CN114250261A - Extraction method of red date kernel polypeptide - Google Patents
Extraction method of red date kernel polypeptide Download PDFInfo
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Abstract
The invention relates to a method for extracting red date seed polypeptide, and belongs to the technical field of polypeptide extraction. The extraction method sequentially comprises the steps of preparing a crude extract, grading salting out of the crude extract, enzymolysis, ultrafiltration, concentration, nanofiltration and vacuum freeze drying, finally red date pit polypeptide powder is prepared, the hydrolysis degree of protein in red date pits is measured by a TCA method, and the content of polypeptide is measured by a Kjeldahl method. The extraction method has the advantages that the protein hydrolysis degree is high, the activity of the prepared polypeptide is high, the extraction time is short, the obtained red date core polypeptide shows better properties than red date protein in the aspects of hygroscopicity, thermal stability, solubility, viscosity and the like, a new way is provided for the extraction of the red date polypeptide, and the extraction method has a stronger industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of polypeptide extraction, and particularly relates to a method for extracting red date seed polypeptide.
Background
Peptides are compounds of alpha-amino acids linked together by peptide bonds, which are intermediates in the hydrolysis of proteins. The compounds obtained by dehydration condensation of two amino acid molecules are called dipeptides, and similarly, tripeptides, tetrapeptides, pentapeptides, and the like are also included. A molecule consisting of three or more amino acid molecules is called a polypeptide. The bioactive peptide is a peptide compound which is formed by dehydration condensation of 10-100 amino acid molecules and has a remarkable effect on regulating the life activities of living organisms or a special physiological function, and is also called as a functional peptide. In general, functional peptides are superior to proteins and amino acids in both nutritional and physiological functions. At present, the application of functional peptides mainly focuses on the aspects of polypeptide drugs, polypeptide drug carriers, tissue engineering materials, polypeptide nutritious foods and the like. Because plant polypeptide has wide sources and low price and has the physiological activity functions of resisting bacteria, resisting oxidation, reducing blood pressure and the like, the plant-derived functional peptide becomes a research hotspot of science and technology workers.
In recent years, the red dates are found to be rich in beneficial components such as various amino acids, proteins, carbohydrates, fats, trace elements, crude fibers, vitamins, organic acids and the like required by human bodies, which are all essential important substances for forming and participating in the life process of the human bodies and are also the material basis of the red dates with various edible and medicinal effects. The red jujube polypeptide has good health care effect and is widely applied. At present, when red date polypeptide is prepared, a protein enzymatic hydrolysis method is often adopted for preparation due to high synthesis cost and low yield and difficulty in separating target active peptide by a fermentation method.
For example, Sunweiyu (research on extraction and biological activity of golden-silk jujube polypeptide) takes jujube protein in concentrated jujube juice ultrafiltration cut-off liquid for producing golden-silk jujube as a raw material, and the optimal process for enzymolysis of jujube protein is researched through a composite enzymolysis process. However, the preparation process still has certain disadvantages: (1) the method mainly aims at the enzymolysis preparation of red date pulp, the research on extracting polypeptide from red date pits is not reported, and the red date pits are usually discarded as waste, so that the waste of resources is caused; (2) when the enzyme dosage reaches a certain amount, the substrate is saturated, excessive enzyme molecules may remain, and the protease is not inactivated after enzymolysis; in the enzymolysis process, more conditions need to be controlled, such as enzyme selection, temperature, substrate concentration, pH, time and the like, and the extraction conditions are relatively strict, so that the conditions are inconvenient to control in actual production, once one of the conditions is changed, the extraction speed and the extraction result are affected, and correspondingly, the performance of the prepared red date polypeptide is also affected, so that the proteolysis degree is low, the product activity is low, and the reaction time is long.
Therefore, the method for extracting the jujube pit polypeptide with excellent performance, simple process and convenient control has extremely important significance.
Disclosure of Invention
The invention aims to provide a method for extracting red date seed polypeptide, which is used for solving the technical problems of red date seed waste, low proteolysis degree, low product activity and long reaction time in the traditional red date polypeptide preparation method.
A method for extracting red date kernel polypeptide comprises the following steps: step (1) preparation of crude extract: removing jujube meat from fresh red dates, drying to obtain a red date pit raw material, crushing the red date pits into powder in a crusher, sieving, dissolving the red date pit powder in ultrapure water, preparing red date pit slurry with different concentration gradients, placing the red date pit slurry in an ultrasonic instrument for ultrasonic oscillation, and centrifuging after the red date pit slurry is fully dissolved to obtain a supernatant, wherein the supernatant is a crude extract;
step (2), fractional salting-out of the crude extract: adding (NH4)2SO4The crystal is subjected to fractional salting-out to obtain crude protein, (NH4)2SO4The ratio of the weight of the crystals to the volume of the crude extract was 20% and 50% (i.e., 20g and 50g of (NH4) were added to 100mL of the crude extract, respectively)2SO4Crystals);
step (3) enzymolysis: adding ultrapure water into the crude protein to fully dissolve the crude protein, then sequentially adding cellulase, pectinase, papain, acid protease and alkaline protease to carry out enzymolysis to prepare polypeptide, after the enzymolysis is finished, inactivating at high temperature, centrifuging to collect supernatant, carrying out vacuum filtration on the supernatant, and collecting filtrate;
step (4), ultrafiltration: performing ultrafiltration on the filtrate obtained in the step (3), and respectively collecting a permeate and a retentate;
and (5) concentrating: concentrating the permeate and the retentate respectively to 5% by mass of concentrated solution by using a rotary evaporator, wherein the concentrated solution of the permeate is concentrated red date kernel juice;
nanofiltration in step (6): sequentially adopting nanofiltration membranes of 1000Da and 500 Da to perform fractional filtration on the concentrated solution of the trapped fluid obtained in the step (5) and collecting filtrate;
step (7) vacuum freeze drying: precooling the filtrate subjected to nanofiltration treatment at 5 ℃, freezing at-80 ℃, carrying out vacuum freeze-drying for 36h at-90 ℃ by using a vacuum freeze-dryer, and drying to obtain the red date core polypeptide powder.
Preferably, the red date pit powder in the step (1) is sieved by a 60-mesh sieve, and after the sieving treatment, the red date pit powder is dissolved in ultrapure water to respectively prepare red date pit serous fluid with the concentration of 20% (W/V), 10% (W/V) and 5% (W/V) (the concentration of 20%, namely 20g of red date pit powder is added with ultrapure water to reach the constant volume of 100 mL); when the centrifugal operation is carried out in the step (1), the rotating speed of the centrifugal machine is 12000r/min, and the centrifugal time is 10 min.
Preferably, the step (2) of fractional salting-out of the crude extract comprises the following specific operation steps: slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the crude extract was 20% (NH4)2SO4Standing at 4 deg.C for 3 h after the crystal is completely dissolved, centrifuging at 12000r/min at 4 deg.C for 30 min, discarding precipitate, collecting supernatant, and slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the supernatant was 50% (NH4)2SO4Standing the crystal completely dissolved at 4 ℃ for 3 h, centrifuging at 12000r/min and 4 ℃ for 30 min, discarding the supernatant to obtain a precipitate, namely crude protein, dissolving the obtained crude protein in 2 times of volume of ultrapure water, centrifuging at 12000r/min and 4 ℃ for 10min, removing insoluble precipitate, collecting the supernatant, dialyzing the supernatant in PBS buffer solution at 4 ℃ and pH 7.5, passing the solution obtained by dialysis through a 0.22 mu m water system membrane, filtering, collecting the filtrate, and storing the filtrate in a 4 ℃ refrigerator for next enzymolysis.
Preferably, the pH value in the enzymolysis process in the step (3) is 7-8, the temperature is 50 ℃, and the enzymolysis time is 5 hours.
Preferably, the enzymolysis process in the step (3) is performed in an ultrasonic instrument, the power of the ultrasonic instrument is below 700W, and the ultrasonic time is 20-40 min.
Preferably, the temperature of high-temperature inactivation in the step (3) is 95 ℃, the inactivation time is 10-20 min, the rotating speed of a centrifuge during the centrifugal operation in the step (3) is 7500 r/min, and the centrifugation time is 10 min.
Preferably, before the ultrafiltration in step (4), granular activated carbon is added into the filtrate obtained in step (3), decolorization and deodorization treatment is carried out, then vacuum filtration is carried out, and the filtrate is collected for next ultrafiltration treatment.
Preferably, the volume ratio of the added weight of the granular activated carbon to the filtrate obtained in the step (3) is 1-3% (namely 1g, 2g and 3g of granular activated carbon are respectively added into 100mL of filtrate), the temperature of the decolorization and deodorization treatment is 60 ℃, and the time is less than 2 h.
Preferably, before the nanofiltration in the step (6), the concentrated solution of the trapped fluid obtained in the step (5) is taken as a sample, the hydrolysis degree of protein in the red date pit is measured by using a TCA method, and the content of the polypeptide is measured by using a Kjeldahl method.
The invention also discloses the red date seed polypeptide extracted by the extraction method of the red date seed polypeptide.
The invention has the beneficial effects that: (1) according to the invention, the red date pit is adopted for extracting the polypeptide, so that the problem of waste resource waste of the red date pit is solved, the utilization rate of the red date is improved, the waste of raw materials is avoided, the production cost is reduced, and the economic benefit of an enterprise is improved; compared with red date protein, the prepared red date seed polypeptide is easier to be absorbed by a human body, has better effect and can enhance the immunity of the human body; in a physicochemical property experiment, the red date core polypeptide shows better properties than red date protein in the aspects of hygroscopicity, thermal stability, solubility, viscosity and the like, namely good hygroscopicity, strong thermal stability, high solubility, low viscosity and the like; (2) the granular active carbon is adopted to decolor and remove odor of the product, so that the bad smell and color generated in the extraction process can be covered, and meanwhile, the solubility of the red date pit polypeptide is increased, so that the red date pit polypeptide is easier to absorb by a human body; (3) centrifuging the enzymolyzed red date pit slurry to obtain supernatant, ultrafiltering the supernatant, and concentrating the permeate to obtain concentrated red date pit juice which can be directly used as a product; the concentrated red date seed juice contains a large amount of beneficial components such as carbohydrate, fat, trace elements, crude fiber, vitamins, amino acids, organic acids and the like, and can be used as a health-care product for improving the immunity of a human body; the interception liquid is a byproduct for producing the jujube pit juice, however, the interception liquid is rich in protein and polysaccharide, the protein in the interception liquid has high nutritive value, the amino acid composition is relatively complete, and the interception liquid is a pure natural plant protein source with rich nutrition, high quality and low price, so that the polypeptide is extracted by using the intercepted liquid after ultrafiltration, the extraction rate is high, and the waste of raw materials is avoided; (4) in the enzymolysis process, an ultrasonic instrument is adopted for assisting, so that the enzymolysis reaction time is shortened, the enzymolysis effect is improved, and a high-efficiency polypeptide product is obtained; (5) the extraction method adopted by the invention is mature, simple and convenient, easy to operate and convenient to control, can obviously improve the economic benefit of red date products, and has a strong industrial application prospect.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
A method for extracting red date kernel polypeptide comprises the following steps: step (1) preparation of crude extract: selecting fresh red dates without plant diseases and insect pests and with complete surfaces, removing date pulp from the red dates, drying to obtain a red date pit raw material, crushing the red date pits into powder in a crusher, sieving the red date pit powder by using a 60-mesh sieve, dissolving the red date pit powder in ultrapure water, and respectively preparing red date pit slurry with the concentration of 20% (W/V), 10% (W/V) and 5% (W/V); (the concentration is 20 percent, namely 20g of red date kernel powder is added with ultrapure water to be constant volume of 100 mL); placing the red date kernel slurry in an ultrasonic instrument for ultrasonic oscillation, carrying out centrifugation treatment after the red date kernel slurry is fully dissolved to obtain supernatant, wherein when the centrifugation operation is carried out, the rotation speed of a centrifuge is 12000r/min, the centrifugation time is 10min, and the supernatant is crude extract;
step (2), fractional salting-out of the crude extract: adding (NH4)2SO4The crystal is subjected to fractional salting-out to obtain crude protein, (NH4)2SO4The weight ratio of the crystal to the volume of the crude extract is 20 percent and 50 percent; the specific operation steps are as follows: slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the crude extract was 20% (NH4)2SO4Standing at 4 deg.C for 3 h after the crystal is completely dissolved, centrifuging at 12000r/min at 4 deg.C for 30 min, discarding precipitate, collecting supernatant, and slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the supernatant was 50% (NH4)2SO4Standing at 4 deg.C for 3 h after completely dissolving crystal, centrifuging at 12000r/min and 4 deg.C for 30 min, discarding supernatant to obtain precipitate as crude protein, dissolving the crude protein in 2 times volume of ultrapure water, centrifuging at 12000r/min and 4 deg.C for 10min, removing insoluble precipitate, collecting supernatant, and concentrating the supernatant in PBS buffer (50 mmol/L KH) with pH of 7.5 at 4 deg.C2PO4-Na2HPO4) Dialyzing, passing the dialyzed solution through a 0.22 μm water system membrane, filtering, collecting filtrate, and storing the filtrate in a refrigerator at 4 deg.C for further enzymolysis;
step (3) enzymolysis: adding ultrapure water into the crude protein to fully dissolve the crude protein, then sequentially adding cellulase, pectinase, papain, acid protease and alkaline protease to carry out enzymolysis to prepare polypeptide, after the enzymolysis is finished, carrying out high-temperature inactivation at the temperature of 95 ℃ for 10-20 min, carrying out centrifugation to collect supernatant, wherein the rotation speed of a centrifuge is 7500 r/min and the centrifugation time is 10min when the centrifugation is carried out, and carrying out vacuum filtration on the supernatant to collect filtrate; the pH value in the enzymolysis process is 7-8, the temperature is 50 ℃, and the enzymolysis time is 5 hours; the enzymolysis process is carried out in an ultrasonic instrument, the power of the ultrasonic instrument is below 700W, and the ultrasonic time is 20-40 min;
step (4), ultrafiltration: performing ultrafiltration on the filtrate obtained in the step (3), adding granular activated carbon into the filtrate obtained in the step (3) before ultrafiltration, performing decolorization and deodorization treatment, then performing vacuum filtration, collecting the filtrate, and performing next ultrafiltration treatment; the volume ratio of the added weight of the granular activated carbon to the filtrate obtained in the step (3) is 1-3% (namely 1g, 2g and 3g of granular activated carbon are respectively added into 100mL of filtrate), the temperature of the decolorization and deodorization treatment is 60 ℃, and the time is less than 2 h; respectively collecting the permeate and the retentate;
and (5) concentrating: concentrating the permeate and the retentate respectively to 5% by mass of concentrated solution by using a rotary evaporator, wherein the concentrated solution of the permeate is concentrated red date kernel juice;
nanofiltration in step (6): sequentially adopting nanofiltration membranes of 1000Da and 500 Da to perform fractional filtration on the concentrated solution of the trapped fluid obtained in the step (5) and collecting filtrate; before nanofiltration in the step (6), taking the concentrated solution of the trapped fluid obtained in the step (5) as a sample, determining the hydrolysis degree of protein in the red date pits by using a TCA method, and determining the content of polypeptide by using a Kjeldahl method;
step (7) vacuum freeze drying: precooling the filtrate subjected to nanofiltration treatment at 5 ℃, freezing at-80 ℃, carrying out vacuum freeze-drying for 36h at-90 ℃ by using a vacuum freeze-dryer, and drying to obtain the red date core polypeptide powder.
The invention also discloses the red date seed polypeptide extracted by the extraction method of the red date seed polypeptide.
Example 1
A method for extracting red date kernel polypeptide comprises the following steps: step (1) preparation of crude extract: selecting high-quality fresh red dates which are ripe, large in size, thick in meat and free of bad fruits, removing date meat from the red dates, drying to obtain a red date pit raw material, crushing red date pits into powder in a crusher, sieving the red date pit powder by using a 60-mesh sieve, adding ultrapure water into 150g of red date pit powder to reach a constant volume of 3000mL, preparing red date pit slurry with the concentration of 5%, placing the red date pit slurry in an ultrasonic instrument for ultrasonic oscillation, performing centrifugal treatment after the red date pit slurry is fully dissolved to obtain a supernatant, wherein when the centrifugal operation is performed, the rotating speed of a centrifugal machine is 12000r/min, the centrifugal time is 10min, and the supernatant is a crude extract;
step (2), fractional salting-out of the crude extract: slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the crude extract was 20% (NH4)2SO4Standing at 4 deg.C for 3 h after the crystal is completely dissolved, centrifuging at 12000r/min at 4 deg.C for 30 min, discarding precipitate, collecting supernatant, and slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the supernatant was 50% (NH4)2SO4Standing at 4 deg.C for 3 h after completely dissolving crystal, centrifuging at 12000r/min and 4 deg.C for 30 min, discarding supernatant to obtain precipitate as crude protein, dissolving the crude protein in 2 times volume of ultrapure water, centrifuging at 12000r/min and 4 deg.C for 10min, removing insoluble precipitate, collecting supernatant, and concentrating the supernatant in PBS buffer (50 mmol/L KH) with pH of 7.5 at 4 deg.C2PO4-Na2HPO4) Dialyzing, passing the dialyzed solution through a 0.22 μm water system membrane, filtering, collecting filtrate, and storing the filtrate in a refrigerator at 4 deg.C for further enzymolysis;
step (3) enzymolysis: adding ultrapure water into crude protein to fully dissolve the crude protein under the conditions that the temperature is 50 ℃ and the pH value is 7, then sequentially adding cellulase, pectinase, papain, acid protease and alkaline protease to carry out enzymolysis to prepare polypeptide, wherein the enzymolysis time is 5h, the enzymolysis process is carried out in an ultrasonic instrument, the power of the ultrasonic instrument is 650W, the ultrasonic time is 20min, after the enzymolysis is finished, high-temperature inactivation is carried out, the high-temperature inactivation temperature is 95 ℃, the inactivation time is 15min, and the supernatant is collected through centrifugal treatment, the rotating speed of a centrifugal machine is 7500 r/min when the centrifugal operation is carried out, the centrifugation time is 10min, and the supernatant is subjected to vacuum filtration operation to collect filtrate;
step (4), ultrafiltration: performing ultrafiltration on the filtrate obtained in the step (3), adding granular activated carbon into the filtrate obtained in the step (3) before ultrafiltration, performing decolorization and deodorization treatment, then performing vacuum filtration, collecting the filtrate, and performing next ultrafiltration treatment; the volume ratio of the weight of the granular activated carbon to the filtrate obtained in the step (3) is 2 percent (namely 2g of granular activated carbon is added into 100mL of filtrate), the temperature of the decolorization and deodorization treatment is 60 ℃, and the time is 1 h; respectively collecting the permeate and the retentate;
and (5) concentrating: concentrating the permeate and the retentate respectively to 5% by mass of concentrated solution by using a rotary evaporator, wherein the concentrated solution of the permeate is concentrated red date kernel juice;
nanofiltration in step (6): sequentially adopting nanofiltration membranes of 1000Da and 500 Da to perform fractional filtration on the concentrated solution of the trapped fluid obtained in the step (5) and collecting filtrate; before nanofiltration in the step (6), taking the concentrated solution of the trapped fluid obtained in the step (5) as a sample, determining the hydrolysis degree of protein in the red date pits by using a TCA method, and determining the content of polypeptide by using a Kjeldahl method; taking 2.5 ml of sample solution, adding 2.5 ml of 10% (W/V) trichloroacetic acid (TCA) aqueous solution, uniformly mixing on a vortex mixer, standing for 10min, and then centrifuging for 15min at 4000r/min to precipitate the protein which is not subjected to enzymolysis; after the precipitation is finished, transferring all the supernatant into a 50ml volumetric flask, fixing the volume to 50ml by using 5% TCA, and shaking up; then 6.0ml of the solution is put into another test tube, 4.0 ml of biuret reagent (sample solution: biuret reagent = 3: 2, V/V) is added, the mixture is uniformly mixed on a vortex mixer, the mixture is kept stand for 10min, the mixture is centrifuged at 2000r/min for 10min, the supernatant is taken to measure the OD value under 540 nm, the polypeptide concentration C (mg/ml) in the sample solution is obtained by contrasting a standard curve, and the polypeptide content in the sample can be further obtained;
step (7) vacuum freeze drying: precooling the filtrate subjected to nanofiltration treatment at 5 ℃, freezing at-80 ℃, carrying out vacuum freeze-drying for 36h at-90 ℃ by using a vacuum freeze-dryer, and drying to obtain the red date core polypeptide powder.
Example 2
A method for extracting red date kernel polypeptide comprises the following steps: step (1) preparation of crude extract: selecting high-quality fresh red dates which are ripe, large in size, thick in meat and free of bad fruits, removing date meat from the red dates, drying to obtain a red date pit raw material, crushing red date pits into powder in a crusher, sieving the red date pit powder by using a 60-mesh sieve, adding ultrapure water into 150g of red date pit powder, fixing the volume to 1500mL, preparing red date pit slurry with the concentration of 10%, placing the red date pit slurry in an ultrasonic instrument for ultrasonic oscillation, performing centrifugal treatment after the red date pit slurry is fully dissolved to obtain a supernatant, wherein when the centrifugal operation is performed, the rotating speed of a centrifugal machine is 12000r/min, the centrifugal time is 10min, and the supernatant is a crude extract;
step (2), fractional salting-out of the crude extract: slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the crude extract was 20% (NH4)2SO4Standing at 4 deg.C for 3 h after the crystal is completely dissolved, centrifuging at 12000r/min at 4 deg.C for 30 min, discarding precipitate, collecting supernatant, and slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the supernatant was 50% (NH4)2SO4Standing at 4 deg.C for 3 h after completely dissolving crystal, centrifuging at 12000r/min and 4 deg.C for 30 min, discarding supernatant to obtain precipitate as crude protein, dissolving the crude protein in 2 times volume of ultrapure water, centrifuging at 12000r/min and 4 deg.C for 10min, removing insoluble precipitate, collecting supernatant, and concentrating the supernatant in PBS buffer (50 mmol/L KH) with pH of 7.5 at 4 deg.C2PO4-Na2HPO4) Dialyzing, passing the dialyzed solution through a 0.22 μm water system membrane, filtering, collecting filtrate, and storing the filtrate in a refrigerator at 4 deg.C for further enzymolysis;
step (3) enzymolysis: adding ultrapure water into crude protein to fully dissolve the crude protein under the conditions that the temperature is 50 ℃ and the pH value is 8, then sequentially adding cellulase, pectinase, papain, acid protease and alkaline protease to carry out enzymolysis to prepare polypeptide, wherein the enzymolysis time is 5h, the enzymolysis process is carried out in an ultrasonic instrument, the power of the ultrasonic instrument is 670W, the ultrasonic time is 30 min, after the enzymolysis is finished, high-temperature inactivation is carried out, the high-temperature inactivation temperature is 95 ℃, the inactivation time is 10min, and the supernatant is collected through centrifugal treatment, the rotating speed of a centrifugal machine is 7500 r/min when the centrifugal operation is carried out, the centrifugation time is 10min, and the supernatant is subjected to vacuum filtration operation to collect filtrate;
step (4), ultrafiltration: performing ultrafiltration on the filtrate obtained in the step (3), adding granular activated carbon into the filtrate obtained in the step (3) before ultrafiltration, performing decolorization and deodorization treatment, then performing vacuum filtration, collecting the filtrate, and performing next ultrafiltration treatment; the volume ratio of the weight of the granular activated carbon to the filtrate obtained in the step (3) is 1 percent (namely 1g of granular activated carbon is added into 100mL of filtrate), the temperature of the decolorization and deodorization treatment is 60 ℃, and the time is 1.5 h; respectively collecting the permeate and the retentate;
and (5) concentrating: concentrating the permeate and the retentate respectively to 5% by mass of concentrated solution by using a rotary evaporator, wherein the concentrated solution of the permeate is concentrated red date kernel juice;
nanofiltration in step (6): sequentially adopting nanofiltration membranes of 1000Da and 500 Da to perform fractional filtration on the concentrated solution of the trapped fluid obtained in the step (5) and collecting filtrate; before nanofiltration in the step (6), taking the concentrated solution of the trapped fluid obtained in the step (5) as a sample, determining the hydrolysis degree of protein in the red date pits by using a TCA method, and determining the content of polypeptide by using a Kjeldahl method; taking 2.5 ml of sample solution, adding 2.5 ml of 10% (W/V) trichloroacetic acid (TCA) aqueous solution, uniformly mixing on a vortex mixer, standing for 10min, and then centrifuging for 15min at 4000r/min to precipitate the protein which is not subjected to enzymolysis; after the precipitation is finished, transferring all the supernatant into a 50ml volumetric flask, fixing the volume to 50ml by using 5% TCA, and shaking up; then 6.0ml of the solution is put into another test tube, 4.0 ml of biuret reagent (sample solution: biuret reagent = 3: 2, V/V) is added, the mixture is uniformly mixed on a vortex mixer, the mixture is kept stand for 10min, the mixture is centrifuged at 2000r/min for 10min, the supernatant is taken to measure the OD value under 540 nm, the polypeptide concentration C (mg/ml) in the sample solution is obtained by contrasting a standard curve, and the polypeptide content in the sample can be further obtained;
step (7) vacuum freeze drying: precooling the filtrate subjected to nanofiltration treatment at 5 ℃, freezing at-80 ℃, carrying out vacuum freeze-drying for 36h at-90 ℃ by using a vacuum freeze-dryer, and drying to obtain the red date core polypeptide powder.
Example 3
A method for extracting red date kernel polypeptide comprises the following steps: step (1) preparation of crude extract: selecting high-quality fresh red dates which are ripe, large in size, thick in meat and free of bad fruits, removing date meat from the red dates, drying to obtain a red date pit raw material, crushing red date pits into powder in a crusher, sieving the red date pit powder by using a 60-mesh sieve, adding ultrapure water into 150g of red date pit powder to reach 1250mL of constant volume, preparing red date pit slurry with the concentration of 20%, placing the red date pit slurry in an ultrasonic instrument for ultrasonic oscillation, performing centrifugal treatment after the red date pit slurry is fully dissolved to obtain a supernatant, wherein when the centrifugal operation is performed, the rotating speed of a centrifugal machine is 12000r/min, the centrifugal time is 10min, and the supernatant is a crude extract;
step (2), fractional salting-out of the crude extract: slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the crude extract was 20% (NH4)2SO4Standing at 4 deg.C for 3 h after the crystal is completely dissolved, centrifuging at 12000r/min at 4 deg.C for 30 min, discarding precipitate, collecting supernatant, and slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the supernatant was 50% (NH4)2SO4Standing at 4 deg.C for 3 h after completely dissolving crystal, centrifuging at 12000r/min and 4 deg.C for 30 min, discarding supernatant to obtain precipitate as crude protein, dissolving the crude protein in 2 times volume of ultrapure water, centrifuging at 12000r/min and 4 deg.C for 10min, removing insoluble precipitate, collecting supernatant, and concentrating the supernatant in PBS buffer (50 mmol/L KH) with pH of 7.5 at 4 deg.C2PO4-Na2HPO4) Filtering the solution with 0.22 μm water system membrane, collecting filtrate, and storing the filtrate in ice at 4 deg.CIn the box, the next enzymolysis operation is carried out;
step (3) enzymolysis: adding ultrapure water into crude protein under the conditions that the temperature is 50 ℃ and the pH value is 7.5 to fully dissolve the crude protein, then sequentially adding cellulase, pectinase, papain, acid protease and alkali protease to carry out enzymolysis to prepare polypeptide, wherein the enzymolysis time is 5h, the enzymolysis process is carried out in an ultrasonic instrument, the power of the ultrasonic instrument is 690W, the ultrasonic time is 40 min, after the enzymolysis is finished, high-temperature inactivation is carried out, the high-temperature inactivation temperature is 95 ℃, the inactivation time is 20min, and the supernatant is collected through centrifugal treatment, the rotating speed of a centrifugal machine is 7500 r/min when the centrifugal operation is carried out, the centrifugation time is 10min, and the supernatant is subjected to vacuum filtration operation to collect filtrate;
step (4), ultrafiltration: performing ultrafiltration on the filtrate obtained in the step (3), adding granular activated carbon into the filtrate obtained in the step (3) before ultrafiltration, performing decolorization and deodorization treatment, then performing vacuum filtration, collecting the filtrate, and performing next ultrafiltration treatment; the volume ratio of the weight of the granular activated carbon to the filtrate obtained in the step (3) is 3 percent (namely 3g of granular activated carbon is added into 100mL of filtrate), the temperature of the decolorization and deodorization treatment is 60 ℃, and the time is 1.8 h; respectively collecting the permeate and the retentate;
and (5) concentrating: concentrating the permeate and the retentate respectively to 5% by mass of concentrated solution by using a rotary evaporator, wherein the concentrated solution of the permeate is concentrated red date kernel juice;
nanofiltration in step (6): sequentially adopting nanofiltration membranes of 1000Da and 500 Da to perform fractional filtration on the concentrated solution of the trapped fluid obtained in the step (5) and collecting filtrate; before nanofiltration in the step (6), taking the concentrated solution of the trapped fluid obtained in the step (5) as a sample, determining the hydrolysis degree of protein in the red date pits by using a TCA method, and determining the content of polypeptide by using a Kjeldahl method; taking 2.5 ml of sample solution, adding 2.5 ml of 10% (W/V) trichloroacetic acid (TCA) aqueous solution, uniformly mixing on a vortex mixer, standing for 10min, and then centrifuging for 15min at 4000r/min to precipitate the protein which is not subjected to enzymolysis; after the precipitation is finished, transferring all the supernatant into a 50ml volumetric flask, fixing the volume to 50ml by using 5% TCA, and shaking up; then 6.0ml of the solution is put into another test tube, 4.0 ml of biuret reagent (sample solution: biuret reagent = 3: 2, V/V) is added, the mixture is uniformly mixed on a vortex mixer, the mixture is kept stand for 10min, the mixture is centrifuged at 2000r/min for 10min, the supernatant is taken to measure the OD value under 540 nm, the polypeptide concentration C (mg/ml) in the sample solution is obtained by contrasting a standard curve, and the polypeptide content in the sample can be further obtained;
step (7) vacuum freeze drying: precooling the filtrate subjected to nanofiltration treatment at 5 ℃, freezing at-80 ℃, carrying out vacuum freeze-drying for 36h at-90 ℃ by using a vacuum freeze-dryer, and drying to obtain the red date core polypeptide powder.
What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept thereof, and these changes and modifications can be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method for extracting red date kernel polypeptide is characterized by comprising the following steps: the method comprises the following steps:
step (1) preparation of crude extract: removing jujube meat from fresh red dates, drying to obtain a red date pit raw material, crushing the red date pits into powder in a crusher, sieving, dissolving the red date pit powder in ultrapure water, preparing red date pit slurry with different concentration gradients, placing the red date pit slurry in an ultrasonic instrument for ultrasonic oscillation, and centrifuging after the red date pit slurry is fully dissolved to obtain a supernatant, wherein the supernatant is a crude extract;
step (2), fractional salting-out of the crude extract: adding (NH4)2SO4The crystal is subjected to fractional salting-out to obtain crude protein, (NH4)2SO4The weight ratio of the crystal to the volume of the crude extract is 20 percent and 50 percent;
step (3) enzymolysis: adding ultrapure water into the crude protein to fully dissolve the crude protein, then sequentially adding cellulase, pectinase, papain, acid protease and alkaline protease to carry out enzymolysis to prepare polypeptide, after the enzymolysis is finished, inactivating at high temperature, centrifuging to collect supernatant, carrying out vacuum filtration on the supernatant, and collecting filtrate;
step (4), ultrafiltration: performing ultrafiltration on the filtrate obtained in the step (3), and respectively collecting a permeate and a retentate;
and (5) concentrating: concentrating the permeate and the retentate respectively to 5% by mass of concentrated solution by using a rotary evaporator, wherein the concentrated solution of the permeate is concentrated red date kernel juice;
nanofiltration in step (6): sequentially adopting nanofiltration membranes of 1000Da and 500 Da to perform fractional filtration on the concentrated solution of the trapped fluid obtained in the step (5) and collecting filtrate;
step (7) vacuum freeze drying: precooling the filtrate subjected to nanofiltration treatment at 5 ℃, freezing at-80 ℃, carrying out vacuum freeze-drying for 36h at-90 ℃ by using a vacuum freeze-dryer, and drying to obtain the red date core polypeptide powder.
2. The extraction method of red date kernel polypeptide according to claim 1, characterized in that: sieving the red date pit powder in the step (1) by a 60-mesh sieve, dissolving the red date pit powder in ultrapure water after sieving, and respectively preparing red date pit serous fluid with the concentration of 20% (W/V), 10% (W/V) and 5% (W/V); when the centrifugal operation is carried out in the step (1), the rotating speed of the centrifugal machine is 12000r/min, and the centrifugal time is 10 min.
3. The extraction method of red date kernel polypeptide according to claim 1, characterized in that: the specific operation steps of the step (2) of fractional salting-out of the crude extract are as follows: slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the crude extract was 20% (NH4)2SO4Standing at 4 deg.C for 3 h after the crystal is completely dissolved, centrifuging at 12000r/min at 4 deg.C for 30 min, discarding precipitate, collecting supernatant, and slowly adding (NH4)2SO4Crystal (NH4)2SO4The ratio of the weight of the crystals to the volume of the supernatant was 50% (NH4)2SO4After the crystal is completely dissolvedStanding at 4 ℃ for 3 h, centrifuging at 12000r/min and 4 ℃ for 30 min, discarding the supernatant to obtain a precipitate, namely crude protein, dissolving the obtained crude protein in 2 times of volume of ultrapure water, centrifuging at 12000r/min and 4 ℃ for 10min, removing the precipitate insoluble in water, collecting the supernatant, dialyzing the supernatant in PBS buffer solution at 4 ℃ and pH 7.5, passing the solution obtained by dialysis through a 0.22 mu m water-based membrane, filtering, collecting the filtrate, and storing the filtrate in a refrigerator at 4 ℃ to be subjected to the next enzymolysis operation.
4. The extraction method of red date kernel polypeptide according to claim 1, characterized in that: and (3) carrying out enzymolysis at the pH value of 7-8 and the temperature of 50 ℃ for 5h in the enzymolysis process.
5. The extraction method of red date kernel polypeptide according to claim 1, characterized in that: and (3) carrying out the enzymolysis process in an ultrasonic instrument, wherein the power of the ultrasonic instrument is below 700W, and the ultrasonic time is 20-40 min.
6. The extraction method of red date kernel polypeptide according to claim 1, characterized in that: the temperature of high-temperature inactivation in the step (3) is 95 ℃, the inactivation time is 10-20 min, the rotating speed of a centrifugal machine during centrifugal operation in the step (3) is 7500 r/min, and the centrifugation time is 10 min.
7. The extraction method of red date kernel polypeptide according to claim 1, characterized in that: and (4) before ultrafiltration in the step (4), adding granular activated carbon into the filtrate obtained in the step (3), carrying out decolorization and deodorization treatment, then carrying out vacuum filtration, collecting the filtrate, and carrying out next ultrafiltration treatment.
8. The extraction method of red date kernel polypeptide according to claim 7, characterized in that: the volume ratio of the added weight of the granular activated carbon to the filtrate obtained in the step (3) is 1-3%, the temperature of the decolorization and deodorization treatment is 60 ℃, and the time is less than 2 hours.
9. The extraction method of red date kernel polypeptide according to claim 1, characterized in that: before the nanofiltration of the step (6), taking the concentrated solution of the trapped fluid obtained in the step (5) as a sample, determining the hydrolysis degree of protein in the red date pit by using a TCA method, and determining the content of polypeptide by using a Kjeldahl method.
10. The red date kernel polypeptide extracted by the extraction method of red date kernel polypeptide according to any one of claims 1-9.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1282022C (en) * | 1986-01-09 | 1991-03-26 | Naoki Higashi | Method for purifying an interferon |
| CN102174625A (en) * | 2010-12-31 | 2011-09-07 | 山东鼎力枣业食品集团有限公司 | Gold thread jujube peptide production method |
| CN105124434A (en) * | 2015-09-09 | 2015-12-09 | 安徽徽普生物科技有限责任公司 | Processing method for wheat puffed food |
| CN105707404A (en) * | 2016-04-05 | 2016-06-29 | 山东鼎力枣业食品集团有限公司 | Production method of golden-silk jujube polypeptides |
| US20170246240A1 (en) * | 2011-10-28 | 2017-08-31 | United Arab Emirates University | Antibacterial Peptide and Method of Treatment Using the Antibacterial Peptide |
| CN107312085A (en) * | 2017-07-06 | 2017-11-03 | 浦江县欧立生物技术有限公司 | A kind of phycocyanin blood sugar lowing polypeptide |
| CN109337952A (en) * | 2018-10-30 | 2019-02-15 | 湖南省锘恩科德生物科技有限公司 | A kind of extracting method of snake slough polypeptide |
| CN110964767A (en) * | 2019-12-30 | 2020-04-07 | 河北中科同创科技发展有限公司 | Method for extracting aster polysaccharide and aster polypeptide |
| CN111560412A (en) * | 2020-05-15 | 2020-08-21 | 大洲新燕(厦门)生物科技有限公司 | Method for simultaneously extracting bird's nest polypeptide and bird's nest polysaccharide from bird's nests |
| CN112458140A (en) * | 2020-12-11 | 2021-03-09 | 武汉轻工大学 | Method for preparing cardamine hirsute selenium polypeptide through continuous enzymolysis and cardamine hirsute selenium polypeptide |
-
2021
- 2021-12-28 CN CN202111622122.0A patent/CN114250261B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1282022C (en) * | 1986-01-09 | 1991-03-26 | Naoki Higashi | Method for purifying an interferon |
| CN102174625A (en) * | 2010-12-31 | 2011-09-07 | 山东鼎力枣业食品集团有限公司 | Gold thread jujube peptide production method |
| US20170246240A1 (en) * | 2011-10-28 | 2017-08-31 | United Arab Emirates University | Antibacterial Peptide and Method of Treatment Using the Antibacterial Peptide |
| CN105124434A (en) * | 2015-09-09 | 2015-12-09 | 安徽徽普生物科技有限责任公司 | Processing method for wheat puffed food |
| CN105707404A (en) * | 2016-04-05 | 2016-06-29 | 山东鼎力枣业食品集团有限公司 | Production method of golden-silk jujube polypeptides |
| CN107312085A (en) * | 2017-07-06 | 2017-11-03 | 浦江县欧立生物技术有限公司 | A kind of phycocyanin blood sugar lowing polypeptide |
| CN109337952A (en) * | 2018-10-30 | 2019-02-15 | 湖南省锘恩科德生物科技有限公司 | A kind of extracting method of snake slough polypeptide |
| CN110964767A (en) * | 2019-12-30 | 2020-04-07 | 河北中科同创科技发展有限公司 | Method for extracting aster polysaccharide and aster polypeptide |
| CN111560412A (en) * | 2020-05-15 | 2020-08-21 | 大洲新燕(厦门)生物科技有限公司 | Method for simultaneously extracting bird's nest polypeptide and bird's nest polysaccharide from bird's nests |
| CN112458140A (en) * | 2020-12-11 | 2021-03-09 | 武汉轻工大学 | Method for preparing cardamine hirsute selenium polypeptide through continuous enzymolysis and cardamine hirsute selenium polypeptide |
Non-Patent Citations (3)
| Title |
|---|
| 冀利;孙曙光;孙玖玉;张新明;黄少华;: "从浓缩枣汁超滤截留液中提取金丝小枣多肽和多糖", 山东食品发酵, no. 01, pages 3 - 10 * |
| 孙久玉;王成忠;孙曙光;冀利;: "金丝小枣多肽功能特性的研究", 山东食品发酵, no. 03, pages 3 - 7 * |
| 王俊;吕广林;田亚平;: "一种枯草芽孢杆菌亮氨酸氨肽酶的提取及部分酶学性质研究", 食品工业科技, no. 01, pages 203 - 206 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024174424A1 (en) * | 2023-02-21 | 2024-08-29 | 宁波大学 | Enzyme digestion product of red date protein, preparation method therefor, and use thereof |
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