillus plantarum*, *Bacillus subtilis*, *Saccharomyces cerevisiae*, and *Aspergillus niger*, followed by thorough mixing. The weight ratio of *Lactobacillus plantarum*, *Bacillus subtilis*, *Saccharomyces cerevisiae*, and *Aspergillus niger* was 2:2:1:2, with the dosage of each agent per 1kg of fermentation substrate being 1g, 1g, 0.5g, and 1g, respectively. Different compound fermentation microbial agents were prepared separately according to the required strains and dosages. The prepared microbial agents were thoroughly mixed with the enzymes and then set aside.

[0053] Table 1. Activation and preparation methods of each enzyme preparation in the compound microbial agent.

[0054]

[0055] 1.7 Addition amount of rapeseed and rapeseed meal to feed at different moisture contents

[0056] Based on the average moisture content of the forage rapeseed plants and rapeseed meal (forage rapeseed: 82.18%, rapeseed meal: 3.20%), the total amount of mixed fermentation substrate was 1 kg, and the addition amounts of the two fermentation substrates were calculated separately. The addition amounts of forage rapeseed and rapeseed meal per kg of fermentation substrate were (55% moisture content): 0.65 kg and 0.35 kg respectively; under 60% moisture content conditions, the addition amounts were 0.72 kg and 0.28 kg respectively; under 65% moisture content conditions, the addition amounts were 0.78 kg and 0.22 kg respectively.

[0057] 1.8 Quality Testing and Analysis of Fermented Feed

[0058] After all fermentation processes were completed, individual samples were taken from each replicate and sent for testing. The contents of five nutritional indicators—crude protein, crude fiber, phytic acid, tannins, and glucosinolates—as well as anti-nutritional factors were determined. The testing standards for each component are as follows: crude protein was determined using the Kjeldahl method (GB / T 6432-2018); crude fiber was determined using the filter bag method (GB / T 6434-2022); phytic acid was determined using GB 5009.153-2016; tannins were determined using spectrophotometry (GB / T 27985-2011); and glucosinolates were determined using the palladium chloride colorimetric method.

[0059] 2. Experimental Methods

[0060] 2.1 Screening test of microbial agents

[0061] The fermentation test for screening microbial agents set up three compound microbial agents and three full-factor tests with different moisture contents.

[0062] Microbial agent A: Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae, Aspergillus niger.

[0063] Microbial agent B: Lactobacillus plantarum, Saccharomyces cerevisiae.

[0064] Microbial agent C: Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae, Aspergillus niger, and cellulase.

[0065] The dosage and preparation method of each microbial agent should refer to "1.6 Inoculation, Cultivation and Preparation of Compound Fermentation Enzymes and Microbial Agents". The substrate moisture content was set at 55%, 60%, and 65%, for a total of 9 treatments with 3 replicates. All treatments are shown in Table 2.

[0066] In each replicate of each treatment, the weight of the mixed fermentation substrate of rapeseed meal and green fodder rapeseed was set at 1 kg. The corresponding weight of fermentation substrate was added according to the requirements of each treatment. The inoculum was sprayed onto the fermentation substrate and thoroughly mixed. The substrate was then packed into 30×40cm silage fermentation bags and vacuum-sealed using an AS-360 vacuum packaging machine. Fermentation was carried out at room temperature for 30 days.

[0067] Table 2 Screening Test Results of Mixed Fermentation Agents for Rapeseed Meal and Feed

[0068] Moisture content Inoculant A Bacterial agent B Inoculant C 55％ A-55％ B-55％ C-55％ 60％ A-60％ B-60％ C-60％ 65％ A-65％ B-65％ C-65％

[0069] 2.2 Results and Analysis of Microbial Agent Screening Tests

[0070] After the fermentation experiment, the contents of crude protein, crude fiber, phytic acid, tannins, and glucosinolates in the fermentation substrate were measured. Specific testing standards are detailed in "1.8 Fermented Feed Quality Detection and Analysis" of this invention. Specific results are available in the table below. Figure 1 . Figure 1 This is a screening diagram of microbial agents for the mixed fermentation of rapeseed meal and feed.

[0071] from Figure 1 It can be seen that the crude protein content was significantly increased in all treatments by co-fermenting rapeseed meal with feed rapeseed. However, the increase was more significant in the treatments with Bacillus subtilis and Aspergillus niger. The percentage increase in crude protein was significantly higher in the treatments with Bacterial Agent A and Bacterial Agent C than in the treatment with Bacterial Agent B, indicating that Bacillus subtilis and Aspergillus niger may have a good effect on increasing crude protein. The "C-65" mode (Bacterial Agent C, 65% moisture content) showed a significant increase of 10.81% in crude protein after fermentation. In all treatment combinations, the two major anti-nutritional factors, tannins and glucosinolates, were reduced to some extent, while the degradation effect on crude fiber was not ideal. Even with the addition of cellulase, Bacterial Agent C still showed poor degradation of crude fiber, but it had a certain advantage over Bacterial Agents A and B without cellulase. Therefore, it can be seen that cellulase alone has limited effect on crude fiber degradation and may need to be combined with other enzyme preparations to achieve the best results.

[0072] Of all the treatments, only inoculant C showed some degradation effect on phytic acid when the substrate moisture content exceeded 60%. This is likely because the addition of Bacillus subtilis and Aspergillus niger to the inoculant releases some phytase during fermentation, thus contributing to the degradation of phytic acid. All other treatments besides inoculant C showed poor phytic acid degradation effects, not only failing to reduce the phytic acid content but also significantly increasing it. Therefore, it is necessary to add a certain amount of phytase in subsequent fermentation experiments to reduce the phytic acid content in the fermentation substrate.

[0073] 2.3 Enzyme Screening Test

[0074] Based on the above-mentioned microbial inoculant screening fermentation experiment, this enzyme preparation screening experiment also set up three moisture content gradients of 55%, 60%, and 65%, using four microbial inoculants and a total of 12 treatments in a comprehensive factorial experiment. Each treatment was replicated three times, and all treatments are shown in Table 3. The following four compound enzyme preparations were selected for this fermentation experiment:

[0075] Enzyme preparation a: phytase, cellulase, hemicellulase, pectinase, xylanase.

[0076] Enzyme preparation b: phytase, cellulase, hemicellulase.

[0077] Enzyme preparation c: phytase, cellulase, hemicellulase, Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae, Aspergillus niger.

[0078] Enzyme preparations d: phytase, cellulase, hemicellulase, pectinase, xylanase, Bacillus subtilis, Lactobacillus plantarum, Saccharomyces cerevisiae, Aspergillus niger.

[0079] Each treatment used 1 kg of fermentation substrate. The experimental procedure was the same as the above-mentioned microbial agent screening experiment. After the fermentation substrate of each treatment was bagged, it was also vacuum sealed and packaged, and fermented at room temperature for 30 days.

[0080] Table 3 Screening Test of Enzyme Preparations for Mixed Fermentation of Rapeseed Meal and Feed

[0081]

[0082]

[0083] 2.4 Results and Analysis of Enzyme Screening Tests

[0084] After fermentation, the contents of crude protein, crude fiber, phytic acid, tannins, and glucosinolates in the fermentation substrate were determined using the same method. Specific results are shown in [link to results]. Figure 2 . Figure 2 This is a graph showing the changes in crude protein and anti-nutritional factors at different water contents during enzyme screening experiments.

[0085] from Figure 2It can be seen that the crude protein content of all treatments was significantly increased, with the highest increase observed in the "d-65%" treatment (enzyme D, 65% moisture content), which showed a 55.95% increase compared to before fermentation. Clearly, the increase in crude protein was higher in treatments with added enzymes c and d than in those with added enzymes a and b. The difference lies in the addition of Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger to enzymes c and d. This further demonstrates that Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger have a significant effect on increasing crude protein content. In enzyme a, which simultaneously added cellulase, hemicellulase, pectinase, and xylanase, the crude fiber content of the substrate decreased by 7.41% and 11.23% after fermentation in the two treatments with 60% and 65% moisture contents, respectively. In all other treatments, except for "d-60%" (enzyme d, 60% moisture content), which showed a 9.24% decrease in crude fiber content, all others showed varying degrees of increase, ranging from 2.49% to 35.52%. The data above reveals that while enzymes b and c contain cellulase and hemicellulase, their degradation effect on crude fiber is not as good as that of enzymes a and d. Inoculant d also shows good degradation effect on crude fiber, reducing it by 9.24% and 0.73% at 60% and 65% moisture contents, respectively. Inoculants a and d show better optimized degradation effects on crude fiber than b and c. This indicates that the degradation of crude fiber by cellulose and hemicellulase requires the assistance of pectinase and xylanase to achieve its maximum effect. This may be due to the high degree of lignification in rapeseed during the flowering period (from full bloom to the end of flowering), resulting in the unique molecular structure of its crude fiber.

[0086] Meanwhile, because phytase was added to all treatments, except for a slight increase in phytic acid content in the "a-55%" treatment (enzyme a, 55% water content), all other treatments showed a significant reduction. Specifically, the "b-55%", "b-60%", "c-60%", "c-65%", "d-55%", and "d-60%" treatments all achieved a 100% degradation rate of phytic acid. Combined with the above microbial agent screening experiments, it is clear that phytase has a very significant effect on the degradation of phytic acid.

[0087] At moisture contents of 55% and 60%, glucosinolate degradation was relatively good, with the highest degradation rate observed in treatment "c-55" at 53.76%. Degradation of glucosinolates was poor at 65% moisture content, with all four microbial agents showing varying degrees of improvement after fermentation, the highest increase reaching 32.62%, indicating that moisture content significantly affects glucosinolate degradation. All treatments showed relatively poor tannin degradation, but all showed varying degrees of improvement after fermentation (42.51%–122.70%), representing a relatively large increase. Subsequent fermentation experiments will consider adding tannin-degrading enzymes to optimize tannin degradation. Furthermore, to further improve the utilization rate of fermented feed and alleviate bloating and diarrhea caused by fermented feed in livestock and poultry, α-galactosidase can be added to the subsequent fermentation feed to improve intestinal health.

[0088] 2.5 Synergistic Fermentation Experiment of Compound Enzyme-Bacterial Preparation

[0089] Based on the aforementioned screening experiments for microbial agents and enzyme preparations, this co-fermentation experiment using a combination of microbial agents and enzyme preparations investigated the optimal substrate moisture content for different moisture content gradients. This study also employed a solid-state fermentation method combining feed rapeseed and rapeseed meal, with the substrate consisting of both. A single-factor experimental design was adopted, with three treatments at substrate moisture contents of 55% (treatment 1), 60% (treatment 2), and 65% (treatment 3), each with three replicates. Corresponding blank controls (CK1, CK2, and CK3) were set at the three moisture contents of 55%, 60%, and 65%, respectively. The control group received no microbial agents. For the other treatments, the fermentation agents selected were phytase, cellulase, hemicellulase, pectinase, tanninase, xylanase, α-galactosidase, and *Lactobacillus plantarum*, *Bacillus subtilis*, *Saccharomyces cerevisiae*, and *Aspergillus niger*. A co-fermentation method was used, and the fermentation time for all treatments was set at 45 days. The dosage and usage of each microorganism and enzyme are as described in "1.6 Inoculation, Cultivation and Preparation of Compound Fermentation Enzymes and Microbial Preparations" of this invention. The fermentation test operation procedure and the detection methods for each nutrient quality are the same as those for the microbial screening test described above.

[0090] 2.6 Results and Analysis of Co-fermentation Experiment of Compound Enzyme-Bacterial Preparation

[0091] By measuring the contents of crude protein, crude fiber, phytic acid, tannins, and glucosinolates in the substrate before and after fermentation, the differences in changes of various nutritional indicators between the treatments before and after mixed fermentation can be identified. For details, please refer to [link to relevant documentation]. Figure 3 . Figure 3 This is a graph showing the changes of five nutritional indicators before and after fermentation in various treatments of the synergistic fermentation experiment of compound enzyme and bacterial preparations.

[0092] from Figure 3It can be seen that the crude protein content increased in all three treatments. Treatment 1 (55% moisture content) showed a 17.31% increase in crude protein, reaching a highly significant level (P < 0.01). Treatment 2 showed a 10.85% increase in crude protein, reaching a significant level (P < 0.05), while Treatment 3 showed no significant increase. Only Treatment 2 effectively reduced crude fiber content, reaching a highly significant level (P < 0.01). Phytic acid content was significantly degraded in all treatments, with a degradation rate exceeding 98%. Tannin content increased in all treatments, with Treatment 2 showing the smallest increase, reaching only 7.45 mg / kg after fermentation. The tannin content in the other two treatments exceeded 10 mg / kg. Treatments 1 and 3 showed increased glucosinolate content (P < 0.05), while only Treatment 2 effectively reduced glucosinolate content, reaching a highly significant level (P < 0.01). The experimental data above show that when the moisture content of the fermentation substrate is around 60%, it has a significant effect on increasing crude protein and reducing anti-nutritional factors such as crude fiber, phytic acid, and glucosinolates. The results are shown in Table 4.

[0093] Table 4. Changes in various nutrient qualities before and after fermentation in Treatment 2 (60% moisture content).

[0094]

[0095] 2.7 Application Analysis as Pig Feed

[0096] Crude protein content is a key indicator of feed quality and is of great significance. As an important component of muscle and organ tissues, protein plays a positive role in pig growth and enhancing disease resistance and immunity. In this experiment, the crude protein content of the substrates increased after fermentation and reached the standard for high-quality pig feed (16%–22%), with treatments 1 and 2 both exceeding 37% crude protein content.

[0097] The standard for phytic acid content in finishing pig feed is no more than 1.2%, and in piglet feed, no more than 0.8%. Excessive phytic acid content can impair the digestion and absorption of other nutrients, reducing digestibility. It can also lead to health problems in pigs such as diarrhea and intestinal microbial imbalance. In this experiment, phytic acid was almost completely degraded in all treatments, with a degradation rate exceeding 98%. The phytic acid content of the fermentation products was 0.1%–0.3%, meeting the requirements for phytic acid content in piglet feed.

[0098] Excessive levels of crude fiber and tannins can also impair the digestion and absorption of other nutrients in pigs, leading to decreased digestibility and daily weight gain. Glucosides, under the action of myrosinase, hydrolyze to form isothiocyanates, oxazolidinyl thiones, and nitrile compounds, which irritate the digestive tract mucosa of pigs, causing ulcers and damaging organs such as the liver. In this experiment, treatment 2 showed that the degradation rates of crude fiber and glucosinolates reached 27.03% and 53.31%, respectively, achieving highly significant results. After fermentation, the crude fiber content of the substrate decreased to 15.31%, the glucosinolate content decreased to 21.27 μmol / g, and the tannin content increased from 5.84 mg / kg to 7.45 mg / kg, a relatively small increase, making it suitable as a feed additive for pig farming.

[0099] 3. Conclusion

[0100] Following the evaluation of three fermentation experiments—"microbial agent screening test," "enzyme preparation screening test," and "synergistic fermentation test of compound enzyme and microbial preparations"—and considering the changes in various nutritional indicators, a mixture of feed rapeseed and rapeseed meal was selected as the fermentation substrate, with a moisture content controlled at approximately 60%. The fermentation agent selected was a compound enzyme and microbial preparation containing phytase, cellulase, hemicellulase, pectinase, tanninase, xylanase, α-galactosidase, and *Lactobacillus plantarum*, *Bacillus subtilis*, *Saccharomyces cerevisiae*, and *Aspergillus niger*. This synergistic fermentation method achieved good fermentation results. While significantly increasing crude protein content, it also significantly reduced the content of anti-nutritional factors such as crude fiber, phytic acid, and glucosinolates, greatly improving the nutritional quality of the fermented feed and providing technical support for the preparation of pig compound feed. Through mixed solid-state fermentation, while improving the nutritional quality of the feed, it also allows for more efficient utilization of rapeseed meal and feed rapeseed. Replacing some of the corn, soybean meal, and wheat bran required for daily pig farming with fermented feed reduces the amount of these concentrates used, lowers enterprise farming costs, and achieves the goal of cost reduction and efficiency improvement.

[0101] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal, characterized in that, Harvest rapeseed from its peak flowering period to its end flowering period, add rapeseed meal and mix well to form a fermentation substrate, so that the moisture content of the fermentation substrate is 55% to 65%. Spray the compound enzyme preparation evenly onto the fermentation substrate, mix thoroughly with a mixer, and finally compact and seal with a packing machine. Pack and store. It can be opened and used gradually after 45 days of fermentation. The compound enzyme preparation includes microbial agents and enzymes; the microbial agents include four types of microbial agents: Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger; the enzymes include phytase, cellulase, hemicellulase, pectinase, tanninase, xylanase, and α-galactosidase.

2. The method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal as described in claim 1, characterized in that, The harvesting of forage rapeseed from its peak flowering period to its end flowering period refers to cutting the rapeseed plants 10-20cm above the ground during this period and then crushing them into 1-2mm coarse pieces using a straw crusher.

3. The method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal as described in claim 1, characterized in that, The fermentation substrate has a water content of 60%.

4. The method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal as described in claim 1, characterized in that, The viable counts of Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger in the microbial agent are 1:1:2:

1.

5. The method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal as described in claim 1, characterized in that, The dosage of the bacterial agent in the compound enzyme preparation is, for example, ≥1.0 × 10⁻⁶ per 1 kg of fermentation substrate, with a live Lactobacillus plantarum count ≥1.0 × 10⁻⁶. 10 CFU / g; Bacillus subtilis viable count ≥1.0 × 10⁻⁶ 10 CFU / g; viable count of Saccharomyces cerevisiae 2.0 × 10⁻⁶ 10 cfu / g; viable Aspergillus niger count ≥1.0×10⁻⁶ 10 cfu / g.

6. The method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal as described in claim 1, characterized in that, The weight ratio of phytase, cellulase, hemicellulase, pectinase, tanninase, xylanase, and α-galactosidase in the enzyme preparation is as follows: phytase 50 mg; cellulase 100 mg; hemicellulase 10 mg; pectinase 200 mg; tanninase 10 mg; xylanase 100 mg; α-galactosidase 50 mg.

7. The method for preparing pig feed by solid-state fermentation of rapeseed and rapeseed meal as described in claim 1, characterized in that, The preparation method of the compound enzyme preparation is as follows, taking the amount required for 1 kg of fermentation substrate as an example: Preparation of microbial agents: Add 50g of brown sugar to 50ml of sterile water, and then add the required Lactobacillus plantarum, Bacillus subtilis, Saccharomyces cerevisiae, and Aspergillus niger in sequence; Enzyme preparation: Weigh 50 mg of phytase with an enzyme activity ≥ 5 U / mg, and bring the volume to 2 ml with sterile water at 50℃, then adjust the pH to 5.

0. Weigh 100 mg of cellulase with an enzyme activity ≥3 U / mg, and bring the volume to 2 ml with sterile water at 40°C, then adjust the pH to 3.

0. Weigh 10 mg of cellulase with an enzyme activity ≥1.5 U / mg, and bring the volume to 1 ml with sterile water at 40°C, adjusting the pH to 5.

0. Weigh 200 mg of pectinase with an enzyme activity ≥40 U / mg, and bring the volume to 2 ml with sterile water at 50°C, then adjust the pH to 4.

0. Weigh 10 mg of tanninase with an enzyme activity ≥0.3 U / mg, and bring the volume to 1 ml with sterile water at 30°C, adjusting the pH to 4.

7. Weigh 100 mg of xylanase with an enzyme activity ≥50 U / mg, and dilute to 1 ml with sterile water at 50°C, then adjust the pH to 5.

0. Weigh 50 mg of α-galactosidase with an enzyme activity ≥2 U / mg, and bring the volume to 1 ml with sterile water at 37°C, adjusting the pH to 5.

5. The enzyme solution is obtained by mixing the enzymes that have been brought to a constant volume. Simply mix the prepared bacterial agent and enzyme agent thoroughly.

8. Pig feed prepared by any one of claims 1-7.

9. The pig feed according to claim 8, characterized in that, When using, add 30% to 40% by weight and mix with 60% to 70% concentrate.

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