Production method of polypeptide with low salt content

Abstract

The invention relates to a low-salt-content polypeptide production method, and belongs to the technical field of polypeptide production. The method comprises the following steps: removing surface meat and grease from cattle bones, sheep bones and pig bones, cleaning, carrying out multi-stage crushing to prepare crushed bones, soaking the crushed bones in a microbial bacteria solution to play a biological treatment role, further adding acid, citric acid, acetic acid and hydrochloric acid with different strengths, carrying out gradient acid pickling, and finally carrying out protease enzymolysis to obtain the high-protein protein powder. And performing spray drying to obtain a finished product of the low-salt-content polypeptide. The low-salt-content polypeptide obtained through recycling of the soup, microbiological treatment, multi-stage pickling and mild enzymolysis is high in quality, low in salt content and sufficient in extraction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of polypeptide production, and particularly relates to a polypeptide production method with low salt content. BACKGROUND

[0002] In recent years, it is believed that the human body absorbs proteins mainly in the form of peptides. Peptides are molecules composed of amino acids, including polypeptides, oligopeptides, micropeptides and small peptides. Polypeptides exist in living organisms, all cells can synthesize polypeptides, and all cells are regulated by polypeptides. Polypeptides have strong activity and diversity, can regulate the physiological functions of various systems and cells in the human body, and activate related enzymes in the body. Therefore, polypeptides are important substances for human health.

[0003] Current polypeptide products usually have a molecular weight of 1000-5000 Da, and are mixed with small molecular impurities such as free amino acids and salts. Therefore, multiple purification technologies such as HPLC and gel filtration are required, which not only increases the cost but also affects the quality. In particular, if the salt content of food-grade polypeptides is too high, it will accumulate through daily intake, causing burden on the cardiovascular system, metabolic system and specific population, increasing the risk of cardiovascular diseases and the metabolic burden on the kidneys. Low-salt polypeptides meet the pursuit of a healthy lifestyle. SUMMARY

[0004] The purpose of the present application is to solve the above technical problems and provide a polypeptide production method with low salt content.

[0005] The present application achieves the above-mentioned purpose through the following technical solutions: The low-salt polypeptide produced by the present application includes the following steps: Step S1: The surface meat and fat of cow bones, sheep bones and pig bones are removed, and the cow bones, sheep bones and pig bones are washed with hot water at 35℃, washed and dried, and then crushed in a crusher. After crushing, the crushed bones are dried and sieved through a 150-180 mesh sieve to obtain a first crushed bone material; Step S2: The first crushed bone material is soaked in a microbial liquid, maintained at a temperature of 30-40℃, stirred at 25r / min for 15-18h, and then sieved through a 150-180 mesh sieve to retain the soup liquid. The first crushed bone material is dried and crushed in a crusher. After crushing, the crushed bones are dried and sieved through a 230-260 mesh sieve to obtain a second crushed bone material; Step S3: The second crushed bone material is soaked in a microbial liquid, maintained at a temperature of 35-45℃, stirred at 15r / min for 15-18h, and then sieved through a 230-260 mesh sieve to retain the soup liquid. The second crushed bone material is dried and crushed in a crusher. After crushing, the crushed bones are dried and sieved through a 320-350 mesh sieve to obtain a third crushed bone material; Step S5, the reserved soup is placed in a 3-5℃ environment for 10-12h, the liquid is filtered out, the filtered liquid is placed in a 1℃ environment for 6-8h, the liquid is filtered out, and a clear soup is obtained; Step S6, the four pieces of crushed aggregate are added to the stirrer, stirred at a temperature of 60-80℃ and a speed of 25r / min, citric acid is added at the beginning of stirring, acetic acid is added after 30min of stirring, hydrochloric acid is added after 45min of stirring, and the stirring is continued for 15min, then the temperature is raised to 130℃ and the pressure is raised to 0.3MPa, the acid hydrolysis is carried out for 6-7h, then the temperature and pressure are lowered, and the tank is discharged, obtaining the five pieces of crushed aggregate; Step S7, 1-1.5 times the volume of water and the clear soup obtained in step S5 are added to the five pieces of crushed aggregate, the pH is adjusted to 7.0-8.0 by using edible alkali, and protease is added at a temperature of 45-65℃, and the enzymolysis is carried out for 5-8h, obtaining a polypeptide solution; Step S8, the polypeptide solution is concentrated to 35% of the original volume by evaporation; Step S9, the concentrated polypeptide solution is introduced into a spray drying tower, the temperature is 130-150℃, and when the moisture content of the low-salt polypeptide powder is reduced to 4%, the drying is stopped, and the low-salt polypeptide is obtained, and the finished product is packaged.

[0006] As a further optimization scheme of the present application, the protease is selected from protease Alcalase 2.4L or protease Alcalase 3.0T.

[0007] As a further optimization scheme of the present application, the preparation raw materials of the microbial liquid by weight include: 5-10 parts of lactococcus lactis strain, 3-6 parts of bacillus strain, 85-90 parts of culture solution; wherein the preparation raw materials of the culture solution include: 15-25 parts of proteose peptone, 40-55 parts of glucose, 5-10 parts of sodium acetate and 60-80 parts of purified water.

[0008] As a further optimization scheme of the present application, the preparation process of the culture solution is as follows: the purified water is heated to 30℃, the proteose peptone, glucose and sodium acetate are added, stirred at a speed of 25r / min for 20-25min, taken out and placed in a high-pressure steam sterilization pot, sterilized at 121℃ and 0.1MPa for 20min, and cooled for standby.

[0009] As a further optimization of the present invention, the preparation process of the microbial culture solution is as follows: Lactococcus lactis strain is added to the culture medium and placed in a constant temperature incubator at 30-37℃ for 18-24 hours to obtain Lactococcus lactis strain solution; Bacillus spp. strain is added to the culture medium and placed in a constant temperature incubator at 28-30℃ with a shaking speed of 150-200 rpm for 12-16 hours to obtain Bacillus spp. strain solution; 10 minutes before soaking the first and second stages of crushed aggregate, the Lactococcus lactis strain solution and the Bacillus spp. strain solution are mixed evenly to obtain the microbial culture solution.

[0010] As a further optimization of the present invention, the mass ratio of cow bone, sheep bone and pig bone is 1:1:0.2.

[0011] As a further optimization of the present invention, in step S2, when the first section of crushed aggregate is soaked in the microbial solution, and in step S3, when the second section of crushed aggregate is soaked in the microbial solution, the microbial solution covers the first section and the second section of crushed aggregate by 3-5 cm.

[0012] As a further optimization of the present invention, the mass ratio of the four-stage crushed aggregate, citric acid, acetic acid and hydrochloric acid is 1:0.08:0.06:0.04.

[0013] As a further optimization of the present invention, the mass ratio of five-stage crushed aggregate to protease is 1:0.2.

[0014] Another technical problem to be solved by the present invention is to provide a low-salt polypeptide obtained by the above method.

[0015] The beneficial effects of this invention are as follows: By recycling the broth, the effective components of beef, sheep, and pork bones are extracted, greatly increasing the extraction yield. The microbial inoculum is used to soak the crushed bone fragments. During this process, lactic acid bacteria produce organic acids that gently dissolve some minerals in the bone fragments, such as calcium salts, exposing collagen fibers and opening pathways for subsequent processing. Bacillus secretes various proteases, accelerating the decomposition of proteins in the bone fragments. The synergistic effect of both achieves biological-level "wall breaking" and preliminary modification of the crushed bone fragments, increasing the total amount of peptides generated by subsequent enzymatic hydrolysis and reducing the salt content. Through stepwise acid hydrolysis, adding acids of different strengths (citric acid, acetic acid, and hydrochloric acid) in stages is gentler and more controllable, and may reduce the generation of harmful byproducts. This invention does not require complex purification equipment, has simple steps, low cost, and good application prospects. Attached Figure Description

[0016] Figure 1 Low-salt peptide test report Detailed Implementation The following further describes the present application in detail, it is necessary to point out here that the following detailed description is only used to further illustrate the present application, and cannot be understood as limiting the scope of protection of the present application, and the skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0017] The method used in the present application is a conventional method known to those skilled in the art, and the reagents and other materials used are commercially available products unless otherwise specified.

[0018] Example 1 The preparation process of the culture solution is as follows: 60 parts of purified water are heated to 30℃, 15 parts of peptone, 40 parts of glucose and 5 parts of sodium acetate are added, and stirred at 25 r / min for 22 min, then taken out and placed in a high-pressure steam sterilization pot, sterilized at 121℃, 0.1 MPa for 20 min, and cooled for standby use; The preparation process of the microbial liquid is as follows: 5 parts of activated Lactococcus lactis is added to 45 parts of culture solution, and placed in a 33℃ constant temperature incubator, cultured for 20 h to obtain Lactococcus lactis strain liquid; 3 parts of activated Bacillus is added to 45 parts of culture solution, and placed in a 28℃ constant temperature incubator, cultured for 13 h at a shaking speed of 180 rpm to obtain Bacillus strain liquid; the Lactococcus lactis strain liquid and the Bacillus strain liquid are mixed uniformly at 10 min before soaking the first and second crushed aggregates, to obtain the microbial liquid; The surface meat and grease of the cow bone, sheep bone and pig bone are removed (the mass ratio of the cow bone, sheep bone and pig bone is 1:1:0.2), the cow bone, sheep bone and pig bone are washed with 35℃ hot water, washed and dried, and then crushed in a crusher, dried and passed through a 165 mesh sieve after crushing, to obtain the first crushed aggregate; The first crushed aggregate is soaked in the microbial liquid (submerged in the microbial liquid by 3 cm), maintained at a temperature of 35℃, stirred at 25 r / min for 16 h, the first crushed aggregate is taken out with a 165 mesh sieve, the soup liquid is reserved, the first crushed aggregate is dried, crushed in a crusher, dried and passed through a 245 mesh sieve after crushing, to obtain the second crushed aggregate; The second crushed aggregate is soaked in the microbial liquid (submerged in the microbial liquid by 3 cm), maintained at a temperature of 40℃, stirred at 15 r / min for 16 h, the second crushed aggregate is taken out with a 245 mesh sieve, the soup liquid is reserved, the second crushed aggregate is dried, crushed in a crusher, dried and passed through a 330 mesh sieve after crushing, to obtain the third crushed aggregate; Put the three pieces of crushed bone into a high-temperature digester, add 1.5 times the volume of water and the soup liquid reserved in steps S2 and S3, and pass high-temperature steam to heat to 115℃, with a gas pressure of 0.4 MPa, and cook for 5 h. The three pieces of crushed bone are taken out using a 330-mesh screen, and the soup liquid is reserved. The three pieces of crushed bone are dried and ground to 460 mesh to obtain four pieces of crushed bone; Put the reserved soup liquid in a 3℃ environment and stand for 10 h. Filter out the liquid, and put the filtered liquid in a 1℃ environment and stand for 6 h. Filter out the liquid to obtain a clear soup liquid; Put the four pieces of crushed bone into a stirrer, stir at a temperature of 60℃ and a speed of 25 r / min. At the beginning of stirring, add citric acid, at 30 min of stirring, add acetic acid, and at 45 min of stirring, add hydrochloric acid (the mass ratio of the four pieces of crushed bone, citric acid, acetic acid, and hydrochloric acid is 1:0.08:0.06:0.04). Continue to stir for 15 min, then increase the temperature to 130℃ and the pressure to 0.3 MPa. After acidolysis for 6 h, reduce the temperature and pressure to take out the tank to obtain five pieces of crushed bone; Add 1.5 times the volume of water and the clear soup liquid obtained in step S5 into the five pieces of crushed bone. Adjust the pH to 7.0 using edible alkali, and add protease Alcalase 2.4L at a temperature of 55℃ (the mass ratio of the five pieces of crushed bone to the protease is 1:0.2). Enzymatic hydrolysis for 6 h to obtain a polypeptide liquid; Concentrate the polypeptide liquid to 35% of the original volume by evaporation; Concentrated polypeptide liquid is passed into a spray drying tower at a temperature of 140℃. When the moisture content of the low-salt polypeptide powder is reduced to 4%, stop drying to obtain a low-salt polypeptide. The inspection report is as shown in Figure 1 The finished product is packaged.

[0019] Example 2 The preparation process of the culture solution is as follows: 70 parts of purified water are heated to 30℃, 20 parts of proteose peptone, 50 parts of glucose, and 8 parts of sodium acetate are added, and stirred at 25 r / min for 22 min. Then, it is taken out and placed in a high-pressure steam sterilization pot, and sterilized at 121℃ and 0.1 MPa for 20 min. After cooling, it is ready for use; The preparation process of the microbial liquid is as follows: 7 parts of activated Lactococcus lactis are added to 45 parts of the culture solution, and placed in a 33℃ constant temperature incubator for 20 h to obtain a Lactococcus lactis strain liquid. 4 parts of activated Bacillus spores are added to 45 parts of the culture solution, and placed in a 28℃ constant temperature incubator. The shaking speed is 180 rpm, and the culture is carried out for 13 h to obtain a Bacillus spores strain liquid. The Lactococcus lactis strain liquid and the Bacillus spores strain liquid are mixed uniformly 10 min before soaking the first and second pieces of crushed bone to obtain a microbial liquid; The surface meat and grease of the beef bone, mutton bone and pork bone are removed (the mass ratio of the beef bone, mutton bone and pork bone is 1:1:0.2), the beef bone, mutton bone and pork bone are cleaned by using 35℃ hot water, and then are dried, broken in a crusher, dried after being broken, and passed through a 165 mesh sieve to obtain a first broken bone material; The first broken bone material is soaked in a microbial bacteria solution (3cm higher than the first broken bone material), kept at 35℃, stirred at 25r / min for 16h, taken out by using a 165 mesh sieve, the soup is reserved, the first broken bone material is dried, broken in a crusher, dried after being broken, and passed through a 245 mesh sieve to obtain a second broken bone material; The second broken bone material is soaked in a microbial bacteria solution (3cm higher than the second broken bone material), kept at 40℃, stirred at 15r / min for 16h, taken out by using a 245 mesh sieve, the soup is reserved, the second broken bone material is dried, broken in a crusher, dried after being broken, and passed through a 330 mesh sieve to obtain a third broken bone material; The third broken bone material is placed in a high-temperature digester, 1.5 times of water and the soup reserved in steps S2 and S3 are added, high-temperature steam is introduced to heat to 115℃, the air pressure reaches 0.4MPa, and the third broken bone material is cooked for 5h, taken out by using a 330 mesh sieve, the soup is reserved, the third broken bone material is dried, and ground to 460 mesh to obtain a fourth broken bone material; The reserved soup is placed in a 3℃ environment for 10h, the liquid is filtered out, the filtered liquid is placed in a 1℃ environment for 6h, the liquid is filtered out, and a clear soup is obtained; The fourth broken bone material is added to a stirrer, stirred at 60℃ and 25r / min, citric acid is added at the beginning of stirring, acetic acid is added after 30min of stirring, hydrochloric acid is added after 45min of stirring (the mass ratio of the fourth broken bone material, citric acid, acetic acid and hydrochloric acid is 1:0.08:0.06:0.04), and then the stirring is continued for 15min, the temperature is increased to 130℃, the pressure is increased to 0.3MPa, and the acid hydrolysis is performed for 6h, and then the temperature and pressure are decreased to obtain a fifth broken bone material; 1.5 times of water and the clear soup obtained in step S5 are added to the fifth broken bone material, the pH is adjusted to 8.0 by using edible alkali, and protease Alcalase 3.0T is added at 50℃ (the mass ratio of the fifth broken bone material and the protease is 1:0.2), and the enzymolysis is performed for 6h to obtain a polypeptide solution; The polypeptide solution is concentrated to 35% of the original volume by using evaporation; The concentrated polypeptide solution is introduced into a spray drying tower, the temperature is 140℃, and the drying is stopped when the water content of the low-salt polypeptide powder is reduced to 4% to obtain a low-salt polypeptide, which is packaged after self-checking.

[0020] Example 3 The preparation process of the culture solution is as follows: 80 parts of purified water is heated to 30℃, 25 parts of proteose peptone, 55 parts of glucose and 10 parts of sodium acetate are added, and stirred at 25 r / min for 22 min, then taken out and placed in a high-pressure steam sterilization pot, sterilized at 121℃, 0.1 MPa for 20 min, and cooled for standby use; The preparation process of the microbial liquid is as follows: 10 parts of activated Lactococcus lactis is added into 45 parts of the culture solution, and placed in a 33℃ constant temperature incubator for 20h to obtain the Lactococcus lactis strain liquid; 6 parts of activated Bacillus is added into 45 parts of the culture solution, and placed in a 28℃ constant temperature incubator, with the shaking speed of 180 rpm, for 13h to obtain the Bacillus strain liquid; the Lactococcus lactis strain liquid and the Bacillus strain liquid are mixed uniformly at 10 min before soaking the first and second crushed aggregates to obtain the microbial liquid; The surface meat and grease of the cow bone, sheep bone and pig bone (the mass ratio of the cow bone, sheep bone and pig bone is 1:1:0.2) are removed, the cow bone, sheep bone and pig bone are washed with 35℃ hot water, washed and dried, and then crushed in a crusher, dried and passed through a 165 mesh sieve after crushing to obtain the first crushed aggregate; The first crushed aggregate is soaked in the microbial liquid (submerged in the microbial liquid by 3 cm), kept at 35℃, stirred at 25 r / min for 16h, then taken out with a 165 mesh sieve, the soup liquid is reserved, the first crushed aggregate is dried, crushed in a crusher, dried and passed through a 245 mesh sieve after crushing to obtain the second crushed aggregate; The second crushed aggregate is soaked in the microbial liquid (submerged in the microbial liquid by 3 cm), kept at 40℃, stirred at 15 r / min for 16h, then taken out with a 245 mesh sieve, the soup liquid is reserved, the second crushed aggregate is dried, crushed in a crusher, dried and passed through a 330 mesh sieve after crushing to obtain the third crushed aggregate; The third crushed aggregate is placed in a high-temperature digester, 1.5 times the volume of water and the soup liquid reserved in steps S2 and S3 are added, high-temperature steam is introduced to heat to 115℃, the gas pressure reaches 0.4 MPa, and the third crushed aggregate is cooked for 5h, then taken out with a 330 mesh sieve, the soup liquid is reserved, the third crushed aggregate is dried and ground to 460 mesh to obtain the fourth crushed aggregate; The reserved soup liquid is placed in a 3℃ environment for 10h, the liquid is filtered out, the filtered liquid is placed in a 1℃ environment for 6h, the liquid is filtered out, and the clear soup liquid is obtained; The four pieces of crushed bone material were added to a stirrer, stirred at a temperature of 60°C and a speed of 25r / min, citric acid was added at the beginning of stirring, acetic acid was added after 30 minutes of stirring, hydrochloric acid was added after 45 minutes of stirring (the mass ratio of the four pieces of crushed bone material, citric acid, acetic acid and hydrochloric acid was 1:0.08:0.06:0.04), and the stirring was continued for 15 minutes, then the temperature was raised to 130°C and the pressure was raised to 0.3MPa, the acidolysis was carried out for 6 hours, then the temperature and pressure were reduced, and the five pieces of crushed bone material were obtained; The water with a volume of 1.5 times and the clear soup obtained in step S5 were poured into the five pieces of crushed bone material, the pH was adjusted to 7.5 by using edible alkali, and the five pieces of crushed bone material were subjected to enzymatic hydrolysis by using protease Alcalase 3.0T (the mass ratio of the five pieces of crushed bone material and the protease was 1:0.2) at a temperature of 65°C for 6 hours, and the polypeptide solution was obtained; The polypeptide solution was concentrated to 35% of the original volume by using evaporation; The concentrated polypeptide solution was introduced into a spray drying tower, the temperature was 140°C, and the drying was stopped when the water content of the polypeptide powder with low salt content was reduced to 4%, and the polypeptide with low salt content was obtained, which was packaged after self-checking.

[0021] Comparative Example 1 The preparation process of the culture solution was as follows: 60 parts of purified water were heated to 30°C, 15 parts of proteose peptone, 40 parts of glucose and 5 parts of sodium acetate were added, and stirred at a speed of 25r / min for 22 minutes, then taken out and placed in a high-pressure steam sterilization pot, sterilized at 121°C and 0.1MPa for 20 minutes, and cooled for standby; The preparation process of the microbial liquid was as follows: 5 parts of activated Lactococcus lactis were added to 45 parts of the culture solution, and placed in a constant temperature incubator at 33°C for 20 hours to obtain the Lactococcus lactis strain liquid; 3 parts of activated Bacillus spores were added to 45 parts of the culture solution, and placed in a constant temperature incubator at 28°C, and the shaking speed was 180rpm, and the culture was carried out for 13 hours to obtain the Bacillus spores strain liquid; the Lactococcus lactis strain liquid and the Bacillus spores strain liquid were mixed uniformly 10 minutes before soaking the first piece of crushed bone material, and the microbial liquid was obtained; The surface meat and fat of the cow bone, sheep bone and pig bone were removed (the mass ratio of the cow bone, sheep bone and pig bone was 1:1:0.2), the cow bone, sheep bone and pig bone were washed with hot water at 35°C, washed and dried, and then crushed in a crusher, dried and passed through a 165 mesh sieve after crushing, and the first piece of crushed bone material was obtained; The first piece of crushed bone material was soaked in the microbial liquid (submerged in the microbial liquid by 3cm), and the temperature was maintained at 35°C, and stirred at a speed of 25r / min for 16 hours, then the first piece of crushed bone material was taken out by using a 165 mesh sieve, the soup was reserved, the first piece of crushed bone material was dried, crushed in a crusher, dried and passed through a 245 mesh sieve after crushing, and the second piece of crushed bone material was obtained; Put the second section of the crushed bone into a high-temperature digester, add 1.5 times the volume of water and the soup liquid reserved in step S2 and step S3, and pass in high-temperature steam to raise the temperature to 115°C and the air pressure to 0.4 MPa, and cook for 5 h. The second section of the crushed bone is taken out using a 330-mesh screen, and the soup liquid is reserved. The second section of the crushed bone is dried and ground to 460 mesh to obtain the third section of the crushed bone; Put the reserved soup liquid into an environment of 3°C and stand for 10 h. Filter out the liquid, and put the filtered liquid into an environment of 1°C and stand for 6 h. Filter out the liquid to obtain the clear soup liquid. Put the third section of the crushed bone into a stirrer, and stir at a temperature of 60°C and a speed of 25 r / min. At the beginning of stirring, add citric acid, at 30 min of stirring, add acetic acid, and at 45 min of stirring, add hydrochloric acid (the mass ratio of the third section of the crushed bone, citric acid, acetic acid, and hydrochloric acid is 1:0.08:0.06:0.04). Continue to stir for 15 min, then raise the temperature to 130°C and the pressure to 0.3 MPa, and acid hydrolyze for 6 h. After cooling and decompression, the tank is discharged to obtain the fourth section of the crushed bone. Add 1.5 times the volume of water and the clear soup liquid obtained in step S5 to the fourth section of the crushed bone, and adjust the pH to 7.0 using edible alkali. At a temperature of 55°C, add protease Alcalase 2.4L (the mass ratio of the fourth section of the crushed bone to the protease is 1:0.2), and enzymatically hydrolyze for 6 h to obtain a polypeptide liquid. Concentrate the polypeptide liquid to 35% of the original volume by evaporation. Pass the concentrated polypeptide liquid into a spray drying tower at a temperature of 140°C. When the water content of the low-salt polypeptide powder is reduced to 4%, stop drying to obtain the low-salt polypeptide, and package the finished product.

[0022] Comparative Example 2 Remove the surface meat and fat from the cow bones, sheep bones, and pig bones (the mass ratio of the cow bones, sheep bones, and pig bones is 1:1:0.2). Wash the cow bones, sheep bones, and pig bones with hot water at 35°C, wash and dry, and put them into a crusher to crush. After crushing, dry and pass through a 165-mesh screen to obtain the first section of the crushed bone. Put the first section of the crushed bone into a high-temperature digester, add 1.5 times the volume of water and the soup liquid reserved in step S2 and step S3, and pass in high-temperature steam to raise the temperature to 115°C and the air pressure to 0.4 MPa, and cook for 5 h. The second section of the crushed bone is taken out using a 330-mesh screen, and the soup liquid is reserved. The second section of the crushed bone is dried and ground to 460 mesh to obtain the third section of the crushed bone; Put the reserved soup liquid into an environment of 3°C and stand for 10 h. Filter out the liquid, and put the filtered liquid into an environment of 1°C and stand for 6 h. Filter out the liquid to obtain the clear soup liquid. The second section of the crushed bone is added to the stirrer, stirred at 60℃ and 25r / min, citric acid is added at the beginning of stirring, acetic acid is added after 30min of stirring, hydrochloric acid is added after 45min of stirring (the mass ratio of the second section of the crushed bone, citric acid, acetic acid and hydrochloric acid is 1:0.08:0.06:0.04), and the stirring is continued for 15min, then the temperature is raised to 130℃ and the pressure is raised to 0.3MPa, the acid hydrolysis is carried out for 6h, and then the temperature and pressure are reduced to take out the tank, to obtain the third section of the crushed bone; The 1.5 times volume of water and the clear soup obtained in step S5 are added to the third section of the crushed bone, the pH is adjusted to 7.0 by adding edible alkali, and the protease Alcalase 3.0T is added at 55℃ (the mass ratio of the third section of the crushed bone and the protease is 1:0.2), and the enzymolysis is carried out for 6h to obtain a polypeptide solution; The polypeptide solution is concentrated to 35% of the original volume by evaporation; The concentrated polypeptide solution is introduced into a spray drying tower, the temperature is 140℃, and the drying is stopped when the moisture content of the low-salt polypeptide powder is reduced to 4% to obtain the low-salt polypeptide, which is packaged as a finished product.

[0023] Comparative Example 3 The preparation process of the culture solution is as follows: 60 parts of purified water are heated to 30℃, 15 parts of proteose peptone, 40 parts of glucose and 5 parts of sodium acetate are added, stirred at 25r / min for 22min, taken out and placed in a high-pressure steam sterilization pot, sterilized at 121℃ and 0.1MPa for 20min, and cooled for standby; The preparation process of the microbial liquid is as follows: 5 parts of activated Lactococcus lactis is added to 45 parts of culture solution, placed in a 33℃ constant temperature incubator, and cultured for 20h to obtain Lactococcus lactis strain liquid; 3 parts of activated Bacillus is added to 45 parts of culture solution, placed in a 28℃ constant temperature incubator, and cultured for 13h at a shaking speed of 180rpm to obtain Bacillus strain liquid; the Lactococcus lactis strain liquid and the Bacillus strain liquid are mixed uniformly 10min before soaking the first section of the crushed bone to obtain the microbial liquid; The surface meat and fat of the cow bone, sheep bone and pig bone are removed (the mass ratio of the cow bone, sheep bone and pig bone is 1:1:0.2), the cow bone, sheep bone and pig bone are washed with 35℃ hot water, washed and dried, crushed in a crusher, dried and passed through a 165 mesh sieve after crushing to obtain the first section of the crushed bone; The first section of the crushed bone is soaked in the microbial liquid (3cm above the first section of the crushed bone), stirred at 25r / min for 16h at 35℃, taken out with a 165 mesh sieve, and the soup is reserved, the first section of the crushed bone is dried, crushed in a crusher, dried and passed through a 245 mesh sieve after crushing to obtain the second section of the crushed bone; The second-stage crushed bone is soaked in water (3 cm higher than the second-stage crushed bone), kept at 40℃, stirred at 15 r / min for 16 h, taken out with a 245-mesh sieve, the soup is reserved, the second-stage crushed bone is dried, put into a crusher, crushed, dried through a 330-mesh sieve, and a third-stage crushed bone is obtained; The third-stage crushed bone is put into a high-temperature digester, 1.5 times of water and the soup reserved in steps S2 and S3 are added, high-temperature steam is introduced to heat to 115℃, the pressure is 0.4 MPa, and the third-stage crushed bone is cooked for 5 h, taken out with a 330-mesh sieve, the soup is reserved, the third-stage crushed bone is dried, ground to 460 mesh, and a fourth-stage crushed bone is obtained; The reserved soup is placed in a 3℃ environment for 10 h, the liquid is filtered out, the filtered liquid is placed in a 1℃ environment for 6 h, the liquid is filtered out, and a clear soup is obtained; The fourth-stage crushed bone is added into a stirrer, stirred at 60℃ and 25 r / min, citric acid is added at the beginning of stirring, acetic acid is added after 30 min of stirring, hydrochloric acid is added after 45 min of stirring (the mass ratio of the fourth-stage crushed bone, citric acid, acetic acid and hydrochloric acid is 1:0.08:0.06:0.04), and the stirring is continued for 15 min, then the temperature is increased to 130℃ and the pressure is increased to 0.3 MPa, the acid hydrolysis is performed for 6 h, and then the temperature and pressure are decreased to obtain a fifth-stage crushed bone; 1.5 times of water and the clear soup obtained in step S5 are added into the fifth-stage crushed bone, edible alkali is used to adjust the pH to 8.0, and protease Alcalase 3.0T is added at 65℃ (the mass ratio of the fifth-stage crushed bone and the protease is 1:0.2), and the enzymolysis is performed for 6 h to obtain a polypeptide solution; The polypeptide solution is concentrated to 35% of the original volume by evaporation; The concentrated polypeptide solution is introduced into a spray drying tower, the temperature is 140℃, and the drying is stopped when the moisture content of the low-salt polypeptide powder is reduced to 4% to obtain a low-salt polypeptide, which is packaged as a finished product.

[0024] Comparative Example 4 The preparation process of the culture solution is as follows: 60 parts of purified water are heated to 30℃, 15 parts of proteose peptone, 40 parts of glucose and 5 parts of sodium acetate are added, stirred at 25 r / min for 22 min, taken out and placed in a high-pressure steam sterilization pot, sterilized at 121℃ and 0.1 MPa for 20 min, and cooled for standby; The preparation process of the microbial liquid is as follows: 5 parts of activated Lactococcus lactis are added into 45 parts of culture solution, and placed in a 33℃ constant temperature incubator for culture for 20 hours to obtain Lactococcus lactis strain liquid; 3 parts of activated Bacillus is placed into 45 parts of culture solution, and placed in a 28℃ constant temperature incubator for culture for 13 hours at a shaking speed of 180 rpm to obtain Bacillus strain liquid; the Lactococcus lactis strain liquid and the Bacillus strain liquid are uniformly mixed at 10 minutes before the second-stage aggregate is soaked to obtain the microbial liquid; The surface meat and grease of the cow bone, sheep bone and pig bone are removed (the mass ratio of the cow bone, sheep bone and pig bone is 1:1:0.2), the cow bone, sheep bone and pig bone are washed by using 35℃ hot water, washed and dried, and then broken in a crusher, dried and passed through a 165-mesh sieve after being broken to obtain first-stage aggregate; The first-stage aggregate is soaked in water (submerged by 3cm), kept at 35℃, stirred at 25r / min for 16 hours, the first-stage aggregate is fished out by using a 165-mesh sieve, the soup liquid is reserved, the first-stage aggregate is dried, broken in a crusher, dried and passed through a 245-mesh sieve after being broken to obtain second-stage aggregate; The second-stage aggregate is soaked in the microbial liquid (submerged by 3cm), kept at 40℃, stirred at 15r / min for 16 hours, the second-stage aggregate is fished out by using a 245-mesh sieve, the soup liquid is reserved, the second-stage aggregate is dried, broken in a crusher, dried and passed through a 330-mesh sieve after being broken to obtain third-stage aggregate; The third-stage aggregate is placed in a high-temperature digester, 1.5 times of water and the soup liquid reserved in steps S2 and S3 are added, high-temperature steam is introduced to heat to 115℃, the air pressure reaches 0.4MPa, and the third-stage aggregate is cooked for 5 hours, the third-stage aggregate is fished out by using a 330-mesh sieve, the soup liquid is reserved, the third-stage aggregate is dried and ground to 460 mesh to obtain fourth-stage aggregate; The reserved soup liquid is placed in a 3℃ environment for 10 hours, the liquid is filtered out, the filtered liquid is placed in a 1℃ environment for 6 hours, the liquid is filtered out to obtain clear soup liquid; The fourth-stage aggregate is added into a stirrer, stirred at 60℃ and 25r / min, citric acid is added at the beginning of stirring, acetic acid is added at 30 minutes of stirring, hydrochloric acid is added at 45 minutes of stirring (the mass ratio of the fourth-stage aggregate, citric acid, acetic acid and hydrochloric acid is 1:0.08:0.06:0.04), and the stirring is continued for 15 minutes, then the temperature is increased to 130℃ and the pressure is increased to 0.3MPa, the acid hydrolysis is carried out for 6 hours, then the temperature and pressure are decreased to take out the tank to obtain fifth-stage aggregate; Add 1.5 times the volume of water and the clear broth obtained in step S5 to the five-piece bone fragments. Adjust the pH to 8.0 using edible alkali. Add 2.4 L of Alcalase (the mass ratio of the five-piece bone fragments to the protease is 1:0.2) at 60°C and enzymatically hydrolyze for 6 hours to obtain a polypeptide solution. The polypeptide solution was concentrated to 35% of its original volume by evaporation. The concentrated polypeptide solution is passed into a spray drying tower at a temperature of 140°C. Drying is stopped when the moisture content of the low-salt polypeptide powder drops to 4%, resulting in low-salt polypeptides, which are then packaged as finished products.

[0025] Comparative Example 5 Remove the surface meat and fat from beef bones, sheep bones and pork bones (the mass ratio of beef bones, sheep bones and pork bones is 1:1:0.2). Wash the beef bones, sheep bones and pork bones with 35℃ hot water, dry them, put them in a crusher to crush them, dry them after crushing, and pass them through a 165-mesh sieve to obtain a piece of crushed bone material. Soak a section of crushed aggregate in water (3cm above the section of crushed aggregate), maintain a temperature of 35℃, stir at 25r / min for 16h, remove the section of crushed aggregate using a 165-mesh sieve, retain the liquid, dry the section of crushed aggregate, place it in a crusher to crush it, dry it after crushing, and pass it through a 245-mesh sieve to obtain two sections of crushed aggregate. Soak the second-stage crushed aggregate in water (3cm above the second-stage crushed aggregate), maintain the temperature at 40℃, stir at 15r / min for 16h, remove the second-stage crushed aggregate using a 245-mesh sieve, retain the broth, dry the second-stage crushed aggregate, place it in a crusher to crush it, dry it after crushing, and pass it through a 330-mesh sieve to obtain the third-stage crushed aggregate. Place the three pieces of bone fragments in a high-temperature steam cooker, add 1.5 times the volume of water and the broth retained in steps S2 and S3, introduce high-temperature steam to raise the temperature to 115°C and the pressure to 0.4MPa, steam for 5 hours, remove the three pieces of bone fragments using a 330-mesh sieve, retain the broth, dry the three pieces of bone fragments, grind them to 460 mesh to obtain four pieces of bone fragments; The reserved broth was left to stand at 3°C ​​for 10 hours, and the liquid was filtered out. The filtered liquid was then left to stand at 1°C for 6 hours, and the liquid was filtered out to obtain clear broth. Four-stage crushed aggregate was added to a mixer and stirred at 25 r / min at 60℃. Citric acid was added at the beginning of stirring, acetic acid was added after 30 min of stirring, and hydrochloric acid was added after 45 min of stirring (the mass ratio of four-stage crushed aggregate, citric acid, acetic acid and hydrochloric acid was 1:0.08:0.06:0.04). After stirring for another 15 min, the pressure was increased to 0.3 MPa at 130℃. After acid hydrolysis for 6 h, the temperature and pressure were reduced and the mixture was discharged from the tank to obtain five-stage crushed aggregate. Add 1.5 times the volume of water and the clear broth obtained in step S5 to the five-piece bone fragments. Adjust the pH to 7.0 using edible alkali. Add 2.4 L of Alcalase (mass ratio of five-piece bone fragments to Alcalase is 1:0.2) at 55°C and enzymatically hydrolyze for 6 hours to obtain a polypeptide solution. The polypeptide solution was concentrated to 35% of its original volume by evaporation. The concentrated polypeptide solution is passed into a spray drying tower at a temperature of 140°C. Drying is stopped when the moisture content of the low-salt polypeptide powder drops to 4%, resulting in low-salt polypeptides, which are then packaged as finished products.

[0026] Performance testing (a) Determination of sodium and ash content in low-salt peptide samples prepared using the methods of Examples 1-3 and Comparative Examples 1-5. According to GB5009.91-2017, Method III, the sodium content in the low-salt polypeptide samples prepared by the methods of Examples 1-3 and Comparative Examples 1-5 was determined; According to Method 1 of GB5009.4-2016, the ash content of low-salt peptide samples prepared by the methods of Examples 1-3 and Comparative Examples 1-5 was determined.

[0027] The test results are shown in Table 1.

[0028] Table 1. Detection of low-salt peptides

[0029] As can be seen from Table 1, the polypeptide samples prepared using the methods in Examples 1-3 have low sodium content and low ash content, which meets the low-salt standard.

[0030] (ii) Following a common method, accurately weigh 5 mg of DPPH powder and mix it with 95% ethanol to a final volume of 100 mL to obtain a DPPH treatment solution with a concentration of 50.0 μg / mL. Take 0.2 g of the peptide sample prepared using the methods of Examples 1-3 and Comparative Examples 1-5, and simultaneously take 2.0 mL of the DPPH treatment solution to mix thoroughly. Incubate at room temperature in the dark for 30 min, and measure the absorbance at a wavelength of 517 nm. Take the average value of the three sets of parallel experimental results and calculate the clearance rate of the peptide sample.

[0031] The test results are shown in Table 2.

[0032] Table 2 DPPH removal rate

[0033] As can be seen from Table 2, the polypeptide samples prepared by the methods of Examples 1-3 have high DPPH scavenging rates, indicating that they can efficiently scavenge excess free radicals generated in the metabolic process of the body, effectively inhibit and repair oxidative damage at the cellular level, and ultimately achieve the health effects of delaying aging, enhancing the body's resistance, and preventing oxidative stress-related diseases.

[0034] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. A method for producing a polypeptide with low salt content, characterized in that, The method comprises the following steps: Step S1, the surface meat and fat of the cattle bone, sheep bone and pig bone are removed, the cattle bone, sheep bone and pig bone are cleaned by using 35℃ hot water, and then washed and dried, and then placed in a crusher for crushing, and then dried and passed through a 150-180 mesh sieve to obtain first crushed bone materials; Step S2, the first crushed bone materials are soaked in a microbial bacteria liquid, kept at a temperature of 30-40℃, stirred at a speed of 25r / min for 15-18h, the first crushed bone materials are taken out by using a 150-180 mesh sieve, the soup liquid is reserved, the first crushed bone materials are dried, placed in a crusher for crushing, dried and passed through a 230-260 mesh sieve to obtain second crushed bone materials; Step S3, the second crushed bone materials are soaked in a microbial bacteria liquid, kept at a temperature of 35-45℃, stirred at a speed of 15r / min for 15-18h, the second crushed bone materials are taken out by using a 230-260 mesh sieve, the soup liquid is reserved, the second crushed bone materials are dried, placed in a crusher for crushing, dried and passed through a 320-350 mesh sieve to obtain third crushed bone materials; Step S4, the third crushed bone materials are placed in a high-temperature cooker, 1-1.5 times of water and the soup liquid reserved in steps S2 and S3 are added, high-temperature steam is introduced to heat to 110-120℃, the air pressure reaches 0.4-0.5MPa, and the third crushed bone materials are cooked for 5-6h, the third crushed bone materials are taken out by using a 320-350 mesh sieve, the soup liquid is reserved, the third crushed bone materials are dried and ground to 440-470 mesh to obtain fourth crushed bone materials; Step S5, the reserved soup liquid is placed in an environment of 3-5℃ for 10-12h, the liquid is filtered out, the filtered liquid is placed in an environment of 1℃ for 6-8h, the liquid is filtered out, and a clear soup liquid is obtained; Step S6, the fourth crushed bone materials are added to a stirrer, stirred at a speed of 25r / min at a temperature of 60-80℃, citric acid is added at the beginning of stirring, acetic acid is added after stirring for 30min, hydrochloric acid is added after stirring for 45min, the stirring is continued for 15min, the temperature is raised to 130℃, the pressure is raised to 0.3MPa, and the acid hydrolysis is carried out for 6-7h, then the temperature and pressure are reduced, and the tank is discharged, to obtain fifth crushed bone materials; Step S7, 1-1.5 times of water and the clear soup liquid obtained in step S5 are added to the fifth crushed bone materials, edible alkali is used to adjust the pH value to 7.0-8.0, and protease is added at a temperature of 45-65℃, and enzymolysis is carried out for 5-8h to obtain a polypeptide liquid; Step S8, the polypeptide liquid is concentrated to 35% of the original volume by evaporation; Step S9, the concentrated polypeptide liquid is introduced into a spray drying tower, the temperature is 130-150℃, and when the water content of the low-salt polypeptide powder is reduced to 4%, the drying is stopped, to obtain a low-salt polypeptide, which is packaged as a finished product.

2. The low-salt polypeptide production method according to claim 1, characterized by, The protease is selected from protease Alcalase 2.4L or protease Alcalase 3.0T.

3. The low-salt polypeptide production method according to claim 1, characterized by, The preparation raw materials of the microbial bacteria liquid include, by weight, 5-10 parts of lactococcus lactis strain, 3-6 parts of bacillus strain, and 85-90 parts of culture solution. The preparation raw materials of the culture solution include 15-25 parts of proteose peptone, 40-55 parts of glucose, 5-10 parts of sodium acetate, and 60-80 parts of purified water.

4. The low-salt polypeptide production method according to claim 3, characterized by, The preparation process of the culture solution is as follows: the purified water is heated to 30℃, and the protein peptone, glucose and sodium acetate are added and stirred at 25 r / min for 20-25 min, and then taken out and sterilized in a high-pressure steam sterilization pot at 121℃ and 0.1 MPa for 20 min, and then cooled for use.

5. The low-salt polypeptide production method according to claim 3, characterized by, The preparation process of the microbial solution is as follows: the lactococcus strain is added into the culture solution and placed in a constant-temperature incubator at 30-37℃ for 18-24 h to obtain the lactococcus strain solution; the bacillus strain is added into the culture solution and placed in a constant-temperature incubator at 28-30℃, and the shaking speed is 150-200 rpm for 12-16 h to obtain the bacillus strain solution; the lactococcus strain solution and the bacillus strain solution are mixed uniformly at 10 min before soaking the first-stage and second-stage crushed aggregates to obtain the microbial solution.

6. The low-salt polypeptide production method according to claim 1, wherein The mass ratio of the cow bone, sheep bone and pig bone is 1:1:0.

2.

7. The method for producing a polypeptide with low salt content according to any one of claims 1 to 6, characterized in that, When the first-stage crushed aggregate is soaked in the microbial solution in the step S2 and the second-stage crushed aggregate is soaked in the microbial solution in the step S3, the microbial solution is 3-5 cm higher than the first-stage and second-stage crushed aggregates.

8. The low-salt polypeptide production method according to claim 7, characterized by, The mass ratio of the four-stage crushed aggregate, citric acid, acetic acid and hydrochloric acid is 1:0.08:0.06:0.

04.

9. The method for producing a polypeptide with low salt content according to any one of claims 1 to 6, characterized in that, The mass ratio of the five-stage crushed aggregate and protease is 1:0.

2.

10. A polypeptide of low salt content, characterized in that, The preparation method is prepared by any one of claims 1-9. The preparation method is prepared by any one of claims 1-9.

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