Preparation method and application of fermented soybean meal

Abstract

The invention provides a preparation method and application of fermented soybean meal, and relates to the technical field of biological feed. The preparation method of the fermented soybean meal comprises the steps of raw material preparation, soybean meal fermentation and finished product preparation. The soybean meal fermentation step comprises the following steps: adding a first strain into soybean meal for primary fermentation, and adding a second strain for secondary fermentation after the primary fermentation is finished; the first-stage fermentation and the second-stage fermentation are preferably anaerobic fermentation; the first strain and the second strain are preferably activated strains; wherein in the first-stage fermentation and / or second-stage fermentation process, volatile substances are monitored, and when the ratio of acetic acid to acetoin in the volatile substances is larger than 1: 1, fermentation is ended. The fermentation process is controlled by monitoring the content of volatile substances, namely acetic acid and acetoin, so that the soybean meal finished product obtained by fermentation has relatively high acidity and acid soluble protein content, the palatability of animals is further improved, the digestion and absorption of the animals are promoted, the microbial environment of the intestinal tracts of the animals is balanced, and the immunity of the animals is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological feed, and particularly relates to a two-stage fermentation soybean meal preparation process and its application. Background Art

[0002] Soybean meal is a by-product obtained by subjecting soybeans to oil extraction, solvent removal, and drying and cooling in the oil processing technology, and then packaging. Because it is rich in protein and has a relatively rich and balanced amino acid composition, it is an indispensable protein source in the animal breeding industry. By using modern bioengineering technology, the protein solubility in soybean meal can be improved to a certain extent through the fermentation of microorganisms, and the digestibility can be increased; at the same time, the antinutritional factors in soybean meal are eliminated, and soybean protein is degraded into high-quality small peptide protein sources.

[0003] The fermentation soybean meal process is closely related to the use of its strains, including the single or compound use of molds, bacilli, lactic acid bacteria, and yeasts, as well as the segmented process in cooperation with the use of strains. Piglets fed a diet supplemented with Aspergillus-fermented soybean meal can increase the weight gain rate and significantly improve the feed conversion rate. Lactic acid bacteria fermentation technology has been widely used in the preparation of dairy products, plants, and silage for many years, and recently has been used in fermented feed, which can significantly reduce the pH value of raw materials and produce a variety of beneficial metabolites represented by lactic acid bacteria to improve the intestinal health of piglets. At the same time, the various organic acids produced by fermentation have a fragrant smell and play a role in flavoring, which can improve the palatability of feed.

[0004] At present, there are many studies on soybean meal fermentation. For example, Chun-Hua Chi et al. studied the effects of using Bacillus amyloliquefaciens, Lactobacillus, and Saccharomyces cerevisiae on the quality of fermented soybean meal, respectively. The results showed that Bacillus amyloliquefaciens showed significant improvement in nutritional quality and biological activity by removing protein- and carbohydrate-based antinutritional factors and allergens. Saccharomyces cerevisiae decomposed carbohydrate-based antinutritional factors but had no obvious effect on protein-based antinutritional factors. Lactobacillus only degraded the trypsin inhibitor based on protein but did not reduce antinutritional factors, resulting in a decrease in the biological activity of the fermented product. It shows that the fermentation characteristics of different strains vary greatly and can play a synergistic role through effective control in the same process.

[0005] CN 114903117 A discloses a method for preparing fermented soybean meal feed by segmented fermentation using compound bacteria. Using soybean meal and other raw materials, Aspergillus niger bacterium agent, Bacillus natto, and Lactobacillus plantarum are added respectively for segmented fermentation. After fermentation, many detection indexes are significantly better than those of soybean meal. For example, the acid-soluble protein is increased by 6 times, the crude protein is increased by 20%-30%, and the stachyose content is reduced from 35.71-36.21 mg / g to 1.74-3.24 mg / g. However, the three-stage fermentation takes 8-12 days in total, which is not conducive to the improvement of large-scale industrialization efficiency, and the long-term segmented fermentation is not conducive to the effective control of the process.

[0006] CN 104012755 A discloses a method for efficiently producing fermented soybean meal by two-step fermentation. Using soybean meal as the main raw material, Bacillus subtilis is used as the main strain for the first shallow pan fermentation. After aerobic fermentation for 1-2 d, fermented soybean meal with high protease activity is obtained. The above shallow pan fermented soybean meal is used as a kind of protease preparation similar to that and mixed with lactic acid bacteria and other unsterilized soybean meal for the second stacking fermentation. The crude protein content of the product obtained after fermentation reaches more than 50%, and the small molecule peptide content is higher than 10%. This invention fully considers the fermentation characteristic requirements of different strains. The segmented process can not only meet the requirements of mixed fermentation of aerobic bacteria and anaerobic bacteria, but also solve the problem that aerobic bacteria are difficult to be applied to solid-state stacking fermentation. However, the production capacity of the shallow pan fermentation during the first fermentation is low and not conducive to industrial application, and the long-term fermentation of 1-2 d is not conducive to the effective control of the process.

[0007] CN 106538823 A discloses a production process of high-acidity fermented soybean meal. Using soybean meal as the raw material, Rhizopus oryzae and lactic acid bacteria are inoculated, and cellulase and glucoamylase are added. By adopting secondary temperature control, finally, fermented soybean meal with an acid content greater than 5% is obtained, and the palatability of the product is better. This invention uses temperature as the basis for process control, but does not consider the temperature influence generated during the fermentation of strains, which is not conducive to effective control.

[0008] The above research mainly elaborates on the differences in fermentation condition requirements of different strains and the differences in segmented fermentation processes. Generally, after inoculating the raw materials, different strains or compound strains use time or temperature as the basis for process control, and adopt the fermentation method of aerobic first and then anaerobic to obtain products. However, there are few reports on the precise control of the process and the process control corresponding to clear technical parameters during fermentation. Summary of the Invention

[0009] The purpose of this application is to provide a method for preparing fermented soybean meal. The finished product of fermented soybean meal prepared by this method has a high acid-soluble protein content and lactic acid content, improves the palatability of animals, promotes the digestion and absorption of animals, balances the intestinal microbial environment of animals, and improves the immunity of animals.

[0010] To achieve the above object, the present application provides a method for preparing fermented soybean meal, comprising the following steps:

[0011] Raw material preparation: Adjust the moisture content of soybean meal;

[0012] Soybean meal fermentation: Add the first strain to the soybean meal for primary fermentation, and add the second strain for secondary fermentation after the primary fermentation ends; The primary fermentation and the secondary fermentation are preferably anaerobic fermentations; The first strain and the second strain are preferably activated strains;

[0013] Finished product preparation: Dry and crush the material obtained after the secondary fermentation to obtain the finished product of fermented soybean meal;

[0014] Among them, during the primary fermentation and / or the secondary fermentation process, volatile substances are monitored, and when acetic acid:acetoin in the volatile substances is >1:1, the fermentation ends.

[0015] In some aspects of the present invention, the moisture content of soybean meal is 40%-50%.

[0016] In some aspects of the present invention, the primary fermentation and the secondary fermentation are anaerobic fermentations.

[0017] In some aspects of the present invention, the temperature of the primary fermentation is 35-45°C.

[0018] In some aspects of the present invention, the temperature of the secondary fermentation is 35-45°C.

[0019] In some aspects of the present invention, the first strain and the second strain are activated strains.

[0020] In some aspects of the present invention, during the primary fermentation process, when acetic acid:acetoin in the volatile substances reaches 2.5-3:1, the fermentation is stopped.

[0021] In some aspects of the present invention, during the secondary fermentation process, when acetic acid:acetoin in the volatile substances reaches 9-12:1, the fermentation is stopped.

[0022] In some aspects of the present invention, the first strain and the second strain include at least one of yeast, lactic acid bacteria, mold, and Bacillus subtilis.

[0023] In some aspects of the present invention, the first strain is yeast, lactic acid bacteria, and mold, or yeast, lactic acid bacteria, and Bacillus subtilis.

[0024] In some aspects of the present invention, the second strain is yeast.

[0025] In some aspects of the present invention, the yeast includes Saccharomyces cerevisiae; the lactic acid bacteria include at least one of Lactobacillus acidophilus, Pediococcus pentosaceus, Pediococcus acidilactici, and Lactobacillus plantarum; the mold includes at least one of Aspergillus niger and Aspergillus oryzae.

[0026] In some aspects of the present invention, a complex enzyme preparation is added during the activation process of the first strain and the second strain.

[0027] The present invention also discloses a fermented soybean meal, which is prepared by any one of the above preparation methods.

[0028] In some aspects of the present invention, the acid-soluble protein in the finished product of fermented soybean meal is ≥16%, and the lactic acid content is ≥6%.

[0029] The present invention also discloses the use of the finished product of fermented soybean meal in the preparation of feed.

[0030] The present invention also discloses an animal feed, which includes the above-mentioned finished product of fermented soybean meal.

[0031] The beneficial effects of the present invention are as follows:

[0032] Through the quantitative detection of volatile substances acetic acid and acetoin, the precise control of the two-stage fermentation process is realized. The anti-nutritional substances in the fermented soybean meal are decomposed, and a large number of low-molecular-weight small peptides are generated. The fermentation of lactic acid bacteria effectively acidifies the material, and the pH decreases. The acid-soluble protein in the obtained product is ≥16%, and the lactic acid content is ≥6%. The high acid-soluble protein content of the finished product prepared by the method of this application indicates that the macromolecular peptides in the soybean meal are decomposed, and the protein solubility increases, which is beneficial to animal digestion and absorption; and the increase in lactic acid content indicates that the content of organic acids in the fermented finished product is high. Organic acids can improve the palatability of feed and can play a role in balancing the intestinal microbial environment of animals, thereby improving animal immunity. Detailed Embodiments

[0033] In the present invention, if not otherwise specified, percentages (%) or parts refer to weight percentages or weight parts relative to the composition.

[0034] In the present invention, if not otherwise specified, the various components or their preferred components involved can be combined with each other to form a new technical solution.

[0035] In the present invention, if not otherwise specified, all the embodiments and preferred embodiments mentioned herein can be combined with each other to form a new technical solution.

[0036] In the present invention, if not otherwise specified, all the technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0037] In the present invention, if there is no contrary description, the sum of the contents of the various components in the composition is 100%.

[0038] In the present invention, unless otherwise specified, the numerical range "a - b" represents an abbreviated representation of any real number combination between a and b, where both a and b are real numbers. For example, the numerical range "0 - 5" means that all real numbers between "0 - 5" are fully listed herein, and "0 - 5" is just an abbreviated representation of these numerical combinations.

[0039] In the present invention, unless otherwise specified, "its combination" refers to a multi-component mixture of the respective components, such as a two-component, three-component, four-component, and up to the maximum possible multi-component mixture.

[0040] If not specifically indicated, the basis for the percentages described in the present invention is the total weight of the composition.

[0041] The "ranges" disclosed herein are in the form of a lower limit and an upper limit. There can be one or more lower limits, and one or more upper limits respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower limit and upper limit define the boundaries of a particular range. All ranges that can be defined in this way are inclusive and combinable, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 - 120 and 80 - 110 are listed for a particular parameter, ranges of 60 - 110 and 80 - 120 are also contemplated. Additionally, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, then the following ranges are all contemplated: 1 - 3, 1 - 4, 1 - 5, 2 - 3, 2 - 4, and 2 - 5.

[0042] In this article, unless otherwise specified, each reaction is carried out at normal temperature and pressure.

[0043] In this article, unless otherwise specified, each reaction step can be carried out sequentially or non-sequentially. For example, other steps can be included between each reaction step, and the reaction steps can also be reversed in order. Preferably, the reaction method in this article is carried out sequentially.

[0044] The present invention provides a method for preparing fermented soybean meal, comprising the following steps:

[0045] Raw material preparation: Adjust the moisture content of the soybean meal;

[0046] Soybean meal fermentation: Add the first strain to the soybean meal for primary fermentation, and add the second strain for secondary fermentation after the primary fermentation ends; the primary fermentation and the secondary fermentation are preferably anaerobic fermentations; the first strain and the second strain are preferably activated strains;

[0047] Finished product preparation: Dry and crush the material obtained after the secondary fermentation ends to obtain the finished product of fermented soybean meal;

[0048] Among them, during the one-stage fermentation and / or two-stage fermentation process, volatile substances are monitored. When acetic acid:acetoin in the volatile substances is >1:1, the fermentation ends.

[0049] In some alternative embodiments, the raw material preparation step is as follows: the finished soybean meal obtained after extracting oil from soybeans is put into an inoculation tank and tap water is added to make the water content 40%-50%, and they are mixed evenly for inoculation preparation.

[0050] In some alternative embodiments, during the one-stage and / or two-stage fermentation process, volatile substances are measured every 6 hours.

[0051] In some alternative embodiments, the volatile substances are taken from above the fermented material.

[0052] In some alternative embodiments, during the one-stage fermentation process, when acetic acid:acetoin in the volatile substances reaches 2.5-3:1, the fermentation is stopped to obtain an intermediate product of soybean meal fermentation.

[0053] In some alternative embodiments, during the two-stage fermentation process, when acetic acid:acetoin in the volatile substances reaches 9-12:1, the fermentation is stopped to obtain soybean meal material.

[0054] In some alternative embodiments, the first strain and the second strain include at least one of yeast, lactic acid bacteria, mold, and Bacillus subtilis.

[0055] In some alternative embodiments, the yeast includes Saccharomyces cerevisiae; the lactic acid bacteria include at least one of Lactobacillus acidophilus, Pediococcus pentosaceus, Pediococcus acidilactici, and Lactobacillus plantarum; the mold includes at least one of Aspergillus niger and Aspergillus oryzae.

[0056] In some alternative embodiments, the above-mentioned first strain is used to prepare an activated bacterial solution.

[0057] In some alternative embodiments, the activated bacterial solution includes the first strain, water, and glucose.

[0058] In some alternative embodiments, the activated bacterial solution includes the first strain, water, glucose, and a complex enzyme preparation.

[0059] In some alternative embodiments, the complex enzyme preparation includes at least one of feed protease, amylase, glucanase, phytase, xylanase, pectinase, cellulase, α-galactosidase, and β-mannanase.

[0060] In some alternative embodiments, the preparation of the activated bacterial solution includes the following steps:

[0061] Add the first strain of bacteria to tap water, then add glucose, and adjust the pH value to 6.2 - 6.5. Mix evenly to obtain the activated bacterial liquid.

[0062] In some alternative embodiments, the activated bacterial liquid further includes a complex enzyme preparation.

[0063] In some alternative embodiments, the preparation of the activated bacterial liquid includes the following steps:

[0064] Add the first strain of bacteria to tap water, then sequentially add glucose and the complex enzyme preparation, and adjust the pH value to 6.2 - 6.5. Mix evenly to obtain the activated bacterial liquid.

[0065] In some alternative embodiments, the weight ratio of the first strain of bacteria, the complex enzyme preparation, tap water, and glucose is 2 - 5:0.3 - 0.5:100:1 - 2.

[0066] In some alternative embodiments, the amount of the activated bacterial liquid added is 5% - 10% of the weight of the soybean meal raw material.

[0067] In some alternative embodiments, the temperature of the first-stage fermentation is 35 - 45°C.

[0068] In some alternative embodiments, both the first-stage fermentation and the second-stage fermentation are anaerobic fermentations.

[0069] In some alternative embodiments, the second strain of bacteria used in the second-stage fermentation includes lactic acid bacteria.

[0070] In some alternative embodiments, lactic acid bacteria are used to prepare the lactic acid bacteria-activated bacterial liquid.

[0071] In some alternative embodiments, the preparation of the lactic acid bacteria liquid includes the following steps:

[0072] Pick lactic acid bacteria and inoculate them into the MRS seed culture medium. Activate them at 37°C for 24h - 48h.

[0073] In some alternative embodiments, the amount of the lactic acid bacteria liquid added is 5% - 10% of the weight of the intermediate product of soybean meal fermentation.

[0074] In some alternative embodiments, the temperature of the second-stage fermentation is 35 - 45°C.

[0075] In some alternative embodiments, the fermentation material obtained after the second-stage fermentation is dried and pulverized to obtain the finished product of fermented soybean meal.

[0076] The following further illustrates the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0077] In the following examples and comparative examples, the enzyme preparation was purchased from Henan Xinyangshao Biotechnology Co., Ltd., with the trade name GPⅠ type dedicated to corn-soybean meal diet; Saccharomyces cerevisiae 1 was purchased from Yinglian (Harbin) Food Additive Co., Ltd., with the trade name Saccharomyces cerevisiae dedicated to livestock and poultry; Lactobacillus acidophilus A (CGMCC No. 11854), Aspergillus oryzae 1 (CGMCC 3.13904) and Bacillus subtilis 1 (CGMCC 1.15792) were purchased from the China General Microbiological Culture Collection Center, and other strains were screened from nature by the laboratory and were all preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation numbers and preservation dates are as follows:

[0078] Aspergillus niger WRD-007: Preservation number CGMCC No. 9069, preservation date April 17, 2014;

[0079] Pediococcus pentosaceus Q: CGMCC No. 25533, preservation date August 15, 2022;

[0080] Bacillus subtilis WBRD00470-B3: CGMCC NO. 7641, preservation date May 24, 2013;

[0081] Pediococcus acidilactici TMM-3: Preservation number CGMCCNo. 21509, preservation date December 21, 2020;

[0082] Saccharomyces cerevisiae WBRD2.12052301: CGMCCNO. 6183, preservation date June 1, 2012.

[0083] In the following examples and comparative examples, the formula of the compound bacterium agent 1 is Saccharomyces cerevisiae 1, Lactobacillus acidophilus A, Aspergillus niger WRD-007; among them, the amount of Saccharomyces cerevisiae 1 is 2.0×10 10 -3.0×10 10 cfu / g, the amount of Lactobacillus acidophilus A is 0.8×10 10 -1.5×10 10 cfu / g, and the amount of Aspergillus niger WRD-007 is 3.0×10 9 -5.5×10 9 cfu / g.

[0084] The formula of compound bacterium agent 2 is Saccharomyces cerevisiae 1, Pediococcus pentosaceus Q, and Aspergillus oryzae 1; among them, the amount of Saccharomyces cerevisiae 1 is 2.0×10 10 -3.0×10 10 cfu / g, the amount of Pediococcus pentosaceus Q is 0.8×10 10 -1.0×10 10 cfu / g, and the amount of Aspergillus oryzae 1 is 3.0×10 9 -5.5×10 9 cfu / g.

[0085] The formula of compound bacterium agent 3 is Saccharomyces cerevisiae 1, Lactobacillus acidophilus A, and Bacillus subtilis WBRD00470 - B3; among them, the amount of Saccharomyces cerevisiae 1 is 2.0×10 10 -3.0×10 10 cfu / g, the amount of Lactobacillus acidophilus A is 0.8×10 10 -1.5×10 10 cfu / g, and the amount of Bacillus subtilis WBRD00470 - B3 is 3.5×10 9 -8.0×10 9 cfu / g.

[0086] The formula of compound bacterium agent 4 is Saccharomyces cerevisiae 1, Pediococcus acidilactici TMM - 3, and Aspergillus niger WRD - 007; among them, the amount of Saccharomyces cerevisiae 1 is 2.0×10 10 -3.0×10 10 cfu / g, the amount of Pediococcus acidilactici TMM - 3 is 0.8×10 10 -1.5×10 10 cfu / g, and the amount of Aspergillus niger WRD - 007 is 3.0×10 9 -5.5×10 9 cfu / g.

[0087] The formula of compound bacterium agent 5 is Saccharomyces cerevisiae 1, Pediococcus acidilactici TMM - 3, and Aspergillus niger WRD - 007; among them, the amount of Saccharomyces cerevisiae 1 is 2.0×10 10 -3.0×10 10 cfu / g, the amount of Pediococcus acidilactici TMM - 3 is 0.8×10 10 -1.5×10 10 cfu / g, and the amount of Aspergillus niger WRD - 007 is 3.0×10 9 -5.5×10 9 cfu / g).

[0088] The formula of the compound microbial agent 6 is Saccharomyces cerevisiae WBRD2.12052301, Lactobacillus acidophilus A, and Aspergillus niger WRD-007; among them, the amount of Saccharomyces cerevisiae WBRD2.12052301 is 2.0×10 10 -3.0×10 10 cfu / g, the amount of Lactobacillus acidophilus A is 0.8×10 10 -1.5×10 10 cfu / g, and the amount of Aspergillus niger WRD-007 is 3.0×10 9 -5.5×10 9 cfu / g.

[0089] The formula of the compound microbial agent 7 is Saccharomyces cerevisiae 1, Lactobacillus acidophilus A, and Bacillus subtilis 1; among them, the amount of Saccharomyces cerevisiae 1 is 2.0×10 10 -3.0×10 10 cfu / g, the amount of Lactobacillus acidophilus A is 0.8×10 10 -1.5×10 10 cfu / g, and the amount of Bacillus subtilis 1 is 3.5×10 9 -8.0×10 9 cfu / g.

[0090] In the following examples and comparative examples, the volatile components were sampled using a gas sampling bottle, and the detection method was GC-MS method: gas chromatography-mass spectrometry, Agilent 6890N / 5973Agilent (American company); gas chromatography conditions: HP-5MS (30×0.25mm×0.25μm film thickness), programmed temperature rise: initial temperature 60°C, hold for 2 min, then rise to 150°C at 5°C / min, hold for 1 min, and then rise to 300°C at 5°C / min, hold for 10 min. The carrier is high-purity He, the column flow rate is 1.0 ml / min, the injection port temperature is 250°C, the injection volume is 1 μl, and the split ratio is 50:1. Among them, the mass spectrometry conditions are: interface temperature 280°C, EI source, ionization voltage 70 eV, ion source temperature 230°C, and the scanning range is 29-400 amu. The mass-to-charge ratio and abundance of the corresponding fragments of each peak were retrieved through the built-in NIST spectral library to obtain the corresponding compounds.

[0091] In the following examples and comparative examples, the index detection methods for the finished products were as follows: moisture was determined by the constant weight method at 105°C; crude protein was determined by the micro-Kjeldahl method (GB / T 5511-2008); acid-soluble protein was determined by the trichloroacetic acid (TCA) method (GB / T 22492-2008); lactic acid was determined by high-performance liquid chromatography HPLC method; pH determination: an appropriate amount of the sample was weighed and added to a certain volume of pure water, shaken evenly, and after standing, it was detected using a pH meter.

[0092] Example 1:

[0093] Two-stage fermentation is carried out with compound bacterial agent 1 as the fermentation strain. The specific steps are as follows:

[0094] (1) Raw material preparation: The soybean meal is crushed into uniform particle size, and tap water is added to make the moisture content 50%, which is used for subsequent fermentation;

[0095] (2) Strain activation: The previously prepared compound bacterial agent 1 for fermented feed is added to tap water, and then glucose is added to adjust the pH value to 6.2. After mixing evenly, the activated bacterial liquid is obtained; among them, the weight ratio of the compound bacterial agent, tap water, and glucose is 2:100:1, and the obtained mixture is called the activated bacterial liquid; then the plant lactic acid bacterium P is activated with MRS medium for 2 d, and then two loops are inoculated into 100 mL of the culture solution and cultured statically at 37 °C for 24 h. The obtained mixture becomes the lactic acid bacterium liquid;

[0096] (3) First-stage fermentation: The above-mentioned activated bacterial liquid is added at 5% of the weight of the soybean meal raw material and mixed evenly. The mixed and inoculated material is placed in a fermentation device and sealed for static fermentation. The fermentation temperature is controlled at 35 °C. The volatile components are taken above the material for detection. When the content ratio of acetic acid to acetoin is 2.5:1, stop and prepare for the second-stage fermentation. At this time, the fermentation time is 18 h;

[0097] (4) Second-stage fermentation: The fermentation intermediate after the first-stage fermentation and the lactic acid bacterium liquid are mixed evenly for inoculation, and the inoculation amount is 5%. Then it is continuously placed in a fermentation device and sealed for static fermentation. The fermentation temperature is controlled at 35 °C. The volatile components are taken above the material for detection. When the content ratio of acetic acid to acetoin is 9:1, stop the fermentation. At this time, the fermentation time is 66 h;

[0098] (5) Finished product preparation: The material after the second-stage fermentation is dried and crushed to obtain the finished product of fermented soybean meal, marked as Product 1, and the main component content is shown in Table 1.

[0099] The obtained finished product of fermented soybean meal, marked as Product 1, and the main component content is shown in Table 1.

[0100] Example 2:

[0101] Two-stage fermentation is carried out with compound bacterial agent 2 as the fermentation strain, and the specific steps are as follows:

[0102] (1) Raw material preparation: The soybean meal is crushed into uniform particle size, and tap water is added to make the moisture content 45%, which is used for subsequent fermentation;

[0103] (2) Strain activation: Add the pre-prepared compound bacterial agent 2 into tap water, then add glucose, adjust the pH value to 6.3, and mix evenly to obtain the activated bacterial liquid; among them, the weight ratio of the compound bacterial agent, tap water, and glucose is 4:100:2, and the obtained mixture is called the activated bacterial liquid. Pediococcus pentosaceus Q is activated with MRS medium for 2 days, then two loops are inoculated into 100 mL of the culture solution and statically cultured at 37 °C for 24 h, and the obtained mixture becomes the lactic acid bacteria liquid;

[0104] (3) First-stage fermentation: Add the above-activated bacterial liquid at 8% of the weight of the soybean meal raw material and mix evenly. Place the mixed and inoculated material in a fermentation equipment and seal it for static fermentation. Control the fermentation temperature at 40 °C, and stop and prepare for the second-stage fermentation when the acetic acid:acetoin content reaches 2.7:1;

[0105] (4) Second-stage fermentation: Mix the fermentation intermediate after the first-stage fermentation and the lactic acid bacteria liquid evenly for inoculation, with an inoculation amount of 8%, and continue to place it in the fermentation equipment for sealed static fermentation. Control the fermentation temperature at 40 °C, and stop the fermentation when the acetic acid:acetoin content reaches 11.6:1;

[0106] (5) Finished product preparation: Dry and crush the material after the second-stage fermentation to obtain the fermented soybean meal finished product, marked as Product 2, and its main component content is shown in Table 1.

[0107] Example 3:

[0108] Perform two-stage fermentation with compound bacterial agent 3 as the fermentation strain, and the specific steps are as follows:

[0109] (1) Raw material preparation: Crush the soybean meal into uniform particle size, add tap water until the moisture content reaches 40% for subsequent fermentation;

[0110] (2) Strain activation: Add the pre-prepared compound bacterial agent 3 into tap water, then add glucose, adjust the pH value to 6.5, and mix evenly to obtain the activated bacterial liquid; among them, the weight ratio of the compound bacterial agent, tap water, and glucose is 5:100:2, and the obtained mixture is called the activated bacterial liquid. Lactobacillus plantarum P is activated with MRS medium for 2 days, then two loops are inoculated into 100 mL of the culture solution and statically cultured at 37 °C for 24 h, and the obtained mixture becomes the lactic acid bacteria liquid;

[0111] (3) First-stage fermentation: Add the above-activated bacterial liquid at 10% of the weight of the soybean meal raw material and mix evenly. Place the mixed and inoculated material in a fermentation equipment and seal it for static fermentation. Control the fermentation temperature at 45 °C, and stop and prepare for the second-stage fermentation when the acetic acid:acetoin content is 3.0:1;

[0112] (4) Secondary fermentation: Mix the fermentation intermediate after the first-stage fermentation and the lactic acid bacteria liquid evenly for inoculation, with an inoculation amount of 10%, and continue to place it in the fermentation equipment for sealed static fermentation. Control the fermentation temperature at 45 °C, and stop fermentation when the acetic acid:acetoin content is 12.0:1;

[0113] (5) Finished product preparation: Dry and crush the material after the second-stage fermentation to obtain the fermented soybean meal finished product, marked as Product 3, and its main component content is shown in Table 1.

[0114] Example 4:

[0115] Perform two-stage fermentation using compound bacterium agent 5 as the fermentation strain. The specific steps are as follows:

[0116] (1) Raw material preparation: Crush the soybean meal into a uniform particle size, add tap water until the moisture content reaches 45% for subsequent fermentation;

[0117] (2) Strain activation: Add the pre-prepared compound bacterium agent 5 to tap water, then add glucose, adjust the pH value to 6.2, and mix evenly to obtain the activated bacterium liquid. Among them, the weight ratio of the compound bacterium agent, tap water, and glucose is 3:100:1.5, and the obtained mixture is called the activated bacterium liquid. Pediococcus acidilactici TMM-3 was screened from nature by the laboratory, activated in MRS medium for 2 days, and then inoculated with two loops into 100 mL of culture solution and cultured statically at 37 °C for 24 h. The obtained mixture becomes the lactic acid bacteria liquid;

[0118] (3) First-stage fermentation: Add the above-activated bacterium liquid at 5% of the weight of the soybean meal raw material and mix evenly. Place the mixed and inoculated material in the fermentation equipment for sealed static fermentation. Control the fermentation temperature at 40 °C, and stop and prepare for the second-stage fermentation when the acetic acid:acetoin content reaches 2.8:1;

[0119] (4) Second-stage fermentation: Mix the fermentation intermediate after the first-stage fermentation and the lactic acid bacteria liquid evenly for inoculation, with an inoculation amount of 5%, and continue to place it in the fermentation equipment for sealed static fermentation. Control the fermentation temperature at 40 °C, and stop fermentation when the acetic acid:acetoin content reaches 10.2:1;

[0120] (5) Finished product preparation: Dry and crush the material after the second-stage fermentation to obtain the fermented soybean meal finished product, marked as Product 4, and its main component content is shown in Table 1.

[0121] Example 5:

[0122] The difference between this example and Example 2 is only that:

[0123] Perform two-stage fermentation using compound bacterium agent 1 as the fermentation strain;

[0124] During the first-stage fermentation, when the acetic acid:acetoin content reaches 2.5:1, stop and prepare for the second-stage fermentation; during the second-stage fermentation, when the acetic acid:acetoin content reaches 8.9:1, 10.7:1, and 12.6:1 respectively, stop the fermentation. Dry and crush the material after the second-stage fermentation to obtain the finished product of fermented soybean meal, and the obtained finished products are respectively labeled as Product 5-1, 5-2, and 5-3. The main component contents are shown in Table 1.

[0125] Example 6:

[0126] The difference between this example and Example 2 is only that:

[0127] When adding glucose during the activation of the strain, add a compound enzyme preparation at the same time, adjust the pH value to 6.5, and mix evenly to obtain the activated bacterial liquid; among them, the weight ratio of the compound bacterium agent, the compound enzyme preparation, tap water, and glucose is 4:0.4:100:2, and the obtained mixture is called the activated bacterial liquid. The compound enzyme preparation includes feed protease, glucanase, phytase, xylanase, glucanase, pectinase, cellulase, and β-mannanase; among them, the amount of feed protease is 50000U / g, the amount of glucanase is 50000U / g, the amount of phytase is 10000U / g, the amount of xylanase is 5000U / g, the amount of pectinase is 5000U / g, the amount of cellulase is 5000U / g, and the amount of β-mannanase is 5000U / g.

[0128] During the first-stage fermentation, when the acetic acid:acetoin content reaches 2.8:1, stop and prepare for the second-stage fermentation; during the second-stage fermentation, when the acetic acid:acetoin content reaches 10.2:1, stop the fermentation. Dry and crush the material after the second-stage fermentation to obtain the finished product of fermented soybean meal, and the obtained finished product is labeled as Product 6. The main component contents are shown in Table 2.

[0129] Example 7:

[0130] The difference between this example and Example 6 is only that:

[0131] The weight ratio of the compound bacterium agent, the compound enzyme preparation, tap water, and glucose is 5:0.2:100:2. At the same time, during the first-stage fermentation, when the acetic acid:acetoin content reaches 2.6:1, stop and prepare for the second-stage fermentation; during the second-stage fermentation, when the acetic acid:acetoin content reaches 7.6:1, 10.1:1, and 12.2:1 respectively, stop the fermentation. Dry and crush the material after the second-stage fermentation to obtain the finished product of fermented soybean meal, and the obtained finished products are respectively labeled as Product 7-1, 7-2, and 7-3. The main component contents are shown in Table 2.

[0132] Example 8:

[0133] The difference between this example and Example 2 is only that:

[0134] Two-stage fermentation was carried out using complex bacterial agent 6 as the fermentation strain;

[0135] In the first-stage fermentation, when the acetic acid:acetoin content reached 2.5:1, the fermentation was stopped and the second-stage fermentation was prepared; in the second-stage fermentation, when the acetic acid:acetoin content reached 12.0:1, the fermentation was stopped. The material after the second-stage fermentation was dried and crushed to obtain the fermented soybean meal product, and the obtained product was labeled as Product 8. The main component contents are shown in Table 2.

[0136] Example 9:

[0137] The difference between this example and Example 2 is only that:

[0138] Two-stage fermentation was carried out using complex bacterial agent 7 as the fermentation strain;

[0139] In the first-stage fermentation, when the acetic acid:acetoin content reached 3.0:1, the fermentation was stopped and the second-stage fermentation was prepared; in the second-stage fermentation, when the acetic acid:acetoin content reached 9.0:1, the fermentation was stopped. The material after the second-stage fermentation was dried and crushed to obtain the fermented soybean meal product, and the obtained product was labeled as Product 9. The main component contents are shown in Table 2.

[0140] Comparative Example 1:

[0141] Two-stage fermentation was carried out using complex bacterial agent 2 as the fermentation strain. The specific steps are as follows:

[0142] (1) Raw material preparation: The soybean meal was crushed into uniform particle size, and tap water was added to make the moisture content 50% for subsequent fermentation;

[0143] (2) Strain activation: The pre-prepared fermented feed complex bacterial agent 2 was added to tap water, and then glucose was added to adjust the pH value to 6.2 and mixed evenly to obtain the activated bacterial liquid; among them, the weight ratio of the complex bacterial agent, tap water, and glucose was 2:100:1, and the obtained mixture was called the activated bacterial liquid; at the same time, Pediococcus pentosaceus Q was activated with MRS medium for 2d, and then two loops were inoculated into 100 mL of the culture solution and cultured statically at 37°C for 24h, and the obtained mixture became the lactic acid bacteria liquid;

[0144] (3) First-stage fermentation: 5% of the above complex bacterial liquid was added based on the weight of the soybean meal raw material and mixed evenly. The mixed and inoculated material was placed in a fermentation device and sealed for static fermentation, and the fermentation temperature was controlled at 35°C; during the first-stage fermentation, when the acetic acid:acetoin content was 2.2:1, the fermentation was stopped and the second-stage fermentation was prepared;

[0145] (4) Second-stage fermentation: The fermentation intermediate after the first-stage fermentation and the lactic acid bacteria liquid were mixed evenly for inoculation, and the inoculation amount was 5%, and it was continuously placed in a fermentation device and sealed for static fermentation, and the fermentation temperature was controlled at 35°C; during the second-stage fermentation, when the acetic acid:acetoin content was 7.4:1, the fermentation was stopped;

[0146] (5) Preparation of finished product: Dry and crush the material after the second-stage fermentation to obtain the finished product of fermented soybean meal, and mark the obtained finished product of fermented soybean meal as Product I. The main component contents are shown in Table 2.

[0147] Comparative Example 2:

[0148] Perform two-stage fermentation using compound bacterium agent 4 as the fermentation strain. The specific steps are as follows:

[0149] (1) Raw material preparation: Crush soybean meal into uniform particle size, add tap water until the moisture content reaches 45%, and use it for subsequent fermentation;

[0150] (2) Strain activation: Add the previously prepared compound bacterium agent 4 into tap water, then add glucose, adjust the pH value to 6.3, and mix evenly to obtain the activated bacterial liquid; among them, the weight ratio of the compound bacterium agent, tap water, and glucose is 4:100:2, and the obtained mixture is called the activated bacterial liquid. Pediococcus pentosaceus Q is activated with MRS medium for 2 days, and then two loops are inoculated into 100 mL of culture solution and cultured statically at 37°C for 24 hours. The obtained mixture becomes the lactic acid bacteria liquid;

[0151] (3) First-stage fermentation: Add the above compound bacterial liquid at 5% of the weight of the soybean meal raw material and mix evenly. Place the mixed and inoculated material in a fermentation device and seal it for static fermentation. Control the fermentation temperature at 35°C. When the acetic acid:acetoin content is 1.3:1, stop and prepare for the second-stage fermentation;

[0152] (4) Second-stage fermentation: Mix the fermentation intermediate after the first-stage fermentation and the lactic acid bacteria liquid evenly for inoculation, with an inoculation amount of 5%, and continue to place it in a fermentation device and seal it for static fermentation. Control the fermentation temperature at 35°C. When the acetic acid:acetoin content is 6.2:1, stop the fermentation;

[0153] (5) Preparation of finished product: Dry and crush the material after the second-stage fermentation to obtain the finished product of fermented soybean meal, and mark the obtained finished product of fermented soybean meal as Product II. The main component contents are shown in Table 3.

[0154] Comparative Example 3:

[0155] The difference between this comparative example and Example 2 is only that:

[0156] Perform two-stage fermentation using compound bacterium agent 1 as the fermentation strain;

[0157] Stop the first-stage fermentation when the acetic acid:acetoin content reaches 1.8:1 and prepare for the second-stage fermentation; in the second-stage fermentation, stop the fermentation when the acetic acid:acetoin content reaches 7.7:1, 11.3:1, and 12.2:1 respectively. Dry and crush the material after the second-stage fermentation to obtain the finished product of fermented soybean meal, and label the obtained finished products as Product III-1, III-2, and III-3 respectively. The main component contents are shown in Table 3.

[0158] Comparative Example 4:

[0159] The difference between this comparative example and Example 7 is only that:

[0160] The weight ratio of the complex bacterial agent, complex enzyme preparation, tap water, and glucose is 2:0.5:100:1. At the same time, stop the first-stage fermentation when the acetic acid:acetoin content reaches 2.2:1 and prepare for the second-stage fermentation; in the second-stage fermentation, stop the fermentation when the acetic acid:acetoin content reaches 8.3:1, 11.0:1, and 13.0:1 respectively. Dry and crush the material after the second-stage fermentation to obtain the finished product of fermented soybean meal, and label the obtained finished products as Product IV-1, IV-2, and IV-3 respectively. The main component contents are shown in Table 3.

[0161] Comparative Example 5:

[0162] Use the complex bacterial agent 1 as the fermentation strain; the specific steps are as follows:

[0163] (1) Raw material preparation: Crush the soybean meal into a uniform particle size, add tap water to a moisture content of 45%, and use it for subsequent fermentation;

[0164] (2) Strain activation: Add the pre-prepared complex bacterial agent 2 to tap water, then add glucose, adjust the pH value to 6.3, and mix evenly to obtain the activated bacterial liquid; among them, the weight ratio of the complex bacterial agent, tap water, and glucose is 4:100:2, and the obtained mixture is called the activated bacterial liquid.

[0165] (3) First-stage fermentation: Add the above-activated bacterial liquid at 8% of the weight of the soybean meal raw material and mix evenly. Place the mixed and inoculated material in a fermentation equipment and seal it for static fermentation. Control the fermentation temperature at 40°C and stop the fermentation when the acetic acid:acetoin content reaches 1.8:1 and 2.5:1 respectively.

[0166] (4) Finished product preparation: Do not carry out the second-stage fermentation. The product obtained from the first-stage fermentation is dried and crushed to obtain the finished product of fermented soybean meal, and label it as Product V-1 and V-2 respectively. The main component contents are shown in Table 3.

[0167] Comparative Example 6

[0168] Use the complex bacterial agent 2 as the fermentation strain for two-stage fermentation. The specific steps are as follows:

[0169] (1) Raw material preparation: Crush soybean meal into uniform particle size, add tap water until the moisture content reaches 50% for subsequent fermentation.

[0170] (2) Strain activation: Add the pre-prepared compound microbial agent for fermented feed 2 into tap water, then add glucose, adjust the pH value to 6.2, and mix evenly to obtain the activated bacterial liquid. Among them, the weight ratio of the compound microbial agent, tap water, and glucose is 2:100:1, and the obtained mixture is called the activated bacterial liquid. At the same time, activate Pediococcus pentosaceus Q with MRS medium for 2 days, then inoculate two loops into 100 mL of culture medium and statically culture at 37 °C for 24 h. The obtained mixture becomes the lactic acid bacteria liquid.

[0171] (3) First-stage fermentation: Add the above compound bacterial liquid at 5% of the weight of the soybean meal raw material and mix evenly. Place the inoculated and mixed materials in a fermentation equipment, seal and statically ferment. Control the fermentation temperature at 35 °C. Stop when the first-stage fermentation time reaches 18 h and prepare for the second-stage fermentation.

[0172] (4) Second-stage fermentation: Mix the fermentation intermediate after the first-stage fermentation and the lactic acid bacteria liquid evenly for inoculation, with an inoculation amount of 5%, and continue to place it in the fermentation equipment, seal and statically ferment. Control the fermentation temperature at 35 °C. Stop fermentation when the second-stage fermentation time reaches 66 h.

[0173] (5) Finished product preparation: Dry and crush the materials after the second-stage fermentation to obtain the finished product of fermented soybean meal. Mark the obtained finished product of fermented soybean meal as Product VI, and its main component content is shown in Table 3.

[0174] Table 1 Main components and contents of the finished product of fermented soybean meal

[0175]

[0176] Table 2 Main components and contents of the finished product of fermented soybean meal

[0177]

[0178]

[0179] Table 3 Main components and contents of the finished product of fermented soybean meal

[0180]

[0181] Tables 1 to 3 list the main components and contents of the fermented products in each example and comparative example. It can be seen from the table that the acid-soluble protein content in the fermented products 1, 2, 3, 4, 5-2, 6, 7-2, 8, and 9 obtained in Examples 1-9 is ≥16%, and the lactic acid content is ≥6%, which is significantly higher than that of soybean meal before fermentation. This shows that the protein solubility of soybean meal increases after fermentation, and the fermentation effectively acidifies the soybean meal. The increase in acid-soluble protein indicates that a large amount of low-molecular-weight peptides are generated after fermentation, which is beneficial to animal digestion and absorption. The increase in lactic acid content indicates that the fermented product contains more organic acids, which can improve the palatability of the feed and is beneficial to balancing the animal intestinal microbial environment, thereby enhancing animal immunity. That is, the fermented soybean meal product prepared by the method of the present application has the effects of good palatability, being beneficial to animal digestion and absorption, improving animal intestinal health, and enhancing immunity.

[0182] By comparing the examples with the comparative examples, it can be seen that during the first-stage fermentation process, when the ratio of acetic acid to acetoin in the volatile substances reaches 2.5-3:1, the fermentation is stopped and the second-stage fermentation is prepared, and the fermentation effect is better. The acid-soluble protein content of the obtained product is ≥16%, and the lactic acid content is ≥6%; during the second-stage fermentation process, when the ratio of acetic acid to acetoin reaches 9-12:1, the fermentation is ended, and the fermentation effect is better. The acid-soluble protein content of the obtained product

[0183] is ≥16%, and the lactic acid content is ≥6%.

[0184] By comparing Example 1 with Comparative Example 6, it can be seen that when the fermentation time is the same, if different strains are replaced, it is impossible to ensure that the acid-soluble protein content and lactic acid content of the fermented product meet the standards, that is, it is impossible to obtain a product with an acid-soluble protein content ≥16% and a lactic acid content ≥6%; therefore, in the prior art, if the fermentation conditions such as strains are changed in actual production, more experiments are needed to explore the optimal fermentation conditions, or more precise control is required to ensure that the fermented product meets the standards. However, it can be seen from each example that even if the fermentation conditions such as strains are changed, by using the method of the present application, by detecting the contents of acetic acid and acetoin and calculating the ratio, and judging the fermentation end point, the fermented product obtained can meet the standards. Therefore, the method of the present application simplifies the fermentation steps and saves labor and material costs.

Claims

1. A preparation method of fermented soybean meal, characterized in that, The preparation method comprises the following steps: Raw material preparation: Adjust the moisture content of soybean meal; Fermentation of soybean meal: Add the first strain to the soybean meal for primary fermentation, and add the second strain for secondary fermentation after the primary fermentation ends; The primary fermentation and the secondary fermentation are preferably anaerobic fermentations; The first strain and the second strain are preferably activated strains; Finished product preparation: Dry and crush the material obtained after the secondary fermentation ends to obtain the finished product of fermented soybean meal; Wherein, during the primary fermentation and / or the secondary fermentation process, volatile substances are monitored, and when acetic acid:acetoin in the volatile substances > 1:1, the fermentation ends.

2. The preparation method according to claim 1, characterized in that, The moisture content of the soybean meal is 40%-50%.

3. The preparation method according to claim 1, characterized in that, During the primary fermentation process, the fermentation temperature is 35-45°C; When acetic acid:acetoin in the volatile substances reaches 2.5-3:1, the fermentation stops.

4. The preparation method according to claim 1, characterized in that, During the secondary fermentation process, the fermentation temperature is 35-45°C; When acetic acid:acetoin in the volatile substances reaches 9-12:1, the fermentation stops.

5. The preparation method according to claim 1, characterized in that, The first strain and the second strain include at least one of yeast, lactic acid bacteria, mold, and Bacillus subtilis; The first strain is preferably yeast, lactic acid bacteria, and mold; Or preferably use yeast, lactic acid bacteria, and Bacillus subtilis; The second strain is preferably lactic acid bacteria.

6. The preparation method according to claim 5, characterized in that, The yeast includes Saccharomyces cerevisiae; The lactic acid bacteria include at least one of Lactobacillus acidophilus, Pediococcus pentosaceus, Pediococcus acidilactici, and Lactobacillus plantarum; The mold includes at least one of Aspergillus niger and Aspergillus oryzae.

7. The preparation method according to claim 5, characterized in that, A complex enzyme preparation is added during the activation process of the first strain; The complex enzyme preparation preferably includes at least one of protease, amylase, glucanase, phytase, xylanase, pectinase, cellulase, α-galactosidase, and β-mannanase.

8. A finished product of fermented soybean meal, characterized in that, The fermented soybean meal is prepared by any one of the preparation methods in claims 1-7.

9. Use of the finished product of fermented soybean meal according to claim 8 in the preparation of feed.

10. An animal feed, characterized in that, The animal feed includes the finished product of the fermented soybean meal described in claim 8.

Images