Method for efficiently preparing active polypeptide through rapeseed meal fermentation

Abstract

The invention provides a method for efficiently preparing active polypeptide by fermenting rapeseed dregs, which comprises the following steps: pretreatment: crushing rapeseed dregs, adding 3-5% of reducing sugar, humidifying and sterilizing; performing mixed bacteria segmented fermentation: inoculating a composite strain consisting of bacillus licheniformis SWJS33 with high yield of aminopeptidase, aspergillus oryzae and lactobacillus plantarum, and fermenting at 30 DEG C or 37 DEG C for 72-84 hours; ultrasonic wall breaking: adding water after fermentation, uniformly mixing, carrying out ultrasonic treatment for 20-30 minutes under the power of 120-200W, and breaking thalli to release intracellular enzyme; enzymolysis: carrying out enzymolysis for 4-6 hours under the condition of 50-60 DEG C; post-treatment: enzyme deactivation, centrifugation and drying are carried out after enzymolysis, and a polypeptide product is obtained. By adding the exogenous reducing sugar, nutrition is enhanced, and thallus proliferation is accelerated; bacillus licheniformis SWJS33 with high yield of aminopeptidase is inoculated, so that the generation of small-molecule active peptide is further promoted; a mixed bacteria composite system and a segmented fermentation technology are adopted, and bacteria cells are crushed in an ultrasonic-assisted manner, so that the enzymolysis efficiency is enhanced, and the yield and activity of the polypeptide are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of polypeptide preparation technology, and specifically to a method for efficiently preparing active polypeptides using rapeseed meal fermentation. Background Technology

[0002] After rapeseed oil is extracted, the remaining product is called rapeseed meal. This product contains a variety of key nutrients, mainly including high-quality protein, dietary fiber, essential amino acids, and various trace elements such as minerals. However, because rapeseed meal has long been underutilized, it not only wastes valuable agricultural resources but also limits its potential application in food, feed, and other fields.

[0003] Currently, the high-value utilization of rapeseed meal mainly relies on enzymatic hydrolysis or fermentation technologies to prepare bioactive peptides, but existing processes have significant bottlenecks. Traditional enzymatic hydrolysis is limited by substrate accessibility, resulting in insufficient exposure of enzymatic sites, leading to low hydrolysis degrees and a wide molecular weight distribution of products. Traditional fermentation methods, such as single-strain solid-state fermentation, can partially degrade anti-nutritional components, but suffer from low peptide conversion rates and insufficient bioactive peptide content. Some strains produce high levels of proteases, resulting in peptides with large molecular weights and low activity after protein degradation. Studies have shown that Bacillus licheniformis SWJS33 produces high levels of aminopeptidase, which can further hydrolyze peptides from the N-terminus into smaller bioactive peptides, effectively improving the yield and bioactivity of bioactive peptides. Pretreatment with exogenous nutrients (such as adding carbon and nitrogen sources) can optimize microbial metabolic activity; mixed-strain fermentation can efficiently degrade large protein molecules through synergistic effects; and ultrasound-assisted enzymatic hydrolysis technology, with its cavitation effect, can directionally expose enzyme cleavage sites, significantly improving enzymatic hydrolysis efficiency. While these methods each have their advantages, their individual application is still insufficient to systematically solve the problem of efficient conversion of rapeseed meal into bioactive peptides. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for efficiently preparing active peptides by fermenting rapeseed meal, which addresses the shortcomings of existing technologies. This method improves the yield and functional activity of active peptides and enhances their potential economic value by using exogenous nutrient regulation, introducing strains that produce high levels of aminopeptidase, and employing a synergistic process of mixed fermentation and ultrasound-assisted enzymatic hydrolysis.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] A method for efficiently preparing bioactive peptides using rapeseed meal fermentation includes the following steps:

[0007] S1. Pre-treat rapeseed meal;

[0008] S2. Add exogenous nutrients to the pretreated rapeseed meal; add mixed microbial inoculum for solid-state fermentation;

[0009] S3. After fermentation, distilled water is added to the fermentation product for ultrasonic treatment. After ultrasonic treatment, the temperature is raised for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated, and the product is separated and dried to obtain the polypeptide product.

[0010] Preferably, in step S1, the pretreatment process includes: crushing the rapeseed meal, sieving it, and then sterilizing it at 120-123℃ for 20 minutes.

[0011] Preferably, in step S2, the exogenous nutrient includes one or a mixture of glucose and peptone, and the amount of the exogenous nutrient added is 3-5 wt% of the dry weight of the rapeseed meal; after adding the exogenous nutrient to the pretreated rapeseed meal, the moisture content of the material is adjusted to 50-70%.

[0012] Preferably, the mixed strain includes a combination of Bacillus licheniformis SWJS33, Lactobacillus plantarum, and Aspergillus oryzae, which produce high levels of aminopeptidase.

[0013] Preferably, the method for adding the mixed bacterial strain is as follows:

[0014] The Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured to obtain a primary seed culture of Bacillus licheniformis SWJS33;

[0015] The *Lactobacillus plantarum* was inoculated into MRS medium and cultured to obtain a primary seed culture of *Lactobacillus plantarum*.

[0016] Bacillus licheniformis SWJS33 primary seed liquid, Lactobacillus plantarum primary seed liquid, and Aspergillus oryzae were directly inoculated into sterilized rapeseed meal substrate in a certain proportion for mixed solid-state fermentation.

[0017] Preferably, the preparation conditions for the primary seed culture of Bacillus licheniformis SWJS33 are: shaking culture at 37℃ and 180rpm for 12h; the preparation conditions for the primary seed culture of Lactobacillus plantarum are: static culture at 37℃ for 12h.

[0018] Preferably, when inoculating rapeseed meal with primary seed culture of Bacillus licheniformis SWJS33, primary seed culture of Lactobacillus plantarum, and Aspergillus oryzae, the inoculation amount of Bacillus licheniformis SWJS33 is 5-8 wt%, the inoculation amount of Lactobacillus plantarum is 1-3 wt%, and the inoculation amount of Aspergillus oryzae is 0.1-0.5 wt%.

[0019] Preferably, the water content of the system during solid-state fermentation is 50-70 wt%, the fermentation temperature is 30 or 37℃, and the fermentation time is 72-84 h.

[0020] Preferably, in step S3, the solid-liquid ratio when the fermentation product is mixed with distilled water is 1:10, the ultrasonic power is 120-200W, and the ultrasonic time is 20-30min.

[0021] Preferably, in step S3, the enzymatic hydrolysis temperature is 50-60℃, the time is 4-6h, and the pH of the system during enzymatic hydrolysis is 6.0-8.0.

[0022] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0023] This invention provides a method for efficiently preparing bioactive peptides using rapeseed meal fermentation. This invention efficiently utilizes inexpensive raw material rapeseed meal to prepare high-value-added bioactive peptides, achieving efficient resource utilization and having significant economic and environmental benefits.

[0024] This invention pretreats and strengthens rapeseed meal by adding exogenous nutrients such as reducing sugars, providing microorganisms with a rapidly available source of carbon and nitrogen and accelerating cell proliferation. Endogenous enzymes generated during fermentation degrade macromolecules, increasing peptide yield. Pretreatment with exogenous nutrients promotes cell metabolism, and combined with mixed-culture fermentation and enzymatic hydrolysis, significantly improving peptide yield and bioactivity. Detailed Implementation

[0025] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0026] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0027] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0028] Example 1

[0029] A method for efficiently preparing bioactive peptides using rapeseed meal fermentation includes the following steps:

[0030] (1) Weigh 10g of rapeseed meal, crush and sieve (60 mesh), add 0.5g of glucose (5% of the rapeseed meal mass), adjust the moisture content to 50%, sterilize at 121℃ for 20min, and cool for later use;

[0031] (2) Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured at 37℃ with shaking (180 rpm) for 12 h to obtain Bacillus licheniformis SWJS33 primary seed culture; Lactobacillus plantarum was inoculated into MRS liquid medium and cultured at 37℃ for 12 h until the medium became turbid to obtain Lactobacillus plantarum primary seed culture. Bacillus licheniformis SWJS33 primary seed culture, Lactobacillus plantarum primary seed culture, and Aspergillus oryzae (in the form of mycelial powder) were directly inoculated into rapeseed meal medium for mixed fermentation. The inoculation ratios of Bacillus licheniformis SWJS33, Lactobacillus plantarum, and Aspergillus oryzae were 5 wt%, 3 wt%, and 0.1 wt%, respectively. The mixed fermentation conditions were: cultured at 37℃ in an incubator for 72 h, with stirring once every 12 h during the period;

[0032] (3) After fermentation, distilled water was added to the fermentation product at a solid-liquid ratio of 1:10 (mass ratio) and mixed well. The mixture was then subjected to ultrasonic treatment (ultrasonic power 200W, ultrasonic time 2min) to destroy the cell wall of the bacteria and release intracellular proteases and unreleased peptides. After ultrasonic treatment, the pH of the system was adjusted to 7.0 and enzymatically hydrolyzed in a water bath at 50℃ for 6h. After enzymatic hydrolysis, the temperature was raised to 80℃ and the enzyme was inactivated in a water bath for 10 minutes. The hydrolysate was then centrifuged (8000rpm, 15min), and the precipitate was dried to obtain the active peptide product.

[0033] Example 2

[0034] A method for efficiently preparing bioactive peptides using rapeseed meal fermentation includes the following steps:

[0035] (1) Weigh 10g of rapeseed meal, crush and sieve (60 mesh), add 0.5g of glucose (5% of the mass of rapeseed meal), adjust the moisture content to 60%, sterilize at 121℃ for 20min, and cool for later use;

[0036] (2) Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured at 37°C with shaking (180 rpm) for 12 h to obtain Bacillus licheniformis SWJS33 primary seed culture. Lactobacillus plantarum was inoculated into MRS liquid medium and cultured at 37°C until the medium became turbid to obtain Lactobacillus plantarum primary seed culture. Bacillus licheniformis SWJS33 primary seed culture, Lactobacillus plantarum primary seed culture, and Aspergillus oryzae (in the form of mycelial powder) were directly inoculated into rapeseed meal medium for mixed fermentation. The inoculation ratios of Bacillus licheniformis SWJS33, Lactobacillus plantarum, and Aspergillus oryzae were 5 wt%, 3 wt%, and 0.2 wt%, respectively. The mixed culture conditions were: cultured at 30°C in an incubator for 84 h, with stirring once every 12 h during the period.

[0037] (3) After fermentation, distilled water was added to the fermentation product at a solid-liquid ratio of 1:10 (mass ratio) and mixed well. The mixture was then subjected to ultrasonic treatment (ultrasonic power 180W, ultrasonic time 25min) to destroy the cell wall of the bacteria and release intracellular proteases and unreleased peptides. After ultrasonic treatment, the pH of the system was adjusted to 6.0 and enzymatically hydrolyzed in a water bath at 60℃ for 4h. After enzymatic hydrolysis, the temperature was raised to 80℃ and the enzyme was inactivated in a water bath for 10 minutes. Finally, the enzymatic hydrolysate was centrifuged (8000rpm, 15min), and the precipitate was dried to obtain the active peptide product.

[0038] Example 3

[0039] A method for efficiently preparing bioactive peptides using rapeseed meal fermentation includes the following steps:

[0040] (1) Weigh 10g of rapeseed meal, crush and sieve (60 mesh), add 0.5g of peptone (5wt% of rapeseed meal mass), adjust the moisture content to 70%, sterilize at 121℃ for 20min, and cool for later use;

[0041] (2) Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured at 37°C with shaking (180 rpm) for 12 h to obtain Bacillus licheniformis SWJS33 primary seed culture. Lactobacillus plantarum was inoculated into MRS liquid medium and cultured at 37°C until the medium became turbid to obtain Lactobacillus plantarum primary seed culture. Bacillus licheniformis SWJS33 primary seed culture, Lactobacillus plantarum primary seed culture, and Aspergillus oryzae (in the form of mycelial powder) were directly inoculated into rapeseed meal medium for mixed fermentation. The inoculation ratios of Bacillus licheniformis SWJS33, Lactobacillus plantarum liquid, and Aspergillus oryzae were 8 wt%, 5 wt%, and 0.3 wt%, respectively. The mixed fermentation conditions were: cultured at 37°C in an incubator for 72 h, with stirring once every 12 h.

[0042] (3) After fermentation, distilled water was added to the fermentation product at a solid-liquid ratio of 1:10 (mass ratio) and mixed well. The mixture was then subjected to ultrasonic treatment (ultrasonic power 160W, ultrasonic time 25min) to destroy the cell wall of the bacteria and release intracellular proteases and unreleased peptides. After ultrasonic treatment, the pH of the fermentation broth was adjusted to 8.0 and enzymatically hydrolyzed in a water bath at 55℃ for 5h. After enzymatic hydrolysis, the temperature was raised to 80℃ and the enzyme was inactivated in a water bath for 10 minutes. The hydrolysate was then centrifuged (8000rpm, 15min), and the precipitate was dried to obtain the active peptide product.

[0043] Example 4

[0044] A method for efficiently preparing bioactive peptides using rapeseed meal fermentation includes the following steps:

[0045] (1) Weigh 10g of rapeseed meal, crush and sieve (60 mesh), add 0.4g of glucose (4% of the rapeseed meal mass), adjust the moisture content to 50%, sterilize at 121℃ for 20min, and cool for later use;

[0046] (2) Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured at 37°C with shaking (180 rpm) for 12 h to obtain Bacillus licheniformis SWJS33 primary seed culture. Lactobacillus plantarum was inoculated into MRS liquid medium and cultured at 37°C until the medium became turbid to obtain Lactobacillus plantarum primary seed culture. Bacillus licheniformis SWJS33 primary seed culture, Lactobacillus plantarum primary seed culture, and Aspergillus oryzae (in the form of mycelial powder) were inoculated into rapeseed meal medium for mixed fermentation. The inoculation ratios of Bacillus licheniformis SWJS33, Lactobacillus plantarum, and Aspergillus oryzae were 5 wt%, 3 wt%, and 0.5 wt%, respectively. The conditions for mixed culture were: cultured at 37°C in an incubator for 72 h, with stirring once every 12 h.

[0047] (3) After fermentation, add distilled water to the fermentation product at a solid-liquid ratio of 1:10 (mass ratio) and mix well. Then, perform ultrasonic treatment in an ultrasonic device (ultrasonic power 120W, ultrasonic time 30min). After ultrasonic treatment, adjust the pH of the fermentation broth to 7.0 and enzymatically hydrolyze it in a water bath at 55℃ for 5h. After enzymatic hydrolysis, raise the temperature to 80℃ and inactivate the enzyme in a water bath for 10 minutes. Then, centrifuge the enzymatic hydrolysate (8000rpm, 15min). After centrifugation and precipitation, dry to obtain the active polypeptide product.

[0048] Example 5

[0049] A method for efficiently preparing bioactive peptides using rapeseed meal fermentation includes the following steps:

[0050] (1) Weigh 10g of rapeseed meal, crush and sieve (60 mesh), add 0.3g of peptone (3% of raw material mass), adjust the moisture content to 60%, adjust the moisture content to 50%, sterilize at 121℃ for 20min, and cool for later use;

[0051] (2) Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured at 37°C with shaking (180 rpm) for 12 h to obtain Bacillus licheniformis SWJS33 primary seed culture. Lactobacillus plantarum was inoculated into MRS liquid medium and cultured at 37°C until the medium became turbid to obtain Lactobacillus plantarum primary seed culture. Bacillus licheniformis SWJS33 primary seed culture, Lactobacillus plantarum primary seed culture, and Aspergillus oryzae (in the form of mycelial powder) were inoculated into rapeseed meal medium for mixed fermentation. The inoculation ratios of Bacillus licheniformis SWJS33, Lactobacillus plantarum, and Aspergillus oryzae were 5 wt%, 5 wt%, and 0.3 wt%, respectively. The conditions for mixed fermentation were: cultured at 30°C in an incubator for 72 h, with stirring once every 12 h.

[0052] (3) After fermentation, add distilled water to the fermentation product at a solid-liquid ratio of 1:10 (mass ratio) and mix well. Then, perform ultrasonic treatment in an ultrasonic device (ultrasonic power 120W, ultrasonic time 30min). After ultrasonic treatment, adjust the pH of the fermentation broth to 7.0 and enzymatically hydrolyze it in a water bath at 50℃ for 6h. After enzymatic hydrolysis, raise the temperature to 80℃ and incubate in a water bath for 10 minutes to inactivate the enzyme. Centrifuge the hydrolysate (8000rpm, 15min), and dry the precipitate after centrifugation to obtain the active polypeptide product.

[0053] Comparative Example 1

[0054] A method for efficiently preparing bioactive peptides using rapeseed meal fermentation includes the following steps:

[0055] (1) Weigh 10g of rapeseed meal, crush and sieve (80 mesh), add 0.4g of peptone (4% of the rapeseed meal mass), adjust the moisture content to 50%, sterilize at 121℃ for 20min, and cool for later use;

[0056] (2) Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured at 37°C with shaking (180 rpm) for 12 h to obtain Bacillus licheniformis SWJS33 primary seed culture; Lactobacillus plantarum was inoculated into MRS liquid medium and cultured at 37°C until the medium became turbid to obtain Lactobacillus plantarum primary seed culture; Bacillus licheniformis SWJS33 primary seed culture, Lactobacillus plantarum primary seed culture, and Aspergillus oryzae (in the form of mycelial powder) were inoculated into rapeseed meal medium for mixed fermentation. The inoculation ratios of Bacillus licheniformis SWJS33, Lactobacillus plantarum, and Aspergillus oryzae were 8 wt%, 5 wt%, and 0.3 wt%, respectively. The mixture was cultured at 30°C for 72 h, with stirring once every 12 h. After fermentation, the product was dried to obtain the active polypeptide product.

[0057] Comparative Example 2

[0058] A method for efficiently preparing bioactive peptides using rapeseed meal fermentation includes the following steps:

[0059] (1) Weigh 10g of rapeseed meal, crush and sieve (60 mesh), add 0.5g of peptone (5% of the rapeseed meal mass) to adjust the moisture content to 60%, sterilize at 121℃ for 20min, and cool for later use;

[0060] (2) Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured at 37℃ with shaking (180 rpm) for 12 h to obtain Bacillus licheniformis SWJS33 primary seed culture; Lactobacillus plantarum was inoculated into MRS liquid medium and cultured at 37℃ for 12 h until the medium became turbid to obtain Lactobacillus plantarum primary seed culture; Bacillus licheniformis SWJS33 primary seed culture, Lactobacillus plantarum primary seed culture, and Aspergillus oryzae (in the form of mycelial powder) were inoculated into rapeseed meal medium for mixed fermentation. The inoculation ratios of Bacillus licheniformis SWJS33, Lactobacillus plantarum, and Aspergillus oryzae were 5 wt%, 5 wt%, and 0.1 wt%, respectively. The conditions for mixed fermentation were: cultured at 37℃ in an incubator for 84 h, with stirring once every 12 h during the period;

[0061] (3) After fermentation, add distilled water to the fermentation product at a solid-liquid ratio of 1:10 (mass ratio) and mix well. Place the mixture in an ultrasonic device for ultrasonic treatment (power 200W, 20min) to destroy the fiber structure of rapeseed meal. After ultrasonic treatment, centrifuge (8000rpm, 15min), and dry the precipitate to obtain the active polypeptide product.

[0062] Comparative Example 3

[0063] A method for efficiently preparing bioactive peptides using rapeseed meal fermentation includes the following steps:

[0064] (1) Weigh 10g of rapeseed meal, crush and sieve (60 mesh), add 0.5g of glucose (5% of the mass of rapeseed meal), adjust the moisture content to 60%, sterilize at 121℃ for 20min, and cool for later use;

[0065] (2) Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured at 37℃ with shaking (180 rpm) for 12 h to obtain Bacillus licheniformis SWJS33 primary seed culture. Lactobacillus plantarum was inoculated into MRS liquid medium and cultured at 37℃ for 12 h until the medium became turbid to obtain Lactobacillus plantarum primary seed culture. Bacillus licheniformis SWJS33 primary seed culture, Lactobacillus plantarum primary seed culture, and Aspergillus oryzae (in the form of mycelial powder) were inoculated into rapeseed meal medium for mixed fermentation. The inoculation ratios of Bacillus licheniformis SWJS33, Lactobacillus plantarum, and Aspergillus oryzae were 5 wt%, 5 wt%, and 0.1 wt%, respectively. The mixture was cultured in an incubator at 37℃ for 72 h, with stirring once every 12 h.

[0066] (3) After fermentation, add distilled water to the fermentation product at a solid-liquid ratio of 1:10 (mass ratio) and mix well. Adjust the pH of the system to 7.0 and enzymatically hydrolyze in a water bath at 50°C for 6 hours. After the enzymatic hydrolysis is completed, inactivate the enzyme by water bath at 80°C for 10 minutes. Centrifuge the hydrolysate (8000 rpm, 15 min), and dry the precipitate to obtain the active polypeptide product.

[0067] The performance of the polypeptide products prepared in the above examples and comparative examples was tested, with untreated rapeseed meal serving as a blank control.

[0068] 1. Determination of polypeptide content

[0069] The biuret method was used for testing.

[0070] 2. Determination of soluble protein content:

[0071] The Coomassie Brilliant Blue method was used for testing.

[0072] 3. Methods for determining antioxidant activity

[0073] The free radical scavenging rate of DPPH and ABTS were respectively measured. + Free radical scavenging rate, hydroxyl radical scavenging rate, Fe 3+ The reducing ability is characterized.

[0074] The test results are shown in Table 1 and Table 2, respectively.

[0075] Table 1

[0076]

[0077] Table 2

[0078]

[0079]

[0080] As can be seen from the test results in Tables 1 and 2, compared with the comparative example and the blank group, the present invention, through the synergistic process of "exogenous nutrient fortification-mixed fermentation-ultrasonic enzymatic hydrolysis" and optimized process parameters, significantly solved the problems of low hydrolysis efficiency and insufficient product activity in the conversion of rapeseed meal into active peptides. The peptide yield of Example 2 reached 141.41 mg / g, and the soluble protein content was 1177.61 μg / g, representing increases of 197.7% and 168.8% respectively compared to the blank group. In Comparative Example 3, the peptide yield was only 112.37 mg / g, and the soluble protein content was 903.47 μg / g. Comparative Example 2 had a soluble protein content of 816.94 μg / g and a peptide yield of less than 100 mg / g, demonstrating the importance of the synergistic effect of ultrasound and enzymatic hydrolysis.

[0081] As shown in Table 2, mixed-culture fermentation significantly enhances the antioxidant activity of bioactive peptides. For example, the DPPH scavenging rate in Example 1 was 99.54% higher than that in the control group. Ultrasonic-assisted enzymatic hydrolysis is the core method for enhancing peptide activity. In Comparative Example 3, which did not undergo ultrasonication, the hydroxyl radical scavenging rate of the obtained peptide was only 38.1%, far lower than that in Example 1 (58.3%). The above test results demonstrate that the peptide yield and functional activity of the present invention meet the standards for industrial application, and the cost advantage is significant, making it suitable for large-scale production in the fields of functional foods and feed additives.

[0082] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of these embodiments are merely to aid in understanding the method and core ideas of the present invention, including the best mode, and to enable any person skilled in the art to practice the present invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims. The scope of protection of this patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements similar to those expressed in the claims, or if they include equivalent structural elements that are not substantially different from those expressed in the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation, characterized in that, Includes the following steps: S1. Pre-treat rapeseed meal; S2. Add exogenous nutrients to the pretreated rapeseed meal; add mixed microbial inoculum for solid-state fermentation; S3. After fermentation, distilled water is added to the fermentation product for ultrasonic treatment. After ultrasonic treatment, the temperature is raised for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the enzyme is inactivated, and the product is separated and dried to obtain the polypeptide product.

2. The method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation according to claim 1, characterized in that: In step S1, the pretreatment process includes: crushing the rapeseed meal, sieving it, and then sterilizing it at 120-123℃ for 20 minutes.

3. The method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation according to claim 1, characterized in that: In step S2, the exogenous nutrient includes one or a mixture of glucose and peptone, and the amount of exogenous nutrient added is 3-5 wt% of the dry weight of rapeseed meal; after adding the exogenous nutrient to the pretreated rapeseed meal, the moisture content of the material is adjusted to 50-70%.

4. The method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation according to claim 1, characterized in that: The mixed strain includes a combination of Bacillus licheniformis SWJS33, Lactobacillus plantarum, and Aspergillus oryzae, which produce high levels of aminopeptidase.

5. The method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation according to claim 4, characterized in that: The method for adding the mixed bacterial strain is as follows: The Bacillus licheniformis SWJS33 was inoculated into LB medium and cultured to obtain a primary seed culture of Bacillus licheniformis SWJS33; The *Lactobacillus plantarum* was inoculated into MRS medium and cultured to obtain a primary seed culture of *Lactobacillus plantarum*. Bacillus licheniformis SWJS33 primary seed liquid, Lactobacillus plantarum primary seed liquid, and Aspergillus oryzae were directly inoculated into sterilized rapeseed meal substrate in a certain proportion for mixed solid-state fermentation.

6. The method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation according to claim 4, characterized in that: The preparation conditions for the primary seed culture of Bacillus licheniformis SWJS33 were: shaking culture at 37℃ and 180 rpm for 12 h; the preparation conditions for the primary seed culture of Lactobacillus plantarum were: static culture at 37℃ for 12 h.

7. The method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation according to claim 5, characterized in that: When inoculating rapeseed meal with primary seed culture of Bacillus licheniformis SWJS33, primary seed culture of Lactobacillus plantarum, and Aspergillus oryzae, the inoculation amount of Bacillus licheniformis SWJS33 is 5-8 wt%, the inoculation amount of Lactobacillus plantarum is 1-3 wt%, and the inoculation amount of Aspergillus oryzae is 0.1-0.5 wt%.

8. The method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation according to claim 1, characterized in that: During solid-state fermentation, the water content of the system is 50-70 wt%, the fermentation temperature is 30 or 37℃, and the fermentation time is 72-84 h.

9. The method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation according to claim 1, characterized in that: In step S3, the solid-liquid ratio of the fermentation product to distilled water is 1:10, the ultrasonic power is 120-200W, and the ultrasonic time is 20-30min.

10. The method for efficiently preparing bioactive polypeptides using rapeseed meal fermentation according to claim 1, characterized in that: In step S3, the enzymatic hydrolysis temperature is 50-60℃, the time is 4-6h, and the pH of the system during enzymatic hydrolysis is 6.0-8.0.

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