Method for improving antioxidant activity of oil cakes through combination of multi-strain fermentation and enzymolysis and application of method

Abstract

The invention discloses a method for improving antioxidant activity of oil cakes through multi-strain fermentation combined with enzymolysis and application. The method comprises the following steps: adding a multi-strain mixed bacterium suspension into sterilized oil cakes, uniformly mixing, performing constant-temperature fermentation at 30 + / -2 DEG C, performing intermittent stirring in the fermentation process, and sequentially performing high-temperature sterilization and freeze-drying treatment after the fermentation is finished, the multi-strain mixed bacterium suspension is a mixed bacterium suspension of aspergillus niger, saccharomyces cerevisiae and bacillus subtilis, and the viable count ratio of the aspergillus niger to the saccharomyces cerevisiae to the bacillus subtilis in the multi-strain mixed bacterium suspension is (1-2): (1-2): (1-2). The application finds that a multi-strain composite fermentation combined enzymolysis technology can realize the synergistic interaction of fermentation and enzymolysis, obviously improves the antioxidant activity of the oil cake meal, and can be applied to preparation of cake meal feed.

Description

Technical Field

[0001] This invention relates to the field of feed processing technology, specifically to a method for improving the antioxidant activity of oilseed cake through multi-strain fermentation combined with enzymatic hydrolysis, and its application. Background Technology

[0002] my country is a major global producer and processor of oilseed crops, with annual production of over ten million tons of oilseed cakes (by-products) from the pressing of woody oil crops such as walnuts, camellia oleifera, and olives. These oilseed cakes are rich in protein (30%-50%), dietary fiber, and polyphenolic active substances, but face three major utilization bottlenecks: 1. High content of anti-nutritional factors: Tannins (3%-10%) bind to proteins to form insoluble complexes, reducing protein digestibility; phytic acid (2%-5%) chelates mineral elements, leading to decreased bioavailability of calcium, zinc, etc.; trypsin inhibitors (1-5 mg / g) inhibit intestinal protease activity, affecting protein absorption. 2. Difficulty in utilizing macromolecules: Proteins are denatured at high temperatures during pressing, resulting in dense peptide chains and low accessibility to enzymatic hydrolysis sites; cellulose (10%-30%) encapsulates nutrients, limiting their release. 3. Poor oxidative stability: Unsaturated fatty acids and polyphenols are easily oxidized during storage, generating aldehydes and ketones, which produce off-flavors and shorten the product's shelf life.

[0003] Single-strain fermentation suffers from limited metabolic capacity and poor stress resistance due to its single enzyme system, making it difficult to efficiently address the complex anti-nutritional factors and structural barriers in oilseed cakes. This results in insufficient nutrient release and limited improvement in functional activity. While multi-strain fermentation can complement enzyme systems (e.g., Aspergillus niger producing amylase and Trichoderma producing cellulase), it still cannot cover all substrates. It relies on the slow secretion of enzymes by microorganisms, leading to long fermentation times, high energy consumption, difficulty in maintaining microbial community balance, and significant batch-to-batch variability. Furthermore, existing processing methods result in oilseed cakes with low antioxidant activity. Summary of the Invention

[0004] This application provides a method and application for improving the antioxidant activity of oilseed cake through multi-strain fermentation combined with enzymatic hydrolysis. This method can significantly improve the antioxidant activity of oilseed cake while ensuring a high degradation rate of anti-nutritional factors, and has good application prospects in the preparation of oilseed cake feed.

[0005] A method for improving the antioxidant activity of oilseed cake through multi-strain fermentation combined with enzymatic hydrolysis includes: Add a multi-strain mixed bacterial suspension to the sterilized oilseed cake, mix well, and ferment at a constant temperature of 30±2 ℃ for 5~6 hours. During the fermentation process, stir intermittently. After the fermentation is completed, the mixture is subjected to high-temperature sterilization and freeze-drying in sequence. Then, it is subjected to tannin enzyme hydrolysis and alkaline protease hydrolysis in sequence. The multi-strain mixed bacterial suspension is a mixed bacterial suspension of Aspergillus niger, Saccharomyces cerevisiae and Bacillus subtilis, and the ratio of the number of viable cells of Aspergillus niger, Saccharomyces cerevisiae and Bacillus subtilis in the multi-strain mixed bacterial suspension is 1~2:1~2:1~2.

[0006] To overcome the bottlenecks of existing technologies, this application proposes a multi-strain solid-state fermentation method using *Aspergillus niger*, *Saccharomyces cerevisiae*, and *Bacillus subtilis*, combined with targeted enzymatic hydrolysis using tanninases and proteases. *Aspergillus niger* primarily secretes acidic proteins, including phytase and cellulase, to hydrolyze phytic acid, debonding minerals and disrupting the cell wall structure to release embedded tannin proteins. *Bacillus subtilis* secretes alkaline and neutral proteases, specifically cleaving the active sites of trypsin inhibitors. *Saccharomyces cerevisiae* lowers the environmental pH to 4.0-5.5 by metabolizing organic acids such as citric acid and malic acid, activating the enzyme system activity of *Aspergillus niger*. Tanninases hydrolyze the ester and glycosidic bonds of tannins, breaking them down into gallic acid and glucose, achieving a tannin degradation rate of 80%-86%. Neutral / alkaline proteases, in conjunction with microbial proteases, further cleave denatured proteins into small peptides, improving solubility and absorption. Microbial fermentation can hydrolyze bound polyphenols such as tannins and lignins, significantly increasing the content of free phenols. Proteolysis produces peptides rich in hydrophobic amino acids such as proline and leucine, enhancing their ability to scavenge DPPH / hydroxyl radicals.

[0007] The method described in this application can achieve an 85% degradation rate of anti-nutritional factors in oilseed cake, increase antioxidant activity by 30%-50%, improve protein bioavailability by 40%, and increase peptide content to 15%-20%. This provides technical support for the application of oilseed cake resources in the field of functional additives for high-protein feeds.

[0008] Several alternative methods are provided below, but they are not intended as additional limitations on the overall solution above. They are merely further additions or optimizations. Provided there are no technical or logical contradictions, each alternative method can be combined individually with respect to the overall solution above, or multiple alternative methods can be combined with each other.

[0009] Optionally, the viable concentration of Aspergillus niger in the multi-strain mixed bacterial suspension is 0.5~2×10⁻⁶. 8 The CFU / mL concentration of Saccharomyces cerevisiae was 0.5~2×10⁻⁶. 8 CFU / mL, viable concentration of Bacillus subtilis is 0.5~2×10⁻⁶. 8 CFU / mL.

[0010] Optionally, the ratio of viable bacteria of Aspergillus niger, Saccharomyces cerevisiae and Bacillus subtilis in the multi-strain mixed bacterial suspension is 1:1:1~2.

[0011] Optionally, the ratio of viable bacteria of Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis in the multi-strain mixed bacterial suspension is 1:1:1 or 1:1:2.

[0012] Furthermore, the viable concentration of Aspergillus niger in the multi-species mixed bacterial suspension is 1×10⁻⁶. 8 CFU / mL, viable concentration of Saccharomyces cerevisiae 1×10⁻⁶ 8 CFU / mL, Bacillus subtilis viable cell concentration 1×10⁻⁶ 8 CFU / mL; or: the viable concentration of Aspergillus niger in the multi-strain mixed bacterial suspension is 0.75 × 10⁻⁶ CFU / mL; 8 The CFU / mL concentration of *Saccharomyces cerevisiae* was 0.75 × 10⁻⁶. 8 The CFU / mL concentration of Bacillus subtilis was 1.5 × 10⁻⁶. 8 CFU / mL.

[0013] Optionally, the amount of the multi-strain mixed bacterial suspension added is 5-8% of the dry weight of the oilseed cake.

[0014] Furthermore, the amount of the multi-strain mixed bacterial suspension added is 6% of the dry weight of the oilseed cake.

[0015] Optionally, during the fermentation process, 5-15% by dry weight of oilseed cake is added as a fermentation auxiliary material. Optionally, the fermentation auxiliary material is wheat bran.

[0016] Optionally, the amount of tanninase used in the tanninase hydrolysis treatment is 0.4-0.6% of the dry weight of the oilseed cake; the amount of alkaline protease used in the alkaline protease hydrolysis treatment is 0.3-0.5% of the dry weight of the oilseed cake. Optionally, the enzyme activity of the tanninase is 450-550 U / g; the enzyme activity of the alkaline protease is 350,000-450,000 U / g.

[0017] Further, in the tanninase enzymatic hydrolysis treatment, the amount of tanninase used is 0.5% of the dry weight of the oilseed cake; in the alkaline protease enzymatic hydrolysis treatment, the amount of alkaline protease used is 0.4% of the dry weight of the oilseed cake. Optionally, the enzyme activity of the tanninase is 500 U / g; the enzyme activity of the alkaline protease is 400,000 U / g. Optionally, the conditions for the tanninase enzymatic hydrolysis treatment are: 50~60℃ for 55~65 min; the conditions for the alkaline protease enzymatic hydrolysis treatment are: 50~60℃ for 5~6 h.

[0018] Optionally, the oilseed cake may be walnut cake, camellia cake, or olive cake.

[0019] Optionally, the antioxidant activity includes at least one of the following: total antioxidant capacity, hydroxyl radical scavenging capacity, DPPH radical scavenging capacity, and flavonoid content.

[0020] This application also provides an oilseed cake prepared by the method described above.

[0021] This application also provides an application of the described method in the preparation of high-protein feed. The oilseed cake prepared in this application can be used as a functional additive for high-protein feed.

[0022] For example, it can be used to prepare pig feed, with soybean meal as the protein source and wheat bran as the carbon source, wherein 2% of the soybean meal is replaced by fermented oilseed meal treated by the method of this application.

[0023] This study found that multi-strain compound fermentation combined with enzymatic hydrolysis can achieve synergistic effects between fermentation and enzymatic hydrolysis, significantly improving the antioxidant activity of oilseed cake, such as total antioxidant capacity, hydroxyl radical scavenging capacity, DPPH radical scavenging capacity, and flavonoid content, which can be applied to the preparation of high-protein feed. Attached Figure Description

[0024] Figure 1 Figure 1 shows the effect of multi-strain fermentation with different mixed bacterial ratios on the content of anti-nutritional factors and in vitro antioxidant activity of walnut cake meal.

[0025] Figure 2 Figure 1 shows the effect of multi-strain fermentation with different mixed bacterial ratios on the content of anti-nutritional factors and in vitro antioxidant activity of camellia seed cake.

[0026] Figure 3 Figure 1 shows the effect of multi-strain co-fermentation with different mixed bacterial ratios on the content of anti-nutritional factors and in vitro antioxidant activity of olive oil cake meal.

[0027] Figure 4 The figure shows the effects of multi-strain fermentation and enzymatic hydrolysis on the content of anti-nutritional factors in walnut cake meal.

[0028] Figure 5 The figure shows the effect of multi-strain compound fermentation and enzymatic hydrolysis on the content of anti-nutritional factors in camellia oil cake.

[0029] Figure 6 The figure shows the effects of multi-strain fermentation and enzymatic hydrolysis on the content of anti-nutritional factors in olive oil cake meal.

[0030] Figure 7 Figure 1 shows the effect of multi-strain fermentation and enzymatic hydrolysis on the in vitro antioxidant activity of walnut cake meal.

[0031] Figure 8 Figure 1 shows the effect of multi-strain compound fermentation and enzymatic hydrolysis on the in vitro antioxidant activity of camellia oil cake.

[0032] Figure 9 Figure 1 shows the effect of multi-strain fermentation and enzymatic hydrolysis on the in vitro antioxidant activity of olive oil cake. Detailed Implementation

[0033] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0035] The Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis used in this application are all commercially available products. The Aspergillus niger (BNCC186380), Saccharomyces cerevisiae (BNCC336054), and Bacillus subtilis (BNCC132865) used in the following examples were all purchased from Beina Biotechnology Co., Ltd.

[0036] The tanninase (enzyme activity: 500 U / g) and alkaline protease (enzyme activity: 400,000 U / g) used were purchased from Nanning Pangbo Biotechnology Co., Ltd., Guangxi Zhuang Autonomous Region.

[0037] The corresponding culture media for each bacterial species are as follows: Aspergillus niger: Potato dextrose agar (PDA medium); Saccharomyces cerevisiae: Yeast extract malt extract medium (YM medium); Bacillus subtilis: Nutrient agar medium (NA medium).

[0038] Example 1 (1) Activation of bacterial strains and preparation of bacterial suspension Commercially available Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis were added to an appropriate amount of sterile water, mixed by pipetting, and then inoculated onto their respective solid culture media for activation. The activated strains were then cultured in a 30 ℃ biochemical incubator until the mycelium or colonies had grown fully.

[0039] Preparation of Aspergillus niger suspension: Aspergillus niger was cultured in solid culture medium (PDA medium), and the Aspergillus niger was washed with sterile water. The solution was then poured into sterile enzyme-free centrifuge tubes, and mycelial residue was filtered off with sterile gauze to obtain an Aspergillus niger suspension. The suspension was mixed with 0.4% trypan blue solution at a volume ratio of 9:1, and the concentration of the Aspergillus niger suspension was adjusted to 3 × 10⁻⁶ by counting with a hemocytometer. 8 CFU / mL, for later use.

[0040] Preparation of Saccharomyces cerevisiae suspension: Wash the Saccharomyces cerevisiae directly with sterile water and pour it into a sterile enzyme-free centrifuge tube. Mix the suspension with 0.4% trypan blue solution at a volume ratio of 9:1. Count the viable cells using a hemocytometer and dilute the suspension to 3 × 10⁻⁶. 8 CFU / mL, for later use.

[0041] Preparation of Bacillus subtilis suspension: Bacillus subtilis was washed directly with sterile water and then transferred to a sterile, enzyme-free centrifuge tube. The bacterial suspension was mixed with 0.4% trypan blue solution at a volume ratio of 9:1. The viable count was performed using a hemocytometer, and the bacterial suspension was diluted to a concentration of 3 × 10⁻⁶. 8 CFU / mL, for later use.

[0042] Mix Aspergillus niger suspension, Saccharomyces cerevisiae suspension, and Bacillus subtilis suspension in volume ratios of 1:1:1, 2:1:1, 1:2:1, and 1:1:2 respectively to prepare mixed bacterial suspensions for later use.

[0043] (2) Solid-state fermentation and sample processing 1) Fermentation Walnut cake meal, camellia oil cake meal, and olive cake meal were crushed separately, passed through a 40-mesh sieve, and dried for later use. 20 g of cake meal (walnut cake meal, camellia oil cake meal, or olive cake meal) and 10% (percentage relative to the dry weight of the cake meal (main ingredient)) of wheat bran were weighed and added to an Erlenmeyer flask, stirred evenly, and the flask mouth was sealed with a breathable sterile sealing film and secured with a rubber band. The Erlenmeyer flask was placed in an autoclave and sterilized at 121℃ for 20 minutes. After sterilization, the Erlenmeyer flask was removed, and after the cake meal mixture cooled, 55% (percentage relative to the dry weight of the cake meal (main ingredient)) of sterile water and 6% (percentage relative to the dry weight of the cake meal (main ingredient)) of mixed bacterial suspension or single bacterial suspension in different mixing ratios were added. After stirring evenly, the Erlenmeyer flask was placed in a 30℃ constant temperature incubator for fermentation, maintaining a certain distance between each flask, and stirring was performed every 24 hours. After fermentation for 6 days, the product is removed and placed in an autoclave for sterilization at 121°C for 20 minutes. After cooling, it is freeze-dried in a freeze dryer and then stored in a freezer at -80°C for later use.

[0044] 2) Tannin-enzymatic hydrolysis of cake meal Take a glass bottle, add 5 g of fermented cake from step 1), the ratio of cake to buffer (phosphate buffer (PBS, pH 7.2-7.4) is 1:7 (g / mL), add 0.5% (0.5% of the dry weight of the fermented cake from step 1, i.e. 25 mg) of tanninase, and enzymatically hydrolyze for 60 min at a temperature of 55℃.

[0045] For experiments that only involve the tannin hydrolysis step, after the hydrolysis is completed, the enzyme is inactivated at 95°C or above for 15 minutes and then freeze-dried for later use.

[0046] For experiments requiring further alkaline protease hydrolysis, alkaline protease is added to the resulting hydrolysate for the next step of hydrolysis.

[0047] 3) Alkaline protease hydrolysis of oilseed cake Add alkaline protease to the enzymatic hydrolysate obtained after tannin hydrolysis in step 2) to continue enzymatic hydrolysis of the cake meal. The enzyme addition amount is 0.4% (0.5% of the dry weight of the cake meal after fermentation in step 1, i.e., 20 mg), the hydrolysis time is 6 h, and the hydrolysis temperature is 55℃. After the hydrolysis is completed, inactivate the enzyme at 95°C or above for 15 minutes, and freeze-dry for later use.

[0048] (3) Determination of anti-nutritional factors a. Tannin content determination Determination of tannin content: GB / T 27985-2011 "Determination of tannins in feed by spectrophotometry".

[0049] b. Phytic acid content determination Phytase can decompose sodium phytate (sodium inositol hexaphosphate) to produce inorganic phosphorus and inositol derivatives. Under acidic conditions, inorganic phosphorus reacts with ammonium molybdate chromogenic reagent to produce a blue molybdenum blue substance with a characteristic absorption peak at 660 nm. The phytic acid content can be calculated by measuring the content of inorganic phosphorus. The determination of inorganic phosphorus content is carried out by the phosphomolybdenum blue spectrophotometric method.

[0050] c. Trypsin inhibitor assay Weigh 0.1 g of the cake meal sample after different treatment steps in step (2), add 0.9 mL of PBS (pH = 7.2-7.4, concentration 0.01 mol / L) for homogenization (sample:PBS = 1:9 mass ratio), and perform homogenization using a tissue homogenizer under ice bath conditions. Centrifuge the homogenate at 5000 rpm for 15 min, and collect the supernatant for testing. The "Plant Trypsin Inhibitor Detection Kit" purchased from Shanghai Huzhen Industrial Co., Ltd. was used, and the operation procedure was performed in accordance with the kit instructions.

[0051] Remove the required strips from the aluminum foil bag after equilibration at room temperature for 20 min. Set up standard and sample wells. Add 50 μL of different concentrations of standard to each standard well; add 10 μL of the test sample to each sample well, followed by 40 μL of sample diluent; do not add any sample or reagent to the blank wells. Except for the blank wells, add 100 μL of horseradish peroxidase-labeled detection antibody to each standard and sample well. Seal the reaction wells with sealing film and incubate at 37 ℃ for 60 min. Discard the reaction solution, pat dry any remaining liquid in the wells with absorbent paper, fill each well with washing buffer, let stand for 1 min, then discard the washing buffer and pat dry again with absorbent paper. Repeat this washing step 5 times. Add 50 μL each of substrate A (peroxidase solution) and substrate B (TMB solution) to each well and incubate at 37 ℃ in the dark for 15 min. Then, add 50 μL of stop solution to each well and measure the OD value of each well at 450 nm within 15 min.

[0052] Plotting the standard curve: In an Excel worksheet, plot the standard concentration on the x-axis and the corresponding OD value on the y-axis to create a linear regression curve for the standard. Calculate the concentration values ​​for each sample based on the curve equation. The resulting standard curve is y = 0.0245x + 0.0745, R². 2 = 0.9990.

[0053] (4) In vitro antioxidant assay a. Total antioxidant capacity Weigh 0.1 g of the cake meal after different treatment steps in step (2), add 1 mL of 80% alcohol solution, and homogenize using a grinder in an ice bath; centrifuge at 10000 g for 10 min at 4 ℃, collect the supernatant, and place it on ice for analysis. In an acidic environment, Fe... 3+ - Tripyridine triazine (Fe 3+ -TPTZ) was reduced to blue Fe 2+ -TPTZ, whose ability reflects the total antioxidant capacity of the sample, has an absorbance value at 593 nm.

[0054] b. Hydroxyl radical scavenging ability Weigh 0.1 g of the cake meal after different treatment steps in step (2), add 1 mL of extraction solution (80% alcohol solution), and homogenize under ice bath conditions; centrifuge the sample at 10000 g at 4 ℃ for 10 min, collect the supernatant, and place it on ice for analysis. The principle of this determination is based on H2O2 / Fe 2+ Hydroxyl radicals are generated via the Fenton reaction; these radicals can ionize phenanthrene-Fe 2+ Fe in aqueous solution 2+ Oxidized to Fe 3+This leads to a decrease in the intensity of its characteristic absorption peak at 536 nm. The degree of suppression of the rate of decrease in absorbance at 536 nm can be used to assess the sample's ability to scavenge hydroxyl radicals.

[0055] c. DPPH free radical scavenging ability Weigh 0.05 g of the cake meal sample after different treatment steps in step (2), add 1 mL of extraction solution (80% alcohol solution), and soak in a 40 ℃ water bath for 30 min; centrifuge the sample at 10000 rpm at 25 ℃ for 10 min, collect the supernatant, and place it on ice for testing. DPPH free radicals have unpaired electrons, and their alcohol solution is purple, exhibiting strong absorption characteristics at 515 nm. When antioxidants are present, DPPH free radicals are scavenged, resulting in a lighter solution color and a decrease in absorbance at 515 nm. Within a certain range, the change in absorbance is proportional to the degree of free radical scavenging. The degree of decrease in absorbance can be used to quantitatively assess the sample's ability to scavenge DPPH free radicals.

[0056] d. Flavonoid content Weigh 0.1 g of the cake meal after different treatment steps in step (2), add 1 mL of extraction solution (80% alcohol solution), and extract using ultrasonic extraction at 60 ℃ for 40 min. Centrifuge at 12000 rpm and 25 ℃ for 10 min, and collect the supernatant for analysis. In alkaline nitrite solution, flavonoids form a red complex with a characteristic absorption peak with aluminum ions. This complex has a characteristic absorption peak at 470 nm, which can be used to accurately calculate the flavonoid content of the sample.

[0057] Experimental results: 1. Effects of different mixed bacterial ratios on the content of antinutritional factors and in vitro antioxidant activity of oilseed cake before and after fermentation.

[0058] (1) Effects of different mixed strain ratios on the content of antinutritional factors and in vitro antioxidant activity of walnut meal before and after fermentation The results are as follows Figure 1 As shown in the figure, A: phytic acid content before and after fermentation with different mixed bacterial ratios; B: trypsin inhibitor content before and after fermentation with different mixed bacterial ratios; C: tannin content before and after fermentation with different mixed bacterial ratios; D: total antioxidant capacity before and after fermentation with different mixed bacterial ratios; E: hydroxyl radical scavenging rate before and after fermentation with different mixed bacterial ratios; F: DPPH radical scavenging rate before and after fermentation with different mixed bacterial ratios; G: flavonoid content before and after fermentation with different mixed bacterial ratios. The horizontal axis of the figure corresponds to samples after fermentation of mixed bacterial suspensions with different mixing ratios in step (2) 1).

[0059] like Figure 1As shown in the AC diagram, compared to before fermentation, the walnut meal fermented with a 1:1:1 ratio of Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis showed the greatest decrease in phytic acid, trypsin inhibitor, and tannin content, and these levels were significantly lower than those of other ratios. Figure 1 As shown in the DG, compared with before fermentation, walnut cake meal fermented with different proportions of Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis showed significantly increased total antioxidant capacity, hydroxyl radical scavenging rate, DPPH radical scavenging rate, and flavonoid content. The 1:1:1 ratio showed the largest increase, followed by 1:1:2. In the 1:1:1 mixed fermentation group, the phytic acid residue rate, trypsin inhibitor (TI) content, and tannin content were the lowest, indicating the best overall effect.

[0060] In summary, the 1:1:1 mixed bacteria ratio for fermenting walnut cake meal resulted in the highest degradation rate of anti-nutritional factors and the highest improvement rate of antioxidant activity, while the 1:2:1 ratio was the least effective.

[0061] (2) Effects of different mixed strain ratios on the content of antinutritional factors and in vitro antioxidant activity of camellia seed cake before and after fermentation The results are as follows Figure 2 As shown in the figure: A: Phytic acid content before and after fermentation with different mixed bacterial ratios; B: Trypsin inhibitor content before and after fermentation with different mixed bacterial ratios; C: Tannin content before and after fermentation with different mixed bacterial ratios; D: Total antioxidant capacity before and after fermentation with different mixed bacterial ratios; E: Hydroxyl radical scavenging rate before and after fermentation with different mixed bacterial ratios; F: DPPH radical scavenging rate before and after fermentation with different mixed bacterial ratios; G: Flavonoid content before and after fermentation with different mixed bacterial ratios. The horizontal axis of the figure corresponds to samples after fermentation of mixed bacterial suspensions with different mixing ratios in step (2) 1). Figure 2 As shown in the AC diagram, compared with before fermentation, the content of phytic acid, trypsin inhibitor, and tannins decreased the most after fermentation of camellia seed cake with a mixture of Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis in ratios of 1:1:1 and 1:1:2, and these levels were significantly lower than those of other ratios. Figure 2 As shown in the DG, compared with before fermentation, the total antioxidant capacity, hydroxyl radical scavenging rate, DPPH radical scavenging rate and flavonoid content of camellia oil cake after fermentation with different ratios of Aspergillus niger, Saccharomyces cerevisiae and Bacillus subtilis were significantly increased. Among them, the 1:1:1 ratio showed the largest increase, followed by the 1:1:2 ratio.

[0062] (3) Effects of different mixed strain ratios on the content of antinutritional factors and in vitro antioxidant activity of olive oil cake meal before and after fermentation The results are as follows Figure 3As shown in the figure: A: Phytic acid content before and after fermentation with different mixed bacterial ratios; B: Trypsin inhibitor content before and after fermentation with different mixed bacterial ratios; C: Tannin content before and after fermentation with different mixed bacterial ratios; D: Total antioxidant capacity before and after fermentation with different mixed bacterial ratios; E: Hydroxyl radical scavenging rate before and after fermentation with different mixed bacterial ratios; F: DPPH radical scavenging rate before and after fermentation with different mixed bacterial ratios; G: Flavonoid content before and after fermentation with different mixed bacterial ratios. The horizontal axis of the figure corresponds to samples after fermentation of mixed bacterial suspensions with different mixing ratios in step (2) 1). Figure 3 As shown in the AC diagram, compared with before fermentation, the content of phytic acid, trypsin inhibitor, and tannins decreased the most after fermentation of olive oil meal in a 1:1:1 ratio of Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis. Furthermore, the content of trypsin inhibitor was significantly lower than in other ratios. Figure 3 As shown in the DG, compared with before fermentation, the total antioxidant capacity, hydroxyl radical scavenging rate, DPPH radical scavenging rate and flavonoid content of olive cake meal were significantly increased after fermentation with different ratios of Aspergillus niger, Saccharomyces cerevisiae and Bacillus subtilis. The 1:1:1 ratio showed the largest increase, followed by the 1:1:2 ratio.

[0063] During fermentation, *Aspergillus niger* secretes cellulase, which facilitates the release of free phytic acid, which can then be hydrolyzed by phytase produced by *Bacillus subtilis*. *Aspergillus niger*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* all produce proteases and cellulases, further increasing the soluble protein content of the fermented oilseed cake and enhancing its nutritional value for feed. Furthermore, the fermentation process, based on the metabolic symbiosis and enzyme activity complementarity of the mixed strains, significantly increases the flavonoid content in the fermented cake, and the antioxidant capacity of the fermentation products is also significantly enhanced.

[0064] 2. Effects of single-strain and mixed-strain fermentation on the content of anti-nutritional factors before and after fermentation and enzymatic hydrolysis of oilseed cake.

[0065] (1) Analysis of anti-nutritional factors content before and after fermentation and combined fermentation and enzymatic hydrolysis of walnut cake meal The results are as follows Figure 4As shown, A: Phytic acid content before and after single-strain fermentation and multi-strain compound fermentation; B: Trypsin inhibitor content before and after single-strain fermentation and multi-strain compound fermentation; C: Tannin content before and after single-strain fermentation and multi-strain compound fermentation; D: Phytic acid content before and after multi-strain compound fermentation and enzymatic hydrolysis; E: Trypsin inhibitor content before and after multi-strain compound fermentation and enzymatic hydrolysis; F: Tannin content before and after multi-strain compound fermentation and enzymatic hydrolysis. In AC, the mixed bacterial solution used for multi-strain compound fermentation is a 1:1:1 mixed bacterial solution; in EF, "before fermentation" refers to oilseed cake raw material, and "after fermentation" refers to fermentation of the 1:1:1 mixed bacterial solution (corresponding to step (2) 1) in Example 1), "fermentation + tanninase" refers to fermentation of the 1:1:1 mixed bacterial solution followed by tanninase hydrolysis (corresponding to step (2) 2) in Example 1), and "fermentation + tanninase + protease" refers to fermentation of the 1:1:1 mixed bacterial solution followed by tanninase hydrolysis and protease hydrolysis (corresponding to step (2) 3) in Example 1). Figures 5-6 Interpreted in the same way.

[0066] like Figure 4 As shown in Figures AC, compared with before fermentation, the contents of phytic acid, trypsin inhibitors, and tannins in walnut meal fermented with single-strain Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis were reduced. Compared with single-strain fermentation, multi-strain fermentation significantly reduced the contents of phytic acid, trypsin inhibitors, and tannins in walnut meal. The results indicate that multi-strain fermentation can significantly reduce the content of anti-nutritional factors in walnut meal than single-strain fermentation, demonstrating the synergistic metabolic advantage among strains.

[0067] like Figure 4 As shown in the DF, the content of phytic acid and tannin in walnut cake was significantly reduced after the addition of tanninase; after the addition of protease, the content of phytic acid, trypsin inhibitor and tannin was further reduced. Tanninase directly degrades tannin, while protease decomposes phytic acid-protein complex and tannin-protein complex. The two work together to achieve deep degradation of phytic acid, trypsin inhibitor and tannin.

[0068] The above results indicate that multi-strain combined fermentation with enzymatic hydrolysis technology can achieve synergistic effects between fermentation and enzymatic hydrolysis, and its degradation capacity for phytic acid, trypsin inhibitors and tannins is much higher than that of single-strain fermentation and simple multi-strain combined fermentation.

[0069] (2) Analysis of the content of anti-nutritional factors before and after fermentation and combined fermentation and enzymatic hydrolysis of camellia oil cake meal The results are as follows Figure 5As shown, A: Phytic acid content before and after single-strain fermentation and multi-strain compound fermentation; B: Trypsin inhibitor content before and after single-strain fermentation and multi-strain compound fermentation; C: Tannin content before and after single-strain fermentation and multi-strain compound fermentation; D: Phytic acid content before and after multi-strain compound fermentation and enzymatic hydrolysis; E: Trypsin inhibitor content before and after multi-strain compound fermentation and enzymatic hydrolysis; F: Tannin content before and after multi-strain compound fermentation and enzymatic hydrolysis.

[0070] like Figure 5 As shown in Figure AC, compared with before fermentation, the contents of phytic acid, trypsin inhibitor, and tannins in camellia oil cake meal decreased after fermentation with single-strain Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis. Compared with single-strain fermentation, multi-strain compound fermentation can significantly reduce the contents of phytic acid, trypsin inhibitor, and tannins in camellia oil cake meal. Figure 5 As shown in the DF, the content of phytic acid and tannin in camellia seed cake decreased significantly after the addition of tanninase; after further addition of protease, the content of phytic acid, trypsin inhibitor and tannin decreased even further.

[0071] The above results indicate that the compound microbial strains degrade anti-nutritional factors more efficiently through metabolic complementarity, exhibiting a synergistic effect. The combined fermentation of the compound microbial strains with enzymatic hydrolysis first decomposes a portion of the anti-nutritional factors through microbial fermentation, and then the subsequent enzymatic treatment specifically decomposes the remaining portion, achieving a synergistic effect between fermentation and enzymatic treatment. This lays the material foundation for converting camellia oil cake into feed.

[0072] (3) Analysis of the content of anti-nutritional factors before and after fermentation and combined fermentation and enzymatic hydrolysis of olive oil cake meal The results are as follows Figure 6 As shown, A: Phytic acid content before and after single-strain fermentation and multi-strain compound fermentation; B: Trypsin inhibitor content before and after single-strain fermentation and multi-strain compound fermentation; C: Tannin content before and after single-strain fermentation and multi-strain compound fermentation; D: Phytic acid content before and after multi-strain compound fermentation and enzymatic hydrolysis; E: Trypsin inhibitor content before and after multi-strain compound fermentation and enzymatic hydrolysis; F: Tannin content before and after multi-strain compound fermentation and enzymatic hydrolysis.

[0073] like Figure 6 As shown in Figures AC, compared to before fermentation, the content of phytic acid, trypsin inhibitors, and tannins in olive oil cake decreased after fermentation with a single strain of *Aspergillus niger*, while the tannin content decreased after fermentation with *Saccharomyces cerevisiae*, and the phytic acid and tannin content decreased after fermentation with *Bacillus subtilis*. Compared with single-strain fermentation, multi-strain combined fermentation significantly reduced the content of phytic acid, trypsin inhibitors, and tannins in olive oil cake. When multiple strains are used in combination, they can cooperate with each other and secrete different enzymes to decompose phytic acid, thus their ability to decompose phytic acid is stronger than that of using a single strain. Furthermore, tannins affect the taste of food and hinder nutrient absorption. Figure 6As shown in Figure C, the mixed strains have the best effect on decomposing tannins, which can improve feed palatability and absorption rate in the gastrointestinal tract.

[0074] like Figure 6 As shown in the DF (Digital Functional Analysis), the content of phytic acid and tannin in olive oil meal significantly decreased after the addition of tanninase; further addition of protease further reduced the content of phytic acid, trypsin inhibitor, and tannin. These results indicate a synergistic effect between fermentation and enzymatic hydrolysis, providing technical support for the high-value utilization of olive oil meal, such as its use as feed and food raw material.

[0075] Mixed microbial strains, through metabolic complementarity (such as secreting different phytase systems), exhibit a significantly stronger ability to degrade phytic acid than single-strain strains. The reduction in phytic acid content further mitigates its inhibitory effect on the activity of various microbial enzymes. The mixed strains secrete multiple proteases that can directly hydrolyze and destroy the active protein structure of trypsin inhibitors. Secondly, the fermentation process alters the pH and temperature of the system, which can also promote the denaturation and inactivation of trypsin inhibitors. Furthermore, regarding tannin degradation, *Aspergillus niger* can secrete tanninases to directly degrade tannins. Simultaneously, the microbial fermentation process produces various organic acids, altering the pH of the fermentation system. Under acidic conditions, tannins are more prone to precipitation or complexation. *Saccharomyces cerevisiae* can further metabolize and transform the phenolic substances produced after tannin degradation, or utilize them as a carbon source, thereby removing them from the system.

[0076] After the mixed-culture fermentation products were treated with tanninase, the exogenous tanninase directly and efficiently decomposed tannins, generating organic acids to reduce the acidity of the system. Simultaneously, the removal of tannins relieved their inhibition of residual phytase and other enzymes in the fermentation products, activating their activity. Furthermore, the acidic environment enhanced the solubility of phytates, thus achieving a secondary, highly efficient degradation of phytates while tannins were deeply removed. Subsequent protease treatment directly catalyzed the degradation of trypsin inhibitors; simultaneously, the protease also disrupted the protein complex encapsulating phytates and tannins, exposing them and making them easier to degrade, achieving a synergistic and deep removal of the three anti-nutritional factors.

[0077] 3. Effects of single-strain and mixed-strain fermentation on the fermentation of oilseed cake and the in vitro antioxidant activity before and after combined fermentation and enzymatic hydrolysis.

[0078] (1) Effects of multi-strain compound fermentation and fermentation combined with enzymatic hydrolysis on the in vitro antioxidant activity of walnut meal The results are as follows Figure 7As shown, A: Total antioxidant capacity before and after single-strain fermentation and multi-strain co-fermentation; B: Hydroxyl radical scavenging rate before and after single-strain fermentation and multi-strain co-fermentation; C: DPPH radical scavenging rate before and after single-strain fermentation and multi-strain co-fermentation; D: Flavonoid content before and after single-strain fermentation and multi-strain co-fermentation; E: Total antioxidant capacity before and after multi-strain co-fermentation and enzymatic hydrolysis; F: Hydroxyl radical scavenging rate before and after multi-strain co-fermentation and enzymatic hydrolysis; G: DPPH radical scavenging rate before and after multi-strain co-fermentation and enzymatic hydrolysis; H: Flavonoid content before and after multi-strain co-fermentation and enzymatic hydrolysis. In AC, the mixed bacterial solution used for multi-strain compound fermentation is a 1:1:1 mixed bacterial solution; in EG, "before fermentation" refers to oilseed cake raw material, and "after fermentation" refers to fermentation of the 1:1:1 mixed bacterial solution (corresponding to step (2) 1) in Example 1), "fermentation + tanninase" refers to fermentation of the 1:1:1 mixed bacterial solution followed by tanninase hydrolysis (corresponding to step (2) 2) in Example 1), and "fermentation + tanninase + protease" refers to fermentation of the 1:1:1 mixed bacterial solution followed by tanninase hydrolysis and protease hydrolysis (corresponding to step (2) 3) in Example 1). Figures 8-9 Interpreted in the same way.

[0079] like Figure 7 As shown in the figure, compared with before fermentation, the total antioxidant capacity, hydroxyl radical scavenging rate, DPPH radical scavenging rate and flavonoid content of walnut cake meal were significantly increased after fermentation with a single strain of Aspergillus niger, Saccharomyces cerevisiae and Bacillus subtilis. After multi-strain compound fermentation, the total antioxidant capacity, hydroxyl radical scavenging rate, DPPH radical scavenging rate and flavonoid content of walnut cake meal were further significantly increased.

[0080] like Figure 7 As shown in the EH, the total antioxidant capacity, hydroxyl radical scavenging rate, DPPH radical scavenging rate and flavonoid content of walnut cake significantly increased after the addition of tanninase; the addition of protease further enhanced these properties.

[0081] (2) Effects of multi-strain compound fermentation and fermentation combined with enzymatic hydrolysis on the in vitro antioxidant activity of camellia oil cake meal The results are as follows Figure 8 As shown, A: Total antioxidant capacity before and after single-strain fermentation and multi-strain co-fermentation; B: Hydroxyl radical scavenging rate before and after single-strain fermentation and multi-strain co-fermentation; C: DPPH radical scavenging rate before and after single-strain fermentation and multi-strain co-fermentation; D: Flavonoid content before and after single-strain fermentation and multi-strain co-fermentation; E: Total antioxidant capacity before and after multi-strain co-fermentation and enzymatic hydrolysis; F: Hydroxyl radical scavenging rate before and after multi-strain co-fermentation and enzymatic hydrolysis; G: DPPH radical scavenging rate before and after multi-strain co-fermentation and enzymatic hydrolysis; H: Flavonoid content before and after multi-strain co-fermentation and enzymatic hydrolysis.

[0082] like Figure 8 As shown in the figure, compared with before fermentation, the total antioxidant capacity, hydroxyl radical scavenging rate, and DPPH radical scavenging rate of camellia seed cake were significantly increased after fermentation with a single strain of Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis. Aspergillus niger significantly increased the flavonoid content of camellia seed cake, while Saccharomyces cerevisiae and Bacillus subtilis had no significant effect. After multi-strain compound fermentation, the total antioxidant capacity, hydroxyl radical scavenging rate, DPPH radical scavenging rate, and flavonoid content of camellia seed cake were further significantly increased.

[0083] like Figure 8 As shown in the EH, the addition of tanninase significantly increased the hydroxyl radical scavenging rate, DPPH radical scavenging rate, and flavonoid content of camellia oil cake; further addition of protease further enhanced the total antioxidant capacity.

[0084] (3) Effects of multi-strain compound fermentation and fermentation combined with enzymatic hydrolysis on the in vitro antioxidant activity of olive oil cake meal The results are as follows Figure 9 As shown, A: Total antioxidant capacity before and after single-strain fermentation and multi-strain co-fermentation; B: Hydroxyl radical scavenging rate before and after single-strain fermentation and multi-strain co-fermentation; C: DPPH radical scavenging rate before and after single-strain fermentation and multi-strain co-fermentation; D: Flavonoid content before and after single-strain fermentation and multi-strain co-fermentation; E: Total antioxidant capacity before and after multi-strain co-fermentation and enzymatic hydrolysis; F: Hydroxyl radical scavenging rate before and after multi-strain co-fermentation and enzymatic hydrolysis; G: DPPH radical scavenging rate before and after multi-strain co-fermentation and enzymatic hydrolysis; H: Flavonoid content before and after multi-strain co-fermentation and enzymatic hydrolysis.

[0085] like Figure 9 As shown in Figure AD, compared with before fermentation, the total antioxidant capacity, hydroxyl radical scavenging rate, DPPH radical scavenging rate, and flavonoid content of olive oil cake significantly increased after fermentation with a single strain of Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis. After multi-strain co-fermentation, the total antioxidant capacity, hydroxyl radical scavenging rate, DPPH radical scavenging rate, and flavonoid content of olive oil cake further increased significantly. Olive oil cake contains abundant phenolic compounds, such as oleuropein and hydroxytyrosol, but these are tightly bound to cellulose. Aspergillus niger can produce enzymes that decompose lignin, specifically releasing these phenolic substances, while yeast can hydrolyze oleuropein into the more antioxidant hydroxytyrosol through esterases.

[0086] like Figure 9As shown in the EH diagram, the addition of tanninase significantly increased the total antioxidant capacity, DPPH free radical scavenging rate, and flavonoid content of olive oil meal; further addition of protease further enhanced the hydroxyl free radical scavenging rate. Polyphenols such as oleuropein and hydroxytyrosol in olive oil meal are often bound to cellulose or protein. Tanninase increases the flavonoid dissolution rate by disrupting their cross-linked structures. Protease dissociates the polyphenol-protein complex, further releasing active sites and generating antioxidant peptides.

[0087] During fermentation, *Aspergillus niger* secretes cellulase and pectinase, which break down cellulose and lignin in oilseed cake, releasing polyphenols and flavonoids bound to them. Simultaneously, *Aspergillus niger*'s own metabolism produces organic acids such as oxalic acid and citric acid, which enhance the antioxidant activity of the substrate. *Saccharomyces cerevisiae* produces ethanol and glutathione through sugar fermentation; ethanol facilitates the dissolution of polyphenols, while glutathione directly scavenge free radicals. *Bacillus subtilis* secretes proteases that break down proteins into small peptides with antioxidant properties; tyrosine-containing peptides are particularly effective at scavenging free radicals. Although these two strains do not significantly increase flavonoid content, they effectively enhance overall antioxidant capacity and free radical scavenging rate through these non-flavonoid components. Compared to single-strain fermentation, mixed-strain fermentation simultaneously disrupts cellulose structure, protein complexes, and the glycosidic bonds of polyphenols. Specifically, the cellulase in *Aspergillus niger* breaks down the cell wall, the β-glucosidase in *Saccharomyces cerevisiae* cleaves the glycosidic bonds of flavonoids, and the protease in *Bacillus subtilis* degrades the protein complex. This synergistic effect significantly improves the release efficiency of bound flavonoids compared to using a single strain.

[0088] After multi-strain fermentation, the tannins produced or released during fermentation may bind with residual proteins in the oilseed cake. Simultaneously, the flavonoids already present in the oilseed cake are not completely separated from the protein or tannin complexes, and the polysaccharides and polyphenols produced during fermentation form micelles, all of which hinder the release of active substances. Tanninases can break down the ester and glycosidic bonds in tannins to produce gallic acid and glucose. Gallic acid is a strong antioxidant, with a DPPH free radical scavenging ability 1.5 times that of tannins. Tanninases can also break down complexes formed by tannins with proteins or flavonoids, thereby releasing the encapsulated flavonoids and free proteins. Proteases can further degrade the protein portion of the "protein-flavonoid" complex, releasing even more free flavonoids. The increase in the total amount of flavonoids directly promotes a further enhancement of DPPH and hydroxyl free radical scavenging capabilities. Compared with methods that directly enzymatically hydrolyze or treat raw materials with a single enzyme, the stepwise treatment strategy of "multi-strain combined enzymatic hydrolysis" adopted in this study can first change the substrate structure through microbial metabolism, and then use enzymes to decompose the complex, thereby achieving a gradual increase in antioxidant activity.

[0089] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for improving the antioxidant activity of oilseed cake through multi-strain fermentation combined with enzymatic hydrolysis, characterized in that, include: Add a multi-strain mixed bacterial suspension to the sterilized oilseed cake, mix well, and ferment at a constant temperature of 30±2 ℃ for 5~6 hours. During the fermentation process, stir intermittently. After the fermentation is completed, the mixture is subjected to high-temperature sterilization and freeze-drying in sequence. Then, it is subjected to tannin enzyme hydrolysis and alkaline protease hydrolysis in sequence. The multi-strain mixed bacterial suspension is a mixed bacterial suspension of Aspergillus niger, Saccharomyces cerevisiae and Bacillus subtilis, and the ratio of the number of viable cells of Aspergillus niger, Saccharomyces cerevisiae and Bacillus subtilis in the multi-strain mixed bacterial suspension is 1~2:1~2:1~2.

2. The method according to claim 1, characterized in that, The ratio of viable Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis in the multi-strain mixed bacterial suspension is 1:1:1~2.

3. The method according to claim 2, characterized in that, The ratio of viable Aspergillus niger, Saccharomyces cerevisiae, and Bacillus subtilis in the multi-strain mixed bacterial suspension is 1:1:1 or 1:1:

2.

4. The method according to claim 1, characterized in that, The viable concentration of Aspergillus niger in the multi-strain mixed bacterial suspension was 0.5~2×10⁻⁶. 8 The CFU / mL concentration of Saccharomyces cerevisiae was 0.5~2×10⁻⁶. 8 CFU / mL, viable concentration of Bacillus subtilis is 0.5~2×10⁻⁶. 8 CFU / mL.

5. The method according to claim 1, characterized in that, The amount of the multi-strain mixed bacterial suspension added is 5-8% of the dry weight of the oilseed cake.

6. The method according to claim 1, characterized in that, The amount of tanninase used in the tanninase hydrolysis treatment is 0.4-0.6% of the dry weight of the oilseed cake; the amount of alkaline protease used in the alkaline protease hydrolysis treatment is 0.3-0.5% of the dry weight of the oilseed cake.

7. The method according to claim 1, characterized in that, The conditions for the tannin enzyme hydrolysis treatment are: hydrolysis at 50~60℃ for 55~65 min; the conditions for the alkaline protease hydrolysis treatment are: hydrolysis at 50~60℃ for 5~6 h.

8. The method according to claim 1, characterized in that, The oilseed cake is walnut cake, camellia cake, or olive cake.

9. The method according to claim 1, characterized in that, The antioxidant activity includes at least one of the following: total antioxidant capacity, hydroxyl radical scavenging capacity, DPPH radical scavenging capacity, and flavonoid content.

10. The application of the method according to any one of claims 1 to 9 in the preparation of high-protein feed, characterized in that, The oilseed cake prepared by the method according to any one of claims 1 to 9 is used as a functional additive for high-protein feed.

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