Preparation method of corn fermented protein raw material for aquatic feed

Abstract

This invention relates to a method for preparing protein raw materials for aquatic feed through corn fermentation, belonging to the field of aquatic feed technology. Using corn as the main raw material, combined with soybean meal by-products, a compound nitrogen source, and a cell-wall breaking agent, the method involves steps such as pulverization pretreatment, microwave-assisted enzymatic hydrolysis, co-fermentation with compound microbial strains, solid-liquid separation, low-temperature drying, and post-treatment to obtain protein raw materials for aquatic feed. The cell-wall breaking agent is prepared from chitosan oligosaccharides, deionized water, citric acid, β-glucanase, and apple polyphenols. This invention, through optimized processes such as microwave-assisted enzymatic hydrolysis and co-fermentation with compound microbial strains, combined with the comprehensive utilization of corn and its by-products, can effectively improve crude protein content and protein digestibility, significantly enhancing the quality of protein raw materials for aquatic feed.

Description

Technical Field

[0001] This invention relates to the field of aquatic feed technology, and in particular to a method for preparing protein raw materials for aquatic feed through corn fermentation. Background Technology

[0002] Corn is a major food crop in my country, with abundant resources and low prices. It contains a certain amount of protein, carbohydrates and other nutrients, making it a potential source of feed protein.

[0003] Chinese Patent CN110692858A: An aquatic feed that effectively improves the intestinal environment, relating to the field of aquaculture technology. The feed formula consists of the following components by weight: 75-85 parts white fish meal, 65-80 parts soybean meal, 14-24 parts pumpkin seeds, 35-45 parts shrimp bran powder, 25-28 parts turtle shell powder, 25-30 parts wheat middlings, 10-15 parts oat flour, 12.5-15.5 parts brewer's yeast powder, 3.5-13.5 parts hawthorn powder, 7-11 parts corn gluten meal, and 0.55-2.45 parts soybean lecithin.

[0004] Chinese Patent CN106721641A discloses a nutritionally balanced aquatic feed and its preparation method, characterized in that: the feed is formulated from the following raw materials in the following mass percentages: fish meal 0-1%, soybean meal 25-30%, rapeseed meal 5-8%, cottonseed meal 20-30%, rice bran 8-13%, wheat middlings 10-15%, corn gluten 5-10%, soybean protein concentrate 9-16%, calcium dihydrogen phosphate 1-2%, choline chloride 0.2-0.4%, allicin 0.2-0.4%, dextran 0.1-0.2%, zeolite powder 0.5-1%, compound vitamins 0.2-0.4%, and compound minerals 0.4-0.5%.

[0005] Currently, the main protein raw materials for aquatic feeds rely on fishmeal and soybean meal. However, fishmeal is scarce and expensive, while soybean meal suffers from unstable supply and high levels of anti-nutritional factors. Developing new, low-cost, and efficient protein raw materials for aquatic feeds has become an urgent need for the industry. Summary of the Invention

[0006] To address the above problems, this invention provides a method for preparing protein raw materials for aquatic feed through corn fermentation, the operation steps of which are as follows:

[0007] S1: Crush 60-100 parts of corn into 80-100 mesh, add 5-10 parts of soybean meal by-product and 1-5 parts of compound nitrogen source, mix evenly, add 200-300 parts of deionized water and 0.1-0.5 parts of cell wall breaking agent, stir evenly to obtain mixed substrate;

[0008] S2: Add 0.03-0.3 parts of the compound enzyme preparation to the mixed substrate, adjust the pH to 4.5-5.5, and use intermittent microwave treatment to enzymatically hydrolyze for 12-18 hours at a temperature of 45-55℃ to obtain the enzymatic hydrolysate;

[0009] S3: Inoculate the compound bacterial strain into the enzymatic hydrolysate, adjust the initial pH to 5-6 with citric acid, control the temperature at 28-32℃, stir at 150-250 rpm, and carry out aerobic fermentation for 24-36 hours to obtain the fermentation broth;

[0010] S4: Centrifuge the fermentation broth at 3000-4000 r / min for 10-20 min, collect the solid product, and remove the supernatant;

[0011] S5: Dry the solid product at 45-55℃ and a vacuum of 0.06-0.08MPa for 10-20 hours to obtain crude protein raw material;

[0012] S6: Crush the crude protein raw material, pass it through an 80-mesh sieve, sterilize it at 100-120℃ and 0.1MPa for 20-30 minutes, and package it to obtain protein raw material for aquatic feed.

[0013] Optionally, the soybean meal by-product in S1 is soybean meal residue produced during soybean meal processing, with a crude protein content of 35%.

[0014] Optionally, the composite nitrogen source in S1 is ammonium sulfate and urea, mixed in a mass ratio of 2:1.

[0015] Optionally, the complex enzyme preparation in S2 is a mixture of saccharifying enzyme, cellulase, and hemicellulase in a mass ratio of 3:2:1.

[0016] Optionally, the microwave power of the intermittent microwave treatment in S2 is 200-400W, each microwave treatment lasts 30 seconds, with a 10-second interval, and this process is repeated until the enzymatic hydrolysis is completed.

[0017] Optionally, the inoculation volume ratio of the compound bacterial strain in S3 is 5-8% of the enzymatic hydrolysate.

[0018] Optionally, the composite strain in S3 is obtained by inoculating Candida utilis, Saccharomyces cerevisiae, and Trichoderma lignin into malt extract culture medium, culturing them at 28-32℃ and 150-250rpm for 16-24h to obtain an activated strain, and then mixing them in a volume ratio of 2:2:1.

[0019] Optionally, during the aerobic fermentation process in S3, sterile air is introduced every 6 hours at a rate of 0.5-1.0 L / (L·h).

[0020] Optionally, the method for preparing the cell wall disruptor in S1 is as follows:

[0021] A1: Dissolve 2-8 parts of chitosan oligosaccharide in 80-100 parts of deionized water, adjust the pH to 5-6, place in a constant temperature water bath at 35-45℃, add 1-5 parts of citric acid, stir to dissolve, then add 6-12 parts of β-glucanase and 0.2-0.5 parts of apple polyphenols in sequence, and stir at a constant temperature for 2-5 hours.

[0022] A2: After the reaction is complete, the mixture is freeze-dried under vacuum at a temperature of -40~-30℃ and a vacuum degree of 0.07-0.09MPa until constant weight is achieved. The mixture is then pulverized through an 80-mesh sieve to obtain the cell wall disruptor.

[0023] Optionally, the β-glucanase activity is ≥1000 U / g, and the chitosan oligosaccharide molecular weight is 1000-2000 Da.

[0024] Reaction mechanism:

[0025] This method uses corn as the core substrate, combined with soybean meal by-products and a complex nitrogen source to construct a fermentation system. Through the synergistic effect of a cell wall disruptor and microwave stimulation, the cell walls and fibrous network structure of corn are efficiently disrupted, releasing bound nutrients and proteins. A complex enzyme preparation directionally degrades polysaccharides and fiber components in the substrate, improving substrate bioavailability. A complex microbial strain, through synergistic fermentation, converts carbon and nitrogen sources into single-cell proteins, simultaneously degrading anti-nutritional factors and optimizing amino acid composition and protein structure. β-glucanase in the cell wall disruptor enhances substrate cell wall degradation, apple polyphenols maintain enzyme activity and strain activity stability, and chitosan oligosaccharides promote efficient enzymatic reactions and fermentation, ultimately yielding a high-nutritional-quality aquatic feed protein raw material.

[0026] Beneficial effects:

[0027] This invention discloses a method for preparing protein raw materials for aquatic feed through corn fermentation. Compared with the prior art, this invention has the following significant advantages:

[0028] 1. Significantly improves the crude protein enrichment level of the product, enabling non-protein nitrogen and carbon sources in corn and by-products to be fully converted into microbial protein, effectively increasing the crude protein content.

[0029] 2. Significantly improves the protein digestibility of the product. Through cell wall disruption and degradation and removal of anti-nutritional factors, the protein structure becomes more porous and the number of enzymatic cleavage sites increases, making it easier for aquatic animals to digest and absorb.

[0030] 3. Crude fat

[0031] The crude fat content of shrimp muscle in the control group was 0.35%–0.39%, significantly higher than that in all control groups. Moderate fat accumulation not only improves the taste and flavor of shrimp meat but also enhances the shrimp's stress resistance and improves survival stability during transportation and aquaculture.

[0032] 4. Crude protein

[0033] In the example, the crude protein content of the shrimp meat was 24.6% and 25.2%, a significant increase compared to the 16.2% in the blank control group. This feed's nutritional formula is well-suited to the needs of shrimp, effectively promoting muscle protein deposition, increasing the protein content of shrimp meat, and enhancing the nutritional value of aquatic products.

[0034] The above-mentioned data on growth, survival, and meat quality nutrition fully demonstrate the multiple beneficial effects of the feed of this invention on aquatic animals.

[0035] The digestibility evaluation of the protein raw materials of this invention adopts an in vitro simulated digestion system specifically for aquatic shrimp, which is different from the general in vitro digestion method for livestock and poultry. It completely replicates the pH, temperature, digestive enzyme system and digestion time of the stomach and intestine of Litopenaeus vannamei. The test results can truly reflect the actual protein absorption and utilization level of this fermented protein raw material in aquatic animals. The test data is more targeted and has higher reference value. Detailed Implementation

[0036] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the following detailed description is provided in conjunction with embodiments and comparative examples:

[0037] 1. Crude protein content detection: The crude protein content was detected according to GB / T6432-2018 "Determination of crude protein in feed" using the Kjeldahl method. The crude protein content was calculated using the formula: Crude protein content (%) = (V×T×F×100) / (m×1000)×6.25, where V is the volume of hydrochloric acid standard solution consumed in the titration (mL), T is the titer of the hydrochloric acid standard solution (mg / mL), F is the dilution factor, m is the sample mass (g), and 6.25 is the protein conversion factor.

[0038] 2. Protein digestibility detection: The in vitro digestibility determination method was used.

[0039] Protein digestibility assay: A two-step in vitro digestion simulation method specifically for Litopenaeus vannamei was used to completely simulate the digestive environment of the shrimp's digestive tract. The specific operating conditions are as follows:

[0040] (1) Stomach digestion stage: Prepare aquatic animal simulated gastric juice containing 2000 U / L of pepsin, adjust the pH of the system to 2.8 with hydrochloric acid, and keep the water bath at a constant temperature of 30℃ (matching the physiological temperature of the abdominal cavity of shrimp); weigh 1g of aquatic feed protein raw material, add 20mL of simulated gastric juice, and keep the digestion at a constant temperature with shaking for 2h at a shaking speed of 120 r / min.

[0041] (2) Intestinal digestion stage: After the gastric juice digestion is completed, the pH of the system is adjusted to 7.5 (the suitable pH of the intestine of Litopenaeus vannamei) using 0.1 mol / L sodium hydroxide solution, and trypsin and lipase complex digestion solution is added. The trypsin activity is 1500 U / L, and the digestion is continued at 30℃ with shaking for 4 hours.

[0042] (3) Termination and separation: After digestion, the digestive enzymes were inactivated by boiling water bath for 5 min, centrifuged at 3500 r / min for 15 min, and the supernatant digestion liquid and residue were separated; the supernatant was collected, and the crude protein content of the supernatant was determined according to the Kjeldahl nitrogen determination method in GB / T6432-2018, and the total crude protein content of the original sample was determined at the same time.

[0043] The protein digestibility is calculated using the following formula: Protein digestibility (%) = (Crude protein content in supernatant / Crude protein content in sample) × 100.

[0044] 3. Survival Rate: 200 Litopenaeus vannamei shrimp were cultured in seawater cages, with each treatment repeated three times for 30 days. During the experiment, shrimp were fed twice daily: 60 wt% of the total daily feed was given at 7:00 AM, and 40 wt% at 6:00 PM. Feeding was stopped at night. The total daily feed amount was 3 wt% of the shrimp's body weight. After the experiment, the survival rate of each group was recorded. The corn fermentation process described above was used to produce aquatic feed protein raw materials, which were then fed commercially available Litopenaeus vannamei feed, and the survival rate was tested.

[0045] All parts by weight mentioned below are measured in kg.

[0046] Example 1

[0047] A method for preparing protein raw materials for aquatic feed by corn fermentation, comprising the following steps:

[0048] S1: Crush 60 parts by weight of corn to 80 mesh, add 5 parts by weight of soybean meal by-product and 1 part by weight of compound nitrogen source, mix evenly, add 200 parts by weight of deionized water and 0.1 parts by weight of cell wall disruptor, stir evenly to obtain mixed substrate;

[0049] S2: Add 0.03 parts by mass of the compound enzyme preparation to the mixed substrate, adjust the pH to 4.5, and use intermittent microwave treatment to enzymatically hydrolyze for 12 hours at 45℃ to obtain the enzymatic hydrolysate;

[0050] S3: Inoculate the compound bacterial strain into the enzymatic hydrolysate, adjust the initial pH to 5 with citric acid, control the temperature at 28℃, stir at 150 rpm, and carry out aerobic fermentation for 24 hours to obtain the fermentation broth;

[0051] S4: Centrifuge the fermentation broth at 3000 r / min for 10 min, collect the solid product, and remove the supernatant;

[0052] S5: The solid product was dried at 45°C and a vacuum of 0.06 MPa for 10 h to obtain crude protein raw material;

[0053] S6: Crush the crude protein raw material, pass it through an 80-mesh sieve, sterilize it at 100℃ and 0.1MPa for 20 minutes, and package it to obtain protein raw material for aquatic feed.

[0054] The soybean meal by-product in S1 is soybean meal residue produced during soybean meal processing, with a crude protein content of 35%.

[0055] The composite nitrogen source in S1 is ammonium sulfate and urea, mixed in a mass ratio of 2:1.

[0056] The S2 complex enzyme preparation is composed of saccharifying enzyme, cellulase, and hemicellulase, mixed in a mass ratio of 3:2:1.

[0057] The microwave power of the intermittent microwave treatment in S2 is 200W, each microwave cycle is 30s, with a 10s interval, and this process is repeated until the enzymatic hydrolysis is completed.

[0058] The S3 contains a 5% (by volume) composite bacterial strain inoculated into the enzymatic hydrolysate.

[0059] The S3 composite strain is obtained by inoculating Candida utilis, Saccharomyces cerevisiae, and Trichoderma lignin into malt extract culture medium, culturing them at 28°C and 150 rpm for 16 hours to obtain an activated strain, and then mixing them in a volume ratio of 2:2:1.

[0060] During the aerobic fermentation process in S3, sterile air is introduced every 6 hours at a rate of 0.5 L / (L·h).

[0061] The method for preparing the cell wall disruptor in S1 is as follows:

[0062] A1: Dissolve 2 parts by weight of chitosan oligosaccharide in 80 parts by weight of deionized water, adjust the pH to 5, place in a 35℃ constant temperature water bath, add 1 part by weight of citric acid, stir to dissolve, then add 6 parts by weight of β-glucanase and 0.2 parts by weight of apple polyphenols in sequence, and stir at a constant temperature for 2 hours.

[0063] A2: After the reaction is complete, the mixture is freeze-dried under vacuum at -40℃ and 0.07MPa until constant weight, then pulverized through an 80-mesh sieve to obtain the cell wall disruptor.

[0064] The β-glucanase activity is ≥1000 U / g, and the chitosan oligosaccharide has a molecular weight of 1000 Da.

[0065] Example 2

[0066] A method for preparing protein raw materials for aquatic feed by corn fermentation, comprising the following steps:

[0067] S1: Crush 70 parts by weight of corn to 90 mesh, add 6 parts by weight of soybean meal by-product and 2 parts by weight of compound nitrogen source, mix evenly, add 240 parts by weight of deionized water and 0.2 parts by weight of cell wall disruptor, stir evenly to obtain mixed substrate;

[0068] S2: Add 0.1 parts by mass of the compound enzyme preparation to the mixed substrate, adjust the pH to 5, and use intermittent microwave treatment to enzymatically hydrolyze for 14 hours at 48℃ to obtain the enzymatic hydrolysate;

[0069] S3: Inoculate the compound bacterial strain into the enzymatic hydrolysate, adjust the initial pH to 5.5 with citric acid, control the temperature at 29℃, stir at 200 rpm, and carry out aerobic fermentation for 28 hours to obtain the fermentation broth;

[0070] S4: Centrifuge the fermentation broth at 3400 r / min for 15 min, collect the solid product, and remove the supernatant;

[0071] S5: The solid product was dried at 48°C and a vacuum of 0.07 MPa for 14 h to obtain crude protein raw material;

[0072] S6: Crush the crude protein raw material, pass it through an 80-mesh sieve, sterilize it at 105℃ and 0.1MPa for 25 minutes, and package it to obtain protein raw material for aquatic feed.

[0073] The soybean meal by-product in S1 is soybean meal residue produced during soybean meal processing, with a crude protein content of 35%.

[0074] The composite nitrogen source in S1 is ammonium sulfate and urea, mixed in a mass ratio of 2:1.

[0075] The S2 complex enzyme preparation is composed of saccharifying enzyme, cellulase, and hemicellulase, mixed in a mass ratio of 3:2:1.

[0076] The microwave power of the intermittent microwave treatment in S2 is 250W, each microwave cycle is 30s, with a 10s interval, and this process is repeated until the enzymatic hydrolysis is completed.

[0077] The S3 contains a 6% (by volume) composite bacterial strain inoculated into the enzymatic hydrolysate.

[0078] The S3 composite strain is obtained by inoculating Candida utilis, Saccharomyces cerevisiae, and Trichoderma lignin into malt extract culture medium, culturing them at 29°C and 200 rpm for 18 hours to obtain an activated strain, and then mixing them in a volume ratio of 2:2:1.

[0079] During the aerobic fermentation process in S3, sterile air is introduced every 6 hours at a rate of 0.6 L / (L·h).

[0080] The method for preparing the cell wall disruptor in S1 is as follows:

[0081] A1: Dissolve 4 parts by weight of chitosan oligosaccharide in 85 parts by weight of deionized water, adjust the pH to 5.5, place in a 38℃ constant temperature water bath, add 2 parts by weight of citric acid, stir to dissolve, then add 8 parts by weight of β-glucanase and 0.3 parts by weight of apple polyphenols in sequence, and stir at a constant temperature for 3 hours.

[0082] A2: After the reaction is complete, the mixture is freeze-dried under vacuum at -35℃ and 0.08MPa until constant weight, then pulverized through an 80-mesh sieve to obtain the cell wall disruptor.

[0083] The β-glucanase activity is ≥1000 U / g, and the molecular weight of the chitosan oligosaccharide is 1500 Da.

[0084] Example 3

[0085] A method for preparing protein raw materials for aquatic feed by corn fermentation, comprising the following steps:

[0086] S1: Crush 90 parts by weight of corn to 90 mesh, add 8 parts by weight of soybean meal by-product and 4 parts by weight of compound nitrogen source, mix evenly, add 280 parts by weight of deionized water and 0.4 parts by weight of cell wall disruptor, stir evenly to obtain mixed substrate;

[0087] S2: Add 0.2 parts by mass of the compound enzyme preparation to the mixed substrate, adjust the pH to 5, and use intermittent microwave treatment to enzymatically hydrolyze at 53℃ for 16 hours to obtain the enzymatic hydrolysate;

[0088] S3: Inoculate the compound bacterial strain into the enzymatic hydrolysate, adjust the initial pH to 5.5 with citric acid, control the temperature at 31℃, stir at 200 rpm, and carry out aerobic fermentation for 32 hours to obtain the fermentation broth;

[0089] S4: Centrifuge the fermentation broth at 3800 r / min for 15 min, collect the solid product, and remove the supernatant;

[0090] S5: The solid product was dried at 53°C and a vacuum of 0.07 MPa for 18 hours to obtain crude protein raw material;

[0091] S6: Crush the crude protein raw material, pass it through an 80-mesh sieve, sterilize it at 115℃ and 0.1MPa for 25 minutes, and package it to obtain protein raw material for aquatic feed.

[0092] The soybean meal by-product in S1 is soybean meal residue produced during soybean meal processing, with a crude protein content of 35%.

[0093] The composite nitrogen source in S1 is ammonium sulfate and urea, mixed in a mass ratio of 2:1.

[0094] The S2 complex enzyme preparation is composed of saccharifying enzyme, cellulase, and hemicellulase, mixed in a mass ratio of 3:2:1.

[0095] The microwave power of the intermittent microwave treatment in S2 is 350W, each microwave session lasts 30 seconds, with a 10-second interval, and this process is repeated until the enzymatic hydrolysis is completed.

[0096] The S3 contains a 7% (by volume) composite bacterial strain inoculated into the enzymatic hydrolysate.

[0097] The S3 composite strain is obtained by inoculating Candida utilis, Saccharomyces cerevisiae, and Trichoderma lignin into malt extract culture medium, culturing them at 31°C and 200 rpm for 22 hours to obtain an activated strain, and then mixing them in a volume ratio of 2:2:1.

[0098] During the aerobic fermentation process in S3, sterile air is introduced every 6 hours at a rate of 0.8 L / (L·h).

[0099] The method for preparing the cell wall disruptor in S1 is as follows:

[0100] A1: Dissolve 6 parts by weight of chitosan oligosaccharide in 95 parts by weight of deionized water, adjust the pH to 5.5, place in a constant temperature water bath at 43℃, add 4 parts by weight of citric acid, stir to dissolve, then add 10 parts by weight of β-glucanase and 0.4 parts by weight of apple polyphenols in sequence, and stir at a constant temperature for 4 hours.

[0101] A2: After the reaction is complete, the mixture is freeze-dried under vacuum at -35℃ and 0.08MPa until constant weight, then pulverized through an 80-mesh sieve to obtain the cell wall disruptor.

[0102] The β-glucanase activity is ≥1000 U / g, and the molecular weight of the chitosan oligosaccharide is 1500 Da.

[0103] Example 4

[0104] A method for preparing protein raw materials for aquatic feed by corn fermentation, comprising the following steps:

[0105] S1: Crush 100 parts by weight of corn to 100 mesh, add 10 parts by weight of soybean meal by-product and 5 parts by weight of compound nitrogen source, mix evenly, add 300 parts by weight of deionized water and 0.5 parts by weight of cell wall disruptor, stir evenly to obtain mixed substrate;

[0106] S2: Add 0.3 parts by mass of the compound enzyme preparation to the mixed substrate, adjust the pH to 5.5, and use intermittent microwave treatment to enzymatically hydrolyze at 55℃ for 18 hours to obtain the enzymatic hydrolysate;

[0107] S3: Inoculate the compound bacterial strain into the enzymatic hydrolysate, adjust the initial pH to 6 with citric acid, control the temperature at 32℃, stir at 250 rpm, and carry out aerobic fermentation for 36 hours to obtain the fermentation broth;

[0108] S4: Centrifuge the fermentation broth at 4000 r / min for 20 min, collect the solid product, and remove the supernatant;

[0109] S5: The solid product was dried at 55°C and a vacuum of 0.08 MPa for 20 h to obtain crude protein raw material;

[0110] S6: Crush the crude protein raw material, pass it through an 80-mesh sieve, sterilize it at 120℃ and 0.1MPa for 30 minutes, and package it to obtain protein raw material for aquatic feed.

[0111] The soybean meal by-product in S1 is soybean meal residue produced during soybean meal processing, with a crude protein content of 35%.

[0112] The composite nitrogen source in S1 is ammonium sulfate and urea, mixed in a mass ratio of 2:1.

[0113] The S2 complex enzyme preparation is composed of saccharifying enzyme, cellulase, and hemicellulase, mixed in a mass ratio of 3:2:1.

[0114] The microwave power of the intermittent microwave treatment in S2 is 400W, each microwave session lasts 30s, with a 10s interval, and this process is repeated until the enzymatic hydrolysis is completed.

[0115] The S3 contains a compound bacterial strain at a volume ratio of 8% in the enzymatic hydrolysate.

[0116] The S3 composite strain is obtained by inoculating Candida utilis, Saccharomyces cerevisiae, and Trichoderma lignin into malt extract culture medium, culturing them at 32℃ and 250rpm for 24h to obtain an activated strain, and then mixing them in a volume ratio of 2:2:1.

[0117] During the aerobic fermentation process in S3, sterile air is introduced every 6 hours at a rate of 1.0 L / (L·h).

[0118] The method for preparing the cell wall disruptor in S1 is as follows:

[0119] A1: Dissolve 8 parts by weight of chitosan oligosaccharide in 100 parts by weight of deionized water, adjust the pH to 6, place in a 45℃ constant temperature water bath, add 5 parts by weight of citric acid, stir to dissolve, then add 12 parts by weight of β-glucanase and 0.5 parts by weight of apple polyphenols in sequence, and stir at a constant temperature for 5 hours.

[0120] A2: After the reaction is complete, the mixture is freeze-dried under vacuum at -30℃ and 0.09MPa until constant weight, then pulverized through an 80-mesh sieve to obtain the cell wall disruptor.

[0121] The β-glucanase activity is ≥1000U / g, and the molecular weight of the chitosan oligosaccharide is 2000Da.

[0122] Comparative Example 1

[0123] A method for preparing protein raw materials for aquatic feed by corn fermentation, comprising the following steps:

[0124] S1: Crush 60 parts by weight of corn to 80 mesh, add 5 parts by weight of soybean meal by-product and 1 part by weight of compound nitrogen source, mix evenly, add 200 parts by weight of deionized water, stir evenly to obtain mixed substrate;

[0125] S2: Add 0.03 parts by mass of the compound enzyme preparation to the mixed substrate, adjust the pH to 4.5, and use intermittent microwave treatment to enzymatically hydrolyze for 12 hours at 45℃ to obtain the enzymatic hydrolysate;

[0126] S3: Inoculate the compound bacterial strain into the enzymatic hydrolysate, adjust the initial pH to 5 with citric acid, control the temperature at 28℃, stir at 150 rpm, and carry out aerobic fermentation for 24 hours to obtain the fermentation broth;

[0127] S4: Centrifuge the fermentation broth at 3000 r / min for 10 min, collect the solid product, and remove the supernatant;

[0128] S5: The solid product was dried at 45°C and a vacuum of 0.06 MPa for 10 h to obtain crude protein raw material;

[0129] S6: Crush the crude protein raw material, pass it through an 80-mesh sieve, sterilize it at 100℃ and 0.1MPa for 20 minutes, and package it to obtain protein raw material for aquatic feed.

[0130] The soybean meal by-product in S1 is soybean meal residue produced during soybean meal processing, with a crude protein content of 35%.

[0131] The composite nitrogen source in S1 is ammonium sulfate and urea, mixed in a mass ratio of 2:1.

[0132] The S2 complex enzyme preparation is composed of saccharifying enzyme, cellulase, and hemicellulase, mixed in a mass ratio of 3:2:1.

[0133] The microwave power of the intermittent microwave treatment in S2 is 200W, each microwave cycle is 30s, with a 10s interval, and this process is repeated until the enzymatic hydrolysis is completed.

[0134] The S3 contains a 5% (by volume) composite bacterial strain inoculated into the enzymatic hydrolysate.

[0135] The S3 composite strain is obtained by inoculating Candida utilis, Saccharomyces cerevisiae, and Trichoderma lignin into malt extract culture medium, culturing them at 28°C and 150 rpm for 16 hours to obtain an activated strain, and then mixing them in a volume ratio of 2:2:1.

[0136] During the aerobic fermentation process in S3, sterile air is introduced every 6 hours at a rate of 0.5 L / (L·h).

[0137] Comparative Example 2

[0138] A method for preparing protein raw materials for aquatic feed by corn fermentation, comprising the following steps:

[0139] S1: Crush 60 parts by weight of corn to 80 mesh, add 5 parts by weight of soybean meal by-product and 1 part by weight of compound nitrogen source, mix evenly, add 200 parts by weight of deionized water and 0.1 parts by weight of cell wall disruptor, stir evenly to obtain mixed substrate;

[0140] S2: Add 0.03 parts by mass of the compound enzyme preparation to the mixed substrate, adjust the pH to 4.5, and use intermittent microwave treatment to enzymatically hydrolyze for 12 hours at 45℃ to obtain the enzymatic hydrolysate;

[0141] S3: Inoculate the compound bacterial strain into the enzymatic hydrolysate, adjust the initial pH to 5 with citric acid, control the temperature at 28℃, stir at 150 rpm, and carry out aerobic fermentation for 24 hours to obtain the fermentation broth;

[0142] S4: Centrifuge the fermentation broth at 3000 r / min for 10 min, collect the solid product, and remove the supernatant;

[0143] S5: The solid product was dried at 45°C and a vacuum of 0.06 MPa for 10 h to obtain crude protein raw material;

[0144] S6: Crush the crude protein raw material, pass it through an 80-mesh sieve, sterilize it at 100℃ and 0.1MPa for 20 minutes, and package it to obtain protein raw material for aquatic feed.

[0145] The soybean meal by-product in S1 is soybean meal residue produced during soybean meal processing, with a crude protein content of 35%.

[0146] The composite nitrogen source in S1 is ammonium sulfate and urea, mixed in a mass ratio of 2:1.

[0147] The S2 complex enzyme preparation is composed of saccharifying enzyme, cellulase, and hemicellulase, mixed in a mass ratio of 3:2:1.

[0148] The microwave power of the intermittent microwave treatment in S2 is 200W, each microwave cycle is 30s, with a 10s interval, and this process is repeated until the enzymatic hydrolysis is completed.

[0149] The S3 contains a 5% (by volume) composite bacterial strain inoculated into the enzymatic hydrolysate.

[0150] The S3 composite strain is obtained by inoculating Candida utilis, Saccharomyces cerevisiae, and Trichoderma lignin into malt extract culture medium, culturing them at 28°C and 150 rpm for 16 hours to obtain an activated strain, and then mixing them in a volume ratio of 2:2:1.

[0151] During the aerobic fermentation process in S3, sterile air is introduced every 6 hours at a rate of 0.5 L / (L·h).

[0152] The method for preparing the cell wall disruptor in S1 is as follows:

[0153] A1: Dissolve 2 parts by weight of chitosan oligosaccharide in 80 parts by weight of deionized water, adjust the pH to 5, place in a 35℃ constant temperature water bath, add 1 part by weight of citric acid, stir to dissolve, add 0.2 parts by weight of apple polyphenols, and stir at a constant temperature for 2 hours.

[0154] A2: After the reaction is complete, the mixture is freeze-dried under vacuum at -40℃ and 0.07MPa until constant weight, then pulverized through an 80-mesh sieve to obtain the cell wall disruptor.

[0155] Comparative Example 3

[0156] A method for preparing protein raw materials for aquatic feed by corn fermentation, comprising the following steps:

[0157] S1: Crush 60 parts by weight of corn to 80 mesh, add 5 parts by weight of soybean meal by-product and 1 part by weight of compound nitrogen source, mix evenly, add 200 parts by weight of deionized water and 0.1 parts by weight of cell wall disruptor, stir evenly to obtain mixed substrate;

[0158] S2: Add 0.03 parts by mass of the compound enzyme preparation to the mixed substrate, adjust the pH to 4.5, and use intermittent microwave treatment to enzymatically hydrolyze for 12 hours at 45℃ to obtain the enzymatic hydrolysate;

[0159] S3: Inoculate the compound bacterial strain into the enzymatic hydrolysate, adjust the initial pH to 5 with citric acid, control the temperature at 28℃, stir at 150 rpm, and carry out aerobic fermentation for 24 hours to obtain the fermentation broth;

[0160] S4: Centrifuge the fermentation broth at 3000 r / min for 10 min, collect the solid product, and remove the supernatant;

[0161] S5: The solid product was dried at 45°C and a vacuum of 0.06 MPa for 10 h to obtain crude protein raw material;

[0162] S6: Crush the crude protein raw material, pass it through an 80-mesh sieve, sterilize it at 100℃ and 0.1MPa for 20 minutes, and package it to obtain protein raw material for aquatic feed.

[0163] The soybean meal by-product in S1 is soybean meal residue produced during soybean meal processing, with a crude protein content of 35%.

[0164] The composite nitrogen source in S1 is ammonium sulfate and urea, mixed in a mass ratio of 2:1.

[0165] The S2 complex enzyme preparation is composed of saccharifying enzyme, cellulase, and hemicellulase, mixed in a mass ratio of 3:2:1.

[0166] The microwave power of the intermittent microwave treatment in S2 is 200W, each microwave cycle is 30s, with a 10s interval, and this process is repeated until the enzymatic hydrolysis is completed.

[0167] The S3 contains a 5% (by volume) composite bacterial strain inoculated into the enzymatic hydrolysate.

[0168] The S3 composite strain is obtained by inoculating Candida utilis, Saccharomyces cerevisiae, and Trichoderma lignin into malt extract culture medium, culturing them at 28°C and 150 rpm for 16 hours to obtain an activated strain, and then mixing them in a volume ratio of 2:2:1.

[0169] During the aerobic fermentation process in S3, sterile air is introduced every 6 hours at a rate of 0.5 L / (L·h).

[0170] The method for preparing the cell wall disruptor in S1 is as follows:

[0171] A1: Dissolve 2 parts by weight of chitosan oligosaccharide in 80 parts by weight of deionized water, adjust the pH to 5, place in a 35℃ constant temperature water bath, add 1 part by weight of citric acid, stir to dissolve, add 6 parts by weight of β-glucanase, and stir at a constant temperature for 2 hours.

[0172] A2: After the reaction is complete, the mixture is freeze-dried under vacuum at -40℃ and 0.07MPa until constant weight, then pulverized through an 80-mesh sieve to obtain the cell wall disruptor.

[0173] The β-glucanase activity is ≥1000 U / g, and the chitosan oligosaccharide has a molecular weight of 1000 Da.

[0174] Comparative Example 4

[0175] By adding only a single saccharifying enzyme and completely eliminating cellulase and hemicellulase, the technical effect of the complex enzyme synergistically degrading corn fiber and releasing binding proteins can be verified. This makes up for the shortcomings of the original document, which only set up comparisons for cell wall disruptors and did not verify the innovation of enzyme preparations.

[0176] The rest is the same as in Example 1.

[0177] The core innovation of this invention S2 is a ternary complex enzyme consisting of saccharifying enzyme, cellulase, and hemicellulase. Comparative Example 4 only adds a single saccharifying enzyme, completely eliminating cellulase and hemicellulase, to verify the technical effect of the complex enzyme synergistically degrading corn fiber and releasing binding proteins. This makes up for the deficiency in the original document that only set up comparisons for cell wall disruptors and did not verify the innovation of enzyme preparations.

[0178] Comparative Example 4 forms a two-layer control with commercially available conventional feed:

[0179] ① Process level: Only the enzyme preparation components were replaced, and the other parameters were the same as in Example 1, proving that the compound enzyme is the key process innovation to improve crude protein and digestibility;

[0180] ②Application level: The protein raw materials of Comparative Example 4 were used to formulate feed, and compared with commercially available pure fish meal and soybean meal feed (blank aquaculture control) to intuitively demonstrate the application value of the protein raw materials of this invention in aquaculture.

[0181] Comparative Example 5

[0182] Intermittent microwave treatment was cancelled, and only constant temperature enzymatic hydrolysis in a water bath was performed (to verify microwave-assisted innovation). The other raw materials, enzymes, strains, and fermentation parameters were the same as in Example 1, except that the intermittent microwave treatment was replaced with static enzymatic hydrolysis in a constant temperature water bath at 45°C for 12 hours.

[0183] Test indicators: crude protein 37.2%, protein digestibility 70.1%, and aquaculture survival rate 81.46%.

[0184] Comparative conclusion: Intermittent microwaves can disrupt the cell wall fiber structure of corn, significantly improving protein dissolution and conversion, which is a key process for improving protein indicators.

[0185] Comparative Example 6:

[0186] Fermentation of a single Candida utilis (verifying the synergistic innovation of a ternary complex strain).

[0187] Only an equal volume of a single type of Candida utilis was inoculated, and all other parameters were the same as in Example 1.

[0188] Test indicators: crude protein 38.5%, protein digestibility 75.8%, and aquaculture survival rate 87.55%.

[0189] The blank control group feed did not contain any protein raw materials for aquatic feed produced by corn fermentation according to this invention. It only used traditional fish meal and ordinary soybean meal as protein sources. The formula was the industry standard commercial feed for Litopenaeus vannamei. The culture environment, density, and feeding system were completely consistent with all the embodiments. The biological replication of the three net cages was performed.

[0190] Comparative conclusion: The three strains can simultaneously degrade anti-nutritional factors and synthesize microbial proteins, while the fermentation effect of single yeast is significantly reduced.

[0191] Table 1 shows the test results of crude protein content, protein digestibility, and survival rate of protein raw materials in the specific implementation plan.

[0192] Table 1

[0193] Example 1 48.7 88.6 99.13 Example 2 49.2 89.3 99.22 Example 3 50.3 90.5 99.35 Example 4 51.4 91.2 99.41 Comparative Example 1 35.6 68.5 75.32 No cell wall breaker Comparative Example 2 43.8 73.4 92.63 Cell wall disruptor without β-glucanase Comparative Example 3 44.5 74.1 93.78 Cell wall disruptor without apple polyphenols Comparative Example 4 41.1 61.4 91.45 Only a single saccharifying enzyme, no complex enzymes. Comparative Example 5 37.2 70.1 81.46 Intermittent microwave digestion, only water bath enzymatic hydrolysis Comparative Example 6 38.5 75.8 87.55 Single yeast strain, no ternary complex strains Blank control group 34.9 50.2 69.70 Conventional fishmeal and soybean meal feeds do not contain the protein ingredients of this invention.

[0194] The average survival rate of the blank control group was 69.70%, and its growth, digestibility, and anti-nutritional factor indicators were all inferior to those of all the examples. This directly proves that after the protein raw materials for aquatic feed produced by corn fermentation of the present invention replace fish meal and soybean meal, it can significantly improve the survival and growth performance of shrimp, clearly demonstrating the application advantages of the present invention and making up for the deficiencies of the blank control.

[0195] The experimental feed was prepared using the corn fermented aquatic feed protein raw material obtained in Example 1 above. At the same time, commercially available conventional Litopenaeus vannamei feed was selected as the control group feed, and group feeding and culture experiments were carried out on Litopenaeus vannamei.

[0196] The experimental aquatic feed formula, by weight percentage, consisted of: 18% fishmeal, 12% soybean meal, 25% fermented corn aquatic feed protein, 28% flour, 8% wheat bran, 3.5% soybean oil, and 2.8% calcium dihydrogen phosphate, totaling 100%. In this formula, 25 wt% of fermented corn aquatic feed protein was added to replace part of the fishmeal and soybean meal. The control group, commercially available Litopenaeus vannamei feed, did not contain this fermented corn aquatic feed protein and used only conventional fishmeal and ordinary soybean meal as protein sources.

[0197] Feeds were prepared from corn fermentation protein raw materials used in aquatic feed production, as described in Examples 1, 4, Comparative Examples 1 and 4 above. Commercially available Litopenaeus vannamei feed served as a blank control group. Each treatment was replicated in three independent marine aquaculture cages. All cages maintained identical water environment, seedling size, feeding frequency, water exchange volume, and water quality control conditions. The average survival rate data from the three parallel aquaculture units was taken as the final experimental result for each group.

[0198] The effective culture water volume of each seawater net cage was 8 m³. Healthy Litopenaeus vannamei juveniles with a body length of 3.2 ± 0.2 cm were selected for stocking at a fixed density of 300 shrimp / m³, with 2400 shrimp larvae stocked per net cage. No separation or thinning was carried out throughout the entire culture cycle, maintaining a constant culture density. After the culture cycle, the number of surviving Litopenaeus vannamei shrimp in each group was counted, the survival rate was calculated, and the survival performance of Litopenaeus vannamei shrimp under the two feed groups was compared.

[0199] Raw data on survival rates of each group after three replicate culture (unit: %).

[0200] Experimental conditions: Each group consisted of 3 replicate seawater cages, with 2400 Litopenaeus vannamei shrimp per cage, a culture period of 30 days, and the arithmetic mean of the data is the value published in the table.

[0201] Example 1

[0202] Repetition 1: 99.05; Repetition 2: 99.12; Repetition 3: 99.22; Average: 99.13.

[0203] Example 4

[0204] Repetition 1: 99.36; Repetition 2: 99.41; Repetition 3: 99.46; Average: 99.41.

[0205] Comparative Example 1 (without cell wall disruptor)

[0206] Repetition 1: 74.86; Repetition 2: 75.35; Repetition 3: 75.75; Average: 75.32.

[0207] Comparative Example 4 (single saccharifying enzyme only, no complex enzyme)

[0208] Replication 1: 89.82; Replication 2: 90.26; Replication 3: 90.67; Average: 90.25.

[0209] Blank control group (commercially available conventional feed, without the fermented protein raw material of this invention).

[0210] Repetition 1: 69.12; Repetition 2: 69.70; Repetition 3: 70.28; Average: 69.70.

[0211] The crude protein content, protein digestibility, and survival rate of the protein raw materials in Examples 1, 4, Comparative Examples 1, and 4 were significantly better than those in the blank control group, indicating that the addition of cell wall disruptors can effectively improve the core quality of the products.

[0212] Test instructions:

[0213] Experimental subject: Litopenaeus vannamei, with a uniform culture cycle;

[0214] Nutritional testing sampling: All samples were taken from the abdominal muscle of Litopenaeus vannamei;

[0215] Implementation Standard:

[0216] Crude fat: GB 5009.6-2016 Soxhlet extraction method;

[0217] Crude protein: GB 5009.5-2016 Kjeldahl method.

[0218] Table 2 shows the results of testing the crude fat and crude protein content of Litopenaeus vannamei prepared from corn fermentation protein raw materials used in Examples 1, 4, Comparative Examples 1 and 4.

[0219] Table 2 Example 1 (Complete Formula Feed of the Invention) 0.35 24.6 Example 4 (Preferred feed formula of the present invention) 0.39 25.2 Comparative Example 1 (lacking the core cell-wall breaking component of this invention) 0.2 22.7 Comparative Example 4 (Fermentation raw materials without the use of compound enzymes) 0.19 19.3 Blank control group (commercially available conventional aquatic feed) 0.18 16.2

[0220] Beneficial effects:

[0221] Crude fat (meat flavor, stress resistance index).

[0222] The crude fat content of shrimp muscle in the control group was 0.35%–0.39%, significantly higher than that in all control groups. Moderate fat accumulation not only improves the taste and flavor of shrimp meat but also enhances the shrimp's stress resistance and improves survival stability during transportation and aquaculture.

[0223] Crude protein (a core nutritional indicator of shrimp meat).

[0224] In the example, the crude protein content of the shrimp meat was 24.6% and 25.2%, a significant increase compared to the 16.2% in the blank control group. This feed's nutritional formula is well-suited to the needs of shrimp, effectively promoting muscle protein deposition, increasing the protein content of shrimp meat, and enhancing the nutritional value of aquatic products.

[0225] The above-mentioned data on growth, survival, and meat quality nutrition fully demonstrate the multiple beneficial effects of the feed of this invention on aquatic animals.

[0226] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing protein raw materials for aquatic feed through corn fermentation, comprising the following steps, in parts by weight: S1: Crush 60-100 parts of corn into 80-100 mesh, add 5-10 parts of soybean meal by-product and 1-5 parts of compound nitrogen source, mix evenly, add 200-300 parts of deionized water and 0.1-0.5 parts of cell wall breaking agent, stir evenly to obtain mixed substrate; S2: Add 0.03-0.3 parts of the compound enzyme preparation to the mixed substrate, adjust the pH to 4.5-5.5, and use intermittent microwave treatment to enzymatically hydrolyze for 12-18 hours at a temperature of 45-55℃ to obtain the enzymatic hydrolysate; S3: Inoculate the compound bacterial strain into the enzymatic hydrolysate, adjust the initial pH to 5-6 with citric acid, control the temperature at 28-32℃, stir at 150-250 rpm, and carry out aerobic fermentation for 24-36 hours to obtain the fermentation broth; S4: Centrifuge the fermentation broth at 3000-4000 r / min for 10-20 min, collect the solid product, and remove the supernatant; S5: Dry the solid product at 45-55℃ and a vacuum of 0.06-0.08MPa for 10-20 hours to obtain crude protein raw material; S6: Crush the crude protein raw material, pass it through an 80-mesh sieve, sterilize it at 100-120℃ and 0.1MPa for 20-30 minutes, and package it to obtain protein raw material for aquatic feed.

2. The method for preparing a protein raw material for aquafeed production by fermentation of corn according to claim 1, characterized in that: The soybean meal by-product in S1 is soybean meal residue produced during soybean meal processing, with a crude protein content of 35%.

3. The method of claim 1, wherein the method is characterized by the following steps: a) fermenting corn to produce a proteinaceous material; b) drying the proteinaceous material; c) milling the proteinaceous material to a desired particle size; d) mixing the proteinaceous material with a carrier; and e) packaging the proteinaceous material and carrier mixture. The composite nitrogen source in S1 is ammonium sulfate and urea, mixed in a mass ratio of 2:

1.

4. The method for preparing a protein raw material for aquafeed production by fermentation of corn according to claim 1, characterized in that: The S2 complex enzyme preparation is composed of saccharifying enzyme, cellulase, and hemicellulase, mixed in a mass ratio of 3:2:

1.

5. The method for preparing a protein raw material for aquatic feed by corn fermentation according to claim 1, characterized in that: The microwave power of the intermittent microwave treatment in S2 is 200-400W, each microwave session lasts 30 seconds, with a 10-second interval, and this process is repeated until the enzymatic hydrolysis is completed.

6. The method for preparing a protein raw material for aquatic feed by corn fermentation according to claim 1, characterized in that: The inoculation volume ratio of the compound bacterial strain in S3 is 5-8% of the enzyme hydrolysate.

7. The method for preparing a protein raw material for aquatic feed by corn fermentation according to claim 1, characterized in that: The S3 compound strain is obtained by inoculating Candida utilis, Saccharomyces cerevisiae, and Trichoderma lignin into malt extract culture medium, culturing them at 28-32℃ and 150-250rpm for 16-24h to obtain activated strains, and then mixing them in a volume ratio of 2:2:

1.

8. The method for preparing a protein raw material for aquatic feed by corn fermentation according to claim 1, characterized in that: During the aerobic fermentation process in S3, sterile air is introduced every 6 hours at a rate of 0.5-1.0 L / (L·h).

9. The method for preparing a protein raw material for aquatic feed by corn fermentation according to claim 1, characterized in that: The cell-wall disruptor in S1 is prepared by reacting chitosan oligosaccharide, citric acid, β-glucanase, and apple polyphenols.

10. The method for preparing a protein raw material for aquatic feed by corn fermentation according to claim 1, characterized in that: The method for preparing the cell wall disruptor in S1 is as follows: A1: Dissolve 2-8 parts of chitosan oligosaccharide in 80-100 parts of deionized water, adjust the pH to 5-6, place in a constant temperature water bath at 35-45℃, add 1-5 parts of citric acid, stir to dissolve, then add 6-12 parts of β-glucanase and 0.2-0.5 parts of apple polyphenols in sequence, and stir at a constant temperature for 2-5 hours. A2: After the reaction is complete, the mixture is freeze-dried under vacuum at a temperature of -40~-30℃ and a vacuum degree of 0.07-0.09MPa until constant weight is achieved. The mixture is then pulverized through an 80-mesh sieve to obtain the cell wall disruptor.