CN114395599A - Preparation method of composite polypeptide - Google Patents
Preparation method of composite polypeptide Download PDFInfo
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Abstract
The invention discloses a preparation method of composite multi-element peptide, which is formed by mixing hydrolyzed protein powder of crayfish by-products and rice bran protein peptide powder; the crawfish byproduct protein hydrolysate is prepared by taking pretreated crawfish byproduct powder as a raw material, and performing irradiation, enzymolysis, separation, concentration and spray drying; the rice bran protein peptide powder is prepared by taking crushed rice bran as a raw material and carrying out ohmic heating treatment, enzymolysis, separation and concentration and spray drying. The invention has simple process, can effectively kill microorganisms and inactivate enzyme activity, has low production cost, high nutritional value, high yield of active substances such as gamma-oryzanol and the like and high DPPH free radical clearance rate.
Description
Technical Field
The invention relates to a preparation method of composite polypeptide, in particular to a compound of procambarus clarkii by-product hydrolyzed protein and rice bran protein active peptide.
Background
The Procambarus clarkii is one of the freshwater shrimps which are widely cultivated and produced in high yield in China, and with the continuous rising of the consumption demand of the Procambarus clarkii and the rapid development of the processing industry, a large amount of shrimp processing byproducts such as minced shrimp, shrimp yellow, washing protein and the like are produced in the processing process. The processing and utilization degree of the byproducts is low, and most of the byproducts are directly discarded, so that the waste of resources is caused. Wherein, the minced shrimp, the shrimp roe and the water-washed protein account for about 5 percent of the weight of the shrimp, contain rich polyunsaturated fatty acids such as EPA, DHA and the like, protein, free amino acid, trace elements and the like, and the extraction of the peptide and the unsaturated fatty acid from the shrimp roe not only can fully utilize resources and improve the added value of the processing of the freshwater shrimp, but also can reduce the environmental burden, and has important significance for promoting the benign development of the processing industry of the freshwater shrimp. However, the shrimp by-products are easily infected by endogenous enzymes and exogenous microorganisms in the process of collection, and the phenomena of freshness reduction, nutritional value reduction and even putrefaction and deterioration are easily caused.
Rice is an important grain crop, the yield of the rice accounts for 25% of the yield of all grain crops, rice bran is a mixture of a milled husk and a small amount of rice embryo and broken rice in the process of milling brown rice, is a byproduct of rice processing, and rice bran resources in China are the first in the world. Although rice bran accounts for only 6-8% of the weight of rice, rice bran contains about 14-20% of protein, and the content of protein far exceeds that of the rice (about 7%). The soluble protein content in the rice bran protein exceeds 70%, has ideal amino acid composition and high digestibility, is a good dietary fiber source, and is a protein with the lowest sensitization in the grains studied so far. The rice bran protein is subjected to enzymolysis under certain conditions to obtain a rice bran protein hydrolysate, namely functional peptide with certain biological activity. Although the rice bran yield is the first in the world in China, the effective utilization rate of the rice bran is very low (less than 20%), most of the rice bran is used as feed, and the resource waste is serious. The reason is that the rice bran has high content of the lipohydrolase and the lipoxygenase and strong activity, so that the acid value is rapidly increased, the rice bran is rapidly rancid, even is heated and mildewed, and the comprehensive utilization is influenced. For example, 108185110A discloses a method for improving functional properties of rice bran protein, which has certain effect of stabilizing rice bran by using twin-screw extrusion, but the high extrusion temperature in the method can cause partial loss of active substances and has complicated operation and higher cost.
Disclosure of Invention
The invention aims to solve the technical problems and provides a preparation method of the composite polypeptide with simple process, capability of effectively killing microorganisms and inactivating enzyme activity, low production cost, high nutritional value, high yield of active substances such as gamma-oryzanol and the like and high DPPH free radical clearance rate.
The compound polypeptide is prepared by mixing a crayfish byproduct protein hydrolysate powder and a rice bran protein peptide powder;
the crawfish byproduct protein hydrolysate is prepared by taking pretreated crawfish byproduct powder as a raw material, and performing irradiation, enzymolysis, separation, concentration and spray drying;
the rice bran protein peptide powder is prepared by taking crushed rice bran as a raw material and carrying out ohmic heating treatment, enzymolysis, separation and concentration and spray drying.
Specifically, the preparation of the crayfish byproduct protein hydrolysate comprises the following steps:
(a1) pretreatment of crayfish by-products: filtering and collecting the crushed crayfish meat, the crayfish egg and/or the water washing protein, drying and crushing to obtain crayfish byproduct powder;
(a2) irradiation: for crayfish byproduct powder60Co-gamma ray irradiation is carried out, the irradiation dose is 3-6 kGy, the irradiation time is 2-4 h, the sample is 40-100 cm away from the ground, the irradiation unevenness is less than 1.0, and the irradiation environment temperature is 18 ℃;
(a3) enzymolysis of crayfish byproduct powder: adding water with the mass being 4-10 times that of the irradiated crawfish byproduct powder, adjusting the pH value of the solution to 7.0-8.5, heating to 45-55 ℃, adding a complex enzyme consisting of porcine pancreatic lipase, flavourzyme, alkaline protease and papain, and carrying out enzymolysis for 1-2 hours at constant temperature; heating the enzymolysis liquid to inactivate enzyme at a temperature of more than or equal to 85 ℃ for more than or equal to 20min after enzymolysis is finished to obtain a crayfish byproduct protein hydrolysate;
(a4) separation and concentration: separating the crayfish byproduct protein hydrolysate by an ultrafiltration method, controlling the ultrafiltration temperature to be 20-25 ℃ and the ultrafiltration pressure to be 0.1-0.4 MPa, concentrating, and spray drying to obtain the crayfish byproduct protein hydrolysate.
In the step (a1), the water content of the crawfish byproduct powder is controlled to be 5-8 wt%.
In the step (a2), the activity of the cobalt source in the irradiation is 1.21 × 1016Bq, and the unit absorbed dose is 2.35-32.28 Gy/min.
In the step (a3), the adding amount of the porcine pancreatic lipase is 3500-4500U/g crawfish byproduct powder, the adding amount of the flavourzyme is 2000-3000U/g crawfish byproduct powder, the adding amount of the alkaline protease is 3000-4000U/g crawfish byproduct powder, and the adding amount of the papain is 3000-4000U/g crawfish byproduct powder.
Specifically, the preparation of the rice bran protein peptide powder comprises the following steps:
(b1) ohmic heating treatment of rice bran: crushing rice bran, carrying out ohmic heating treatment by taking water as a medium and the rice bran concentration of 10-30% (w/V) at the electric field frequency of 50Hz and the electric field intensity of 60-70V/cm until the highest temperature of rice bran liquid is 50-60 ℃, preserving the heat for 1-3 min, and then stopping the treatment and cooling;
(b2) extracting rice bran protein by rice bran enzymolysis: adjusting the pH value of the rice bran solution to 4.5-6.5, adding cellulase at the temperature of 45-55 ℃, performing constant-temperature enzymolysis for 0.5-1.5 h, adjusting the pH value to 10-12, and centrifuging at 6000r/min for 10min to obtain rice bran protein liquid;
(b3) and (3) rice bran protein enzymolysis extraction: adding water into the rice bran protein liquid, controlling the concentration of a substrate to be 1.0-5.0 mg/mL, fully stirring uniformly, adjusting the pH value to be 7.5-10, adding trypsin, carrying out constant-temperature water bath at 35-45 ℃ for 1-2 h, heating and enzyme deactivation treatment on an enzymolysis liquid after enzymolysis is finished, carrying out enzyme deactivation at the temperature of 94-96 ℃ for 15-25 min, and carrying out centrifugal separation on the enzymolysis liquid to obtain a rice bran protein peptide liquid;
(b4) separation and concentration: separating the rice bran protein peptide liquid by adopting an ultrafiltration method, controlling the ultrafiltration operation temperature to be 20-25 ℃ and the ultrafiltration operation pressure to be 0.1-0.4 MPa, and then concentrating and spray drying to obtain the rice bran protein peptide powder.
And (b1) crushing the rice bran to 80-100 meshes.
In the step (b2), the enzyme activity of the cellulase is 1 x 105～2×105U/g, the addition amount is 3.5-4.5% of the rice bran.
In the step (b3), the enzyme activity of the trypsin is 4 x 104～5×104U/g, the addition amount is 5-8% of the rice bran.
The ratio of the crayfish byproduct hydrolyzed protein powder to the rice bran protein peptide powder is 1: 1.5-3.5, and the particle size is required to be more than or equal to 120 meshes.
In view of the problems in the background art, the inventor makes the following improvements:
one) selection60And irradiating the pretreated crayfish byproduct powder by using a Co-gamma ray. After the food is irradiated by a certain dose, pathogenic bacteria, putrefying bacteria and other harmful microorganisms in the food can be effectively killed, the shelf life of the food is prolonged, the problems of chemical residue, radioactive pollution and the like do not exist, and the method is carried out at normal temperature. Aiming at the crayfish byproduct powder, the irradiation dose is controlled to be 3-6 kGy, the irradiation time is 2-4 h, unacceptable irradiation odor can be generated due to overhigh irradiation dose, the eating taste is influenced, and effective sterilization is difficult due to overlow irradiation dose. The water content of the crawfish byproduct powder is further controlled to be 5-8 wt%, too high water content is subjected to radiolysis to generate too much free radicals, crawfish yellow protein and fat are more easily oxidized, and too low water content can generate too high production energy consumption.
And secondly) carrying out enzymolysis on the crayfish byproduct powder by adopting a compound enzyme consisting of the porcine pancreatic lipase, the flavourzyme, the alkaline protease and the papain, wherein the porcine pancreatic lipase can hydrolyze fat in the crayfish byproduct, the porcine pancreatic lipase is non-specific lipase, ester bonds of all sites in triglyceride can be hydrolyzed to a greater extent, more free fatty acids are released, the flavourzyme, the alkaline protease and the papain can hydrolyze proteins, but sites of different proteases acting on the proteins are different, and the aim of improving the enzymolysis efficiency and effect can be achieved by compounding the compound enzyme. The enzymolysis temperature is further controlled to be 45-55 ℃, and the activity of protease is influenced by overhigh temperature and overlow temperature, so that the enzymolysis effect is influenced.
And II) carrying out stabilizing treatment on the rice bran by selecting ohmic heating treatment. The ohmic heating treatment has a non-thermoelectric field effect and a strong oxidation effect generated by ionization of a liquid medium, and can cause the permeability of microorganisms and rice bran cell membranes to be increased and even to be broken, and biological macromolecules to be dissociated, so that the aims of sterilizing inactive enzyme and improving the yield of an active substance gamma-oryzanol are fulfilled. In addition, ohmic heating utilizes the dielectric property of rice bran liquid, when current passes through, causes the rice bran liquid temperature to rise, further reaches the purpose of inactive enzyme of disinfecting, has the advantage such as the intensification is fast, and the heating is even, and is pollution-free, heat utilization rate height. According to the invention, aiming at the characteristics that the rice bran is easily rancid under the enzymatic action of high-activity lipase and lipoxygenase and active substances in the rice bran which are easily oxidized are easily subjected to oxidation reaction at a higher temperature so as to be damaged, when ohmic heating treatment is carried out, the electric field frequency is controlled to be 50Hz, the electric field intensity is 60-70V/cm, the temperature is kept for 1-3 min at the highest temperature of 50-60 ℃, and through controlling the parameters, the purposes of sterilizing and inactivating enzymes are ensured, the rice bran is stabilized, and the loss of the active substances in the rice bran which are easily oxidized can be effectively reduced.
Thirdly), performing two-step hydrolysis on the rice bran on the basis of sterilization inactive enzyme, degrading the rice bran cell walls by using cellulase to release more protein, increasing the protein extraction rate, and obtaining rice bran protein liquid, wherein the pH value of the rice bran solution is controlled to be 4.5-6.5, the gelatinization of starch is intensified at the temperature of 45-55 ℃, the viscosity of the rice bran solution is increased, and the protein is not easy to dissolve out; too low can affect the enzymatic activity of the cellulase; then adding trypsin for further enzymolysis, so that the rice bran protein is degraded into polypeptide micromolecules with different chain lengths, and obtaining the rice bran protein peptide liquid. The ohmic heating treatment is used for leading the cell walls and cell membranes of the rice bran to gather charges and form pores under the influence of an electric field, the cell contents can be better dissolved out through the enzymolysis of cellulase, the content of gamma-oryzanol in the final product is higher, more bioactive peptides can be obtained through the second step of enzymolysis, and the health care function of the product is improved.
Has the advantages that: the irradiation is utilized to sterilize the crayfish byproduct powder, the ohmic heating treatment is utilized to stabilize the rice bran fine powder, microorganisms are killed, the enzyme activity is inactivated, the crayfish byproduct and the crayfish byproduct hydrolyzed protein peptide which can be directly absorbed by the human body in the rice bran are extracted, the unsaturated fatty acid such as the rice bran protein active peptide, EPA, DHA and the like, the gamma-oryzanol, the amino acid and the dietary fiber are complementary and synergistic, the immune function is improved, the oxidation resistance, the blood pressure reduction and the blood fat reduction are facilitated, and the essential amino acid, the EPA and the DHA of the human body are supplemented, which cannot be compared with any single peptide. The invention has the advantages of simple process, easy control, low production cost, energy conservation, balanced and rich nutrition, high nutritive value, high yield of active substances such as gamma-oryzanol and the like and high DPPH free radical clearance rate, and has important significance for promoting high-valued utilization of aquatic product byproducts and rice byproducts in China and transformation upgrading of processing technology.
Detailed Description
Example 1:
preparing a crayfish byproduct protein hydrolysate:
(a1) filtering and collecting crayfish by-product, cleaning, blowing at 50 deg.C for drying, pulverizing to obtain crayfish by-product powder with water content of 8 wt%,
(a2) use of60Co-gamma ray irradiation is carried out, the irradiation dose is 6kGy, the irradiation time is 4 hours, the sample is 100cm away from the ground, the irradiation unevenness is less than 1.0, and the irradiation environment temperature is 18 ℃.
(a3) Adding 10 times of water by mass into the crayfish byproduct powder, adjusting the pH value of the solution to 8.5, heating to 55 ℃, and adding a complex enzyme consisting of porcine pancreatic lipase, flavourzyme, alkaline protease and papain, wherein the adding amount of the porcine pancreatic lipase is 4500U/g of the crayfish byproduct powder, the adding amount of the flavourzyme is 3000U/g of the crayfish byproduct powder, the adding amount of the alkaline protease is 4000U/g of the crayfish byproduct powder, and the adding amount of the papain is 4000U/g of the crayfish byproduct powder, and carrying out constant-temperature enzymolysis for 2 hours. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme.
(a4) Respectively separating the hydrolyzed protein liquid of the crayfish by-product by adopting an ultrafiltration method, controlling the temperature to be 20-25 ℃, controlling the ultrafiltration operation pressure to be 0.1-0.4 MPa, concentrating the hydrolysate by utilizing multi-effect concentration equipment, respectively drying and screening the concentrated hydrolysate by spray drying, and performing spray drying at the air inlet temperature of 200 ℃ and the air outlet temperature of 105 ℃ to obtain the hydrolyzed protein powder of the crayfish by-product.
Preparation of rice bran protein peptide powder
(b1) Pulverizing fresh rice bran to 100 mesh, spraying water to make rice bran concentration 30 wt%, ohmic heating at electric field frequency of 50Hz, electric field strength of 70V/cm and temperature of 60 deg.C for 3min, stopping heating, and cooling for 10 min;
(b2) adjusting pH of rice bran solution to 6.5, adding cellulase when temperature reaches 55 deg.C, enzyme activity is 2 × 105U/g, adding 4.5 wt% of enzyme, performing enzymolysis at constant temperature for 1.5h, adjusting pH to 12, and centrifuging at 6000r/min for 10min to obtain rice bran protein solution.
(b3) Adding water into rice bran protein solution, controlling substrate concentration at 5.0mg/mL, stirring well, adjusting pH to 10, adding 8 wt% trypsin with enzyme activity of 5 × 104U/g. Water bath is carried out for 2h at the constant temperature of 45 ℃. Heating the enzymolysis solution for enzyme deactivation at 96 deg.C for 25min, and centrifuging to obtain testa oryzae protein peptide solution.
(b4) Separating the hydrolyzed protein liquid of the crayfish byproduct and the rice bran protein peptide liquid by an ultrafiltration method, controlling the temperature to be 20-25 ℃, and controlling the ultrafiltration pressure to be 0.1-0.4 MPa. Concentrating the hydrolysate by using a multi-effect concentration device, and drying and screening the concentrated hydrolysate by spray drying respectively at an air inlet temperature of 200 ℃ and an air outlet temperature of 105 ℃ to obtain the rice bran protein peptide powder.
In the embodiment, 50g of crawfish byproduct hydrolyzed protein powder and 75g of rice bran protein peptide powder are compounded to obtain the composite polypeptide powder, wherein the moisture content of the polypeptide powder is 2.3 wt%, the amino acid content is 357.29mg/g, the soluble dietary fiber content is 5.37g/100g, the EPA and DHA content is 2.35mg/g, the gamma-oryzanol content is 4.38mg/g, and the DPPH free radical scavenging rate is 93.65%.
Example 2
Preparing a crayfish byproduct protein hydrolysate:
(a1) in the same manner as in example 1, crawfish byproduct powder having a moisture content of 6 wt% was obtained.
(a2) The crawfish byproduct powder with water content of 6 wt% is used60Co-gamma ray irradiation is carried out, the irradiation dose is 5kGy, and the irradiation time is 3 h.
(a3) Adding 6 times of water by mass into the crayfish by-product powder, adjusting the pH value to 8, heating to 45 ℃, adding a complex enzyme, wherein the adding amount of the porcine pancreatic lipase is 4000U/g crayfish by-product powder, the adding amount of the flavourzyme is 2500U/g crayfish by-product powder, the adding amount of the alkaline protease is 3500U/g crayfish by-product powder, and the adding amount of the papain is 3500U/g crayfish by-product powder, and carrying out enzymolysis for 1.5h at constant temperature. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme.
(a4) In the same manner as in example 1, a hydrolyzed protein powder of a crawfish byproduct was obtained.
Preparation of rice bran protein peptide powder
(b1) Pulverizing fresh rice bran into 80 mesh, spraying water to make rice bran concentration be 22 wt%, and ohmic heating at 60 deg.C and 60V/cm electric field intensity for 2min to obtain rice bran solution.
(b2) Adjusting the pH value of the rice bran solution to 5.5, adding cellulase when the temperature is 40 ℃, adding 4 wt% of enzyme, performing enzymolysis for 1h at constant temperature, adjusting the pH value to 11, and centrifuging to obtain the rice bran protein solution.
(b3) Adding water into the rice bran protein solution, controlling the substrate concentration to be 2.5mg/mL, fully and uniformly stirring, adjusting the pH to be 8.5, adding 6.5 wt% of trypsin, and carrying out constant-temperature water bath at 40 ℃ for 1.5 h. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme, and centrifugally separating the enzymolysis liquid to obtain the rice bran protein peptide liquid.
(b4) In the same manner as in example 1, rice bran protein peptide powder was obtained.
In the embodiment, 50g of crawfish byproduct hydrolyzed protein powder and 125g of rice bran protein peptide powder are compounded to obtain the composite polypeptide powder, wherein the moisture content of the polypeptide powder is 2.9 wt%, the amino acid content is 332.12mg/g, the soluble dietary fiber content is 5.44g/100g, the EPA and DHA content is 2.17mg/g, the gamma-oryzanol content is 4.57mg/g, and the DPPH free radical scavenging rate is 91.85%.
Example 3
Preparing a crayfish byproduct protein hydrolysate:
(a1) in the same manner as in example 1, crawfish byproduct powder having a moisture content of 5 wt% was obtained.
(a2) The crawfish byproduct powder with water content of 5 wt% is used60Co-gamma ray irradiation is carried out, the irradiation dose is 3kGy, and the irradiation time is 2 h.
(a3) Adding water with the mass being 4 times of that of the crawfish byproduct powder, adjusting the pH value to be 7, heating to 45 ℃, adding a complex enzyme, wherein the adding amount of the porcine pancreatic lipase is 3500U/g of the crawfish byproduct powder, the adding amount of the flavourzyme is 2000U/g of the crawfish byproduct powder, the adding amount of the alkaline protease is 3000U/g of the crawfish byproduct powder, and the adding amount of the papain is 3000U/g of the crawfish byproduct powder, and carrying out enzymolysis for 1h at constant temperature. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme.
(a4) In the same manner as in example 1, a hydrolyzed protein powder of a crawfish byproduct was obtained.
Preparation of rice bran protein peptide powder
(b1) Pulverizing fresh rice bran into 80 mesh, spraying water to make rice bran solution with rice bran concentration of 10 wt% undergo ohmic heating treatment, electric field strength 60V/cm, temperature 50 deg.C, and heat-insulating for 1 min.
(b2) Adjusting pH of the rice bran solution to 4.5, adding cellulase when the temperature reaches 45 ℃, adding 3.5 wt% of enzyme, performing enzymolysis at constant temperature for 0.5h, adjusting pH to 10, and centrifuging to obtain rice bran protein solution.
(b3) Adding water into the rice bran protein solution, controlling the substrate concentration to be 1.0mg/mL, fully and uniformly stirring, adjusting the pH to be 7.5, adding 5 wt% of trypsin, and carrying out constant-temperature water bath at 35 ℃ for 1 h. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme, and centrifugally separating the enzymolysis liquid to obtain the rice bran protein peptide liquid.
(b4) In the same manner as in example 1, rice bran protein peptide powder was obtained.
In the embodiment, 50g of crawfish byproduct hydrolyzed protein powder and 175g of rice bran protein peptide powder are compounded to obtain the composite polypeptide powder, wherein the moisture content of the polypeptide powder is 3.4 wt%, the amino acid content is 314.57mg/g, the soluble dietary fiber content is 5.91g/100g, the EPA and DHA content is 1.97mg/g, the gamma-oryzanol content is 4.51mg/g, and the DPPH free radical scavenging rate is 89.38%.
Example 4
Preparing a crayfish byproduct protein hydrolysate:
(a1) in the same manner as in example 1, crawfish byproduct powder having a moisture content of 7 wt% was obtained.
(a2) The crawfish byproduct powder with water content of 7 wt% is used60Co-gamma ray irradiation is carried out, the irradiation dose is 4kGy, and the irradiation time is 2.5 h.
(a3) Adding water with the mass being 8 times of that of the crawfish byproduct powder into the crawfish byproduct powder, adjusting the pH value to be 7.5, heating to 45 ℃, adding complex enzyme, wherein the adding amount of the porcine pancreatic lipase is 3500U/g of the crawfish byproduct powder, the adding amount of the flavourzyme is 2000U/g of the crawfish byproduct powder, the adding amount of the alkaline protease is 3500U/g of the crawfish byproduct powder, and the adding amount of the papain is 3000U/g of the crawfish byproduct powder, and carrying out enzymolysis for 1.5 hours at constant temperature. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme.
(a4) In the same manner as in example 1, a hydrolyzed protein powder of a crawfish byproduct was obtained.
Preparation of rice bran protein peptide powder
(b1) Pulverizing fresh rice bran to 100 mesh, spraying water to make rice bran concentration 20 wt%, and ohmic heating at 55 deg.C and electric field strength 65V/cm for 3 min.
(b2) Adjusting pH of the rice bran solution to 5, adding cellulase when the temperature reaches 50 ℃, adding 4 wt% of enzyme, performing enzymolysis at constant temperature for 0.5h, adjusting pH to 10, and centrifuging to obtain rice bran protein solution.
(b3) Adding water into the rice bran protein solution, controlling the substrate concentration to be 2.0mg/mL, fully and uniformly stirring, adjusting the pH to be 8.0, adding 5 wt% of trypsin, and carrying out constant-temperature water bath at 35 ℃ for 1 h. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme, and centrifugally separating the enzymolysis liquid to obtain the rice bran protein peptide liquid.
(b4) In the same manner as in example 1, rice bran protein peptide powder was obtained.
In the embodiment, 50g of crawfish byproduct hydrolyzed protein powder and 125g of rice bran protein peptide powder are compounded to obtain the composite polypeptide powder, wherein the moisture content of the polypeptide powder is 2.7 wt%, the amino acid content is 320.16mg/g, the soluble dietary fiber content is 5.63g/100g, the EPA and DHA content is 2.05mg/g, the gamma-oryzanol content is 4.49mg/g, and the DPPH free radical scavenging rate is 89.74%.
Example 5
Preparing a crayfish byproduct protein hydrolysate:
(a1) as in example 1, crawfish byproduct powder having a moisture content of 6.5 wt% was obtained.
(a2) The crawfish byproduct powder with water content of 6.5 wt% is used60Co-gamma ray irradiation is carried out, the irradiation dose is 4.5kGy, and the irradiation time is 3 h.
(a3) Adding water with the mass being 7 times of that of the crawfish byproduct powder into the crawfish byproduct powder, adjusting the pH value to be 8, heating to 50 ℃, adding a complex enzyme, wherein the adding amount of the porcine pancreatic lipase is 4000U/g of the crawfish byproduct powder, the adding amount of the flavourzyme is 2500U/g of the crawfish byproduct powder, the adding amount of the alkaline protease is 3500U/g of the crawfish byproduct powder, and the adding amount of the papain is 3500U/g of the crawfish byproduct powder, and carrying out enzymolysis for 1.5h at constant temperature. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme.
(a4) In the same manner as in example 1, a hydrolyzed protein powder of a crawfish byproduct was obtained.
Preparation of rice bran protein peptide powder
(b1) Pulverizing fresh rice bran to 90 mesh, spraying water to make rice bran concentration 20 wt%, and performing ohmic heating treatment at electric field intensity of 65V/cm and temperature of 55 deg.C for 2 min.
(b2) Adjusting the pH value of the rice bran solution to 5.5, adding cellulase when the temperature reaches 50 ℃, adding 4 wt% of enzyme, performing enzymolysis for 1h at constant temperature, adjusting the pH value to 11, and centrifuging to obtain the rice bran protein solution.
(b3) Adding water into the rice bran protein solution, controlling the substrate concentration to be 3.0mg/mL, fully and uniformly stirring, adjusting the pH to be 9.0, adding 7 wt% of trypsin, and carrying out constant-temperature water bath at 40 ℃ for 1.5 h. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme, and centrifugally separating the enzymolysis liquid to obtain the rice bran protein peptide liquid.
(b4) In the same manner as in example 1, rice bran protein peptide powder was obtained.
In the embodiment, 50g of crawfish byproduct hydrolyzed protein powder and 100g of rice bran protein peptide powder are compounded to obtain the composite polypeptide powder, wherein the moisture content of the polypeptide powder is 2.8 wt%, the amino acid content is 327.48mg/g, the soluble dietary fiber content is 5.71g/100g, the EPA and DHA content is 2.13mg/g, the gamma-oryzanol content is 4.62mg/g, and the DPPH free radical scavenging rate is 90.41%.
Example 6
Preparing a crayfish byproduct protein hydrolysate:
(a1) as in example 1, crawfish byproduct powder having a moisture content of 7.5 wt% was obtained.
(a2) The crawfish byproduct powder with water content of 7.5 wt% is used60Co-gamma ray irradiation is carried out, the irradiation dose is 4kGy, and the irradiation time is 2.5 h.
(a3) Adding water with the mass being 7 times of that of the crawfish byproduct powder into the crawfish byproduct powder, adjusting the pH value to be 7.5, heating to 50 ℃, adding complex enzyme, wherein the adding amount of the porcine pancreatic lipase is 3500U/g of the crawfish byproduct powder, the adding amount of the flavourzyme is 2500U/g of the crawfish byproduct powder, the adding amount of the alkaline protease is 3500U/g of the crawfish byproduct powder, and the adding amount of the papain is 3000U/g of the crawfish byproduct powder, and carrying out enzymolysis for 1.5 hours at constant temperature. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme.
(a4) In the same manner as in example 1, a hydrolyzed protein powder of a crawfish byproduct was obtained.
Preparation of rice bran protein peptide powder
(b1) Pulverizing fresh rice bran to 90 mesh, spraying water to make rice bran concentration be 25 wt%, and ohmic heating at 50 deg.C and electric field strength of 70V/cm for 3 min.
(b2) Adjusting pH of the rice bran solution to 5.5, adding cellulase when the temperature reaches 50 ℃, adding 4.5 wt% of enzyme, performing enzymolysis for 1h at constant temperature, adjusting pH to 11, and centrifuging to obtain rice bran protein solution.
(b3) Adding water into the rice bran protein solution, controlling the substrate concentration to be 3.0mg/mL, fully and uniformly stirring, adjusting the pH to be 8.5, adding 7 wt% of trypsin, and carrying out constant-temperature water bath at 40 ℃ for 1 h. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme, and centrifugally separating the enzymolysis liquid to obtain the rice bran protein peptide liquid.
(b4) In the same manner as in example 1, rice bran protein peptide powder was obtained.
In the embodiment, 50g of crawfish byproduct hydrolyzed protein powder and 75g of rice bran protein peptide powder are compounded to obtain the composite polypeptide powder, wherein the moisture content of the polypeptide powder is 3.2 wt%, the amino acid content is 343.18mg/g, the soluble dietary fiber content is 5.47g/100g, the EPA and DHA content is 2.39mg/g, the gamma-oryzanol content is 4.53mg/g, and the DPPH free radical scavenging rate is 91.25%.
Example 7
Preparing a crayfish byproduct protein hydrolysate:
(a1) as in example 1, crawfish byproduct powder having a moisture content of 6.5 wt% was obtained.
(a2) The crawfish byproduct powder with water content of 6.5 wt% is used60Co-gamma ray irradiation is carried out, the irradiation dose is 3kGy, and the irradiation time is 3 h.
(a3) Adding water with the mass being 9 times of that of the crawfish byproduct powder, adjusting the pH value to be 7, heating to 55 ℃, adding a complex enzyme, wherein the adding amount of the porcine pancreatic lipase is 4500U/g of the crawfish byproduct powder, the adding amount of the flavourzyme is 2500U/g of the crawfish byproduct powder, the adding amount of the alkaline protease is 3500U/g of the crawfish byproduct powder, and the adding amount of the papain is 3500U/g of the crawfish byproduct powder, and carrying out enzymolysis for 1h at constant temperature. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme.
(a4) In the same manner as in example 1, a hydrolyzed protein powder of a crawfish byproduct was obtained.
Preparation of rice bran protein peptide powder
(b1) Pulverizing fresh rice bran into 80 mesh, spraying water to make rice bran concentration 15 wt%, and performing ohmic heating treatment at electric field intensity of 65V/cm and temperature of 50 deg.C for 2 min.
(b2) Adjusting pH of the rice bran solution to 4.5, adding cellulase when the temperature reaches 55 ℃, adding 4 wt% of enzyme, performing enzymolysis at constant temperature for 1.5h, adjusting pH to 12, and centrifuging to obtain rice bran protein solution.
(b3) Adding water into the rice bran protein solution, controlling the substrate concentration to be 2.5mg/mL, fully and uniformly stirring, adjusting the pH to be 9.5, adding 6 wt% of trypsin, and carrying out constant-temperature water bath at 45 ℃ for 1.5 h. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme, and centrifugally separating the enzymolysis liquid to obtain the rice bran protein peptide liquid.
(b4) In the same manner as in example 1, rice bran protein peptide powder was obtained.
In the embodiment, 50g of crawfish byproduct hydrolyzed protein powder and 112.5g of rice bran protein peptide powder are compounded to obtain the composite polypeptide powder, wherein the moisture content of the polypeptide powder is 2.6 wt%, the amino acid content is 367.39mg/g, the soluble dietary fiber content is 5.58g/100g, the EPA and DHA content is 2.26mg/g, the gamma-oryzanol content is 4.62mg/g, and the DPPH free radical scavenging rate is 89.84%.
Example 8
Preparing a crayfish byproduct protein hydrolysate:
(a1) as in example 1, crawfish byproduct powder having a moisture content of 7.5 wt% was obtained.
(a2) The crawfish byproduct powder with water content of 7.5 wt% is used60Co-gamma ray irradiation is carried out, the irradiation dose is 6kGy, and the irradiation time is 2 h.
(a3) Adding water with the mass being 9 times of that of the crawfish byproduct powder into the crawfish byproduct powder, adjusting the pH value to be 8, heating to 50 ℃, adding a complex enzyme, wherein the adding amount of the porcine pancreatic lipase is 3500U/g of the crawfish byproduct powder, the adding amount of the flavourzyme is 2500U/g of the crawfish byproduct powder, the adding amount of the alkaline protease is 4000U/g of the crawfish byproduct powder, and the adding amount of the papain is 4000U/g of the crawfish byproduct powder, and carrying out enzymolysis for 1.5 hours at constant temperature. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme.
(a4) In the same manner as in example 1, a hydrolyzed protein powder of a crawfish byproduct was obtained.
Preparation of rice bran protein peptide powder
(b1) Pulverizing fresh rice bran to 100 mesh, spraying water to make rice bran concentration 15 wt%, and ohmic heating at 60 deg.C and electric field strength 70V/cm for 3 min.
(b2) Adjusting pH of the rice bran solution to 5.5, adding cellulase when the temperature reaches 45 ℃, adding 4.5 wt% of enzyme, performing enzymolysis at constant temperature for 0.5h, adjusting pH to 10, and centrifuging to obtain rice bran protein solution.
(b3) Adding water into the rice bran protein solution, controlling the substrate concentration to be 3.5mg/mL, fully and uniformly stirring, adjusting the pH to be 8.5, adding 5 wt% of trypsin, and carrying out constant-temperature water bath at 40 ℃ for 2 h. And after the enzymolysis is finished, heating the enzymolysis liquid to inactivate enzyme, and centrifugally separating the enzymolysis liquid to obtain the rice bran protein peptide liquid.
(b4) In the same manner as in example 1, rice bran protein peptide powder was obtained.
In the embodiment, 50g of shrimp yellow hydrolyzed protein powder and 150g of rice bran protein peptide powder are compounded to obtain the composite polypeptide powder, wherein the moisture content of the polypeptide powder is 2.9 wt%, the amino acid content is 362.15mg/g, the soluble dietary fiber content is 5.49g/100g, the EPA and DHA content is 2.33mg/g, the gamma-oryzanol content is 4.49mg/g, and the DPPH free radical clearance rate is 91.16%.
Comparative examples of irradiation treatment:
placing the pretreated crayfish byproduct powder in a polyethylene plastic sealing bag, sealing the sealing bag and then carrying out60Co-gamma ray irradiation, cobalt source activity 1.21X 1016Bq and dynamic dose rate is 2.35-32.28 Gy/min. And (3) treating the crayfish with different irradiation doses of 0, 1, 2, 3, 4, 5, 6 and 7kGy, and measuring the total number of colonies of the crayfish byproduct powder treated with different irradiation doses. The actual absorbed dose of the sample is calibrated by a silver dichromate dosimeter (a self-made glass ampoule dosimeter, wherein the low dose range contains 0.35mmol/L of silver dichromate, and the high dose range contains 2.5mmol/L of silver dichromate), the dosimeter is stored at normal temperature, and dose detection is carried out after the sample is irradiated, so that the actual absorbed dose is respectively 0, 1.25, 2.35, 3.32, 4.17, 5.30, 6.11 and 7.24 kGy.
TABLE 1 Total number of colonies at different irradiation doses
As can be seen from the results in Table 1, the irradiation treatment was effective in killing microorganisms. When the irradiation dose is 3-7 kGy, the total number of bacterial colonies in the crayfish byproduct powder is gradually reduced along with the increase of the irradiation dose, but the irradiation dose of 7kGy can generate unacceptable irradiation odor, so 3-6 kGy is selected as the irradiation dose for stabilizing the crayfish byproduct powder.
Ohmic heating treatment comparative experimental example:
the rice bran sample is uniformly mixed with the medium by spraying, the mixture is hermetically packaged in an ohmic heating treatment chamber, adjustable parameters comprise power, frequency and temperature, when the rice bran sample reaches the required parameters, a power supply is turned off, the rice bran sample is kept at the set temperature for a period of time, the rice bran sample is taken out of the treatment chamber, and the activity of the lipoxygenase is measured. Ohmic heating treatment is carried out on the rice bran solution by adopting different heating temperatures of 30, 40, 50, 60 and 70 ℃, different heat preservation times of 1, 2, 3 and 4min and different electric field strengths of 30, 40, 50, 60 and 70V/cm.
TABLE 2 Lipase Activity at different treatment temperatures (electric field intensity 60V/cm, incubation time 2min)
TABLE 3 Lipase Activity at different incubation times (field strength 60V/cm, treatment temperature 50 ℃ C.)
TABLE 4 Lipase Activity at different electric field strengths (treatment temperature 50 ℃ C., incubation time 2min)
As can be seen from the results in tables 2, 3 and 4, the enzyme activity was effectively inactivated by ohmic heating treatment. Under the same other conditions, the enzyme activity is reduced along with the increase of the treatment temperature, but the temperature has little influence on the enzyme activity after 50 ℃. When other conditions are the same, the ohmic heating treatment time hardly influences the activity of the inactivated enzyme, and as the process is mainly heat preservation, the influence of a simple heat effect on the activity of the inactivated enzyme is small. When other conditions are the same, the enzyme activity is reduced along with the increase of the electric field intensity, and the current is increased and the current density is enhanced due to the increase of the voltage, so that the enzyme activity passivation is accelerated.
Claims (10)
1. A preparation method of compound multi-peptide is characterized in that the compound multi-peptide is prepared by mixing hydrolyzed protein powder of crayfish by-products and rice bran protein peptide powder;
the crawfish byproduct protein hydrolysate is prepared by taking pretreated crawfish byproduct powder as a raw material, and performing irradiation, enzymolysis, separation, concentration and spray drying;
the rice bran protein peptide powder is prepared by taking crushed rice bran as a raw material and carrying out ohmic heating treatment, enzymolysis, separation and concentration and spray drying.
2. The method for preparing the complex polypeptide as claimed in claim 1, wherein the preparation of the crayfish byproduct protein hydrolysate powder comprises the following steps:
(a1) pretreatment of crayfish by-products: filtering and collecting the crushed crayfish meat, the crayfish egg and/or the water washing protein, drying and crushing to obtain crayfish byproduct powder;
(a2) irradiation: for crayfish byproduct powder60Co-gamma ray irradiation is carried out, the irradiation dose is 3-6 kGy, the irradiation time is 2-4 h, the sample is 40-100 cm away from the ground, the irradiation unevenness is less than 1.0, and the irradiation environment temperature is 18 ℃;
(a3) enzymolysis of crayfish byproduct powder: adding water with the mass being 4-10 times that of the irradiated crawfish byproduct powder, adjusting the pH value of the solution to 7.0-8.5, heating to 45-55 ℃, adding a complex enzyme consisting of porcine pancreatic lipase, flavourzyme, alkaline protease and papain, and carrying out enzymolysis for 1-2 hours at constant temperature; heating the enzymolysis liquid to inactivate enzyme at a temperature of more than or equal to 85 ℃ for more than or equal to 20min after enzymolysis is finished to obtain a crayfish byproduct protein hydrolysate;
(a4) separation and concentration: separating the crayfish byproduct protein hydrolysate by an ultrafiltration method, controlling the ultrafiltration temperature to be 20-25 ℃ and the ultrafiltration pressure to be 0.1-0.4 MPa, concentrating, and spray drying to obtain the crayfish byproduct protein hydrolysate.
3. The method for producing a complex polypeptide as claimed in claim 2, wherein in the step (a1), the water content of the crayfish by-product powder is controlled to 5 to 8 wt%.
4. The method of claim 2, wherein in the step (a2), the activity of the cobalt source in the irradiation is 1.21 x 1016Bq, and the unit absorbed dose is 2.35-32.28 Gy/min.
5. The method for preparing complex polypeptide according to claim 2, wherein in the step (a3), the amount of the porcine pancreatic lipase added is 3500 to 4500U/g crayfish byproduct powder, the amount of the flavourzyme added is 2000 to 3000U/g crayfish byproduct powder, the amount of the alkaline protease added is 3000 to 4000U/g crayfish byproduct powder, and the amount of the papain added is 3000 to 4000U/g crayfish byproduct powder.
6. The method for preparing the complex polypeptide of any one of claims 1 to 5, wherein the rice bran protein peptide powder is prepared by the steps of:
(b1) ohmic heating treatment of rice bran: crushing rice bran, carrying out ohmic heating treatment by taking water as a medium and the rice bran concentration of 10-30% (w/V) at the electric field frequency of 50Hz and the electric field intensity of 60-70V/cm until the highest temperature of rice bran liquid is 50-60 ℃, preserving the heat for 1-3 min, and then stopping the treatment and cooling;
(b2) extracting rice bran protein by rice bran enzymolysis: adjusting the pH value of the rice bran solution to 4.5-6.5, adding cellulase at the temperature of 45-55 ℃, performing constant-temperature enzymolysis for 0.5-1.5 h, adjusting the pH value to 10-12, and centrifuging at 6000r/min for 10min to obtain rice bran protein liquid;
(b3) and (3) rice bran protein enzymolysis extraction: adding water into the rice bran protein liquid, controlling the concentration of a substrate to be 1.0-5.0 mg/mL, fully stirring uniformly, adjusting the pH value to be 7.5-10, adding trypsin, carrying out constant-temperature water bath at 35-45 ℃ for 1-2 h, heating and enzyme deactivation treatment on an enzymolysis liquid after enzymolysis is finished, carrying out enzyme deactivation at the temperature of 94-96 ℃ for 15-25 min, and carrying out centrifugal separation on the enzymolysis liquid to obtain a rice bran protein peptide liquid;
(b4) separation and concentration: separating the rice bran protein peptide liquid by adopting an ultrafiltration method, controlling the ultrafiltration operation temperature to be 20-25 ℃ and the ultrafiltration operation pressure to be 0.1-0.4 MPa, and then concentrating and spray drying to obtain the rice bran protein peptide powder.
7. The method for producing a complex polypeptide according to claim 5, wherein in the step (b1), the rice bran is pulverized to 80 to 100 mesh.
8. The method for preparing the complex polypeptide of claim 5, wherein the enzymatic activity of the cellulase in the step (b2) is 1 x 105～2×105U/g, the addition amount is 3.5-4.5% of the rice bran.
9. The method of claim 5, wherein in the step (b3), the trypsin has an enzymatic activity of 4X 104～5×104U/g, the addition amount is 5-8% of the rice bran.
10. The preparation method of the composite polypeptide as claimed in claim 1, wherein the ratio of the hydrolyzed protein powder of the crayfish byproduct to the rice bran protein peptide powder is 1: 1.5-3.5 by mass, and the particle size is not less than 120 meshes.
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| WO2020228713A1 (en) * | 2019-05-14 | 2020-11-19 | 湖南贝贝昇生物科技有限公司 | Method for extracting useful substances from shrimp shells |
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