Method for segmented fermentation of soybean meal feed by using bacillus coagulans

Abstract

The invention discloses a method for segmented fermentation of soybean meal feed by using bacillus coagulans, and belongs to the technical field of feed processing. The preparation method comprises the following steps: preparing a bacillus coagulans seed solution, inoculating a soybean meal raw material with the bacillus coagulans seed solution, and carrying out first-stage and second-stage fermentation. The bacillus coagulans strain adopted by the invention still has good adaptability and fermentation capacity in a complex feed matrix environment, and through first-stage fermentation, it is ensured that an initial flora quickly occupies a micro-ecological site in the initial stage of fermentation, so that the growth of infectious microbes is inhibited, the primary degradation reaction of a substrate is accelerated, and the degradation rate of the substrate is increased; in the first stage fermentation, effective degradation of anti-nutritional factors and preliminary improvement of raw material structures are ensured, the second stage fermentation strengthens metabolic conversion of substrates and accumulation of beneficial products and promotes synthesis accumulation of beneficial metabolites such as lactic acid and short-chain fatty acid, and staged fermentation not only realizes deep degradation of the anti-nutritional factors, but also promotes accumulation of the beneficial metabolites, so that the anti-nutritional factors can be effectively degraded. The functionality and the nutritional value of the soybean meal feed are obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of feed processing, in particular to a method for fermenting soybean meal feed by using Bacillus coagulans in stages. Background Art

[0002] Soybean meal is a commonly used raw material in plant-based protein feeds. Its high protein content, wide availability, and reasonable price make it widely used in livestock, poultry, and aquaculture. However, unprocessed soybean meal contains anti-nutritional factors such as trypsin inhibitors, phytic acid, and antigenic proteins, which not only reduce its nutritional value but also may adversely affect the animal's digestive system, affecting feed utilization. Therefore, effectively degrading the anti-nutritional factors in soybean meal and improving its digestibility and palatability are currently key areas of technological research and development in the feed industry. Microbial fermentation, due to its low cost, environmental friendliness, and high conversion efficiency, has become an important means of improving the nutritional quality of soybean meal. Fermentation not only effectively degrades anti-nutritional factors but also synthesizes a variety of beneficial metabolites, enhancing the functionality of soybean meal. However, existing soybean meal fermentation methods often utilize a single bacterial strain and a single-stage fermentation process. This leads to problems such as low fermentation efficiency, insufficient bacterial metabolic capacity, and poor product stability, making it difficult to fully optimize the nutritional quality of soybean meal. Summary of the Invention

[0003] The object of the present invention is to provide a method for fermenting soybean meal feed in stages using Bacillus coagulans to solve the problems raised in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solution: a method for fermenting soybean meal feed in stages using Bacillus coagulans, comprising:

[0005] pre-treating soybean meal raw material and applying a conditioning solution, amplifying and culturing a Bacillus coagulans strain using a culture medium, collecting the bacterial cells by centrifugation and resuspending them in a physiological buffer to obtain a Bacillus coagulans seed solution;

[0006] The soybean meal raw material is inoculated with the Bacillus coagulans seed liquid required for the first stage fermentation to carry out the first stage fermentation, so as to allow the Bacillus coagulans to initially grow and metabolize and partially degrade the anti-nutritional factors in the soybean meal. After the first stage fermentation reaches the predetermined indicators, additional nutrients and regulating liquid are added to the fermented soybean meal, and the Bacillus coagulans seed liquid is inoculated again to carry out the second stage fermentation.

[0007] When the fermented soybean meal reaches the set biochemical index, the fermentation process is terminated, and the fermented soybean meal is dried and crushed, the crushed fermented soybean meal is homogenized, and the quality of the prepared fermented soybean meal is tested.

[0008] Furthermore, pre-treating the soybean meal raw material and applying a conditioning liquid specifically includes the following steps:

[0009] Use a crusher to physically crush the soybean meal raw material and control the particle size to 0.5-2.0mm;

[0010] Prepare a regulating liquid and evenly spray the regulating liquid at 30% of the mass of the soybean meal raw material, adjust the moisture content of the soybean meal raw material to 40%-50%, and mix the regulating liquid and soybean meal by a stirring device during spraying;

[0011] The soybean meal raw material sprayed with the regulating liquid is mechanically stirred using a mixer for 5-15 minutes.

[0012] Furthermore, the regulating solution comprises the following substances in parts by weight: 1-3 parts of glucose, 0.2-0.6 parts of calcium lactate, 0.1-0.3 parts of sodium chloride, 0.05-0.2 parts of dipotassium hydrogen phosphate, 0.01-0.05 parts of polyoxyethylene stearate, and the rest is deionized water, and the pH of the regulating solution is adjusted to 5.5-6.5.

[0013] Furthermore, the Bacillus coagulans strain is amplified and cultured in a culture medium, the cells are collected by centrifugation and resuspended in a physiological buffer to obtain a Bacillus coagulans seed solution, which specifically includes the following steps:

[0014] A Bacillus coagulans strain with high protease activity, high stress resistance and high reproductive capacity is selected, the strain is inoculated on a slant culture medium, and the culture is placed in a 37° C. incubator for 16-24 hours to obtain activated colonies;

[0015] The activated bacterial colonies were inoculated into a preculture medium for preculture, wherein the preculture medium comprises the following components in weight percentages: 1.0-2.0 parts of tryptone, 0.3-0.8 parts of yeast extract, 0.5-1.0 parts of sodium chloride, 0.5-2.0 parts of glucose, and the remainder being deionized water, and the pH value of the culture medium was adjusted to 6.5-7.2;

[0016] The pre-culture temperature is 37°C, the shaker speed is 180 rpm, and the culture time is 8-12 hours, until the bacterial concentration reaches OD600 of 0.8-1.2;

[0017] The bacterial liquid obtained from the pre-culture is inoculated into a liquid culture medium for expanded culture at an inoculum volume of 10%. The components of the liquid culture medium are the same as those of the pre-culture medium. The culture temperature for the expanded culture is 37°C, the shaker speed is 180 rpm, and the culture time is 8-16 hours, until the bacterial concentration reaches an OD600 of 1.5-2.5. During the culture process, the dissolved oxygen level is controlled to be no less than 20%;

[0018] After the expansion culture is completed, the bacterial liquid is collected by centrifugation, and the precipitated bacteria are resuspended in a physiological buffer solution to obtain the Bacillus coagulans seed liquid.

[0019] Furthermore, the first stage of fermentation specifically includes the following steps:

[0020] The pretreated soybean meal raw material is transferred to a fermentation container, which is a stainless steel horizontal fermentation tank or a tank fermentation device. Bacillus coagulans seed liquid is evenly sprayed into the soybean meal raw material at a ratio of 6% to 10% of the wet weight of the soybean meal raw material. During the inoculation process, the soybean meal raw material and the Bacillus coagulans seed liquid are stirred and mixed;

[0021] During the first stage of fermentation, the fermentation temperature is controlled at 35-40° C., the relative humidity is controlled at 60%-70%, the fermentation time is controlled at 24-48 hours, and the soybean meal raw material is turned over intermittently during the fermentation process.

[0022] Furthermore, the first stage of fermentation further includes the following steps:

[0023] During the first stage of fermentation, fermented soybean meal samples were sampled and tested every 6-12 hours. Test items included pH value, temperature, moisture content, protease activity, and bacterial count.

[0024] When the pH value is stable in the range of 6.0-6.5, the protease activity reaches the preset threshold, and the bacterial count reaches 1.0×10 8 When the content of phytic acid or trypsin inhibitor in the original soybean meal is significantly reduced, it is determined that the first stage fermentation has reached the predetermined index and the first stage fermentation is completed.

[0025] Furthermore, the second stage fermentation specifically includes the following steps:

[0026] After the first stage of fermentation is completed, the fermented soybean meal is retained in the original fermentation container, and a nutrient solution is added at a rate of 5% to 15% of the wet weight of the fermented soybean meal. The nutrients in the nutrient solution are preferably glucose, corn steep liquor, yeast extract, and amino acids.

[0027] At the same time, add a regulating liquid at 10%-30% of the wet weight of the fermented soybean meal and stir to mix the fermented soybean meal to adjust the overall moisture content of the fermented soybean meal to 45%-55%;

[0028] After the addition operation is completed, the Bacillus coagulans seed liquid is inoculated at 3%-6% of the wet weight of the fermented soybean meal. After the inoculation is completed, the fermentation temperature is maintained at 35-38°C and the relative humidity is maintained at 60%-75%. A suitable oxygen supply environment is maintained through intermittent ventilation, and the ventilation rate is controlled at 0.1-0.5vvm. The fermentation time is 24-48 hours.

[0029] Furthermore, the second stage fermentation further includes the following steps:

[0030] During the second stage of fermentation, fermentation samples were collected every 8 hours for testing. Testing items included pH value, temperature, moisture content, bacterial count, phytic acid residue, and lactic acid and short-chain fatty acid content.

[0031] When the test results show that the bacterial count is not less than 1.0×10 8 When the pH value is stable in the range of 6.0-6.5, the phytic acid degradation rate is greater than 80%, and a certain amount of lactic acid and short-chain fatty acids are detected, it is determined that the second stage fermentation has reached the termination standard and the second stage fermentation is completed.

[0032] Furthermore, the fermented soybean meal is dried and crushed, and the crushed fermented soybean meal is homogenized, which specifically includes the following steps:

[0033] After the second stage of fermentation is completed, the fermented soybean meal that has been confirmed to have completed fermentation is evenly spread in a drying device. The drying device is a hot air circulation drying oven. The drying temperature is controlled at 45-55°C and the drying time is controlled at 4-12 hours until the moisture content of the fermented soybean meal drops to 10%-12%.

[0034] The drying process adopts a layered turning method, and after drying is completed, the fermented soybean meal is cooled and dried to room temperature.

[0035] Furthermore, the fermented soybean meal is dried and crushed, and the crushed fermented soybean meal is homogenized, which specifically includes the following steps:

[0036] The dried fermented soybean meal is fed into a grinder for grinding, and the operating parameters of the grinder are controlled so that the particle size of the obtained powder is controlled between 200-500 μm;

[0037] The crushed fermented soybean meal powder is homogenized by a spiral mixer. The stirring time is set to 10-20 minutes according to the batch weight, and the stirring speed is set to 60-120 rpm. Anti-caking aid is added during the homogenization process.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] 1. The Bacillus coagulans strain used in the present invention has high protease activity, high stress resistance and high reproductive capacity, ensuring good adaptability and fermentation capacity in a complex feed matrix environment. Initial activation through slant culture can restore the physiological metabolic state of the bacteria and improve its subsequent expansion efficiency. During the pre-culture stage, the bacterial activity is rapidly increased and the expression of its metabolic enzymes is promoted. The bacterial concentration is accurately controlled to provide a uniform and highly active seed liquid for subsequent fermentation. The use of a physiological buffer to resuspend the bacteria can effectively maintain cell activity and provide a stable seed liquid delivery medium, ensuring the stability and efficiency of the fermentation starting bacterial population.

[0040] 2. The present invention effectively ensures that the starting flora quickly occupies the microecological site in the early stage of fermentation by setting the first stage fermentation, inhibits the growth of miscellaneous bacteria, and accelerates the initial degradation reaction of the substrate. Under controlled temperature and humidity, Bacillus can be promoted to rapidly reproduce and secrete key metabolic enzymes such as proteases, thereby effectively destroying anti-nutritional factors such as trypsin inhibitors and phytic acid in soybean meal, ensuring the effective degradation of anti-nutritional factors and the initial improvement of the raw material structure. The second stage fermentation further strengthens the metabolic conversion of the substrate and the accumulation of beneficial products on the basis of the first stage fermentation, promotes the synthesis and accumulation of beneficial metabolites such as lactic acid and short-chain fatty acids, further improves the feed flavor and the animal intestinal probiotic environment, and maintains suitable microenvironmental conditions. It effectively improves the activity and metabolic intensity of the bacteria in the fermentation system, and the staged fermentation not only achieves the deep degradation of anti-nutritional factors, but also promotes the accumulation of various beneficial metabolites, which significantly improves the functionality and nutritional value of the soybean meal feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic flow chart of the staged fermentation method of the present invention. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] See also Figure 1 , the present invention provides the following technical solutions:

[0044] The method for fermenting soybean meal feed in stages using Bacillus coagulans comprises:

[0045] pre-treating soybean meal raw material and applying a conditioning solution, amplifying and culturing a Bacillus coagulans strain using a culture medium, collecting the bacterial cells by centrifugation and resuspending them in a physiological buffer to obtain a Bacillus coagulans seed solution;

[0046] The soybean meal raw material is inoculated with the Bacillus coagulans seed liquid required for the first stage fermentation to carry out the first stage fermentation, so as to allow the Bacillus coagulans to initially grow and metabolize and partially degrade the anti-nutritional factors in the soybean meal. After the first stage fermentation reaches the predetermined indicators, additional nutrients and regulating liquid are added to the fermented soybean meal, and the Bacillus coagulans seed liquid is inoculated again to carry out the second stage fermentation.

[0047] When the fermented soybean meal reaches the set biochemical index, the fermentation process is terminated, and the fermented soybean meal is dried and crushed, the crushed fermented soybean meal is homogenized, and the quality of the prepared fermented soybean meal is tested.

[0048] Use a crusher to physically crush the soybean meal raw material and control the particle size to 0.5-2.0mm;

[0049] Prepare a conditioning liquid and evenly spray the conditioning liquid at 30% of the mass of the soybean meal raw material, adjust the moisture content of the soybean meal raw material to 40%-50%, and mix the conditioning liquid and soybean meal through a stirring device during spraying;

[0050] The soybean meal raw material sprayed with the regulating liquid is mechanically stirred using a mixer for 5-15 minutes.

[0051] The regulating solution comprises the following substances in parts by weight: 1-3 parts of glucose, 0.2-0.6 parts of calcium lactate, 0.1-0.3 parts of sodium chloride, 0.05-0.2 parts of dipotassium hydrogen phosphate, 0.01-0.05 parts of polyoxyethylene stearate, and the rest is deionized water. The pH of the regulating solution is adjusted to 5.5-6.5.

[0052] In the above embodiment, the soybean meal raw material is physically crushed and the particle size is controlled to increase its specific surface area, which is more conducive to the attachment and growth of microorganisms in the subsequent fermentation process, thereby improving the fermentation efficiency. The spraying of the regulating liquid effectively regulates the initial moisture content of the soybean meal raw material, so that it is in a moisture environment suitable for the growth and reproduction of microorganisms, and avoids excessively high or low moisture content affecting the activity of the bacteria. At the same time, the components such as glucose, calcium lactate, sodium chloride, dipotassium hydrogen phosphate, polyoxyethylene stearate in the regulating liquid not only provide a basic energy source, minerals and buffer system for microorganisms, but also have the functions of regulating osmotic pressure, improving soybean meal texture and promoting beneficial metabolism. The pH of the regulating liquid is controlled within the range of 5.5-6.5, which can create a more stable acid-base environment for subsequent fermentation, reduce the initial strain stress response, improve its activity, and provide sufficient preparation in terms of physical and chemical conditions for the subsequent microbial fermentation process.

[0053] A Bacillus coagulans strain with high protease activity, high stress resistance and high reproductive capacity is selected, the Bacillus coagulans strain is inoculated on a slant culture medium, and cultured in a 37°C incubator for 16-24 hours to obtain activated colonies;

[0054] The activated bacterial colonies were inoculated into a preculture medium for preculture. The preculture medium included the following components in weight percentages: 1.0-2.0 parts of tryptone, 0.3-0.8 parts of yeast extract, 0.5-1.0 parts of sodium chloride, 0.5-2.0 parts of glucose, and the remainder was deionized water. The pH value of the culture medium was adjusted to 6.5-7.2.

[0055] The pre-culture temperature is 37°C, the shaker speed is 180 rpm, and the culture time is 8-12 hours, until the bacterial concentration reaches OD600 of 0.8-1.2;

[0056] The bacterial liquid obtained from the pre-culture is inoculated into a liquid culture medium for expansion culture at an inoculum volume of 10%. The components of the liquid culture medium are the same as those of the pre-culture medium. The expansion culture temperature is 37°C, the shaker speed is 180 rpm, and the culture time is 8-16 hours, until the bacterial concentration reaches OD600 of 1.5-2.5. During the culture process, the dissolved oxygen level is controlled to be not less than 20%;

[0057] After the expansion culture is completed, the bacterial liquid is collected by centrifugation, and the precipitated bacteria are resuspended in a physiological buffer solution to obtain the Bacillus coagulans seed liquid.

[0058] In the above examples, the Bacillus coagulans strain employed exhibits high protease activity, strong stress resistance, and high reproductive capacity, ensuring its excellent adaptability and fermentation capacity in complex feed matrix environments. Initial activation through slant culture restores the physiological metabolic state of the bacteria and improves the efficiency of subsequent expansion. During the pre-culture stage, the use of a culture medium containing readily metabolizable carbon and nitrogen sources such as tryptone, yeast extract, and glucose, combined with appropriate pH and temperature conditions, rapidly enhances bacterial activity and promotes the expression of metabolic enzymes. Through staged culture and OD600 monitoring, precise control of bacterial concentration is achieved, providing a uniform and highly active seed solution for subsequent fermentation. In particular, maintaining the dissolved oxygen level at no less than 20% during expansion culture maintains the aerobic metabolic efficiency of the Bacillus, preventing a decrease in bacterial yield or premature entry into the dormant phase due to oxygen deficiency. Furthermore, resuspending the bacteria in a physiological buffer effectively maintains cell activity and provides a stable seed solution delivery medium, ensuring the stability and efficiency of the fermentation starting population.

[0059] The pretreated soybean meal raw material is transferred to a fermentation container, which is a stainless steel horizontal fermentation tank or a tank fermentation device. Bacillus coagulans seed liquid is evenly sprayed into the soybean meal raw material at a ratio of 6% to 10% of the wet weight of the soybean meal raw material. During the inoculation process, the soybean meal raw material and the Bacillus coagulans seed liquid are stirred and mixed;

[0060] During the first stage of fermentation, the fermentation temperature is controlled at 35-40°C, the relative humidity is controlled at 60%-70%, the fermentation time is controlled at 24-48 hours, and the soybean meal raw material is turned intermittently during the fermentation process;

[0061] During the first stage of fermentation, fermented soybean meal samples were sampled and tested every 6-12 hours. Test items included pH value, temperature, moisture content, protease activity, and bacterial count.

[0062] When the pH value is stable in the range of 6.0-6.5, the protease activity reaches the preset threshold, and the bacterial count reaches 1.0×10 8 When the content of phytic acid or trypsin inhibitor in the original soybean meal is significantly reduced, it is determined that the first stage fermentation has reached the predetermined index and the first stage fermentation is completed.

[0063] After the first stage of fermentation is completed, the fermented soybean meal is retained in the original fermentation container, and a nutrient solution is added at a rate of 5% to 15% of the wet weight of the fermented soybean meal. The nutrients in the nutrient solution are preferably glucose, corn steep liquor, yeast extract, and amino acids.

[0064] At the same time, add a regulating liquid at 10%-30% of the wet weight of the fermented soybean meal and stir to mix the fermented soybean meal to adjust the overall moisture content of the fermented soybean meal to 45%-55%;

[0065] After the addition operation is completed, the Bacillus coagulans seed solution is inoculated at 3%-6% of the wet weight of the fermented soybean meal. After the inoculation is completed, the fermentation temperature is maintained at 35-38°C and the relative humidity is maintained at 60%-75%. A suitable oxygen supply environment is maintained by intermittent ventilation with the ventilation rate controlled at 0.1-0.5 vvm. The fermentation time is 24-48 hours.

[0066] During the second stage of fermentation, fermentation samples were collected every 8 hours for testing. Testing items included pH value, temperature, moisture content, bacterial count, phytic acid residue, and lactic acid and short-chain fatty acid content.

[0067] When the test results show that the bacterial count is not less than 1.0×10 8 When the pH value is stable in the range of 6.0-6.5, the phytic acid degradation rate is greater than 80%, and a certain amount of lactic acid and short-chain fatty acids are detected, it is determined that the second stage fermentation has reached the termination standard and the second stage fermentation is completed.

[0068] In the above embodiment, by setting the first stage fermentation, a 6%-10% ratio of Bacillus coagulans seed liquid is used for inoculation, which effectively ensures that the starting flora quickly occupies the microecological site in the early stage of fermentation, inhibits the growth of miscellaneous bacteria, and accelerates the initial degradation reaction of the substrate. Under controlled temperature and humidity, Bacillus can be promoted to multiply rapidly and secrete key metabolic enzymes such as protease, thereby effectively destroying anti-nutritional factors such as trypsin inhibitors and phytic acid in soybean meal. Through intermittent stirring operation, poor ventilation and uneven heating caused by raw material accumulation are avoided, so that the bacteria are evenly distributed and the growth environment is stable. When the fermentation end standard is established as protease activity, bacterial count and phytic acid degradation rate are up to standard, a good foundation can be laid for the second stage fermentation, ensuring the effective degradation of anti-nutritional factors and the preliminary improvement of raw material structure, which is one of the key links of the segmented fermentation method.

[0069] In the above embodiment, the design of the second stage fermentation further strengthens the metabolic conversion of the substrate and the accumulation of beneficial products on the basis of the first stage fermentation. By adding nutrient solution and regulating liquid, the content of available carbon source and nitrogen source in the fermentation matrix can be significantly improved to meet the subsequent high-density growth and metabolic needs of microorganisms. The components of the supplementary liquid can stimulate the secondary metabolic potential of Bacillus, promote the synthesis and accumulation of beneficial metabolites such as lactic acid and short-chain fatty acids, and further improve the feed flavor and animal intestinal probiotic environment. At the same time, by precisely controlling the moisture content and fermentation conditions, including inoculation amount, temperature, humidity and ventilation rate, etc., maintaining suitable microenvironmental conditions, effectively improving the activity and metabolic intensity of the bacteria in the fermentation system. The second stage fermentation not only achieves the deep degradation of anti-nutritional factors, but also promotes the accumulation of a variety of beneficial metabolites, significantly improving the functionality and nutritional value of soybean meal feed, which is an important manifestation of the process of the present invention being superior to conventional single-stage fermentation.

[0070] After the second stage of fermentation is completed, the fermented soybean meal is evenly spread in a drying device. The drying device is a hot air circulation drying oven. The drying temperature is controlled at 45-55°C and the drying time is controlled at 4-12 hours until the moisture content of the fermented soybean meal drops to 10%-12%.

[0071] The drying process adopts the layered turning method, and the fermented soybean meal is cooled and dried to room temperature after drying.

[0072] The dried fermented soybean meal is fed into a grinder for grinding, and the operating parameters of the grinder are controlled so that the particle size of the obtained powder is controlled between 200-500 μm;

[0073] The crushed fermented soybean meal powder is homogenized by a spiral mixer. The stirring time is set to 10-20 minutes according to the batch weight, and the stirring speed is set to 60-120 rpm. Anti-caking aid is added during the homogenization process.

[0074] The technical solution of the present invention is further illustrated by the following control group and application examples:

[0075] Control group: The operation was carried out according to the technical plan specified in the instructions without any variable adjustment;

[0076] Application Example 1: Based on the technical solution specified in the instructions, the strain was replaced with a Bacillus subtilis strain, and the culture medium formula remained the same as the original solution; the culture conditions such as culture temperature, pH, and shaker speed remained unchanged; the inoculum size, nutrient addition strategy, and moisture content adjustment used in the fermentation stages (first and second stages) were the same as those of the control group;

[0077] Application Example 2: Based on the control group, the inoculation amount of Bacillus coagulans seed liquid in the first stage of fermentation was adjusted to 3%;

[0078] Application Example 3: Based on the control group, the inoculation amount of Bacillus coagulans seed liquid in the second stage fermentation was adjusted to 8%;

[0079] The soybean meal feed was fermented and produced according to different processes of the control group, application example 1, application example 2 and application example 3, and after production, the fermented soybean meal feed in the control group, application example 1, application example 2 and application example 3 was sampled and tested, and the bacterial count, pH value, phytic acid residue, phytic acid degradation rate, lactic acid content, short-chain fatty acid content, crude protein content and moisture content were determined. The specific test results are shown in Table 1:

[0080]

[0081] Table 1

[0082] As shown in Table 1, the control group performed best in all test indicators. The phytic acid degradation rate of the fermented soybean meal reached 90%, the accumulation of lactic acid and short-chain fatty acids was high, and the crude protein content was significantly improved. This proves that the Bacillus coagulans seed solution formula, two-stage fermentation inoculation ratio and nutritional control method used in the standard process have synergistic advantages and can achieve the optimization of soybean meal fermentation efficiency and quality. In Application Example 2, although the fermentation process can be completed by replacing the strain, its degradation efficiency and metabolic activity are slightly reduced compared with the standard Bacillus coagulans, indicating that the strain selection has a significant effect on the fermentation effect, verifying the importance of selecting a specific Bacillus coagulans strain in the present invention. In Application Example 3, the inoculation amount in the first stage was reduced. Due to the insufficient number of bacteria in the early stage, the fermentation start-up efficiency was low and the degradation of anti-nutritional factors was insufficient, which ultimately affected the overall fermentation quality. This shows that the inoculation amount in the first stage is crucial to bacterial growth and preliminary substrate processing. In Application Example 4, the inoculation amount in the second stage was increased, which increased the amount of fermentation products produced in the later stage to a certain extent, but failed to significantly exceed the standard process. There is an optimal range for the inoculation amount in the second stage, and blindly increasing it cannot proportionally improve the fermentation effect.

[0083] By using the method of the present invention to ferment soybean meal feed in stages using Bacillus coagulans and strictly controlling the strain, inoculation amount and fermentation conditions, the degradation rate of anti-nutritional factors in soybean meal can be significantly improved, the metabolic activity and nutritional value of the fermentation products can be enhanced, and the feed quality can be effectively improved. The method has clear process advantages and promotion value.

[0084] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for fermenting soybean meal feed by segmentation using Bacillus coagulans, characterized in that: include: pre-treating soybean meal raw material and applying a conditioning solution, amplifying and culturing a Bacillus coagulans strain using a culture medium, collecting the bacterial cells by centrifugation and resuspending them in a physiological buffer to obtain a Bacillus coagulans seed solution; The soybean meal raw material is inoculated with the Bacillus coagulans seed liquid required for the first stage fermentation to carry out the first stage fermentation, so as to allow the Bacillus coagulans to initially grow and metabolize and partially degrade the anti-nutritional factors in the soybean meal. After the first stage fermentation reaches the predetermined indicators, additional nutrients and regulating liquid are added to the fermented soybean meal, and the Bacillus coagulans seed liquid is inoculated again to carry out the second stage fermentation. When the fermented soybean meal reaches the set biochemical index, the fermentation process is terminated, and the fermented soybean meal is dried and crushed, the crushed fermented soybean meal is homogenized, and the quality of the prepared fermented soybean meal is tested.

2. The method for fermenting soybean meal feed using Bacillus coagulans in stages according to claim 1, wherein: Pre-treating the soybean meal raw material and applying the conditioning liquid specifically includes the following steps: Use a crusher to physically crush the soybean meal raw material and control the particle size to 0.5-2.0mm; Prepare a conditioning liquid and evenly spray the conditioning liquid at 30% of the mass of the soybean meal raw material, adjust the moisture content of the soybean meal raw material to 40%-50%, and mix the conditioning liquid and soybean meal by a stirring device during spraying; The soybean meal raw material sprayed with the regulating liquid is mechanically stirred using a mixer for 5-15 minutes.

3. The method for fermenting soybean meal feed by segmented fermentation using Bacillus coagulans according to claim 2, characterized in that: The regulating solution comprises the following substances in parts by weight: 1-3 parts of glucose, 0.2-0.6 parts of calcium lactate, 0.1-0.3 parts of sodium chloride, 0.05-0.2 parts of dipotassium hydrogen phosphate, 0.01-0.05 parts of polyoxyethylene stearate, and the remainder is deionized water. The pH of the regulating solution is adjusted to 5.5-6.

5.

4. The method for fermenting soybean meal feed by segmentation using Bacillus coagulans according to claim 1, wherein: The Bacillus coagulans strain is amplified and cultured in a culture medium, and the cells are collected by centrifugation and resuspended in a physiological buffer to obtain a Bacillus coagulans seed solution, which specifically includes the following steps: A Bacillus coagulans strain with high protease activity, high stress resistance and high reproductive capacity is selected, the strain is inoculated on a slant culture medium, and the culture is placed in a 37° C. incubator for 16-24 hours to obtain activated colonies; The activated bacterial colonies were inoculated into a preculture medium for preculture, wherein the preculture medium comprises the following components in weight percentages: 1.0-2.0 parts of tryptone, 0.3-0.8 parts of yeast extract, 0.5-1.0 parts of sodium chloride, 0.5-2.0 parts of glucose, and the remainder being deionized water, and the pH value of the culture medium was adjusted to 6.5-7.2; The pre-culture temperature is 37°C, the shaker speed is 180 rpm, and the culture time is 8-12 hours, until the bacterial concentration reaches OD600 of 0.8-1.2; The bacterial liquid obtained from the pre-culture is inoculated into a liquid culture medium for expanded culture at an inoculum volume of 10%. The components of the liquid culture medium are the same as those of the pre-culture medium. The culture temperature for the expanded culture is 37°C, the shaker speed is 180 rpm, and the culture time is 8-16 hours, until the bacterial concentration reaches an OD600 of 1.5-2.

5. During the culture process, the dissolved oxygen level is controlled to be no less than 20%; After the expansion culture is completed, the bacterial liquid is collected by centrifugation, and the precipitated bacteria are resuspended in a physiological buffer solution to obtain the Bacillus coagulans seed liquid.

5. The method for fermenting soybean meal feed by segmentation using Bacillus coagulans according to claim 1, wherein: The first stage of fermentation specifically includes the following steps: The pretreated soybean meal raw material is transferred to a fermentation container, which is a stainless steel horizontal fermentation tank or a tank fermentation device. Bacillus coagulans seed liquid is evenly sprayed into the soybean meal raw material at a ratio of 6% to 10% of the wet weight of the soybean meal raw material. During the inoculation process, the soybean meal raw material and the Bacillus coagulans seed liquid are stirred and mixed; During the first stage of fermentation, the fermentation temperature is controlled at 35-40° C., the relative humidity is controlled at 60%-70%, the fermentation time is controlled at 24-48 hours, and the soybean meal raw material is turned over intermittently during the fermentation process.

6. The method for fermenting soybean meal feed by segmentation using Bacillus coagulans according to claim 5, characterized in that: The first stage of fermentation also includes the following steps: During the first stage of fermentation, fermented soybean meal samples were sampled and tested every 6-12 hours. Test items included pH value, temperature, moisture content, protease activity, and bacterial count. When the pH value is stable in the range of 6.0-6.5, the protease activity reaches the preset threshold, and the bacterial count reaches 1.0×10 8 When the content of phytic acid or trypsin inhibitor in the original soybean meal is significantly reduced, it is determined that the first stage fermentation has reached the predetermined index and the first stage fermentation is completed.

7. The method for fermenting soybean meal feed by segmentation using Bacillus coagulans according to claim 1, wherein: The second stage fermentation specifically includes the following steps: After the first stage of fermentation is completed, the fermented soybean meal is retained in the original fermentation container, and a nutrient solution is added at a rate of 5% to 15% of the wet weight of the fermented soybean meal. The nutrients in the nutrient solution are preferably glucose, corn steep liquor, yeast extract, and amino acids. At the same time, add a regulating liquid at 10%-30% of the wet weight of the fermented soybean meal and stir to mix the fermented soybean meal to adjust the overall moisture content of the fermented soybean meal to 45%-55%; After the addition operation is completed, the Bacillus coagulans seed liquid is inoculated at 3%-6% of the wet weight of the fermented soybean meal. After the inoculation is completed, the fermentation temperature is maintained at 35-38°C and the relative humidity is maintained at 60%-75%. A suitable oxygen supply environment is maintained through intermittent ventilation, and the ventilation rate is controlled at 0.1-0.5vvm. The fermentation time is 24-48 hours.

8. The method for fermenting soybean meal feed by segmentation using Bacillus coagulans according to claim 7, characterized in that: The second stage fermentation specifically further comprises the following steps: During the second stage of fermentation, fermentation samples were collected every 8 hours for testing. Testing items included pH value, temperature, moisture content, bacterial count, phytic acid residue, and lactic acid and short-chain fatty acid content. When the test results show that the bacterial count is not less than 1.0×10 8 When the pH value is stable in the range of 6.0-6.5, the phytic acid degradation rate is greater than 80%, and a certain amount of lactic acid and short-chain fatty acids are detected, it is determined that the second stage fermentation has reached the termination standard and the second stage fermentation is completed.

9. The method for fermenting soybean meal feed by segmentation using Bacillus coagulans according to claim 1, wherein: The fermented soybean meal is dried and crushed, and the crushed fermented soybean meal is homogenized, specifically comprising the following steps: After the second stage of fermentation is completed, the fermented soybean meal that has been confirmed to have completed fermentation is evenly spread in a drying device. The drying device is a hot air circulation drying oven. The drying temperature is controlled at 45-55°C and the drying time is controlled at 4-12 hours until the moisture content of the fermented soybean meal drops to 10%-12%. The drying process adopts a layered turning method, and after drying is completed, the fermented soybean meal is cooled and dried to room temperature.

10. The method for fermenting soybean meal feed by segmentation using Bacillus coagulans according to claim 9, characterized in that: The fermented soybean meal is dried and crushed, and the crushed fermented soybean meal is homogenized, which specifically includes the following steps: The dried fermented soybean meal is fed into a grinder for grinding, and the operating parameters of the grinder are controlled so that the particle size of the obtained powder is controlled between 200-500 μm; The crushed fermented soybean meal powder is homogenized by a spiral mixer. The stirring time is set to 10-20 minutes according to the batch weight, and the stirring speed is set to 60-120 rpm. Anti-caking aid is added during the homogenization process.

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