Highly stable and easily absorbed soybean meal fermentation liquor, preparation method and application thereof

Abstract

The application discloses a kind of high-stability easy-absorbed soybean meal fermentation liquor, preparation method and application, it is related to the technical field of fermented feed and functional protein hydrolysate, including the following steps: step one: after defatted soybean meal is crushed, mixed with water, wet heat swelling treatment is carried out, then pre-enzymolysis is carried out by adding composite enzyme, and soybean meal pre-enzymolysis slurry is obtained.Step two: inoculate bacillus and aspergillus in soybean meal pre-enzymolysis slurry and carry out first-stage aerobic fermentation, and first fermentation liquor is obtained.Step three: after the first fermentation liquor is cooled, inoculate lactic acid bacteria and yeast and carry out second-stage micro-aerobic fermentation, and second fermentation liquor is obtained;Through wet heat swelling and composite enzyme pre-enzymolysis treatment, the cell wall structure of soybean meal is loose, the action efficiency of subsequent microorganism and protease to soybean meal protein is improved, and the settlement caused by large particle residue in later period is reduced.

Description

Technical Field

[0001] This invention relates to the field of fermented feed and functional protein hydrolysate technology, specifically to a highly stable and easily absorbed soybean meal fermentation liquid, its preparation method, and its application. Background Technology

[0002] Soybean meal is a commonly used plant protein source in animal feed, with advantages such as wide availability, high protein content, and relatively low cost. However, ordinary soybean meal contains macroglobulins, β-conglycinin, trypsin inhibitors, phytic acid, oligosaccharides, and some indigestible cell wall components. These components can affect protein digestibility and utilization, and may also cause digestive burden in young animals or aquatic animals.

[0003] Fermented soybean meal is a type of protein feed that reduces anti-nutritional factors and increases the content of small peptides and free amino acids through microbial fermentation and enzymatic hydrolysis. Currently, most fermented soybean meal products are powdered products formed after solid-state fermentation and drying, suitable for addition to compound feeds, but not convenient for addition to drinking water, liquid feeding, spray mixing, or aquatic application. To improve ease of use, some companies are exploring the preparation of soybean meal into liquid fermentation broth or concentrated fermentation broth.

[0004] However, liquid soybean meal fermentation broth presents a specific and easily overlooked problem in actual production and storage: even with increased small-molecule peptide content, a certain amount of large-molecule proteins, hydrophobic peptides, cell wall polysaccharides, trace lipids, and microbial fragments remain in the system. These components are prone to association, flocculation, and sedimentation during acid regulation, sterilization, concentration, and room-temperature storage, leading to turbidity, stratification, bottom sedimentation, and fluctuations in the effective absorbable components of the fermentation broth. This problem is particularly pronounced in products intended for liquid feeding, drinking water supplementation, and aquatic application.

[0005] In existing technologies, methods such as simple filtration, adding preservatives, increasing acidity, or adding thickeners are commonly used to improve the stability of soybean meal fermentation broth. However, simple filtration can lead to the loss of some nutrients; excessive acidification may affect palatability in animals; and while thickeners can slow sedimentation, they may not solve the problem of protein peptide aggregation after heat treatment and may also affect the flowability of drinking water systems. Therefore, there is an urgent need for a method to prepare soybean meal fermentation broth that can both increase the proportion and absorption potential of small peptides and maintain low sedimentation, low turbidity, and high centrifugal stability after heat treatment and storage at room temperature. Summary of the Invention

[0006] The purpose of this invention is to provide a highly stable and easily absorbed soybean meal fermentation liquid, its preparation method, and its application, in order to solve the problem that residual macromolecular proteins, hydrophobic peptides, and cell fragments in liquid soybean meal fermentation liquid are prone to turbidity and sedimentation during heat treatment and storage, resulting in fluctuations in effective small peptide components and insufficient stability in end use.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing highly stable and easily absorbed soybean meal fermentation broth, comprising the following steps: Step 1: After grinding the defatted soybean meal, mix it with water and perform a wet heat swelling treatment. Then, add a compound enzyme for pre-enzymatic hydrolysis to obtain soybean meal pre-enzymatic hydrolysate.

[0008] Step 2: Inoculate Bacillus and Aspergillus into the pre-enzymatic hydrolysate of soybean meal to carry out the first stage of aerobic fermentation and obtain the first fermentation mash.

[0009] Step 3: After cooling the first fermentation mash, inoculate it with lactic acid bacteria and yeast to carry out the second stage of microaerobic fermentation, and obtain the second fermentation mash.

[0010] Step 4: Separate the second fermentation mash into solid and liquid phases, take the liquid phase for enzymatic hydrolysis, cleavage and membrane fractionation, and collect the soybean meal peptide permeate with a molecular weight of less than 5000 Da.

[0011] Step 5: Add a stabilization system consisting of soluble soybean polysaccharide, L-arginine and trehalose to the soybean meal peptide permeate, adjust the pH to 4.2-4.8, and then homogenize under high pressure and sterilize at low temperature to obtain a highly stable and easily absorbed soybean meal fermentation broth.

[0012] Furthermore, the preparation steps for the pre-enzymatic hydrolysate of soybean meal are as follows: Blend defatted soybean meal to 40-80 mesh, mix defatted soybean meal and water at a mass ratio of 1:4-8, soak and swell at 55-70℃ for 30-60 minutes, adjust the pH to 6.0-7.2, then add neutral protease, cellulase and phytase, and enzymatically hydrolyze at 45-55℃ for 1-3 hours to obtain pre-enzymatically hydrolyzed soybean meal slurry.

[0013] Furthermore, the amounts of neutral protease, cellulase, and phytase added were 0.3-1.2%, 0.05-0.3%, and 0.02-0.15% of the weight of defatted soybean meal, respectively.

[0014] Furthermore, in step two, the Bacillus is either Bacillus subtilis or Bacillus belye, and the Aspergillus is Aspergillus oryzae; the total inoculum for the first-stage aerobic fermentation is 2-6% of the mass of the soybean meal pre-enzymatic hydrolysate, with the ratio of viable Bacillus to Aspergillus being 1:0.5-2; the temperature for the first-stage aerobic fermentation is 32-38℃, the aeration rate is 0.2-0.8 vvm, the stirring speed is 80-180 r / min, and the fermentation time is 12-24 h.

[0015] Furthermore, in step three, the lactic acid bacteria are Lactobacillus plantarum or Lactobacillus casei, and the yeast is Saccharomyces cerevisiae; the total inoculum for the second stage of microaerobic fermentation is 1-5% of the mass of the first fermentation mash, wherein the ratio of live lactic acid bacteria to live yeast is 1:0.2-1; the temperature for the second stage of microaerobic fermentation is 30-37℃, the fermentation time is 16-36h, and the final pH of the fermentation is 4.0-4.8.

[0016] Furthermore, the specific steps of enzymatic digestion and membrane fractionation in step four are as follows: Centrifuge or press filter the second fermentation mash, take the liquid phase and add alkaline protease or flavor protease, and perform enzymatic hydrolysis at 45-55℃ and pH 6.5-8.0 for 0.5-2 hours. Then raise the temperature to 85-95℃ and keep it at 5-15 minutes to inactivate the enzyme. After cooling, pass the mash through a microfiltration membrane with a pore size of 0.1-0.5μm and an ultrafiltration membrane with a molecular weight cutoff of 5000Da in sequence. Collect the ultrafiltration permeate to obtain soybean meal small peptide permeate.

[0017] Furthermore, in step five, the mass ratio of soluble soybean polysaccharide, L-arginine, and trehalose in the stabilization system is 1:0.2-0.8:1-4; the total amount added to the stabilization system is 0.3-2.0% of the mass of the soybean meal peptide permeate, and the pH is adjusted to 4.2-4.8 by lactic acid, citric acid, or a combination thereof.

[0018] Furthermore, in step five, the pressure for high-pressure homogenization is 20-60 MPa, and the number of homogenization cycles is 1-3; low-temperature sterilization is performed at 65-75℃ for 15-30 min, or at 80-90℃ for 5-10 min; after low-temperature sterilization, the fermentation liquid is cooled to below 25℃ and bottled, and the soluble solids content of the highly stable and easily absorbed soybean meal fermentation liquid before bottling is 8-20 wt%.

[0019] This invention also provides a highly stable and easily absorbed soybean meal fermentation broth, prepared by the above-described method. The highly stable and easily absorbed soybean meal fermentation broth comprises soybean meal peptides, free amino acids, organic acids, soluble soybean polysaccharides, L-arginine, and trehalose; its pH is 4.2-4.8, its soluble solids content is 8-20 wt%, peptides with a molecular weight less than 1000 Da account for 45-75% of the total peptide mass, and peptides with a molecular weight less than 5000 Da account for more than 80% of the total peptide mass.

[0020] The present invention also provides the application of the above-mentioned highly stable and easily absorbed soybean meal fermentation liquid in the preparation of animal feed additives, which are used in compound feeds, liquid feeds or drinking water supplements for weaned piglets, broilers, laying hens, calves, fish or shrimp.

[0021] Compared with the prior art, the present invention provides a highly stable and easily absorbable soybean meal fermentation liquid, its preparation method and its application, which has the following beneficial effects: through moist heat swelling and compound enzyme pre-enzymatic hydrolysis treatment, the cell wall structure of soybean meal is loosened, which improves the efficiency of subsequent microorganisms and proteases on soybean meal protein and reduces sedimentation caused by large particle residue in the later stage.

[0022] The first-stage aerobic fermentation using Bacillus and Aspergillus can utilize the protease produced by Bacillus and the complex enzyme system of protease, cellulase and phytase produced by Aspergillus to initially degrade macromolecular proteins and antinutritional factors in soybean meal.

[0023] The study employed lactic acid bacteria and yeast for the second stage of microaerobic fermentation. The lactic acid bacteria produced organic acids, causing a slow decrease in the system's pH, which helped inhibit unwanted microorganisms and improve the fermentation flavor. The soluble nutrients produced by the yeast's metabolism contributed to improving the palatability of the fermentation broth. Compared to direct strong acid regulation, staged microaerobic fermentation reduced the rapid flocculation of protein peptides caused by acid shock.

[0024] By incorporating enzymatic hydrolysis and fractionation with a 5000Da ultrafiltration membrane after fermentation, the proportion of residual large molecular weight proteins and hydrophobic large peptides can be reduced, while the proportion of peptides with a molecular weight of less than 5000Da can be increased, thereby improving the absorbability and storage stability of the fermentation broth.

[0025] A stabilization system consisting of soluble soybean polysaccharide, L-arginine, and trehalose was adopted. Soluble soybean polysaccharide provides steric hindrance and hydrophilic protection, L-arginine improves the dissolution and dispersion of some hydrophobic peptides, and trehalose reduces the probability of association between small peptides and residual proteins during heat treatment and storage. The three components work synergistically to reduce turbidity and sedimentation. Attached Figure Description

[0026] Figure 1 The images are scanning electron microscope images of the freeze-dried solids of soybean meal fermentation broth obtained in Example 3 and Comparative Example 1.

[0027] Figure 2 The image shows the molecular weight distribution of the soybean meal fermentation broth obtained in Example 3 and Comparative Example 4, obtained by gel permeation chromatography.

[0028] Figure 3 The images show SDS-PAGE electrophoresis diagrams of the soybean meal fermentation broths obtained in Example 3, Comparative Example 1, and Comparative Example 2. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Please see Figures 1-3 This invention provides a technical solution for a highly stable and easily absorbed soybean meal fermentation broth, its preparation method, and its application: In the following examples, the defatted soybean meal was commercially available feed-grade defatted soybean meal with a crude protein content of 46.8%; the neutral protease activity was 50,000 U / g; the cellulase activity was 10,000 U / g; the phytase activity was 5,000 U / g; the alkaline protease activity was 100,000 U / g; the flavor protease activity was 20,000 U / g; and the Bacillus subtilis, Bacillus belye, Aspergillus oryzae, Lactobacillus plantarum, Lactobacillus casei, and Saccharomyces cerevisiae were all commercially available microbial agents for food or feed processing, and were activated according to the product instructions before inoculation.

[0031] Test methods 1. Crude protein content: Determined according to GB / T 6432-2018 "Determination of crude protein in feed - Kjeldahl method". This standard is a current recommended national standard and is applicable to the determination of crude protein in feed ingredients, compound feed, concentrated feed, concentrate supplements and additive premixes.

[0032] 2. Peptide molecular weight distribution: Referring to the requirements for the quality and inspection of soybean peptide products in GB / T 22492-2008 "Soybean Peptide Powder", the proportion of peptides with different molecular weight ranges was determined by gel permeation chromatography.

[0033] 3. Free amino acid content: Determined according to GB / T 18246-2019 "Determination of Amino Acids in Feed". This standard specifies the methods for determining total amino acids and free amino acids in feed.

[0034] 4. pH Measurement: The glass electrode pH meter method was used, referring to GB 5009.237-2016 "National Food Safety Standard - Determination of pH Value in Food". This standard specifies the principle of pH measurement using a glass electrode as the indicating electrode.

[0035] 5. Residual activity of trypsin inhibitor: determined according to GB 5009.224-2016 "National Food Safety Standard - Determination of Trypsin Inhibitor Activity in Soybean Products"; this standard is applicable to the determination of trypsin inhibitor activity in soybean products.

[0036] 6. Total bacterial count, coliform bacteria and salmonella: Total bacterial count shall be determined according to GB / T 13093-2023 "Determination of total bacterial count in feed"; coliform bacteria shall be determined according to GB / T 18869-2019 "Determination of coliform bacteria in feed"; salmonella shall be determined according to GB / T 13091-2018 "Determination of salmonella in feed".

[0037] 7. Sedimentation rate: Place 100 mL of sample in a stoppered graduated cylinder and let it stand at 25℃ for 30 days. Record the volume of the bottom sedimentation layer, V1, and the total sample volume, V0. The sedimentation rate is calculated as V1 / V0 × 100%. Each group is measured in triplicate, and the average value is taken. This index is used to evaluate the stratification and sedimentation tendency of liquid products during storage.

[0038] 8. Centrifugation stability: Take 50g of sample, centrifuge at 3000r / min for 10min, decant the supernatant and weigh it. Centrifugation stability is calculated as supernatant mass / sample mass before centrifugation × 100%. Each group is measured in triplicate, and the average value is taken.

[0039] 9. Turbidity Change After Heat Treatment: The sample was treated at 85℃ for 10 min and then cooled to 25℃. Using distilled water as a blank, the absorbance OD600 was measured at 600 nm. The sample was then allowed to stand at 25℃ for 30 days, and the measurement was repeated. ΔOD600 was calculated. The smaller the ΔOD600, the smaller the turbidity change after heat treatment.

[0040] 10. In vitro protein digestibility: A two-step in vitro digestion method using pepsin-trypsin was adopted. First, pepsin was added at pH 2.0 for 2 hours, and then the pH was adjusted to 7.5 before adding trypsin for 4 hours. After digestion, the proportion of soluble nitrogen to total nitrogen was measured to calculate the in vitro protein digestibility.

[0041] Example 1: A method for preparing a highly stable and easily absorbed soybean meal fermentation broth, comprising the following steps: S1: Grind defatted soybean meal to 40 mesh, mix defatted soybean meal and water at a mass ratio of 1:4, soak and swell at 55℃ for 30 min, adjust pH to 6.0, then add neutral protease, cellulase and phytase, and enzymatically hydrolyze at 45℃ for 1 h to obtain soybean meal pre-enzymatic hydrolysate; wherein the added amounts of neutral protease, cellulase and phytase are 0.3%, 0.05% and 0.02% of the mass of defatted soybean meal, respectively.

[0042] S2: Inoculate the soybean meal pre-enzymatic hydrolysate with Bacillus subtilis and Aspergillus oryzae, with a total inoculation amount of 2% of the soybean meal pre-enzymatic hydrolysate mass and a live bacteria ratio of Bacillus subtilis to Aspergillus oryzae of 1:0.5; and aerobic fermentation for 12 h at 32℃, aeration rate of 0.2 vvm and stirring speed of 80 r / min to obtain the first fermentation mash.

[0043] S3: Cool the first fermentation mash to 30°C, inoculate with Lactobacillus plantarum and Saccharomyces cerevisiae, with a total inoculation amount of 1% of the mass of the first fermentation mash and a live count ratio of Lactobacillus plantarum to Saccharomyces cerevisiae of 1:0.2; ferment at 30°C under microaerobic conditions for 16 hours, with the fermentation endpoint pH being 4.8, to obtain the second fermentation mash.

[0044] S4: The second fermentation mash was pressure filtered, and alkaline protease was added to the liquid phase. The enzyme was further digested at 45℃ and pH 6.5 for 0.5 h. Then the temperature was raised to 85℃ and kept at 5 min to inactivate the enzyme. After cooling, the mixture was passed through a microfiltration membrane with a pore size of 0.5 μm and an ultrafiltration membrane with a molecular weight cutoff of 5000 Da. The ultrafiltration permeate was collected to obtain soybean meal small peptide permeate.

[0045] S5: Add a stabilization system to the soybean meal peptide permeate. The mass ratio of soluble soybean polysaccharide, L-arginine and trehalose in the stabilization system is 1:0.2:1. The total amount of the stabilization system added is 0.3% of the mass of the soybean meal peptide permeate. Adjust the pH to 4.8 with lactic acid, homogenize once at 20 MPa, then treat at 65℃ for 30 min, and cool to below 25℃ to obtain a highly stable and easily absorbed soybean meal fermentation broth with a soluble solids content of 8.4 wt%.

[0046] Example 2, a method for preparing a highly stable and easily absorbed soybean meal fermentation broth, is basically the same as in Example 1, except that: In S1, defatted soybean meal is pulverized to 50 mesh, the mass ratio of defatted soybean meal to water is 1:5, the swelling temperature is 58℃, the swelling time is 38 min, the pH is 6.3, the addition amounts of neutral protease, cellulase and phytase are 0.5%, 0.10% and 0.05% respectively, the enzymatic hydrolysis temperature is 48℃ and the enzymatic hydrolysis time is 1.4 h.

[0047] In S2, the total inoculum was 3%, the ratio of live Bacillus subtilis to Aspergillus oryzae was 1:0.8, the fermentation temperature was 34℃, the aeration rate was 0.35 vvm, the stirring speed was 100 r / min, and the fermentation time was 15 h.

[0048] In S3, the total inoculum of Lactobacillus plantarum and Saccharomyces cerevisiae was 2%, the ratio of live bacteria was 1:0.4, the fermentation temperature was 32℃, the fermentation time was 20h, and the final pH of the fermentation was 4.6.

[0049] In S4, the supplementary enzymatic hydrolysis temperature was 48℃, the pH was 7.0, the supplementary enzymatic hydrolysis time was 0.8h, the enzyme inactivation conditions were 88℃ for 8min, and the microfiltration membrane pore size was 0.4μm.

[0050] In S5, the mass ratio of soluble soybean polysaccharide, L-arginine and trehalose in the stabilization system was 1:0.3:1.5, the total amount added was 0.6%, the pH was adjusted to 4.6, the homogenization pressure was 30 MPa, the homogenization was performed once, the low temperature sterilization conditions were 70℃ for 25 min, and the soluble solids content was 10.6 wt%.

[0051] Example 3, a method for preparing a highly stable and easily absorbed soybean meal fermentation broth, is basically the same as in Example 1, except that: In S1, defatted soybean meal is pulverized to 60 mesh, the mass ratio of defatted soybean meal to water is 1:6, the swelling temperature is 62℃, the swelling time is 45 min, the pH is 6.6, the addition amounts of neutral protease, cellulase and phytase are 0.8%, 0.18% and 0.08% respectively, the enzymatic hydrolysis temperature is 50℃ and the enzymatic hydrolysis time is 2 h.

[0052] In S2, the total inoculum was 4%, the ratio of live Bacillus subtilis to Aspergillus oryzae was 1:1, the fermentation temperature was 36℃, the aeration rate was 0.5 vvm, the stirring speed was 130 r / min, and the fermentation time was 18 h.

[0053] In S3, the total inoculum of Lactobacillus plantarum and Saccharomyces cerevisiae was 3%, the ratio of live bacteria was 1:0.6, the fermentation temperature was 34℃, the fermentation time was 26h, and the final pH of the fermentation was 4.5.

[0054] In S4, after taking the liquid phase, flavor protease was added, and the enzyme was further digested at 50℃ and pH 7.2 for 1.2 h. Then, the temperature was raised to 90℃ and kept at 10 min to inactivate the enzyme. After cooling, the solution was passed through a microfiltration membrane with a pore size of 0.3 μm and an ultrafiltration membrane with a molecular weight cutoff of 5000 Da in sequence, and the ultrafiltration permeate was collected.

[0055] In S5, the mass ratio of soluble soybean polysaccharide, L-arginine and trehalose in the stabilization system was 1:0.5:2.5, and the total amount added was 1.0%. The pH was adjusted to 4.5 with lactic acid and citric acid, homogenized twice at 40 MPa, and then treated at 85℃ for 8 min. After cooling, a highly stable and easily absorbed soybean meal fermentation broth with a soluble solids content of 14.2 wt% was obtained.

[0056] Example 4, a method for preparing a highly stable and easily absorbed soybean meal fermentation broth, is basically the same as in Example 1, except that: In S1, defatted soybean meal is pulverized to 65 mesh, the mass ratio of defatted soybean meal to water is 1:6.5, the swelling temperature is 65℃, the swelling time is 50 min, the pH is 6.8, the addition amounts of neutral protease, cellulase and phytase are 0.9%, 0.22% and 0.10% respectively, the enzymatic hydrolysis temperature is 52℃ and the enzymatic hydrolysis time is 2.4 h.

[0057] In S2, the total inoculum was 4.5%, the ratio of viable Bacillus belyss to Aspergillus oryzae was 1:1.2, the fermentation temperature was 36℃, the aeration rate was 0.6 vvm, the stirring speed was 150 r / min, and the fermentation time was 20 h.

[0058] In S3, the total inoculum of Lactobacillus casei and Saccharomyces cerevisiae was 3.5%, the ratio of viable cells was 1:0.7, the fermentation temperature was 35℃, the fermentation time was 30h, and the final pH of the fermentation was 4.4.

[0059] In S4, after taking the liquid phase, flavor protease was added, and the enzyme was further digested at 52℃ and pH 7.5 for 1.5 h. The enzyme inactivation conditions were 92℃ for 10 min, and the microfiltration membrane pore size was 0.2 μm.

[0060] In S5, the mass ratio of soluble soybean polysaccharide, L-arginine and trehalose in the stabilization system was 1:0.6:3, and the total amount added was 1.3%. The pH was adjusted to 4.4, homogenized twice at 45 MPa, and then treated at 85℃ for 8 min. After cooling, a highly stable and easily absorbed soybean meal fermentation broth with a soluble solids content of 16.1 wt% was obtained.

[0061] Example 5, a method for preparing a highly stable and easily absorbed soybean meal fermentation broth, is basically the same as in Example 1, except that: In S1, defatted soybean meal is pulverized to 70 mesh, the mass ratio of defatted soybean meal to water is 1:7, the swelling temperature is 68℃, the swelling time is 55 min, the pH is 7.0, the addition amounts of neutral protease, cellulase and phytase are 1.0%, 0.25% and 0.12% respectively, the enzymatic hydrolysis temperature is 54℃ and the enzymatic hydrolysis time is 2.6 h.

[0062] In S2, the total inoculum was 5%, the ratio of viable Bacillus belyss to Aspergillus oryzae was 1:1.5, the fermentation temperature was 37℃, the aeration rate was 0.7 vvm, the stirring speed was 165 r / min, and the fermentation time was 22 h.

[0063] In S3, the total inoculum of Lactobacillus plantarum and Saccharomyces cerevisiae was 4%, the ratio of live bacteria was 1:0.8, the fermentation temperature was 36℃, the fermentation time was 32h, and the final pH of the fermentation was 4.3.

[0064] In S4, after taking the liquid phase, flavor protease was added, and the enzyme was digested for 1.8 h at 54 ℃ and pH 7.8. The enzyme inactivation conditions were 95 ℃ for 12 min, and the microfiltration membrane pore size was 0.2 μm.

[0065] In S5, the mass ratio of soluble soybean polysaccharide, L-arginine and trehalose in the stabilization system was 1:0.7:3.5, and the total amount added was 1.6%. The pH was adjusted to 4.3, homogenized three times at 50 MPa, and then treated at 90℃ for 5 min. After cooling, a highly stable and easily absorbed soybean meal fermentation broth with a soluble solids content of 18.0 wt% was obtained.

[0066] Example 6, a method for preparing a highly stable and easily absorbed soybean meal fermentation broth, is basically the same as that in Example 1, except that: In S1, defatted soybean meal is pulverized to 80 mesh, the mass ratio of defatted soybean meal to water is 1:8, the swelling temperature is 70℃, the swelling time is 60 min, the pH is 7.2, the addition amounts of neutral protease, cellulase and phytase are 1.2%, 0.3% and 0.15% respectively, the enzymatic hydrolysis temperature is 55℃ and the enzymatic hydrolysis time is 3 h.

[0067] In S2, the total inoculum was 6%, the ratio of viable Bacillus belyss to Aspergillus oryzae was 1:2, the fermentation temperature was 38℃, the aeration rate was 0.8 vvm, the stirring speed was 180 r / min, and the fermentation time was 24 h.

[0068] In S3, the total inoculum of Lactobacillus casei and Saccharomyces cerevisiae was 5%, the ratio of live bacteria was 1:1, the fermentation temperature was 37℃, the fermentation time was 36h, and the final pH of the fermentation was 4.0.

[0069] In S4, after taking the liquid phase, alkaline protease was added, and the enzyme was digested for 2 hours at 55℃ and pH 8.0. Then, the temperature was raised to 95℃ and kept for 15 minutes to inactivate the enzyme. After cooling, the solution was passed through a microfiltration membrane with a pore size of 0.1 μm and an ultrafiltration membrane with a molecular weight cutoff of 5000 Da in sequence, and the ultrafiltration permeate was collected.

[0070] In S5, the mass ratio of soluble soybean polysaccharide, L-arginine and trehalose in the stabilization system was 1:0.8:4, and the total amount added was 2.0%. The pH was adjusted to 4.2 with citric acid, homogenized three times at 60 MPa, and then treated at 90℃ for 10 min. After cooling, a highly stable and easily absorbed soybean meal fermentation broth with a soluble solids content of 19.5 wt% was obtained.

[0071] In Comparative Example 1, neutral protease, cellulase and phytase were not added for pre-enzymatic hydrolysis in step S1. Only the same wet heat swelling treatment at the same temperature and time was performed. The other raw materials, dosages and process conditions were the same as in Example 3.

[0072] In Comparative Example 2, Aspergillus oryzae was not inoculated in step S2; only Bacillus subtilis was inoculated, and the total inoculation amount was kept at 4%. The remaining raw materials, amounts, and process conditions were the same as in Example 3.

[0073] In Comparative Example 3, Lactobacillus plantarum was not inoculated in step S3, only Saccharomyces cerevisiae was inoculated, and the pH was adjusted to 4.5 with lactic acid after fermentation. The remaining raw materials, dosages and process conditions were the same as in Example 3.

[0074] In Comparative Example 4, the 5000Da ultrafiltration membrane fractionation was not performed in step S4. The filtrate was collected directly after 0.3μm microfiltration. The other raw materials, dosages and process conditions were the same as in Example 3.

[0075] In Comparative Example 5, L-arginine was not added to the stabilization system in step S5, and the missing mass was made up by trehalose, so that the total amount added to the stabilization system was still 1.0%. The other raw materials, amounts and process conditions were the same as in Example 3.

[0076] Comparative Example 6: High-pressure homogenization was not performed in step S5, and the remaining raw materials, amounts, and process conditions were the same as in Example 3.

[0077] Table 1

[0078] Table 2

[0079] Table 3

[0080] Table 4

[0081] Table 5

[0082] As shown in the table, in the soybean meal fermentation broth obtained in Examples 1-6, the proportion of peptides with a molecular weight less than 5000 Da was higher than 80%, and the proportion of peptides with a molecular weight less than 1000 Da was 47.2-73.8%, indicating that the present invention can obtain a high proportion of small molecule peptides at both ends and in the middle region of the scope defined by the claims. Examples 3 and 4 showed a better balance between the proportion of small peptides, sedimentation rate, centrifugal stability and in vitro protein digestibility; Compared with Example 3, Comparative Example 1 did not undergo pre-enzymatic hydrolysis with a compound enzyme, resulting in a decrease in the proportion of small peptides, an increase in the residual activity of trypsin inhibitor, and an increase in the sedimentation rate from 0.7% to 5.9%. This indicates that pre-enzymatic hydrolysis not only promotes protein degradation but also reduces coarse particles and sedimentation sources in the subsequent fermentation broth.

[0083] Compared with Example 3, in Comparative Example 2 without inoculation with Aspergillus oryzae, the proportion of peptides with a molecular weight less than 1000 Da decreased from 64.6% to 51.2%, and the in vitro protein digestibility decreased from 95.2% to 87.9%. This indicates that the complex enzyme system formed by Aspergillus oryzae and Bacillus helps to further degrade soybean meal protein and cell wall components, and increase the proportion of easily absorbed small molecule components.

[0084] Compared with Example 3, Comparative Example 3 did not inoculate with lactic acid bacteria but only adjusted the acidity at the endpoint. Although the final pH was the same, the sedimentation rate increased to 3.2% and ΔOD600 increased to 0.104. This indicates that the slow acidification and metabolic environment formed during the microaerobic fermentation of lactic acid bacteria helps to reduce the aggregation of protein peptides caused by acid shock.

[0085] Compared with Example 3, Comparative Example 4 did not undergo 5000Da membrane fractionation. The proportion of peptides with a molecular weight of less than 5000Da was only 65.8%, the sedimentation rate increased to 9.6%, and the centrifugal stability decreased to 76.5%. This shows that membrane fractionation is a key step in removing large molecular proteins and large peptides and improving the storage stability of fermentation broth.

[0086] Compared with Example 3, the stabilization system of Comparative Example 5 did not add L-arginine, and the proportion of small peptides did not change much, but the sedimentation rate increased from 0.7% to 5.1%, and ΔOD600 increased from 0.041 to 0.176. This indicates that the main contribution of L-arginine is not to increase the degree of hydrolysis, but to improve the dispersion stability of hydrophobic peptides and residual proteins.

[0087] Compared with Example 3, Comparative Example 6 did not undergo high-pressure homogenization, resulting in a sedimentation rate of 4.4% and a centrifugal stability of 87.2%. This indicates that high-pressure homogenization can refine cell fragments, trace lipids, and peptide aggregates, thereby improving the dispersion uniformity of the liquid fermentation broth. After low-temperature sterilization, Example 3 and all comparative examples showed good microbial control results, indicating that the differences in sedimentation rate, centrifugal stability, and turbidity in Table 2 mainly stemmed from the influence of key technical features on the colloidal stability and peptide composition of the fermentation broth, rather than abnormal stratification caused by microbial contamination.

Claims

1. A method for preparing a highly stable and easily absorbed soybean meal fermentation broth, characterized in that, The steps include the following: Step 1: After grinding the defatted soybean meal, mix it with water and perform a wet heat swelling treatment. Then add a compound enzyme for pre-enzymatic hydrolysis to obtain soybean meal pre-enzymatic hydrolysate. Step 2: Inoculate the soybean meal pre-enzymatic hydrolysate with Bacillus and Aspergillus to carry out the first stage of aerobic fermentation to obtain the first fermentation mash; Step 3: After cooling the first fermentation mash, inoculate it with lactic acid bacteria and yeast to carry out the second stage of microaerobic fermentation, and obtain the second fermentation mash; Step 4: The second fermentation mash is subjected to solid-liquid separation. The liquid phase is subjected to enzymatic hydrolysis, cleavage, and membrane fractionation. The permeate of soybean meal small peptides with a molecular weight of less than 5000 Da is collected. Step 5: Add a stabilization system consisting of soluble soybean polysaccharide, L-arginine and trehalose to the soybean meal peptide permeate, adjust the pH to 4.2-4.8, and then homogenize under high pressure and sterilize at low temperature to obtain a highly stable and easily absorbed soybean meal fermentation broth.

2. The method for preparing a highly stable and easily absorbed soybean meal fermentation broth according to claim 1, characterized in that, The preparation steps of the soybean meal pre-enzymatic hydrolysis slurry described in step one are as follows: Defatted soybean meal is pulverized to 40-80 mesh and mixed with water at a mass ratio of 1:4-8. The mixture is soaked and swelled at 55-70℃ for 30-60 minutes, and the pH is adjusted to 6.0-7.

2. Then, neutral protease, cellulase and phytase are added, and the mixture is enzymatically hydrolyzed at 45-55℃ for 1-3 hours to obtain pre-enzymatically hydrolyzed soybean meal slurry. The amounts of neutral protease, cellulase, and phytase added are 0.3-1.2%, 0.05-0.3%, and 0.02-0.15% of the weight of defatted soybean meal, respectively.

3. The method for preparing a highly stable and easily absorbed soybean meal fermentation broth according to claim 1, characterized in that, The Bacillus mentioned in step two is Bacillus subtilis or Bacillus belesiensis, and the Aspergillus is Aspergillus oryzae. The total inoculum for the first stage of aerobic fermentation is 2-6% of the mass of the soybean meal pre-enzymatic hydrolysate, with the ratio of viable Bacillus and Aspergillus being 1:0.5-2. The temperature for the first stage of aerobic fermentation is 32-38℃, the aeration rate is 0.2-0.8 vvm, the stirring speed is 80-180 r / min, and the fermentation time is 12-24 h.

4. The method for preparing a highly stable and easily absorbed soybean meal fermentation broth according to claim 1, characterized in that, The lactic acid bacteria mentioned in step three are Lactobacillus plantarum or Lactobacillus casei, and the yeast is Saccharomyces cerevisiae; The total amount of inoculum for the second stage of microaerobic fermentation is 1-5% of the mass of the first fermentation mash, wherein the ratio of live lactic acid bacteria to live yeast is 1:0.2-1; The temperature for the second stage of microaerobic fermentation is 30-37℃, the fermentation time is 16-36h, and the final pH of the fermentation is 4.0-4.

8.

5. The method for preparing a highly stable and easily absorbed soybean meal fermentation broth according to claim 1, characterized in that, The specific steps of the enzymatic digestion and membrane fractionation process described in step four are as follows: Centrifuge or press filter the second fermentation mash, take the liquid phase and add alkaline protease or flavor protease, and perform enzymatic hydrolysis at 45-55℃ and pH 6.5-8.0 for 0.5-2 hours. Then raise the temperature to 85-95℃ and keep it at 5-15 minutes to inactivate the enzyme. After cooling, pass the mash through a microfiltration membrane with a pore size of 0.1-0.5μm and an ultrafiltration membrane with a molecular weight cutoff of 5000Da in sequence. Collect the ultrafiltration permeate to obtain soybean meal small peptide permeate.

6. The method for preparing a highly stable and easily absorbed soybean meal fermentation broth according to claim 1, characterized in that, In the stabilization system described in step five, the mass ratio of soluble soybean polysaccharide, L-arginine, and trehalose is 1:0.2-0.8:1-4. The total amount of the stabilization system added is 0.3-2.0% of the mass of the soybean meal peptide permeate, and the pH is adjusted to 4.2-4.8 by lactic acid, citric acid or a combination thereof.

7. The method for preparing a highly stable and easily absorbed soybean meal fermentation broth according to claim 1, characterized in that, The pressure for high-pressure homogenization in step five is 20-60 MPa, and the number of homogenization cycles is 1-3. The low-temperature sterilization is performed at 65-75℃ for 15-30 minutes, or at 80-90℃ for 5-10 minutes. After low-temperature sterilization, the fermentation liquid is cooled to below 25°C and then bottled. Before bottling, the soluble solids content of the highly stable and easily absorbed soybean meal fermentation liquid is 8-20 wt%.

8. A highly stable and easily absorbed soybean meal fermentation liquid, characterized in that, Prepared by the preparation method according to any one of claims 1-7; The highly stable and easily absorbed soybean meal fermentation broth includes soybean meal peptides, free amino acids, organic acids, soluble soybean polysaccharides, L-arginine, and trehalose. The highly stable and easily absorbed soybean meal fermentation broth has a pH of 4.2-4.8, a soluble solids content of 8-20 wt%, peptides with a molecular weight of less than 1000 Da accounting for 45-75% of the total peptide mass, and peptides with a molecular weight of less than 5000 Da accounting for more than 80% of the total peptide mass.

9. The highly stable and easily absorbed soybean meal fermentation liquid according to claim 8, characterized in that: The highly stable and easily absorbed soybean meal fermentation broth, after standing at 25℃ for 30 days, has a sedimentation rate of no more than 3.0%, a centrifugal stability of no less than 90%, a total acid content (calculated as lactic acid) of 0.5-2.5 wt%, and a free amino acid content of 1.0-5.0 wt%. The centrifugal stability is defined as the percentage of the supernatant mass relative to the sample mass before centrifugation after centrifuging the fermentation broth at 3000 r / min for 10 min.

10. The application of the highly stable and easily absorbed soybean meal fermentation broth according to claim 8 or 9 in the preparation of animal feed additives, characterized in that: The animal feed additives are used in compound feeds, liquid feeds or drinking water supplements for weaned piglets, broilers, laying hens, calves, fish or shrimp; When used in compound feed, the amount of the highly stable and easily absorbed soybean meal fermented liquid added is 0.5-5% of the mass of the compound feed; when used as a drinking water supplement, the amount of the highly stable and easily absorbed soybean meal fermented liquid added is 0.1-2% of the volume of drinking water.

Images