Cottonseed protein extraction method based on enzymolysis and fermentation

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a cottonseed protein extraction method based on enzymolysis and fermentation. The method comprises the following steps: (1) pretreatment: performing hull-kernel separation on cotton seeds to obtain cotton kernels and cotton hulls, and performing softening, embryo pressing and drying on the cotton kernels in sequence to obtain pretreated cotton kernels; (2) degreasing: degreasing the pretreated cottonseed kernels to obtain degreased cottonseed meal; (3) leaching: carrying out secondary leaching extraction on the degreased cottonseed meal by using a methanol aqueous solution to obtain a leached material; draining the leached material, desolventizing and drying to obtain crude cottonseed protein; and (4) adding water into the coarse cottonseed protein to obtain a fermented material, adding compound bacteria and neutral protease into the fermented material, fermenting, and drying, crushing and sieving after fermentation to obtain the cottonseed protein based on enzymolysis and fermentation. The cottonseed protein disclosed by the invention has obvious effects of relieving fatty liver of large yellow croaker and preventing and treating liver and gall diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a cottonseed protein extraction method based on enzymatic hydrolysis and fermentation. Background Art

[0002] Cottonseed is a byproduct of cotton processing. Cottonseed protein is a nutritious protein source with a well-balanced amino acid profile, including a reasonable ratio of essential to non-essential amino acids. When cottonseed protein concentrate replaces part of the fermented soybean meal in feed, it can significantly improve the digestibility of crude protein, crude fat, and acid detergent fiber in the diet. Therefore, cottonseed protein can be used as an alternative feed protein source and has great potential as a high-quality feed protein source. Cottonseed protein not only meets the nutritional needs of animal growth, but also has high utilization efficiency and economic value.

[0003] Free gossypol in cottonseed is toxic. If animals ingest gossypol at levels exceeding safety limits, it accumulates in the body and is excreted slowly, leading to chronic poisoning and damage to internal organs, including genitals. Existing biological methods for detoxifying free gossypol are effective, but reducing its content significantly improves the safety and nutritional value of cottonseed protein, enabling its wider application in food, feed, and functional products. This not only broadens the application range of cottonseed protein but also enhances its market competitiveness and economic value.

[0004] Large yellow croaker ( Pseudosciaena crocea ) is one of the important marine aquaculture fish in China. Its aquaculture value is significant because of its delicious meat, high nutritional value and strong market demand. However, due to the widespread use of high-fat feed, a large amount of fat is deposited in the fish body, especially in the liver, resulting in a high incidence of hepatobiliary syndrome and intestinal diseases such as fatty liver, and once the disease occurs, it often causes large-scale deaths. Chinese patent CN 106071106 B provides a method for fermenting cottonseed protein and preparing fermented cottonseed protein by solid-state aerobic fermentation. The method is rich in growth factors and peptides after enzyme treatment and fermentation, which can improve the quality of meat, eggs, etc. and improve the quality of agricultural products. However, the free phenol content of the cottonseed protein prepared by this method cannot meet the current market demand, and the effect of reducing fatty liver of large yellow croaker and preventing and treating the occurrence of liver and gallbladder diseases is not ideal.

[0005] Therefore, there is an urgent need for a cottonseed protein extraction method based on enzymatic hydrolysis and fermentation. Summary of the Invention

[0006] The purpose of the present invention is to provide a cottonseed protein extraction method based on enzymatic hydrolysis and fermentation.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: A cottonseed protein extraction method based on enzymatic hydrolysis and fermentation comprises the following steps: (1) Pretreatment: After the cotton seeds are separated from the shell and kernel, the cotton kernel and cotton hull are obtained. The cotton kernel is softened, pressed and dried in sequence to obtain the pretreated cotton kernel; (2) Degreasing: Degreasing the pre-treated cottonseed to obtain defatted cottonseed meal; (3) Leaching: The defatted cottonseed meal is subjected to secondary leaching extraction using a methanol-water solution to obtain a leached material; the leached material is then drained, desolventized, and dried to obtain crude cottonseed protein; (4) Add water to the crude cottonseed protein and mix well to obtain a fermentation material. Add composite bacteria and neutral protease to the fermentation material, and ferment it aerobically at a constant temperature of 35-38°C for 50-55 hours. After the fermentation is completed, dry it at 65-70°C, crush it, and pass it through a 40-mesh sieve to obtain cottonseed protein based on enzymatic hydrolysis and fermentation.

[0008] The present invention uses a specific type of composite bacteria and enzymes for mixed fermentation to improve the content of free phenol in cottonseed protein. Free phenol has potential harm to organs such as the liver and kidneys of fish, and may particularly affect the growth and immune function of large yellow croaker. The present invention significantly reduces the content of free gossypol in cottonseed protein through fermentation, thereby improving the safety of feed and reducing the negative impact on the health of large yellow croaker. In addition, many countries and regions have strict restrictions on the content of gossypol in feed. Reducing the free gossypol content helps ensure that feed products comply with more relevant regulatory requirements and expand the scope of market application. At the same time, feeding large yellow croaker with the cottonseed protein of the present invention can improve the liver-to-body ratio and abdominal fat rate of large yellow croaker.

[0009] Furthermore, the pretreatment conditions in step (1) are: softening temperature is 70-80°C and drying temperature is 70-80°C.

[0010] Furthermore, the thickness of the pressed embryo in step (1) is 0.3-0.5 mm.

[0011] Furthermore, the specific steps of degreasing in step (2) include: pre-treating the cottonseeds to extract oil using No. 6 solvent oil, the temperature is 50-55°C, the weight ratio of No. 6 solvent oil to pre-treated cottonseeds is (1.2-1.5):1, the leaching time is 120-150 minutes, and the defatted cottonseed meal is obtained.

[0012] Furthermore, in step (3), each stage of leaching extraction is stirred at 40-45 r / min for 15-20 min, and the concentration of the methanol aqueous solution is 85-90 (v / v)%; Furthermore, in the step (3), the weight ratio of the defatted cottonseed meal to the methanol aqueous solution is 1:(1-2).

[0013] Furthermore, in step (4), the weight ratio of water to crude cottonseed protein is (0.7-0.9):1.

[0014] Furthermore, the composite bacteria in step (4) includes Bacillus subtilis and Candida tropicalis.

[0015] Candida tropicalis, Beina Chuanglian Biotechnology Co., Ltd., number: BNCC140189.

[0016] Bacillus subtilis, Beina Chuanglian Biotechnology Co., Ltd., number: BNCC188062.

[0017] Furthermore, the amount of Bacillus subtilis was 1×10 6 -3×10 6 CFU / g crude cottonseed protein; and the amount of tropical Candida was 4×10 6 -6×10 6 CFU / g crude cottonseed protein.

[0018] Furthermore, the dosage of the neutral protease is 1500-2000 U / g crude cottonseed protein.

[0019] The present invention uses specific types of composite bacteria and enzymes for mixed fermentation, which can improve the composition of active components such as amino acids in cottonseed protein, improve the liver-to-body ratio and abdominal fat rate of large yellow croaker, and also affect the content of free phenols.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are: 1. The present invention improves the free phenol content in cottonseed protein by using specific species and dosages of composite bacteria and enzymes for mixed fermentation. This significantly reduces the free gossypol content in cottonseed protein through fermentation, improving feed safety and reducing negative health effects on large yellow croaker. Many countries and regions have strict limits on gossypol content in feed. Reducing free gossypol content helps ensure that feed products comply with more relevant regulations and expands market applications.

[0021] 2. The present invention utilizes specific types and amounts of composite bacteria and enzymes for mixed fermentation to improve the composition of active components in cottonseed protein, such as amino acids, and thereby improve the liver-to-body ratio and abdominal fat percentage of large yellow croaker. The liver-to-body ratio is an important indicator of liver burden in fish. An excessively high liver-to-body ratio can lead to excessive metabolic stress in the liver or fat accumulation. By optimizing the composition of cottonseed protein through fermentation, the present invention improves the metabolic function of the large yellow croaker liver, reduces fat accumulation in the liver, and thus lowers the liver-to-body ratio, protecting liver health. After fermentation, the amino acid and other components of the cottonseed protein of the present invention further promote normal lipid metabolism in large yellow croaker, reduce abnormal fat accumulation, and mitigate the risk of diseases caused by impaired fat metabolism. Excessively high abdominal fat percentages can affect the aesthetic appearance and market acceptance of large yellow croaker. The amino acids and other active ingredients in the fermented cottonseed protein of the present invention can regulate fat distribution in large yellow croaker, reduce abdominal fat accumulation, and thus lower the abdominal fat percentage, enhancing product value. They also help promote muscle protein synthesis while reducing excessive fat deposition, resulting in a firmer, more tender meat that meets consumers' demand for high-quality aquatic products. DETAILED DESCRIPTION

[0022] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0023] The raw materials used in the following examples of the present invention are all commercially available commodities: Candida tropicalis, Beina Chuanglian Biotechnology Co., Ltd., number: BNCC140189.

[0024] Bacillus subtilis, Beina Chuanglian Biotechnology Co., Ltd., number: BNCC188062.

[0025] Neutral protease, activity 100 U / mg. Shanghai Yuanye Biotechnology Co., Ltd.

[0026] Papain, enzyme activity 800 U / mg. Shanghai Yuanye Biotechnology Co., Ltd.

[0027] Example 1 This embodiment provides a cottonseed protein extraction method based on enzymatic hydrolysis and fermentation, comprising the following steps: (1) Pretreatment: After the cotton seeds are separated from the hulls, the cotton seeds and hulls are obtained. The cotton seeds are softened, pressed and dried in sequence to obtain pretreated cotton seeds. The softening temperature is 75°C and the drying temperature is 75°C. The pressed seeds have a thickness of 0.4 mm.

[0028] (2) Degreasing: The pretreated cottonseeds were degreased and the oil was extracted from the pretreated cottonseeds using No. 6 solvent oil at a temperature of 52°C. The weight ratio of No. 6 solvent oil to pretreated cottonseeds was 1.3:1. The extraction time was 130 min to obtain defatted cottonseed meal.

[0029] (3) Leaching: The defatted cottonseed meal is subjected to secondary leaching extraction using a methanol-water solution, with each stage of leaching extraction being stirred at 40 r / min for 20 min, and the concentration of the methanol-water solution being 90 (v / v)%; the weight ratio of the defatted cottonseed meal to the methanol-water solution being 1:1.5; obtaining the leached material; and then draining, desolventizing, and drying the leached material to obtain crude cottonseed protein; (4) Add water to the crude cottonseed protein, with a weight ratio of water to crude cottonseed protein of 0.8:1; mix them evenly to obtain a fermentation material, add composite bacteria and neutral protease to the fermentation material, and ferment it aerobically at a constant temperature of 37°C for 52 hours. After the fermentation is completed, dry it, crush it, and pass it through a 40-mesh sieve to obtain cottonseed protein based on enzymatic hydrolysis and fermentation.

[0030] The composite bacteria include Bacillus subtilis and Candida tropicalis. The dosage of Bacillus subtilis is 2×10 6 CFU / g crude cottonseed protein; the dosage of tropical Candida was 5×10 6 CFU / g crude cottonseed protein.

[0031] The dosage of neutral protease was 1800 U / g crude cottonseed protein.

[0032] Example 2 This embodiment provides a cottonseed protein extraction method based on enzymatic hydrolysis and fermentation, comprising the following steps: (1) Pretreatment: After the cotton seeds are separated from the hulls, the cotton seeds and hulls are obtained. The cotton seeds are softened, pressed and dried in sequence to obtain pretreated cotton seeds. The softening temperature is 80°C and the drying temperature is 80°C. The pressed seeds have a thickness of 0.4 mm.

[0033] (2) Degreasing: The pretreated cottonseeds were degreased and the oil was extracted from the pretreated cottonseeds using No. 6 solvent oil at a temperature of 55°C. The weight ratio of No. 6 solvent oil to pretreated cottonseeds was 1.2:1. The extraction time was 150 min to obtain the defatted cottonseed meal.

[0034] (3) Leaching: The defatted cottonseed meal is subjected to secondary leaching extraction using a methanol-water solution, with each stage of leaching extraction being stirred at 45 r / min for 15 min, and the concentration of the methanol-water solution being 85 (v / v)%; the weight ratio of the defatted cottonseed meal to the methanol-water solution being 1:2; obtaining the leached material; and then draining, desolventizing, and drying the leached material to obtain crude cottonseed protein; (4) Add water to the crude cottonseed protein, with a weight ratio of water to crude cottonseed protein of 0.9:1; mix them evenly to obtain a fermentation material, add composite bacteria and neutral protease to the fermentation material, and ferment it aerobically at a constant temperature of 38°C for 50 hours. After the fermentation is completed, dry, crush, and pass through a 40-mesh sieve to obtain cottonseed protein based on enzymatic hydrolysis and fermentation.

[0035] The composite bacteria include Bacillus subtilis and Candida tropicalis. The dosage of Bacillus subtilis is 1×10 6 CFU / g crude cottonseed protein; the dosage of tropical Candida was 6×10 6 CFU / g crude cottonseed protein.

[0036] The dosage of neutral protease was 1500 U / g crude cottonseed protein.

[0037] Comparative Example 1 The difference between this comparative example and Example 1 is that the composite bacteria include Aspergillus niger and Candida utilis. The dosage of Aspergillus niger is 1×10 6 CFU / g crude cottonseed protein; the dosage of Candida utilis was 6×10 6 CFU / g crude cottonseed protein.

[0038] Candida utilis, Beina Chuanglian Biotechnology Co., Ltd., No.: BNCC336517.

[0039] Aspergillus niger, Beina Chuanglian Biotechnology Co., Ltd., number: BNCC142643.

[0040] Comparative Example 2 The difference between this comparative example and Example 1 is that the neutral protease is replaced by papain, and the amount of papain used is 1500 U / g crude cottonseed protein.

[0041] Comparative Example 3 The difference between this comparative example and Example 1 is that the amount of Bacillus subtilis used is 5×10 6 CFU / g crude cottonseed protein; the dosage of tropical Candida was 2×10 6 CFU / g crude cottonseed protein.

[0042] Comparative Example 4 The difference between this comparative example and Example 1 is that the amount of neutral protease used is 1000 U / g crude cottonseed protein.

[0043] Comparative Example 5 The difference between this comparative example and Example 1 is that the amount of neutral protease used is 2500 U / g crude cottonseed protein.

[0044] Performance Testing 1. Referring to the national standard GB / T13086-2020 for the determination of free gossypol in feed, the content of free phenol in the enzymatically hydrolyzed and fermented cottonseed proteins prepared in Examples 1-2 and Comparative Examples 1-5 was determined. The results are shown in Table 1.

[0045] Table 1 Free phenol test results (unit: mg / kg)

[0046] As shown in Table 1, the free phenol content of Examples 1-2 is less than 210 mg / kg. In contrast, the free phenol content of the products produced by the patented fermented cottonseed protein and solid-state aerobic fermentation methods for preparing fermented cottonseed protein is 276-329 mg / kg, significantly higher than that of the products of the present invention. Furthermore, the present invention does not require the addition of corn flour as a fermentation feedstock, resulting in a simpler raw material composition.

[0047] In Comparative Examples 1 and 2, the types of bacteria and enzymes changed, and the free phenol content increased, indicating that the bacteria and enzymes used in the present invention work synergistically to reduce the free phenol content.

[0048] In Comparative Examples 3-5, the dosage of bacteria and enzymes were changed, and the free phenol content increased to varying degrees, indicating that only under specific dosage conditions can the three play a better synergistic role.

[0049] 2. Large yellow croaker breeding experiment: carried out at the breeding base in Fuding City, Fujian Province.

[0050] Healthy large yellow croaker had an average body weight of (90.5 ± 4.0) g. The fish were randomly divided into four replicates per group, with 60 fish per replicate. During the experimental period, the fish were fed twice daily (8:00 AM and 5:00 PM) until full. The culture period lasted for 8 weeks. The feed formula for each group included the following ingredients by weight: 42 parts fish meal, 10 parts cottonseed protein prepared in the present invention, 20 parts flour, 7 parts fish oil, 11 parts microcrystalline cellulose, 1.7 parts soy lecithin, 1 part vitamin premix, and 0.7 part mineral premix.

[0051] High-fat group: The cottonseed protein prepared in the present invention was replaced with commercially available soybean meal purchased from Jize County Tiande Biological Feed Co., Ltd.

[0052] Control group: fish oil was replaced with 6 servings.

[0053] Prior art group: The cottonseed protein prepared in the present invention is replaced by the fermented cottonseed protein prepared in Example 5 of the method for preparing fermented cottonseed protein by solid-state aerobic fermentation, which is provided in reference to Chinese patent CN 106071106 B.

[0054] At the end of the breeding experiment, the fish were fasted for 24 hours. The number of fish in each cage was counted and the total weight was measured. The fish were anesthetized and dissected. The hepatopancreas and abdominal fat were separated and weighed, and the liver-to-body ratio and abdominal fat percentage were calculated.

[0055] Liver-to-body ratio = liver weight / whole fish weight × 100%; Abdominal fat rate = abdominal fat weight / total fish weight × 100%.

[0056] The results are shown in Table 2.

[0057] Table 2 Test results of large yellow croaker (%)

[0058] As shown in Table 2, the liver-to-body ratio and abdominal fat rate of large yellow croaker fed the feed supplemented with the cottonseed protein of the present invention were lower than those of the high-fat group, while the effect of the conventional group was less satisfactory. Analysis suggests that the active substances contained in the cottonseed protein of the present invention, such as the specific types and amounts of amino acids, help activate the mTOR signaling pathway in large yellow croaker and regulate liver lipid metabolism.

[0059] The types of bacteria and enzymes in Comparative Examples 1 and 2 changed, and the liver-to-body ratio and abdominal fat rate were higher than those in Example 1, indicating that the specific types of bacteria and enzymes used in the present invention and the components produced by fermentation are helpful in regulating the liver lipid metabolism of large yellow croaker.

[0060] In comparative examples 3-5, the dosage of bacteria and enzymes was changed, and the liver-to-body ratio and abdominal fat rate were higher than those in Example 1, indicating that only under specific dosage conditions can the content of effective components produced be higher, which is more helpful in regulating the liver lipid metabolism of large yellow croaker.

[0061] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A cottonseed protein extraction method based on enzymatic hydrolysis and fermentation, characterized in that: The following steps are involved: (1) Pretreatment: After the cotton seeds are separated from the shell and kernel, the cotton kernel and cotton hull are obtained. The cotton kernel is softened, pressed and dried in sequence to obtain the pretreated cotton kernel; (2) Degreasing: Degreasing the pre-treated cottonseed to obtain defatted cottonseed meal; (3) Leaching: The defatted cottonseed meal is subjected to secondary leaching extraction using a methanol-water solution to obtain a leached material; the leached material is then drained, desolventized, and dried to obtain crude cottonseed protein; (4) Add water to the crude cottonseed protein and mix well to obtain a fermentation material. Add composite bacteria and neutral protease to the fermentation material and ferment it aerobically at a constant temperature of 35-38°C for 50-55 hours. After the fermentation is completed, dry, crush, and sieve to obtain cottonseed protein based on enzymatic hydrolysis and fermentation.

2. The cottonseed protein extraction method based on enzymatic hydrolysis and fermentation according to claim 1, characterized in that: In the step (1), the softening temperature is 70-80°C and the drying temperature is 70-80°C.

3. The cottonseed protein extraction method based on enzymatic hydrolysis and fermentation according to claim 1, characterized in that: The thickness of the pressed embryo in step (1) is 0.3-0.5 mm.

4. The cottonseed protein extraction method based on enzymatic hydrolysis and fermentation according to claim 1, characterized in that: The specific steps of degreasing in step (2) include: pre-treating cottonseeds with solvent oil to extract oil, the temperature is 50-55°C, the weight ratio of solvent oil to pre-treated cottonseeds is (1.2-1.5):1, the leaching time is 120-150 minutes, and the defatted cottonseed meal is obtained.

5. The cottonseed protein extraction method based on enzymatic hydrolysis and fermentation according to claim 1, characterized in that: In step (3), each stage of leaching extraction is stirred at 40-45 r / min for 15-20 min, and the concentration of the methanol aqueous solution is 85-90 (v / v)%.

6. The cottonseed protein extraction method based on enzymatic hydrolysis and fermentation according to claim 1, characterized in that: The weight ratio of the defatted cottonseed meal to the methanol aqueous solution in step (3) is 1:(1-2).

7. The cottonseed protein extraction method based on enzymatic hydrolysis and fermentation according to claim 1, characterized in that: In the step (4), the weight ratio of water to crude cottonseed protein is (0.7-0.9):

1.

8. The cottonseed protein extraction method based on enzymatic hydrolysis and fermentation according to claim 1, characterized in that: The composite bacteria in step (4) include Bacillus subtilis and Candida tropicalis.

9. The cottonseed protein extraction method based on enzymatic hydrolysis and fermentation according to claim 8, characterized in that: The dosage of Bacillus subtilis was 1×10 6 -3×10 6 CFU / g crude cottonseed protein; the dosage of tropical Candida was 4×10 6 -6×10 6 CFU / g crude cottonseed protein.

10. The cottonseed protein extraction method based on enzymatic hydrolysis and fermentation according to claim 1, characterized in that: The dosage of neutral protease is 1500-2000U / g crude cottonseed protein.

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