Preparation method of marine shellfish polypeptide
Through the combination of frozen and low-temperature vacuum microwave treatment combined with enzymatic decomposition technology, the problem of insufficient development of marine shellfish protein resources has been solved, the yield and biological activity of peptides have been improved, and the efficient utilization of marine shellfish resources and the realization of economic value has been achieved.
Patent Information
- Application Number
- CN202411491844.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-24
- Publication Date
- 2025-01-03
AI Technical Summary
In the prior art, marine shellfish protein resources are not fully developed and there is a lack of effective small molecule shellfish polypeptide extraction methods, which leads to poor competitiveness in the international market, and the processing method is mainly human-powered, and the development is stagnant.
The freezing treatment is combined with low-temperature vacuum microwave treatment, and the enzymatic decomposition is successively enzymatic in acidic and alkaline environments, and the enzymatic decomposition efficiency is improved through the compatibility of complex enzymes, including the use of Aspergillus oryzae protease, papain, lipase, Novo protease, trypsin, and blue The complex of enzymes such as copper peptides controls the enzymatic parameters to improve the yield and biological activity of the peptide.
It significantly improves the yield and biological activity of peptides, reduces production costs, improves the purity and safety of products, is suitable for large-scale industrial production, and makes full use of marine biological resources, enhancing the added value of marine shellfish resources.
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Figure CN119242747A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of polypeptide extraction, and in particular to a method for preparing marine shellfish polypeptides. Background Art
[0002] Marine organisms live in extreme environments such as high pressure, high / low temperature and high salt for a long time. Marine biological proteins are very different from terrestrial biological proteins in terms of both amino acid composition and sequence. Marine shellfish have high nutritional value and rich and balanced amino acid content, making them a high-quality food protein source. At present, marine biological protein resources have not been well developed. In addition to being eaten directly, existing marine biological proteins in my country are often made into food and enter the market only through simple processing technology, which makes the products less competitive in the international market.
[0003] In my country, apart from fresh sales, shellfish processing is still mainly manual, and its development is stagnant at an early stage. The enzymatic hydrolysates of marine shellfish are not only rich in functional nutrients such as polypeptides, oligopeptides, and amino acids, but can also produce some bioactive peptides with special physiological functions, and have broad application prospects.
[0004] At present, the biotechnology extraction of marine biological protein resources, the use of microbial enzymes to catalyze hydrolysis into small molecule peptides, and the improvement of digestion conversion rate have become a hot spot for development. Shellfish is the most common marine mollusk. At present, there are few high-value-added products using shellfish as raw materials, and effective methods for the extraction of small molecule shellfish peptides have not yet been developed. How to increase the added value of marine shellfish resources, make full use of them, and fully realize their economic value has excellent research prospects. Summary of the invention
[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for preparing marine shellfish polypeptides.
[0006] A method for preparing marine shellfish polypeptides comprises the following steps:
[0007] S1. Clean the marine shellfish, steam them with water for 10-20 minutes, remove the shells and internal organs, grind them, sieve them, freeze them at -40 to -60°C for 1-4 hours, adjust the absolute pressure of the system to 70-80Pa, perform microwave treatment under the heat and pressure maintenance state, the effective microwave time is 5-10 minutes, warm them to room temperature, adjust the water content to 20-30%, and obtain a slurry;
[0008] S2. Add water to the slurry, let it stand at room temperature for 5-10 hours, adjust the pH value of the system to 6-6.5, add complex enzyme a, enzymolyze at 40-50℃ for 3-6 hours, heat to 90-95℃, keep warm for 5-10 minutes, and cool to room temperature to obtain enzymolysis material;
[0009] S3. Adjust the pH value of the enzymatic hydrolysis system to 7.8-8.2, add complex enzyme b, perform enzymatic hydrolysis at 50-60℃ for 1-2h, raise the temperature to 90-100℃, keep warm for 10-20min, cool to room temperature, and remove the precipitate by centrifugation; precipitate the hydrolyzate with ethanol, and centrifuge again to remove the precipitate; separate through filter membrane, collect the retained product, and spray dry.
[0010] Preferably, in S1, the marine shellfish is at least one of scallops, pearl oysters, oysters, clams and razor clams.
[0011] Preferably, in S1, saturated steam with a temperature of 100-105°C is used for steaming.
[0012] Preferably, in S1, the microwave power is 100-200 W, and the specific operation of the microwave treatment is as follows: after the frozen ground slurry is microwaved for 5-10 seconds, the microwave treatment is suspended, the material temperature is cooled to -40 to -60°C again, and then the above operation is repeated until the actual microwave treatment time reaches 5-10 minutes.
[0013] Preferably, in S2, the complex enzyme a comprises: Aspergillus oryzae protease, papain, lipase, and lecithin; wherein the mass ratio of Aspergillus oryzae protease, papain, lipase, and lecithin is 1-3:2-6:1-5:1-2.
[0014] The compound enzyme is a combination of Aspergillus oryzae protease, papain and lipase. Its activity is greatly enhanced under the action of lecithin. It can quickly enzymolyze protein macromolecules in the slurry with high degradation efficiency.
[0015] Preferably, in S2, in the complex enzyme a, the specific activity of Aspergillus oryzae protease is 7000-9000 U / g, the specific activity of papain is 6000-8000 U / g, and the specific activity of lipase is 5000-9000 U / g.
[0016] Preferably, in S2, the mass ratio of slurry, water and complex enzyme a is 50-100:200-300:1-2.
[0017] Preferably, in S3, the mass ratio of the enzymatic hydrolysis material to the complex enzyme b is 280-400:1-2.
[0018] Preferably, in S3, the complex enzyme b comprises: Novo protease, trypsin, and blue copper peptide; wherein the mass ratio of Novo protease, trypsin, and blue copper peptide is 1-2:1-3:0.01-0.1.
[0019] Compound enzyme b is compounded with Novo protease and trypsin, so that the resulting polypeptide has high biological activity, but it is highly irritating to the skin. The present invention further uses blue copper peptide to cooperate, which not only greatly enhances the enzyme activity, greatly increases the proportion of polypeptides with a molecular weight of less than 500 Daltons, but also significantly reduces the irritation effect on the skin, and greatly enhances the safety of the product.
[0020] Preferably, in S3, in the complex enzyme b, the specific activity of Novo protease is 8000-14000 U / g, and the specific activity of trypsin is 6000-10000 U / g.
[0021] Preferably, in S3, the pore size of the filter membrane is 30 nm; and the inlet air temperature of the spray drying is 200-210°C.
[0022] A marine shellfish polypeptide is prepared by adopting the marine shellfish polypeptide preparation method.
[0023] Beneficial effects:
[0024] The present invention adopts freezing treatment in combination with low-temperature vacuum microwave treatment, which can not only better preserve the biological activity of shellfish, but also can cooperate with the subsequent enzymatic hydrolysis process, not only improve the hydrolysis degree of protein polypeptides, significantly increase the polypeptide yield, but also can significantly shorten the enzymatic hydrolysis time and reduce costs.
[0025] In the enzymatic hydrolysis process of the present invention, enzymatic hydrolysis is carried out in acidic and alkaline environments in turn, and the enzymatic hydrolysis parameters are controlled, so that the obtained shellfish polypeptides are not only of high purity, but also of extremely high biological activity. It is found through experiments that among the marine shellfish polypeptides obtained by the present invention, polypeptides with a molecular weight of less than 1000 Daltons account for 97.2%, and polypeptides with a molecular weight of less than 500 Daltons account for 52.4%, so that it not only has significant antioxidant activity, strong reducing ability, excellent free radical scavenging ability, but also has low biological toxicity, low skin irritation, and is safe and reliable.
[0026] The invention greatly improves the conversion rate and biological activity of the product by controlling the enzymatic hydrolysis process and time; and the process conditions are easy to control, suitable for large-scale industrial production, and at the same time fully utilizes marine biological resources and improves the added value of marine shellfish resources. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a comparison chart of the percentages of the peak areas of polypeptides below 1 kDa and polypeptides below 0.5 kDa in the oyster polypeptides obtained in Example 5 and Comparative Examples 1-3.
[0028] Figure 2 The RC of the oyster polypeptide obtained in Example 5 and Comparative Examples 1-3 after chorioallantoic membrane vascular assay (CAMVA) 50 Comparison chart.
[0029] Figure 3 This is a comparison chart of the free radical scavenging and reducing abilities of the oyster polypeptides obtained in Example 5 and Comparative Examples 1-3. DETAILED DESCRIPTION
[0030] The present invention will be further explained below in conjunction with specific embodiments.
[0031] Example 1
[0032] A method for preparing marine shellfish polypeptides comprises the following steps:
[0033] S1. Clean the scallops, add them into a cooking pot, steam them at 100°C for 10 min under normal pressure, remove the shells and internal organs, send them into a pulping machine for pulping, pass through an 80-mesh sieve, freeze them at -40°C for 1 h, and adjust the system absolute pressure to 70 Pa;
[0034] Then, the frozen ground material was subjected to microwave treatment at a microwave power of 100 W for 5 seconds, and then the microwave treatment was suspended, and the material temperature was cooled to -40°C again, and the above operation was repeated until the actual microwave treatment time reached 5 minutes.
[0035] The mixture was then heated to room temperature and the water content was adjusted to 20% to obtain a slurry.
[0036] S2. Add 200 g of water to 50 g of slurry, let stand at room temperature for 5 h, adjust the pH value of the system to 6-6.5, add 1 g of complex enzyme a, hydrolyze at 40 ° C for 3 h, heat to 90 ° C, keep warm for 5 min, cool to room temperature, and obtain enzymolysis material.
[0037] The compound enzyme a is composed of Aspergillus oryzae protease, papain, lipase and lecithin in a mass ratio of 1:2:1:1. In the compound enzyme a, the specific activity of Aspergillus oryzae protease is 7000U / g, the specific activity of papain is 6000U / g, and the specific activity of lipase is 5000U / g.
[0038] S3. Adjust the pH value of the system of 280g enzymatic hydrolysis material to 7.8-8.2, add 1g of complex enzyme b, enzymolyze at a temperature of 50°C for 1h, raise the temperature to 90°C, keep warm for 10min, cool to room temperature, and centrifuge to remove the precipitate; precipitate the hydrolyzate with ethanol, and centrifuge again to remove the precipitate; separate through a filter membrane with a pore size of 30nm, collect the retained product, and spray dry (the inlet air temperature is 200°C).
[0039] Compound enzyme b is composed of Novo protease, trypsin and blue copper peptide in a mass ratio of 1:1:0.01. In compound enzyme b, the specific activity of Novo protease is 8000U / g, and the specific activity of trypsin is 6000U / g.
[0040] Example 2
[0041] A method for preparing marine shellfish polypeptides comprises the following steps:
[0042] S1. Clean the pearl oysters, add them into a cooking pot, steam them at 105°C for 20 min at normal pressure, remove the shells and internal organs, send them into a pulping machine for pulping, pass through an 80-mesh sieve, freeze them at -60°C for 4 h, and adjust the system absolute pressure to 80 Pa;
[0043] Then, the frozen ground material was subjected to microwave treatment at a microwave power of 200 W for 10 seconds, and then the microwave treatment was suspended, and the material temperature was cooled to -60°C again, and the above operation was repeated until the actual microwave treatment time reached 10 minutes.
[0044] The mixture was then heated to room temperature and the water content was adjusted to 30% to obtain a slurry.
[0045] S2. Add 300 g of water to 100 g of slurry, let stand at room temperature for 10 h, adjust the pH value of the system to 6-6.5, add 2 g of complex enzyme a, hydrolyze at 50 ° C for 6 h, heat to 95 ° C, keep warm for 10 min, cool to room temperature, and obtain enzymolysis material.
[0046] The compound enzyme a is composed of Aspergillus oryzae protease, papain, lipase and lecithin in a mass ratio of 3:6:5:2. In the compound enzyme a, the specific activity of Aspergillus oryzae protease is 9000U / g, the specific activity of papain is 8000U / g, and the specific activity of lipase is 9000U / g.
[0047] S3. Adjust the pH value of the system of 400g enzymatic hydrolysis material to 7.8-8.2, add 2g of complex enzyme b, enzymolyze at 60℃ for 1-2h, raise the temperature to 100℃, keep warm for 20min, cool to room temperature, and centrifuge to remove the precipitate; precipitate the hydrolyzate with ethanol, centrifuge again to remove the precipitate; separate through a filter membrane with a pore size of 30nm, collect the retained product, and spray dry (the inlet air temperature is 210℃).
[0048] Compound enzyme b is composed of Novo protease, trypsin and blue copper peptide in a mass ratio of 2:3:0.1. In compound enzyme b, the specific activity of Novo protease is 14000U / g, and the specific activity of trypsin is 10000U / g.
[0049] Example 3
[0050] A method for preparing marine shellfish polypeptides comprises the following steps:
[0051] S1. Clean the four-cornered clams, add them into a cooking pot, steam them at 101°C for 18 min under normal pressure, remove the shells and internal organs, send them into a pulping machine for pulping, pass through an 80-mesh sieve, freeze them at -45°C for 3 h, and adjust the system absolute pressure to 73 Pa;
[0052] Then, the frozen ground material was subjected to microwave treatment at a microwave power of 180 W for 7 seconds, and then the microwave treatment was suspended, and the material temperature was cooled to -45°C again, and the above operation was repeated until the actual microwave treatment time reached 9 minutes.
[0053] The temperature was then raised to room temperature and the water content was adjusted to 22% to obtain a slurry.
[0054] S2. Add 220 g of water to 90 g of the slurry, let it stand at room temperature for 9 h, adjust the pH value of the system to 6-6.5, add 1.3 g of complex enzyme a, hydrolyze at 47 ° C for 4 h, heat to 94 ° C, keep warm for 7 min, cool to room temperature, and obtain enzymolysis material.
[0055] The compound enzyme a is composed of Aspergillus oryzae protease, papain, lipase and lecithin in a mass ratio of 2.5:3:4:1.3. In the compound enzyme a, the specific activity of Aspergillus oryzae protease is 8500U / g, the specific activity of papain is 6500U / g, and the specific activity of lipase is 8000U / g.
[0056] S3. Adjust the pH value of the system of 300g enzymatic hydrolysis material to 7.8-8.2, add 1.7g complex enzyme b, enzymolyze at a temperature of 52°C for 100min, raise the temperature to 92°C, keep warm for 18min, cool to room temperature, and centrifuge to remove the precipitate; precipitate the hydrolyzate with ethanol, centrifuge again to remove the precipitate; separate through a filter membrane with a pore size of 30nm, collect the retained product, and spray dry (the inlet air temperature is 202°C).
[0057] Compound enzyme b is composed of Novo protease, trypsin and blue copper peptide in a mass ratio of 2:1:0.1. In compound enzyme b, the specific activity of Novo protease is 8000U / g, and the specific activity of trypsin is 10000U / g.
[0058] Example 4
[0059] A method for preparing marine shellfish polypeptides comprises the following steps:
[0060] S1. Clean the razor clams, add them into a cooking pot, steam them at 104°C for 12 min at normal pressure, remove the shells and internal organs, send them into a pulping machine for pulping, pass through an 80-mesh sieve, freeze them at -55°C for 2 h, and adjust the system absolute pressure to 77 Pa;
[0061] Then, the frozen ground material was subjected to microwave treatment at a microwave power of 120 W for 9 seconds, and then the microwave treatment was suspended, and the material temperature was cooled to -55°C again, and the above operation was repeated until the actual microwave treatment time reached 7 minutes.
[0062] The temperature was then raised to room temperature and the water content was adjusted to 28% to obtain a slurry.
[0063] S2. Add 280 g of water to 70 g of the slurry, let it stand at room temperature for 7 h, adjust the pH value of the system to 6-6.5, add 1.7 g of complex enzyme a, hydrolyze at 43 ° C for 5 h, heat to 91 ° C, keep warm for 9 min, cool to room temperature, and obtain enzymolysis material.
[0064] The compound enzyme a is composed of Aspergillus oryzae protease, papain, lipase and lecithin in a mass ratio of 1.5:5:2:1.7. In the compound enzyme a, the specific activity of Aspergillus oryzae protease is 7500U / g, the specific activity of papain is 7500U / g, and the specific activity of lipase is 6000U / g.
[0065] S3. Adjust the pH value of the system of 360g enzymatic hydrolysis material to 7.8-8.2, add 1.3g complex enzyme b, enzymolyze at a temperature of 58°C for 80min, raise the temperature to 98°C, keep warm for 12min, cool to room temperature, and centrifuge to remove the precipitate; precipitate the hydrolyzate with ethanol, and centrifuge again to remove the precipitate; separate through a filter membrane with a pore size of 30nm, collect the retained product, and spray dry (the inlet air temperature is 208°C).
[0066] Compound enzyme b is composed of Novo protease, trypsin and blue copper peptide in a mass ratio of 1:3:0.01. In compound enzyme b, the specific activity of Novo protease is 14000U / g, and the specific activity of trypsin is 6000U / g.
[0067] Example 5
[0068] A method for preparing marine shellfish polypeptides comprises the following steps:
[0069] S1. Clean the oysters, add them into a cooking pot, steam them at 103°C for 15 min at normal pressure, remove the shells and internal organs, send them into a pulping machine for pulping, pass through an 80-mesh sieve, freeze them at -50°C for 2.5 h, and adjust the system absolute pressure to 75 Pa;
[0070] Then, the frozen ground material was subjected to microwave treatment at a microwave power of 150 W for 8 seconds, and then the microwave treatment was suspended, and the material temperature was cooled to -50°C again, and the above operation was repeated until the actual microwave treatment time reached 8 minutes.
[0071] The mixture was then heated to room temperature and the water content was adjusted to 25% to obtain a slurry.
[0072] S2. Add 250 g of water to 80 g of the slurry, let it stand at room temperature for 8 h, adjust the pH value of the system to 6-6.5, add 1.5 g of complex enzyme a, hydrolyze at 45 ° C for 4.5 h, heat to 93 ° C, keep warm for 8 min, cool to room temperature, and obtain enzymolysis material.
[0073] The compound enzyme a is composed of Aspergillus oryzae protease, papain, lipase and lecithin in a mass ratio of 2:4:3:1.5. In the compound enzyme a, the specific activity of Aspergillus oryzae protease is 8000U / g, the specific activity of papain is 7000U / g, and the specific activity of lipase is 7000U / g.
[0074] S3. Adjust the pH value of the system of 320g enzymatic hydrolysis material to 7.8-8.2, add 1.5g complex enzyme b, enzymolyze at a temperature of 55°C for 90min, raise the temperature to 95°C, keep warm for 15min, cool to room temperature, and centrifuge to remove the precipitate; precipitate the hydrolyzate with ethanol, and centrifuge again to remove the precipitate; separate with a filter membrane with a pore size of 30nm, collect the retained product, and spray dry (the inlet air temperature is 205°C).
[0075] Compound enzyme b is composed of Novo protease, trypsin and blue copper peptide in a mass ratio of 1.5:2:0.05. In compound enzyme b, the specific activity of Novo protease is 11000U / g, and the specific activity of trypsin is 8000U / g.
[0076] Comparative Example 1
[0077] A method for preparing marine shellfish polypeptides comprises the following steps:
[0078] S1. Clean the oysters, add them into a cooking pot, steam them at 103°C for 15 min at normal pressure, remove the shells and internal organs, send them into a pulping machine for pulping, pass through an 80-mesh sieve, freeze them at -50°C for 2.5 h, and adjust the absolute pressure of the system to 75 Pa; then warm them to room temperature and adjust the water content to 25% to obtain a slurry.
[0079] S2. Add 250 g of water to 80 g of the slurry, let it stand at room temperature for 8 h, adjust the pH value of the system to 6-6.5, add 1.5 g of complex enzyme a, hydrolyze at 45 ° C for 4.5 h, heat to 93 ° C, keep warm for 8 min, cool to room temperature, and obtain enzymolysis material.
[0080] The compound enzyme a is composed of Aspergillus oryzae protease, papain, lipase and lecithin in a mass ratio of 2:4:3:1.5. In the compound enzyme a, the specific activity of Aspergillus oryzae protease is 8000U / g, the specific activity of papain is 7000U / g, and the specific activity of lipase is 7000U / g.
[0081] S3. Adjust the pH value of the system of 320g enzymatic hydrolysis material to 7.8-8.2, add 1.5g complex enzyme b, enzymolyze at a temperature of 55°C for 90min, raise the temperature to 95°C, keep warm for 15min, cool to room temperature, and centrifuge to remove the precipitate; precipitate the hydrolyzate with ethanol, and centrifuge again to remove the precipitate; separate with a filter membrane with a pore size of 30nm, collect the retained product, and spray dry (the inlet air temperature is 205°C).
[0082] Compound enzyme b is composed of Novo protease, trypsin and blue copper peptide in a mass ratio of 1.5:2:0.05. In compound enzyme b, the specific activity of Novo protease is 11000U / g, and the specific activity of trypsin is 8000U / g.
[0083] Comparative Example 2
[0084] A method for preparing marine shellfish polypeptides comprises the following steps:
[0085] S1. Clean the oysters, add them into a cooking pot, steam them at 103°C for 15 minutes at normal pressure, remove the shells and internal organs, send them into a pulper for grinding, pass through an 80-mesh sieve, freeze them at -50°C for 2.5 hours, and adjust the absolute pressure of the system to 75 Pa; then treat them with a low-temperature microwave for 8 minutes at a microwave power of 150 W; then warm them to room temperature and adjust the water content to 25% to obtain a slurry.
[0086] S2. Add 250 g of water to 80 g of the slurry, let it stand at room temperature for 8 h, adjust the pH value of the system to 6-6.5, add 1.5 g of complex enzyme a, hydrolyze at 45 ° C for 4.5 h, heat to 93 ° C, keep warm for 8 min, cool to room temperature, and obtain enzymolysis material.
[0087] The compound enzyme a is composed of Aspergillus oryzae protease, papain, lipase and lecithin in a mass ratio of 2:4:3:1.5. In the compound enzyme a, the specific activity of Aspergillus oryzae protease is 8000U / g, the specific activity of papain is 7000U / g, and the specific activity of lipase is 7000U / g.
[0088] S3. Adjust the pH value of the system of 320g enzymatic hydrolysis material to 7.8-8.2, add 1.5g complex enzyme b, enzymolyze at a temperature of 55°C for 90min, raise the temperature to 95°C, keep warm for 15min, cool to room temperature, and centrifuge to remove the precipitate; precipitate the hydrolyzate with ethanol, and centrifuge again to remove the precipitate; separate with a filter membrane with a pore size of 30nm, collect the retained product, and spray dry (the inlet air temperature is 205°C).
[0089] Compound enzyme b is composed of Novo protease, trypsin and blue copper peptide in a mass ratio of 1.5:2:0.05. In compound enzyme b, the specific activity of Novo protease is 11000U / g, and the specific activity of trypsin is 8000U / g.
[0090] Comparative Example 3
[0091] A method for preparing marine shellfish polypeptides comprises the following steps:
[0092] S1. Clean the oysters, add them into a cooking pot, steam them at 103°C for 15 min at normal pressure, remove the shells and internal organs, send them into a pulping machine for pulping, pass through an 80-mesh sieve, freeze them at -50°C for 2.5 h, and adjust the system absolute pressure to 75 Pa;
[0093] Then, the frozen ground material was subjected to microwave treatment at a microwave power of 150 W for 8 seconds, and then the microwave treatment was suspended, and the material temperature was cooled to -50°C again, and the above operation was repeated until the actual microwave treatment time reached 8 minutes.
[0094] The mixture was then heated to room temperature and the water content was adjusted to 25% to obtain a slurry.
[0095] S2. Add 250 g of water to 80 g of the slurry, let it stand at room temperature for 8 h, adjust the pH value of the system to 6-6.5, add 1.5 g of complex enzyme a, hydrolyze at 45 ° C for 4.5 h, heat to 93 ° C, keep warm for 8 min, cool to room temperature, and obtain enzymolysis material.
[0096] The compound enzyme a is composed of Aspergillus oryzae protease, papain, lipase and lecithin in a mass ratio of 2:4:3:1.5. In the compound enzyme a, the specific activity of Aspergillus oryzae protease is 8000U / g, the specific activity of papain is 7000U / g, and the specific activity of lipase is 7000U / g.
[0097] S3. Adjust the pH value of the system of 320g enzymatic hydrolysis material to 7.8-8.2, add 1.5g complex enzyme b, enzymolyze at a temperature of 55°C for 90min, raise the temperature to 95°C, keep warm for 15min, cool to room temperature, and centrifuge to remove the precipitate; precipitate the hydrolyzate with ethanol, and centrifuge again to remove the precipitate; separate with a filter membrane with a pore size of 30nm, collect the retained product, and spray dry (the inlet air temperature is 205°C).
[0098] Compound enzyme b is composed of Novo protease and trypsin at a mass ratio of 1.5:2. In compound enzyme b, the specific activity of Novo protease is 11000U / g, and the specific activity of trypsin is 8000U / g.
[0099] With reference to GB / T 22729-2008 "Marine Fish Oligopeptide Powder", the molecular weight distribution of the oyster polypeptides obtained in Example 5 and Comparative Examples 1-3 was detected by high performance liquid chromatography, and the percentage of the peak area below 1 kDa and below 0.5 kDa was plotted as a bar graph for comparison.
[0100] like Figure 1 As shown, in the oyster polypeptides obtained in Example 5, the percentage of polypeptides below 1 kDa and below 0.5 kDa is the highest, which is better than that of Comparative Examples 1-3 (P < 0.05). In the oyster polypeptides obtained in Comparative Example 3, the percentage of polypeptides below 1 kDa is higher than that of Comparative Examples 1-2 (P < 0.05), but the percentage of polypeptides below 0.5 kDa is less than that of Comparative Examples 1-2 (P < 0.05), which confirms that the blue copper peptide can assist Novo protease and trypsin, enhance the enzymatic activity of complex enzyme b, and further degrade the enzymatic material.
[0101] The chorioallantoic membrane vascular test (CAMVA) was used to verify the irritation of the oyster polypeptides obtained in Example 5 and Comparative Examples 1-3, as follows:
[0102] The incubation stage of fertilized chicken eggs was carried out under normal temperature and humidity conditions, and the number of experimental chicken embryos in each group was 15. 0.1mol / L NaOH solution was used as a positive control. A silicone rubber ring was placed on the chorioallantoic membrane of each chicken embryo, and 35mg of each group of polypeptide samples were added. The skylight was sealed with transparent tape and placed in a 37.5±0.5℃ incubator. 40 minutes after adding the sample, the chicken embryo was taken out of the incubator, and the transparent tape and the eggshell around the skylight were removed for observation. Use a magnifying glass lamp to observe the condition of the silicone rubber ring and the chorioallantoic membrane of the chicken embryo. Chicken embryos with abnormal conditions outside the ring will be eliminated, and the RC 50 Conduct skin irritation test.
[0103] In the CAMVA trial classification, RC 50 <1.0%, the sample is judged as mild stimulation or above; RC 50 1.0-20.0%, the sample is judged as slightly irritated; RC 50 >20.0%, the sample is judged to be non-irritating.
[0104] like Figure 2 As shown, the positive control RC 50 <1.0%, with strong stimulation; while the RC of comparative example 3 50 <2.0%, indicating that the oyster polypeptide obtained in Comparative Example 3 is irritating. Example 5 and Comparative Example 1-2RC 50 >20.0%, indicating that the oyster polypeptides obtained by the three methods are non-irritating and have good safety.
[0105] The free radical scavenging ability of the oyster polypeptides obtained in Example 5 and Comparative Examples 1-3 was further determined by the ABTS method (the concentration of each group was 0.5 mg / mL), with Trolox as the standard. The reducing ability of the oyster polypeptides obtained in Example 5 and Comparative Examples 1-3 was determined by the FRAP method (the concentration of each group was 6 mg / mL).
[0106] like Figure 3 As shown, the free radical scavenging ability and reducing ability of the oyster polypeptide obtained in Example 5 are both the highest, which are better than those of Comparative Examples 1-3 (P<0.05).
[0107] The applicant believes that this is because the present invention adopts freezing treatment in combination with low-temperature vacuum microwave treatment, which can not only better retain the biological activity of shellfish, but also can be coordinated with subsequent fermentation processes, not only improve the hydrolysis degree of protein polypeptides, significantly increase polypeptide yield, but also significantly shorten the enzymolysis time and reduce costs. In the enzymolysis process, the present invention performs enzymolysis in acidic and alkaline environments in turn, and controls the enzymolysis parameters. The obtained shellfish polypeptides are not only of high purity, but also of extremely high biological activity, and have significant antioxidant activity, strong reducing ability, and excellent ability to scavenge free radicals. The present invention further adopts blue copper peptides in combination with Novo protease and trypsin, which not only greatly enhances enzyme activity, greatly increases the proportion of polypeptides with a molecular weight of less than 500 Daltons, and significantly reduces the irritation effect on the skin, and greatly enhances the safety of the product.
[0108] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A method for preparing a marine shellfish polypeptide, characterized in that: The steps include: S1. Clean the marine shellfish, steam them with water for 10-20 minutes, remove the shells and internal organs, grind them, sieve them, freeze them at -40 to -60°C for 1-4 hours, adjust the absolute pressure of the system to 70-80Pa, perform microwave treatment under the heat and pressure maintenance state, the effective microwave time is 5-10 minutes, warm them to room temperature, adjust the water content to 20-30%, and obtain a slurry; S2. Add water to the slurry, let it stand at room temperature for 5-10 hours, adjust the pH value of the system to 6-6.5, add complex enzyme a, enzymolyze at 40-50℃ for 3-6 hours, heat to 90-95℃, keep warm for 5-10 minutes, and cool to room temperature to obtain enzymolysis material; S3. Adjust the pH value of the enzymatic hydrolysis system to 7.8-8.2, add complex enzyme b, perform enzymatic hydrolysis at 50-60℃ for 1-2h, raise the temperature to 90-100℃, keep warm for 10-20min, cool to room temperature, and remove the precipitate by centrifugation; precipitate the hydrolyzate with ethanol, and centrifuge again to remove the precipitate; separate through filter membrane, collect the retained product, and spray dry.
2. The method for preparing marine shellfish polypeptide according to claim 1, characterized in that: In S1, the marine shellfish is at least one of scallops, pearl shells, oysters, clams, and razor clams.
3. The method for preparing marine shellfish polypeptide according to claim 1, characterized in that: In S1, saturated steam with a temperature of 100-105°C is used for steaming.
4. The method for preparing marine shellfish polypeptide according to claim 1, characterized in that: In S1, the microwave power is 100-200W, and the specific operation of the microwave treatment is as follows: after the frozen ground material is microwaved for 5-10s, the microwave treatment is suspended, the material temperature is cooled to -40 to -60°C again, and then the above operation is repeated until the actual microwave treatment time reaches 5-10min.
5. The method for preparing marine shellfish polypeptide according to claim 1, characterized in that: In S2, the complex enzyme a includes: Aspergillus oryzae protease, papain, lipase, and lecithin; wherein the mass ratio of Aspergillus oryzae protease, papain, lipase, and lecithin is 1-3:2-6:1-5:1-2.
6. The method for preparing marine shellfish polypeptide according to claim 5, characterized in that: In S2, in the complex enzyme a, the specific activity of Aspergillus oryzae protease is 7000-9000 U / g, the specific activity of papain is 6000-8000 U / g, and the specific activity of lipase is 5000-9000 U / g.
7. The method for preparing marine shellfish polypeptide according to claim 5, characterized in that: In S2, the mass ratio of slurry, water and complex enzyme a is 50-100:200-300:1-2.
8. The method for preparing marine shellfish polypeptide according to claim 1, characterized in that: In S3, the complex enzyme b includes: Novo protease, trypsin, and blue copper peptide; wherein the mass ratio of Novo protease, trypsin, and blue copper peptide is 1-2:1-3:0.01-0.
1.
9. The method for preparing marine shellfish polypeptide according to claim 1, characterized in that: In S3, the pore size of the filter membrane is 30 nm; the inlet air temperature of the spray drying is 200-210°C.
10. A marine shellfish polypeptide, characterized in that: The marine shellfish polypeptide is prepared by the method for preparing the marine shellfish polypeptide according to any one of claims 1 to 9.