Preparation method of soybean meal through synergistic fermentation of bacteria and enzymes

Abstract

The invention provides a preparation method of bacterium-enzyme synergistic fermented soybean meal, and relates to the technical field of biologication.The preparation method comprises the following steps that S1, soybean meal and alpha-galactosidase are added for enzymolysis; s2, adding fig protease to carry out enzymolysis; s3, performing aerobic fermentation on the material obtained in the step S2, saccharomycetes and bacillus; s4, performing anaerobic fermentation on the material obtained in the step S3, enterococcus avium, curcumin and lactic acid bacteria; and S5, performing drying treatment to obtain the fermented soybean meal. The fermented soybean meal obtained by the invention is low in raffinose and stachyose content, obviously improved in acid-soluble protein content, good in palatability, high in nutritional value and relatively good in safety.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing soybean meal through cooperative fermentation of bacteria and enzymes. Background Art

[0002] Soybean meal is a by-product produced during the production and extraction of soybean oil. Due to its high plant protein content and balanced essential amino acid composition, it has long been considered an important and inexpensive source of plant protein in food and animal feed. However, the presence of off-flavor, poor palatability, and a large number of anti-nutritional factors, such as allergenic antigenic proteins, trypsin inhibitors, and flatulence factors such as stachyose and raffinose, seriously affects the digestion and absorption of soybean meal nutrients by livestock and poultry, resulting in excessively low bioavailability of the nutrients in soybean meal, limiting its application in the food and feed industries. Due to processing technology limitations, traditional soybean meal has two major technical bottlenecks: low acid-soluble protein content and incomplete degradation of raffinose, which seriously restrict its nutritional value and functional properties.

[0003] First, the protein in soybean meal exists primarily in the form of large aggregates, with acid-soluble proteins (small peptides soluble in dilute acid) comprising less than 5%. This lack of acid-soluble proteins significantly reduces digestion and absorption in acidic environments, such as gastric juice. While existing physical or chemical treatments (such as high-temperature cooking and acid-base hydrolysis) can increase the acid-soluble protein content, they can easily disrupt the amino acid structure and produce anti-nutritional factors.

[0004] Secondly, soybean meal contains oligosaccharides such as raffinose, which can reach as high as 5-8%. Raffinose is difficult to digest and ferments in the intestines, producing gas and causing abdominal distension and other discomfort. This severely impacts livestock and poultry's ability to digest and absorb soybean meal nutrients, resulting in low bioavailability of soybean meal nutrients and limiting its application in the food and feed industries.

[0005] Currently, this problem is partially addressed by treating soybean meal using physical, chemical, enzymatic, and microbial fermentation methods. However, these methods often result in reduced protein quality, residual chemicals, excessive reliance on environmental conditions, long fermentation times, high energy consumption, and high processing costs, limiting the application and development of soybean meal-based feeds.

[0006] In comparison, fermenting soybean meal using bacterial and enzyme synergistic fermentation technology can significantly reduce the content of anti-nutritional factors and increase the content of crude protein, crude fat, and organic acids in soybean meal. The large-scale proliferation of beneficial bacteria and the digestive enzymes produced by fermentation can convert the large molecular proteins in soybean meal into active small peptides, free amino acids, fatty acids and other nutrients, effectively improving the nutritional value and functional properties of soybean meal, significantly reducing the digestive stress of young animals, protecting animal health, promoting the growth of livestock and poultry, and enhancing the body's immunity. At the same time, the development of efficient and green preparation technology for functional soybean meal produced by bacterial and enzyme synergistic fermentation also has the advantages of safety, short fermentation time, high energy efficiency, and low cost. Therefore, the development of efficient and green preparation technology for functional soybean meal produced by bacterial and enzyme synergistic fermentation is a key technical issue that needs to be solved for the large-scale and industrial application of soybean meal. Summary of the Invention

[0007] In view of this, the present invention proposes a preparation method of soybean meal by synergistic fermentation of bacteria and enzymes.

[0008] The technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides a method for preparing soybean meal by bacterial enzyme synergistic fermentation, comprising the following steps: S1, adding soybean meal and α-galactosidase for enzymatic hydrolysis; S2, adding ficin for enzymatic hydrolysis; S3, aerobic fermentation of the material obtained in step S2 with yeast and Bacillus; S4, performing anaerobic fermentation on the material obtained in step S3, Enterococcus avium, curcumin, and lactic acid bacteria; S5, performing a drying process to obtain the fermented soybean meal; Wherein, the mass of the α-galactosidase is 0.3‰~0.5‰ of the mass of the soybean meal; The mass of the ficin is 0.1% to 0.3% of the mass of the soybean meal; The yeast is Kluyveromyces marxianus, the Bacillus is Bacillus subtilis, and the lactic acid bacteria is Lactobacillus plantarum.

[0009] The present invention uses α-galactosidase and ficin to enzymatically hydrolyze soybean meal. α-galactosidase can effectively remove oligosaccharides such as raffinose and stachyose from the soybean meal. Ficin can specifically hydrolyze large molecular proteins in the soybean meal, breaking them down into small molecular peptides and amino acids. The solubility of the small molecular proteins after enzymatic hydrolysis is significantly improved under acidic conditions. It can also partially degrade anti-nutritional factors such as trypsin inhibitors in the soybean meal.

[0010] The fermentation process of the present invention includes an aerobic fermentation stage and an anaerobic fermentation stage. The aerobic stage includes yeast and bacillus, which is conducive to the rapid proliferation of yeast and the utilization of inorganic nutrients by bacillus.

[0011] During the anaerobic fermentation stage, the present invention utilizes Enterococcus avium, curcumin, and lactic acid bacteria for anaerobic fermentation. Enterococcus avium can hydrolyze raffinose into galactose, glucose, and fructose, significantly reducing the raffinose content. Curcumin can inhibit the activity of amino acid decarboxylase, thereby inhibiting the catalytic conversion of amino acids to biogenic amines. Furthermore, curcumin, as a potent antioxidant, can enhance antioxidant activity. Lactic acid bacteria fermentation of soybean meal can produce organic acids, soluble polypeptides, and other substances, which are highly palatable and nutritious.

[0012] Bacillus subtilis secretes large amounts of neutral and alkaline proteases, effectively hydrolyzing soy protein into peptides and free amino acids. It also produces bacteriocins and organic acids, which effectively inhibit the growth of harmful bacteria like E. coli during fermentation, providing active probiotics, ensuring fermentation safety, and contributing to animal intestinal health.

[0013] Lactobacillus plantarum fermentation can produce a large amount of lactic acid, which can improve palatability, inhibit the growth of putrefactive bacteria and pathogens, enhance the body's immunity, and promote nutrient absorption.

[0014] Kluyveromyces marxianus can promote the degradation of raffinose and stachyose, and produce volatile flavor substances such as alcohols and esters (such as ethanol and ethyl acetate) during the metabolism process, which can mask the unpleasant odor of soybean meal, give the product a special fermentation aroma, and significantly improve palatability.

[0015] The synergistic effect of Enterococcus avium, Bacillus subtilis, Lactobacillus plantarum and Kluyveromyces marxianus can improve the protein solubility of soybean meal, increase the acid-soluble protein content, and reduce the content of oligosaccharides such as raffinose. Probiotics can be produced during the fermentation process, inhibit harmful bacteria and pathogens, improve the unpleasant odor of soybean meal, and impart fragrance and good palatability.

[0016] On the basis of the above technical solution, step S4 further includes performing a first anaerobic fermentation on the material obtained in step S3 and lactic acid bacteria, then adding avian enterococci for a second anaerobic fermentation, and finally adding curcumin for a third anaerobic fermentation.

[0017] On the basis of the above technical solution, further, the time for the first anaerobic fermentation, the second anaerobic fermentation, and the third anaerobic fermentation is 15 hours.

[0018] On the basis of the above technical solution, further, in step S1, the conditions for enzymatic hydrolysis include: temperature of 50°C to 55°C, pH of 6.0 to 6.5, and enzymatic hydrolysis time of 2 to 3 hours.

[0019] After the enzymatic hydrolysis in step S1 is completed, a high-temperature enzyme inactivation treatment is performed.

[0020] On the basis of the above technical solution, further, in step S2, the conditions for enzymatic hydrolysis include: temperature of 50°C to 55°C, pH of 6.0 to 6.5, and enzymatic hydrolysis time of 2 to 3 hours.

[0021] After the enzymatic hydrolysis in step S2 is completed, a high-temperature enzyme inactivation treatment is performed.

[0022] Based on the above technical solution, preferably, the enzyme activity of the α-galactosidase is 3000 IU / g.

[0023] On the basis of the above technical solution, further, the enzymatic activity of the ficin is 200,000 IU / g to 300,000 IU / g.

[0024] On the basis of the above technical solution, further, the effective viable bacteria count of the avian enterococcus is 10 3 CFU / g~10 5 CFU / g.

[0025] On the basis of the above technical solution, further, the conditions of the aerobic fermentation include: temperature between 30° C. and 37° C., and pH between 6.5 and 7.0.

[0026] On the basis of the above technical solution, further, in the good fermentation stage, molasses is added in an amount of 0.2% to 0.5% of the mass of the soybean meal.

[0027] On the basis of the above technical solution, further, the conditions of the anaerobic fermentation include: temperature between 35° C. and 37° C., and pH between 6.5 and 7.0.

[0028] On the basis of the above technical solution, further, the effective viable counts of Kluyveromyces marxianus, Bacillus subtilis and Lactobacillus plantarum are 1×10 9 CFU / g, 2×10 9 CFU / g, 6×10 9 CFU / g.

[0029] On the basis of the above technical solution, further, the drying treatment includes drying using a spray dryer, and the spray drying conditions are: inlet air temperature is 200°C~250°C, and outlet air temperature is 90°C~95°C.

[0030] In the second aspect, the fermented soybean meal is prepared using the above preparation method.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The fermented soybean meal obtained by the invention has low contents of raffinose and stachyose, significantly improved content of acid-soluble protein, good palatability, high nutritional value and good safety. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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.

[0033] In the following specific embodiments, α-galactosidase and ficin are both commercially available products, and Enterococcus avium, Kluyveromyces marxianus, Bacillus subtilis, and Lactobacillus plantarum are all independently preserved by our company.

[0034] In the following specific embodiments, the high temperature enzyme inactivation treatment is performed by heating the temperature to 85° C. to 90° C. for 20 min to 30 min.

[0035] Example 1 This embodiment provides a method for preparing soybean meal by fermentation with bacteria and enzymes, comprising the following steps: S1, add 10kg soybean meal, crush it, add 2 times the mass of water, mix well, add 0.4‰ of the mass of soybean meal α-galactosidase for enzymolysis, and perform high temperature enzyme inactivation after enzymolysis; S2, adding 0.2% of the mass of the soybean meal of ficin to perform enzymolysis, and then performing high-temperature enzyme inactivation treatment after the enzymolysis is completed; S3, aerobic fermentation of the material obtained in step S2 with Kluyveromyces marxianus, Bacillus subtilis, and molasses with a mass of 0.4% of the mass of soybean meal, wherein the effective viable counts of Kluyveromyces marxianus and Bacillus subtilis are 1×10 9 CFU / g, 2×10 9 CFU / g, aerobic fermentation conditions are: temperature at 35°C, pH at 6.7, and fermentation time for 22 h; S4, the material obtained in step S3 is subjected to the first anaerobic fermentation with plant lactobacillus, and then avian enterococcus is added to carry out the second anaerobic fermentation, and finally curcumin is added to carry out the third anaerobic fermentation; the effective viable counts of avian enterococcus and plant lactobacillus are 10 4 CFU / g, 6×10 9 CFU / g, the conditions for anaerobic fermentation were: temperature at 36°C, pH at 6.7; S5. Using a spray dryer to perform drying treatment to obtain the fermented soybean meal.

[0036] Example 2 This embodiment provides a method for preparing soybean meal by fermentation with bacteria and enzymes, comprising the following steps: S1. Add 10 kg of soybean meal, crush it, add 2 times the mass of water, mix well, add 0.3‰ of the mass of soybean meal of α-galactosidase for enzymolysis, and perform high temperature enzyme inactivation after enzymolysis; S2, adding 0.1% of the mass of the soybean meal of ficin to perform enzymolysis, and then performing high-temperature enzyme inactivation treatment after the enzymolysis is completed; S3, aerobic fermentation of the material obtained in step S2 with Kluyveromyces marxianus, Bacillus subtilis, and molasses with a mass of 0.2% of the mass of soybean meal, wherein the effective viable counts of Kluyveromyces marxianus and Bacillus subtilis are 1×10 9 CFU / g, 2×10 9 CFU / g, aerobic fermentation conditions are: temperature at 30℃, pH at 6.5, and fermentation time for 20h; S4, the material obtained in step S3 is subjected to the first anaerobic fermentation with Lactobacillus plantarum, and then Enterococcus avium is added to carry out the second anaerobic fermentation, and finally curcumin is added to carry out the third anaerobic fermentation; the effective viable counts of Enterococcus avium and Lactobacillus plantarum are 10 3 CFU / g, 6×10 9 CFU / g, the conditions for anaerobic fermentation were: temperature at 35°C, pH at 6.5; S5. Using a spray dryer to perform drying treatment to obtain the fermented soybean meal.

[0037] Example 3 This embodiment provides a method for preparing soybean meal by fermentation with bacteria and enzymes, comprising the following steps: S1. Add 10 kg of soybean meal, crush it, add 2 times the mass of water, mix well, add 0.5‰ of α-galactosidase based on the mass of soybean meal for enzymolysis, and perform high temperature enzyme inactivation after enzymolysis. S2, adding 0.3% of the mass of the soybean meal of ficin to perform enzymolysis, and then performing high-temperature enzyme inactivation treatment after the enzymolysis is completed; S3, aerobic fermentation of the material obtained in step S2 with Kluyveromyces marxianus, Bacillus subtilis, and molasses with a mass of 0.5% of the mass of soybean meal, wherein the effective viable counts of Kluyveromyces marxianus and Bacillus subtilis are 1×10 9 CFU / g, 2×10 9 CFU / g, aerobic fermentation conditions are: temperature at 37°C, pH at 7.0, and fermentation time for 24 h; S4, the material obtained in step S3 is subjected to the first anaerobic fermentation with Lactobacillus plantarum, and then Enterococcus avium is added to carry out the second anaerobic fermentation, and finally curcumin is added to carry out the third anaerobic fermentation; the effective viable counts of Enterococcus avium and Lactobacillus plantarum are 105 CFU / g, 6×10 9 CFU / g, the conditions for anaerobic fermentation were: temperature at 37°C, pH at 7.0; S5. Using a spray dryer to perform drying treatment to obtain the fermented soybean meal.

[0038] Comparative Example 1 The difference between this comparative example and Example 1 is that it does not contain ficin.

[0039] Comparative Example 2 The difference between this comparative example and Example 1 is that it does not contain avian Enterococcus.

[0040] Comparative Example 3 The difference between this comparative example and Example 1 is that it does not contain ficin and avian Enterococcus.

[0041] Comparative Example 4 The difference between this comparative example and Example 1 is that this comparative example provides a commercially available ordinary fermented soybean meal.

[0042] Comparative Example 5 The difference between this comparative example and Example 1 is that it does not contain curcumin.

[0043] Comparative Example 6 The difference between this comparative example and Example 1 is that the third anaerobic fermentation is not performed, and curcumin and Enterococcus avium are added in the second anaerobic fermentation.

[0044] Performance testing 1. According to the test method specified in the industry standard NY / T 2218-2022, conventional performance tests were performed on the fermented soybean meals prepared in Examples 1-3 and Comparative Examples 1-4. The test results are shown in Table 1.

[0045] As shown in Table 1, the acid-soluble protein content in Examples 1-3 of the present invention was significantly higher than that in Comparative Examples 1-4. This is because ficin can specifically hydrolyze the macromolecular proteins in soybean meal, breaking them down into small peptides and amino acids. The solubility of the enzymatically hydrolyzed small peptides and amino acids is significantly increased under acidic conditions, thereby increasing the acid-soluble protein content of the fermented soybean meal.

[0046] Table 1 Nutritional component test results

[0047] Detection of raffinose and stachyose content The fermented soybean meal prepared in Examples 1-3 and Comparative Examples 1-6 was tested for raffinose and stachyose content using the SXA03-FB-017 method. The test results are shown in Table 2.

[0048] As shown in Table 2, the contents of raffinose and stachyose in Examples 1-3 decreased significantly, indicating that the degradation rate of raffinose and stachyose in the preparation method of the present invention using bacterial enzyme-cooperative fermentation of soybean meal is better than that of conventional fermented soybean meal.

[0049] Comparative Example 5 does not contain curcumin, but the contents of raffinose and stachyose are still low, indicating that not using curcumin does not affect the reduction of the contents of raffinose and stachyose.

[0050] In Comparative Example 6, curcumin and Enterococcus avium were subjected to a second anaerobic fermentation. Curcumin had a certain inhibitory effect on Enterococcus avium, thereby affecting the degradation of raffinose and stachyose by Enterococcus avium.

[0051] Table 2 Detection results of raffinose and stachyose content

[0052] 3. Biogenic amine content detection The fermented soybean meals prepared in Examples 1-3 and Comparative Example 5 were tested for biogenic amines according to GB / T 40258-2021. The test results are shown in Table 3.

[0053] As shown in Table 3, the biogenic amine contents in Examples 1-3 are all within the industry's required range, indicating good safety. However, in Comparative Example 5, both histamine and tyramine contents are relatively high, likely due to the lack of curcumin, a material that inhibits amino acid decarboxylase.

[0054] Table 3 Results of biogenic amine content detection

[0055] 4. Mycotoxin test results As shown in Table 4, no common mycotoxins were detected in Examples 1-3, indicating good safety.

[0056] Table 4 Mycotoxin test results

[0057] In summary, the method for preparing soybean meal by fermentation with bacteria and enzymes provided by the present invention can significantly increase the content of acid-soluble protein, while significantly reducing the contents of raffinose and stachyose, and has good safety.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing soybean meal by fermentation with bacterial enzymes, characterized in that: The steps include: S1, adding soybean meal and α-galactosidase for enzymatic hydrolysis; S2, adding ficin for enzymatic hydrolysis; S3, aerobic fermentation of the material obtained in step S2 with yeast and Bacillus; S4, anaerobic fermentation of the material obtained in step S3 with avian Enterococcus, curcumin and lactic acid bacteria; S5, performing a drying process to obtain the fermented soybean meal; Wherein, the mass of the α-galactosidase is 0.3‰~0.5‰ of the mass of the soybean meal; The mass of the ficin is 0.1% to 0.3% of the mass of the soybean meal; The yeast is Kluyveromyces marxianus, the Bacillus is Bacillus subtilis, and the lactic acid bacteria is Lactobacillus plantarum.

2. The method for preparing soybean meal by bacterial enzyme cooperative fermentation according to claim 1, characterized in that: Step S4 includes subjecting the material obtained in step S3 to a first anaerobic fermentation with lactic acid bacteria, then adding avian enterococci to perform a second anaerobic fermentation, and finally adding curcumin to perform a third anaerobic fermentation.

3. The method for preparing soybean meal by bacterial enzyme cooperative fermentation according to claim 1, characterized in that: The enzyme activity of the α-galactosidase is 3000 IU / g.

4. The method for preparing soybean meal by bacterial enzyme cooperative fermentation according to claim 1, wherein: The enzymatic activity of the ficin is 200,000 IU / g to 300,000 IU / g.

5. The method for preparing soybean meal by bacterial enzyme cooperative fermentation according to claim 1, characterized in that: The effective viable bacteria count of the avian enterococcus is 10 3 CFU / g~10 5 CFU / g.

6. The method for preparing soybean meal by bacterial enzyme cooperative fermentation according to claim 1, characterized in that: The conditions for aerobic fermentation include: temperature between 30° C. and 37° C., and pH between 6.5 and 7.

0.

7. The method for preparing soybean meal by bacterial enzyme cooperative fermentation according to claim 1, characterized in that: The conditions for the anaerobic fermentation include: a temperature of 35-37° C. and a pH of 6.5-7.

0.

8. The fermented soybean meal prepared by the preparation method according to any one of claims 1 to 7.

Images