Small molecular selenium-rich walnut peptide and preparation method thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a micromolecular selenium-rich walnut peptide and a preparation method thereof. The preparation method of the micromolecular selenium-rich walnut peptide sequentially comprises the following steps: selecting selenium-rich walnuts, crushing walnut kernels, performing enzymolysis twice, filtering, purifying, concentrating and drying, packaging, detecting and warehousing. According to the method disclosed by the invention, the selenium-rich walnut protein is decomposed into small molecule peptides by an enzymolysis technology with the extraction rate of greater than or equal to 60% and is obviously higher than the extraction rate (less than or equal to 55%) of a traditional acid method through an alkali-solution and acid-isolation method, so that the solubility and bioavailability of the selenium-rich walnut protein are obviously improved. The double-enzyme step-by-step enzymolysis improves the peptide yield to 75%-80%, not only improves the absorption efficiency of the selenium-rich walnut protein, but also endows the selenium-rich walnut protein with unique functional activity, such as enhancement of antioxidant ability, promotion of immune regulation, improvement of mineral absorption of human bodies and the like. In addition, the introduction of the selenium-rich element further enhances the biological activity of the product, so that the product is more excellent in the aspects of oxidation resistance, immunity improvement and health maintenance.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a small-molecule selenium-enriched walnut peptide and a preparation method thereof. Background Art

[0002] Walnut is a nut with high nutritional value, rich in protein, unsaturated fatty acids, vitamins (such as vitamin E), minerals (such as zinc, magnesium, potassium) and dietary fiber. Walnuts have a high protein content, and their main component is high-quality plant protein, which is rich in essential amino acids and beneficial to human health. The oil content in walnuts can reach 60%-70%, of which the content of unsaturated fatty acids is more than 90%. The protein content of selenium-rich walnuts is 14%-17%, and the digestibility is 87.2%. Although traditional walnut products (such as walnut kernels, walnut oil, and walnut powder) have high nutritional value, their absorption and utilization rates are low due to their large protein molecular weight. Selenium is an essential trace element for the human body and has important biological functions, such as anti-oxidation, improving immunity, protecting the cardiovascular system, and preventing certain cancers. Selenium deficiency may lead to decreased immune function, cardiovascular disease, and thyroid problems. The enzymatic hydrolysis technology can decompose selenium-rich walnut protein into small molecule peptides, which can not only improve its solubility and bioavailability, but also give it special functional activities (such as anti-oxidation, immunomodulation, etc.).

[0003] In recent years, walnuts have been increasingly used in daily life and industrial production, but they also face some obvious problems. For example, the existing enzymatic hydrolysis process is single, resulting in a wide distribution of peptide molecular weight (1000-10000Da), and a bioavailability of less than 50%. There are dozens of walnut oil manufacturers in my country, but in the production process, a large amount of defatted walnut residue is used as feed, fertilizer or directly discarded, resulting in resource waste and environmental pollution, and low added value. Defatted walnut residue is a by-product of walnut oil extraction. According to the 2021 data from the Journal of the Chinese Cereals and Oils Association, the protein content of defatted walnut residue is 32.5%-47.8%, which is mainly composed of albumin, globulin, alcohol-soluble protein and glutenin, accounting for 6.81%, 17.57%, 5.33% and 70.11% of the total selenium-rich walnut protein, respectively.

[0004] Studies have shown that the protein ingested by humans is not absorbed mainly in the form of amino acids, but in the form of peptides. Some peptides not only provide necessary nutrition for human growth and development, but also have a variety of physiological functions, such as promoting the absorption of minerals, antibacterial and antiviral, improving immunity, anti-oxidation, lowering cholesterol, anti-cancer, and removing free radicals and anti-aging. Domestic and foreign studies have also confirmed that selenium-rich walnut protein can be obtained by bio-enzymatic hydrolysis to obtain walnut peptides, which have significant immunomodulatory, antioxidant and anti-cancer activities.

[0005] Based on this, we proposed a small molecule selenium-rich walnut peptide and its preparation method, hoping to solve the shortcomings of the existing technology. Summary of the invention

[0006] The purpose of the present invention is to provide a small molecule selenium-enriched walnut peptide and a preparation method thereof in view of the existing problems.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for preparing small molecule selenium-rich walnut peptides, comprising the following steps in sequence: selecting selenium-rich walnuts and crushing walnut kernels, twice enzymolysis, filtering, purifying, concentrating and drying, packaging and testing for storage;

[0009] The two enzymatic hydrolysis processes are specifically as follows: adding alkaline protease with a mass of 1%-3% of selenium-rich walnut protein to the selenium-rich walnut protein extract extracted in the protein extraction process for a first enzymatic hydrolysis, and the pH value of the first enzymatic hydrolysis reaction is 8-9; after the first enzymatic hydrolysis is completed, the enzyme is inactivated; the enzyme hydrolyzate that has been inactivated is cooled, and a neutral protease equivalent to the mass of 1%-3% of the selenium-rich walnut protein is added for a second enzymatic hydrolysis, and the pH value of the second enzymatic hydrolysis reaction is 6-7; after the enzymatic hydrolysis is completed, the enzyme is inactivated.

[0010] Further preferably, the preparation process of the selenium-rich walnut protein extract is as follows: selenium-rich walnuts are squeezed to obtain defatted walnut residues, the defatted walnut residues are crushed, deionized water is added in an amount of 5 to 8 times the mass of the walnut residues, alkali is added to adjust the pH to 8 to 9, and after soaking for 1 h to 2 h, the lower precipitate is removed by centrifugation; acid is added to adjust the pH of the centrifuge to 4, the centrifuge is separated after standing for 1 h to 3 h, the lower precipitate is removed and washed with water until neutral, to obtain a selenium-rich walnut protein extract.

[0011] Small molecule peptides are prepared by making full use of the by-product of walnut oil extraction - defatted walnut residue, which is low-cost. Selenium-rich walnut protein is extracted using alkali dissolution and acid precipitation methods, with an extraction rate of more than 67%.

[0012] Further preferably, during the pH adjustment, the base added is a sodium hydroxide solution, and the acid added is a citric acid solution.

[0013] Further preferably, the filtration process is: adding activated carbon to the enzymatic hydrolyzate after two enzymatic hydrolysis, the amount of activated carbon added is 2%-4% of the mass of selenium-rich walnut protein, and heating to 100°C, maintaining for 5min-10min, and then cooling to 80°C and standing for 0.5h-2h to remove the color and odor in the enzymatic hydrolyzate.

[0014] By using the enzymatic hydrolysis technology of the present invention, selenium-enriched walnut protein is decomposed into small molecule peptides, which significantly improves the solubility and bioavailability of selenium-enriched walnut protein. The absorption efficiency of selenium-enriched walnut protein is improved, and unique functional activity is given to it to ensure that each enzyme can play an enzymatic hydrolysis role under the most suitable conditions, so that the content of small molecule selenium-enriched walnut peptides is high.

[0015] Further preferably, the purification and concentration process is: centrifuging the filtered small molecule selenium-rich walnut peptide solution to remove solid residues, filtering the obtained clear liquid with an ultrafiltration membrane with a molecular weight of 2000 Daltons-5000 Daltons to remove large molecular impurities in the clear liquid, and then using a nanofiltration membrane with a molecular weight of 200 Daltons to remove inorganic salts and impurities, and concentrating the filtered clear liquid to a solid content of 20%-30%, thereby obtaining a small molecule selenium-rich walnut peptide solution.

[0016] By adopting a centrifugal coordinated multi-stage membrane separation system to purify small molecule selenium-rich walnut peptides, the impurity removal, desalination and concentration effects are good. Low-temperature nanofiltration concentration ensures the activity of the product. At the same time, the product has a low salt content and is suitable for large-scale industrial production.

[0017] Further preferably, the membrane pressure of the ultrafiltration membrane is 0.6MPa-1.5MPa, and the ultrafiltration temperature is 25°C-35°C; the membrane pressure of the nanofiltration membrane is 1.5MPa-2.5MPa, and the nanofiltration temperature is 25°C-40°C.

[0018] Further preferably, the drying process is: spray drying the purified and concentrated small molecule selenium-rich walnut peptide solution, the outlet air temperature of the spray drying is 70℃-90℃, the inlet air temperature is 150℃-180℃, and the small molecule selenium-rich walnut peptide powder is obtained after drying.

[0019] Further preferably, the enzyme inactivation treatment process is: heating the primary enzymatic hydrolysis solution or the secondary enzymatic hydrolysis solution obtained after the primary enzymatic hydrolysis or the secondary enzymatic hydrolysis to 80° C. and maintaining the temperature for 10 min-20 min for enzyme inactivation treatment.

[0020] More preferably, the primary enzymolysis temperature is 30°C-60°C, and the time is 3h-6h; the secondary enzymolysis temperature is 40°C-60°C, and the time is 3h-6h.

[0021] A small molecule selenium-rich walnut peptide, wherein the color of the small molecule selenium-rich walnut peptide is graded Y10-Y20 according to the GB / T22460-2008 colorimetric standard.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. The small molecule selenium-enriched walnut peptide in the present invention not only retains the excellent functions of walnut peptide, but also enhances its biological activity through selenium-enriched technology. The small molecule selenium-enriched walnut peptide shows more significant effects in improving human immunity, anti-oxidation and other physiological functions, providing a higher value-added and environmentally friendly innovative solution for the functional food field.

[0024] 2. The present invention uses an enzymatic hydrolysis technology with an alkali-soluble acid precipitation extraction rate of ≥60%, which is significantly higher than the traditional acid extraction rate (≤55%), to decompose selenium-rich walnut protein into small molecule peptides, significantly improving the solubility and bioavailability of selenium-rich walnut protein. This double-enzyme step-by-step enzymatic hydrolysis increases the peptide yield to 75%-80%, which is 20%-30% higher than the single enzyme method. It not only improves the absorption efficiency of selenium-rich walnut protein, but also gives it unique functional activities, such as enhancing antioxidant capacity, promoting immune regulation and improving human mineral absorption. In addition, the introduction of selenium-rich elements further enhances the biological activity of the product, making it more excellent in anti-oxidation, immunity enhancement and health maintenance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure is a flow chart for the preparation of the small molecule selenium-enriched walnut peptide of the present invention. DETAILED DESCRIPTION

[0026] In order to further explain the present invention, it is described below in conjunction with the following specific embodiments.

[0027] Example 1

[0028] A method for preparing a small molecule selenium-enriched walnut peptide comprises the following steps:

[0029] 1) Protein extraction: selenium-rich walnuts are selected, and the walnuts are defatted to obtain defatted walnut residues. After the defatted selenium-rich walnut residues are crushed, deionized water 5 times the mass of the walnut residues is added, and sodium hydroxide solution is added to adjust the pH to 9. After soaking for 2 hours, the lower layer of precipitation is removed by a centrifuge; citric acid solution is added to adjust the pH of the centrifuge to 4, and the centrifuge is performed after standing for 3 hours, and the lower layer of precipitation is removed and washed with water until neutral to obtain a selenium-rich walnut protein extract. The quality of the selenium-rich walnut protein in the selenium-rich walnut protein extract is detected by a Baume meter or a Kjeldahl nitrogen determination method;

[0030] This embodiment makes full use of the byproduct of walnut oil extraction, defatted walnut residue, to prepare small molecule peptides, which is low in cost. The alkali dissolution and acid precipitation method is used to extract selenium-rich walnut protein, and the extraction rate is greater than 67%;

[0031] 2) Two enzymolysis: The two enzymolysis processes are to add an alkaline protease equivalent to 3% of the mass of selenium-rich walnut protein to the selenium-rich walnut protein extract for one enzymolysis, the temperature of the first enzymolysis is 50°C, the pH value of the first enzymolysis is 9, and the time of the first enzymolysis is 3h; after the first enzymolysis is completed, the temperature of the first enzymolysis solution is raised to 40°C-60°C, and the temperature is kept constant for 10min to inactivate the enzyme, and the residual enzyme activity is detected after the enzyme inactivation treatment. ≤5U / g (refer to GB 5009.224-2016); the first enzymolysis solution after the enzyme inactivation treatment is cooled to 20°C, and a neutral protease equivalent to 3% of the mass of selenium-rich walnut protein is added for a second enzymolysis, the temperature of the second enzymolysis is 45°C, the pH value of the second enzymolysis reaction is 7, and the time of the second enzymolysis is 3h; after the second enzymolysis is completed, the temperature of the second enzymolysis solution is raised to 80°C, and the temperature is kept constant for 20min to inactivate the enzyme, and the residual enzyme activity is detected after the enzyme inactivation treatment. ≤5U / g (refer to GB 5009.224-2016);

[0032] The enzymatic hydrolysis technology used in the present invention decomposes selenium-rich walnut protein into small molecule peptides, which significantly improves the solubility and bioavailability of selenium-rich walnut protein. It improves the absorption efficiency of selenium-rich walnut protein and also gives it unique functional activity to ensure that each enzyme can play an enzymatic hydrolysis role under the most suitable conditions, so that the content of small molecule selenium-rich walnut peptides obtained is relatively high;

[0033] 3) Filtration: The filtration process is to add activated carbon to the enzymatic hydrolyzate after two enzymatic hydrolysis, the amount of activated carbon added is 2% of the mass of selenium-enriched walnut protein, and heat it to 100°C, keep it for 8 minutes, then cool it to 80°C and let it stand for 2 hours to remove the color and odor in the enzymatic hydrolyzate;

[0034] 4) Purification and concentration: The filtered small molecule selenium-enriched walnut peptide solution is centrifuged to remove solid residues, and the obtained clear liquid is filtered with an ultrafiltration membrane with a molecular weight of 3500 Daltons to remove macromolecular impurities in the clear liquid, the membrane pressure of the ultrafiltration membrane is 0.6 MPa, and the ultrafiltration temperature is 30°C; then, a nanofiltration membrane with a molecular weight of 200 Daltons is used to remove inorganic salts and small molecule impurities, the membrane pressure of the nanofiltration membrane is 1.5 MPa, and the nanofiltration temperature is 30°C; the filtered clear liquid is concentrated to a solid content of 30%, that is, a small molecule selenium-enriched walnut peptide solution is obtained;

[0035] The centrifugal synergistic multi-stage membrane separation system is used to purify small molecule selenium-rich walnut peptides, which has good impurity removal, desalination and concentration effects. Low-temperature nanofiltration concentration ensures the activity of the product. At the same time, the product has a low salt content and is suitable for large-scale industrial production.

[0036] 5) Drying: The purified and concentrated small molecule selenium-rich walnut peptide solution is spray-dried, and the outlet air temperature of the spray drying is 80° C. and the inlet air temperature is 160° C. After drying, the small molecule selenium-rich walnut peptide powder is obtained.

[0037] Example 2

[0038] A method for preparing a small molecule selenium-enriched walnut peptide comprises the following steps:

[0039] 1) Protein extraction: selenium-rich walnuts are selected, and the walnuts are defatted to obtain defatted walnut residues. After the defatted selenium-rich walnut residues are crushed, deionized water 8 times the mass of the walnut residues is added, and sodium hydroxide solution is added to adjust the pH to 8.5. After soaking for 1.5 hours, the lower layer of precipitation is removed by a centrifuge; citric acid solution is added to adjust the pH of the centrifuge to 4, and the centrifuge is separated after standing for 3 hours. The lower layer of precipitation is removed and washed with water until neutral to obtain a selenium-rich walnut protein extract, and the quality of the selenium-rich walnut protein in the selenium-rich walnut protein extract is detected by a Baume meter or a Kjeldahl nitrogen determination method;

[0040] This embodiment makes full use of the byproduct of walnut oil extraction, defatted walnut residue, to prepare small molecule peptides, which is low in cost. The alkali dissolution and acid precipitation method is used to extract selenium-rich walnut protein, and the extraction rate is greater than 67%;

[0041] 2) Two enzymolysis: The two enzymolysis processes are to add an alkaline protease equivalent to 3% of the mass of selenium-rich walnut protein to the selenium-rich walnut protein extract for one enzymolysis, the temperature of the first enzymolysis is 60°C, the pH value of the first enzymolysis is 9, and the time of the first enzymolysis is 3h; after the first enzymolysis is completed, the temperature of the first enzymolysis solution is raised to 40°C-60°C, and the temperature is kept constant for 10min to inactivate the enzyme, and the residual enzyme activity is detected after the inactivation treatment is ≤5U / g (refer to GB 5009.224-2016); the first enzymolysis solution after the enzyme inactivation treatment is cooled to 20°C, and a neutral protease equivalent to 1% of the mass of selenium-rich walnut protein is added for a second enzymolysis, the temperature of the second enzymolysis is 55°C, the pH value of the second enzymolysis reaction is 6.5, and the time of the second enzymolysis is 6h; after the second enzymolysis is completed, the temperature of the second enzymolysis solution is raised to 80°C, and the temperature is kept constant for 20min to inactivate the enzyme, and the residual enzyme activity is detected after the inactivation treatment is ≤5U / g (refer to GB 5009.224-2016);

[0042] 3) Filtration: The filtration process is to add activated carbon to the enzymatic hydrolyzate after two enzymatic hydrolysis, the amount of activated carbon added is 2% of the mass of selenium-enriched walnut protein, and heat it to 100°C, keep it for 5 minutes, then cool it to 80°C and let it stand for 1 hour to remove the color and odor in the enzymatic hydrolyzate;

[0043] 4) Purification and concentration: The filtered small molecule selenium-enriched walnut peptide solution is centrifuged to remove solid residues, and the obtained clear liquid is filtered with an ultrafiltration membrane with a molecular weight of 4000 Daltons to remove macromolecular impurities in the clear liquid, the membrane pressure of the ultrafiltration membrane is 0.8MPa, and the ultrafiltration temperature is 30°C; then, a nanofiltration membrane with a molecular weight of 200 Daltons is used to remove inorganic salts and small molecule impurities, the membrane pressure of the nanofiltration membrane is 2MPa, and the nanofiltration temperature is 30°C; the filtered clear liquid is concentrated to a solid content of 30%, that is, a small molecule selenium-enriched walnut peptide solution is obtained;

[0044] 5) Drying: The purified and concentrated small molecule selenium-rich walnut peptide solution is spray-dried, and the outlet air temperature of the spray drying is 70° C. and the inlet air temperature is 180° C. After drying, the small molecule selenium-rich walnut peptide powder is obtained.

[0045] Example 3

[0046] A method for preparing a small molecule selenium-enriched walnut peptide comprises the following steps:

[0047] 1) Protein extraction: selenium-rich walnuts are selected, and the walnuts are defatted to obtain defatted walnut residues. After the defatted selenium-rich walnut residues are crushed, deionized water 8 times the mass of the walnut residues is added, and sodium hydroxide solution is added to adjust the pH to 9. After soaking for 1 hour, the lower layer of precipitation is removed by a centrifuge; citric acid solution is added to adjust the pH of the centrifuge to 4, and the centrifuge is performed after standing for 3 hours, and the lower layer of precipitation is removed and washed with water until neutral to obtain a selenium-rich walnut protein extract. The quality of the selenium-rich walnut protein in the selenium-rich walnut protein extract is detected by a Baume meter or a Kjeldahl nitrogen determination method;

[0048] This embodiment makes full use of the byproduct of walnut oil extraction, defatted walnut residue, to prepare small molecule peptides, which is low in cost. The alkali dissolution and acid precipitation method is used to extract selenium-rich walnut protein, and the extraction rate is greater than 67%;

[0049] 2) Two enzymolysis: The two enzymolysis processes are to add an alkaline protease equivalent to 2% of the mass of selenium-rich walnut protein to the selenium-rich walnut protein extract for one enzymolysis, the temperature of the first enzymolysis is 40°C, the pH value of the first enzymolysis is 9, and the time of the first enzymolysis is 4h; after the first enzymolysis is completed, the temperature of the first enzymolysis solution is raised to 40°C-60°C, and the temperature is kept constant for 10min to inactivate the enzyme, and the residual enzyme activity is detected after the enzyme inactivation treatment. ≤5U / g (refer to GB 5009.224-2016); the first enzymolysis solution after the enzyme inactivation treatment is cooled to 20°C, and a neutral protease equivalent to 2% of the mass of selenium-rich walnut protein is added for a second enzymolysis, the temperature of the second enzymolysis is 45°C, the pH value of the second enzymolysis reaction is 6.5, and the time of the second enzymolysis is 6h; after the second enzymolysis is completed, the temperature of the second enzymolysis solution is raised to 80°C, and the temperature is kept constant for 20min to inactivate the enzyme, and the residual enzyme activity is detected after the enzyme inactivation treatment. ≤5U / g (refer to GB 5009.224-2016);

[0050] 3) Filtration: The filtration process is to add activated carbon to the enzymatic hydrolyzate after two enzymatic hydrolysis, the amount of activated carbon added is 3% of the mass of selenium-enriched walnut protein, and heat it to 100° C., keep it for 5 minutes, then cool it to 80° C. and let it stand for 1 hour to remove the color and odor in the enzymatic hydrolyzate;

[0051] 4) Purification and concentration: The filtered small molecule selenium-enriched walnut peptide solution is centrifuged to remove solid residues, and the obtained clear liquid is filtered with an ultrafiltration membrane with a molecular weight of 4000 Daltons to remove macromolecular impurities in the clear liquid, the membrane pressure of the ultrafiltration membrane is 0.8 MPa, and the ultrafiltration temperature is 25°C; then, a nanofiltration membrane with a molecular weight of 200 Daltons is used to remove inorganic salts and small molecule impurities, the membrane pressure of the nanofiltration membrane is 1.5 MPa, and the nanofiltration temperature is 25°C; the filtered clear liquid is concentrated to a solid content of 30%, that is, a small molecule selenium-enriched walnut peptide solution is obtained;

[0052] 5) Drying: The purified and concentrated small molecule selenium-rich walnut peptide solution is spray-dried, and the outlet air temperature of the spray drying is 80° C. and the inlet air temperature is 160° C. After drying, the small molecule selenium-rich walnut peptide powder is obtained.

[0053] Example 4

[0054] A method for preparing a small molecule selenium-enriched walnut peptide comprises the following steps:

[0055] 1) Protein extraction: selenium-rich walnuts are selected, and the walnuts are defatted to obtain defatted walnut residues. After the defatted selenium-rich walnut residues are crushed, deionized water 7 times the mass of the walnut residues is added, and sodium hydroxide solution is added to adjust the pH to 8.5. After soaking for 2 hours, the lower layer of precipitation is removed by a centrifuge; citric acid solution is added to adjust the pH of the centrifuge to 4, and the centrifuge is performed after standing for 3 hours, and the lower layer of precipitation is removed and washed with water until neutral to obtain a selenium-rich walnut protein extract. The quality of the selenium-rich walnut protein in the selenium-rich walnut protein extract is detected by a Baume meter or a Kjeldahl nitrogen determination method;

[0056] This embodiment makes full use of the byproduct of walnut oil extraction, defatted walnut residue, to prepare small molecule peptides, which is low in cost. The alkali dissolution and acid precipitation method is used to extract selenium-rich walnut protein, and the extraction rate is greater than 67%;

[0057] 2) Two enzymolysis: The two enzymolysis processes are to add an alkaline protease equivalent to 3% of the mass of selenium-rich walnut protein to the selenium-rich walnut protein extract for one enzymolysis, the temperature of the first enzymolysis is 50°C, the pH value of the first enzymolysis is 8.5, and the time of the first enzymolysis is 6h; after the first enzymolysis is completed, the temperature of the first enzymolysis solution is raised to 40°C-60°C, and the temperature is kept constant for 10min to inactivate the enzyme, and the residual enzyme activity is detected after the enzyme inactivation treatment. ≤5U / g (refer to GB 5009.224-2016); the first enzymolysis solution after the enzyme inactivation treatment is cooled to 20°C, and a neutral protease equivalent to 2.5% of the mass of selenium-rich walnut protein is added for a second enzymolysis, the temperature of the second enzymolysis is 40°C, the pH value of the second enzymolysis reaction is 6, and the time of the second enzymolysis is 3h; after the second enzymolysis is completed, the temperature of the second enzymolysis solution is raised to 80°C, and the temperature is kept constant for 20min to inactivate the enzyme, and the residual enzyme activity is detected after the enzyme inactivation treatment. ≤5U / g (refer to GB 5009.224-2016);

[0058] 3) Filtration: The filtration process is to add activated carbon to the enzymatic hydrolyzate after two enzymatic hydrolysis, the amount of activated carbon added is 4% of the mass of selenium-enriched walnut protein, and heat it to 100° C., keep it for 8 minutes, then cool it to 80° C. and let it stand for 1.5 hours to remove the color and odor in the enzymatic hydrolyzate;

[0059] 4) Purification and concentration: The filtered small molecule selenium-enriched walnut peptide solution is centrifuged to remove solid residues, and the obtained clear liquid is filtered with an ultrafiltration membrane with a molecular weight of 5000 Daltons to remove macromolecular impurities in the clear liquid, the membrane pressure of the ultrafiltration membrane is 1MPa, and the ultrafiltration temperature is 30°C; then, a nanofiltration membrane with a molecular weight of 200 Daltons is used to remove inorganic salts and small molecule impurities, the membrane pressure of the nanofiltration membrane is 2MPa, and the nanofiltration temperature is 40°C; the filtered clear liquid is concentrated to a solid content of 30%, that is, a small molecule selenium-enriched walnut peptide solution is obtained;

[0060] 5) Drying: The purified and concentrated small molecule selenium-rich walnut peptide solution is spray-dried, and the outlet air temperature of the spray drying is 80° C. and the inlet air temperature is 160° C. After drying, the small molecule selenium-rich walnut peptide powder is obtained.

[0061] Example 5

[0062] A method for preparing a small molecule selenium-enriched walnut peptide comprises the following steps:

[0063] 1) Protein extraction: selenium-rich walnuts are selected, and the walnuts are defatted to obtain defatted walnut residues. After the defatted selenium-rich walnut residues are crushed, deionized water 7 times the mass of the walnut residues is added, and sodium hydroxide solution is added to adjust the pH to 8.5. After soaking for 1.5 hours, the lower layer of precipitation is removed by a centrifuge; citric acid solution is added to adjust the pH of the centrifuge to 4, and the centrifuge is separated after standing for 3 hours. The lower layer of precipitation is removed and washed with water until neutral to obtain a selenium-rich walnut protein extract, and the quality of the selenium-rich walnut protein in the selenium-rich walnut protein extract is detected by a Baume meter or a Kjeldahl nitrogen determination method;

[0064] This embodiment makes full use of the byproduct of walnut oil extraction, defatted walnut residue, to prepare small molecule peptides, which is low in cost. The alkali dissolution and acid precipitation method is used to extract selenium-rich walnut protein, and the extraction rate is greater than 67%;

[0065] 2) Two enzymolysis: The two enzymolysis processes are to add an alkaline protease equivalent to 1% of the mass of selenium-rich walnut protein to the selenium-rich walnut protein extract for one enzymolysis, the temperature of the first enzymolysis is 60°C, the pH value of the first enzymolysis is 8.5, and the time of the first enzymolysis is 6h; after the first enzymolysis is completed, the temperature of the first enzymolysis solution is raised to 40°C-60°C, and the temperature is kept constant for 10min to inactivate the enzyme, and the residual enzyme activity is detected after the enzyme inactivation treatment. ≤5U / g (refer to GB 5009.224-2016); the first enzymolysis solution after the enzyme inactivation treatment is cooled to 20°C, and a neutral protease equivalent to 1% of the mass of selenium-rich walnut protein is added for a second enzymolysis, the temperature of the second enzymolysis is 60°C, the pH value of the second enzymolysis reaction is 7, and the time of the second enzymolysis is 5h; after the second enzymolysis is completed, the temperature of the second enzymolysis solution is raised to 80°C, and the temperature is kept constant for 20min to inactivate the enzyme, and the residual enzyme activity is detected after the enzyme inactivation treatment. ≤5U / g (refer to GB 5009.224-2016);

[0066] 3) Filtration: The filtration process is to add activated carbon to the enzymatic hydrolyzate after two enzymatic hydrolysis, the amount of activated carbon added is 3% of the mass of selenium-enriched walnut protein, and heat it to 100° C., keep it for 10 minutes, then cool it to 80° C. and let it stand for 1 hour to remove the color and odor in the enzymatic hydrolyzate;

[0067] 4) Purification and concentration: The filtered small molecule selenium-enriched walnut peptide solution is centrifuged to remove solid residues, and the obtained clear liquid is filtered with an ultrafiltration membrane with a molecular weight of 2000 Daltons to remove macromolecular impurities in the clear liquid, the membrane pressure of the ultrafiltration membrane is 1.5 MPa, and the ultrafiltration temperature is 35°C; then, a nanofiltration membrane with a molecular weight of 200 Daltons is used to remove inorganic salts and small molecule impurities, the membrane pressure of the nanofiltration membrane is 2.5 MPa, and the nanofiltration temperature is 40°C; the filtered clear liquid is concentrated to a solid content of 30%, that is, a small molecule selenium-enriched walnut peptide solution is obtained;

[0068] 5) Drying: The purified and concentrated small molecule selenium-rich walnut peptide solution is spray-dried, and the outlet air temperature of the spray drying is 80° C. and the inlet air temperature is 160° C. After drying, the small molecule selenium-rich walnut peptide powder is obtained.

[0069] Experimental testing

[0070] (1) Determination of the extraction rate of selenium-enriched walnut protein

[0071] The protein mass M in the selenium-enriched walnut powder was determined by the Kjeldahl nitrogen method, and the extracted walnut protein mass m was determined by the Coomassie brilliant blue method. The walnut protein extraction rate = m / M × 100%.

[0072] After measurement, the walnut protein extraction rates of Examples 1-5 are shown in Table 1:

[0073] Table 1 Extraction rate of small molecule selenium-enriched walnut protein

[0074]

[0075]

[0076] As can be seen from Table 1, the extraction rate of selenium-enriched walnut protein in Example 1 is the highest, reaching 67.66%.

[0077] (2) Determination of nutritional components of small molecule selenium-rich walnut peptides

[0078] GB 5009.5-2010 was used to test the contents of selenium, sodium, energy, protein, fat, carbohydrates, etc. of the small molecule selenium-enriched walnut peptide prepared by the method described in Example 1. The test results are shown in Table 2.

[0079] Table 2 Selenium, sodium, energy, protein, fat and carbohydrate content of small molecule selenium-enriched walnut peptides of the present invention

[0080] project Per 100g selenium 45.0ug sodium 135mg energy 1946.25kJ protein 10.375g Fat 12.625g carbohydrate 76.625g

[0081] As can be seen from Table 2, the yield of small molecule selenium-enriched walnut peptide prepared by the method described in Example 1 is 45.0 ug / 100 g, and the selenium-enriched effect is significant.

[0082] (3) Antioxidant activity detection

[0083] According to GB / T 38165-2019, the DPPH radical scavenging rate of the product of Example 1 was 86.7%, and the hydroxyl radical scavenging rate was 78.2%.

[0084] (4) Molecular weight distribution detection

[0085] According to HPLC-MALDI-TOF analysis, the molecular weight of the product of Example 1 is concentrated in the range of 200-1500 Daltons, accounting for ≥90%.

[0086] The small molecule selenium-rich walnut peptide prepared by the above method is light yellow and has no odor. The production process does not require the addition of organic solvents, and the process is simple, efficient and energy-saving, low cost, and high hydrolysis efficiency. In addition, the product obtained by spray drying has the characteristics of stable structure and excellent performance.

[0087] The measurement methods and instruments not described in detail above are conventional techniques and will not be described in detail here.

[0088] 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 small molecule selenium-enriched walnut peptide, characterized in that: The following steps are included in sequence: Selection of selenium-rich walnuts, crushing of walnut kernels, double enzymatic hydrolysis, filtration, purification, concentration and drying, packaging, testing and storage; The two enzymatic hydrolysis processes are specifically as follows: adding alkaline protease with a mass of 1%-3% of selenium-rich walnut protein to the selenium-rich walnut protein extract extracted in the protein extraction process for a first enzymatic hydrolysis, and the pH value of the first enzymatic hydrolysis reaction is 8-9; after the first enzymatic hydrolysis is completed, the enzyme is inactivated; the enzyme hydrolyzate that has been inactivated is cooled, and a neutral protease equivalent to the mass of 1%-3% of the selenium-rich walnut protein is added for a second enzymatic hydrolysis, and the pH value of the second enzymatic hydrolysis reaction is 6-7; after the enzymatic hydrolysis is completed, the enzyme is inactivated.

2. The method for preparing a small molecule selenium-enriched walnut peptide according to claim 1, characterized in that: The preparation process of the selenium-rich walnut protein extract is as follows: selenium-rich walnuts are squeezed to obtain defatted walnut residues, the defatted walnut residues are crushed, deionized water of 5 to 8 times the mass of the walnut residues is added, alkali is added to adjust the pH to 8 to 9, the solution is soaked for 1 to 2 hours, and the lower precipitate is removed by centrifugation; acid is added to adjust the pH of the centrifuge to 4, the solution is allowed to stand for 1 to 3 hours, and the lower precipitate is removed and washed with water until neutral, so as to obtain the selenium-rich walnut protein extract.

3. A method for preparing a small molecule selenium-enriched walnut peptide according to claim 1 or 2, characterized in that: During the pH adjustment, the added alkali is a sodium hydroxide solution, and the added acid is a citric acid solution.

4. The method for preparing a small molecule selenium-enriched walnut peptide according to claim 1, characterized in that: The filtering process is as follows: adding activated carbon to the enzymatic hydrolyzate after two enzymatic hydrolysis, wherein the amount of activated carbon added is 2%-4% of the mass of selenium-rich walnut protein, and heating to 100°C, maintaining for 5min-10min, and then cooling to 80°C and standing for 0.5h-2h to remove the color and odor in the enzymatic hydrolyzate.

5. The method for preparing a small molecule selenium-enriched walnut peptide according to claim 1, characterized in that: The purification and concentration process is as follows: the filtered small molecule selenium-enriched walnut peptide solution is centrifuged to remove solid residues, the obtained clear liquid is filtered with an ultrafiltration membrane with a molecular weight of 2000 Daltons to 5000 Daltons to remove macromolecular impurities in the clear liquid, and then the inorganic salts and impurities are removed with a nanofiltration membrane with a molecular weight of 200 Daltons, and the filtered clear liquid is concentrated to a solid content of 20% to 30%, thereby obtaining a small molecule selenium-enriched walnut peptide solution.

6. The method for preparing a small molecule selenium-enriched walnut peptide according to claim 5, characterized in that: The membrane pressure of the ultrafiltration membrane is 0.6MPa-1.5MPa, and the ultrafiltration temperature is 25°C-35°C; the membrane pressure of the nanofiltration membrane is 1.5MPa-2.5MPa, and the nanofiltration temperature is 25°C-40°C.

7. The method for preparing a small molecule selenium-enriched walnut peptide according to claim 1, characterized in that: The drying process is as follows: spray drying the purified and concentrated small molecule selenium-rich walnut peptide solution, the outlet air temperature of the spray drying is 70°C-90°C, the inlet air temperature is 150°C-180°C, and the small molecule selenium-rich walnut peptide powder is obtained after drying.

8. The method for preparing a small molecule selenium-enriched walnut peptide according to claim 1, characterized in that: The enzyme inactivation treatment process is: heating the primary enzymatic hydrolysis solution or the secondary enzymatic hydrolysis solution obtained after the primary enzymatic hydrolysis or the secondary enzymatic hydrolysis to 80° C. and maintaining the temperature for 10 min-20 min to perform enzyme inactivation treatment.

9. The method for preparing a small molecule selenium-enriched walnut peptide according to claim 1, characterized in that: The primary enzymolysis temperature is 30°C-60°C, and the time is 3h-6h; the secondary enzymolysis temperature is 40°C-60°C, and the time is 3h-6h.

10. A small molecule selenium-enriched walnut peptide, characterized in that: The small molecule selenium-enriched walnut peptide is prepared by the preparation method according to any one of claims 1 to 9, wherein the color of the small molecule selenium-enriched walnut peptide is graded Y10-Y20 according to the GB / T 22460-2008 colorimetric standard.

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