CN110714046B - Preparation method of polypeptide for hydrolyzing pork protein of suckling pig - Google Patents
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Abstract
The invention discloses a method for preparing polypeptide of hydrolyzed pork protein of a suckling pig, which relates to the technical field of pork protein hydrolysis, and the technical scheme is characterized by comprising the following steps: preparation of glycosylated pork protein: dispersing pork protein in a phosphoric acid buffer solution with the pH value of 7.0, and uniformly stirring to obtain a pork protein dispersion liquid with the mass fraction of 8%; adding alginate-derived oligosaccharide into pork protein dispersion liquid for glycosylation, and freeze drying to obtain glycosylated pork protein; hydrolysis of pork protein: dissolving glycosylated pork protein in water, and adjusting the feed-liquid ratio to be 1: 20 obtaining a dissolved solution; adding hydrolase with the mass of 1-2% of the glycosylated pork protein into the dissolved solution for enzymolysis reaction, boiling for 10min after enzymolysis for 2-3h, centrifuging and collecting the supernatant to obtain an enzymolysis solution. The invention solves the problem that the hydrolysis efficiency of hydrolase is influenced due to poor pork protein solubility, and achieves the effect of improving pork protein solubility.
Description
Technical Field
The invention relates to the technical field of pork protein hydrolysis, in particular to a preparation method of polypeptide for hydrolyzing pork protein of a suckling pig.
Background
At present, the piglets which are not weaned are called as the suckling pigs, the influence on the pork of the suckling pigs in the process of the acquired breeding is lacking, and the intake of toxic and harmful substances is less.
In the prior art, reference is made to a Chinese patent with publication number CN110150449A, which discloses the use of functional polypeptide of pork, including the use of functional polypeptide of pork in preparing health products, and the health products comprise the following components in parts by weight: 8-10 parts of common clubmoss herb, 8-12 parts of garden balsam stem, 5-8 parts of diamond grass, 1-3 parts of pork functional polypeptide, 4-6 parts of schizophragma integrifolia, 7-9 parts of lobelia chinensis and 4-7 parts of setaria viridis.
However, in the process of enzymolysis of the pig protein, distilled water is directly added into pork, and due to poor solubility of the pork protein, a large amount of suspended matter which is insoluble in water remains in the mixed solution after the pork protein is mixed with water, and when the hydrolase is added into the mixed solution, the hydrolysis efficiency of the hydrolase is affected.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of polypeptide for hydrolyzing pork protein of a suckling pig, which has the advantage of improving the solubility of the pork protein.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for preparing polypeptide of hydrolyzed pork protein of porket comprises the following steps:
preparation of glycosylated pork protein: dispersing pork protein in a phosphoric acid buffer solution with the pH value of 7.0, and uniformly stirring to obtain a pork protein dispersion liquid with the mass fraction of 8%; adding alginate-derived oligosaccharide into the pork protein dispersion liquid for glycosylation, and freeze-drying after reaction to obtain glycosylated pork protein;
hydrolysis of pork protein: dissolving the glycosylated pork protein in water, and adjusting the feed-liquid ratio to be 1: 20 obtaining a dissolved solution; adding hydrolase with the mass of 1-2% of the glycosylated pork protein into the dissolved solution for enzymolysis reaction, boiling for 10min after enzymolysis for 2-3h, centrifuging and collecting the supernatant to obtain an enzymolysis solution.
By adopting the technical scheme, because the amino acid residues in the protein contain a large number of hydrophobic groups, the pork protein is glycosylated by the alginate-derived oligosaccharides, and the alginate-derived oligosaccharides and the amino acid residues on the protein form glycosidic bonds, so that the solubility of the pork protein in water is enhanced, and the effect of protease on the pork protein in the hydrolysis process is improved.
The invention is further configured to: in the preparation process of the glycosylated pork protein, the addition amount of the alginate oligosaccharide is 0.2-0.3g/g pork, the reaction temperature is 70-80 ℃, and the reaction time is 4-6 h.
By adopting the technical scheme, the alginate-derived oligosaccharide has good hydrophilicity, and the pork protein is immersed in the alginate-derived oligosaccharide for glycosylation reaction, so that the hydrophilicity of the pork protein is enhanced, the solubility of the pork protein is improved conveniently, and when the addition amount of the alginate-derived oligosaccharide is 0.2-0.3g/g pork, the solubility of the pork protein is the best, the addition amount of the alginate-derived oligosaccharide is increased continuously, and the solubility change of the pork protein is not great.
The invention is further configured to: in the process of hydrolyzing the pork protein, the hydrolase is papain, the reaction pH is 7.0, and the reaction temperature is 50 ℃.
By adopting the technical scheme, the optimum pH value of the papain is 6-7, the optimum reaction temperature is 50-65 ℃, and the hydrolysis effect of the papain on pork protein is improved by selecting the pH value of 7.0 and the reaction temperature of 50 ℃.
The invention is further configured to: in the process of hydrolyzing the pork protein, the hydrolase is trypsin, the reaction pH is 8.0, and the reaction temperature is 37 ℃.
By adopting the technical scheme, the optimum pH value of the trypsin is 7.8-8.5, the optimum reaction temperature is 37 ℃, and the hydrolysis effect of the trypsin on pork protein is increased by selecting the pH value of 8.0 and the reaction temperature of 37 ℃.
The invention is further configured to: in the process of hydrolyzing the pork protein, the hydrolase is neutral protease, the reaction pH is 7.0, and the reaction temperature is 45 ℃.
By adopting the technical scheme, the optimal pH value of the neutral protease is 6.0-7.0, the optimal reaction temperature is 45-55 ℃, and the hydrolysis effect of the neutral protease on pork protein is increased by selecting the pH value of 7.0 and the reaction temperature of 45 ℃.
The invention is further configured to: the preparation method of the alginate jelly oligosaccharide comprises the following steps: preparing seaweed gel into a seaweed gel solution with the mass fraction of 1%, adjusting the pH to 7.0, adding seaweed gel lyase into the seaweed gel solution for hydrolysis reaction, boiling after hydrolysis is finished, centrifuging and collecting supernatant; adjusting the pH value of the supernatant to 2.85, centrifuging and collecting a centrifugate; evaporating and concentrating the centrifugate to obtain a concentrated solution, adding 90% ethanol into the concentrated solution, centrifuging after the reaction is finished, collecting the precipitate, and freeze-drying to obtain the trehalose oligosaccharide.
By adopting the technical scheme, after the seaweed gel oligosaccharide is subjected to enzymolysis, other impurities are separated out by adjusting the pH value; centrifuging to remove impurities, adding ethanol into the concentrated solution to separate out oligosaccharide, and centrifuging to collect precipitate.
The invention is further configured to: in the enzymolysis process of the seaweed gel, the addition amount of the seaweed gel lyase is 5-10U/mL, the reaction temperature is 35-45 ℃, and the reaction time is 4-6 h.
By adopting the technical scheme, the optimal reaction temperature of the seaweed gel lyase is 35-45 ℃, and the enzymolysis efficiency of the seaweed gel lyase on seaweed gel is improved by carrying out enzymolysis at the optimal temperature.
The invention is further configured to: separating the enzymolysis liquid by an ultrafiltration membrane with the molecular weight cutoff of 5ku, wherein the sample loading amount is 25mL, the nitrogen pressure is 0.02Mpa, and collecting the cutoff liquid.
By adopting the technical scheme, the polypeptide is purified by the ultrafiltration membrane after the polypeptide is hydrolyzed, and components with the molecular weight of more than 5ku are removed.
In summary, compared with the prior art, the invention has the following beneficial effects:
1. the pork protein is glycosylated before hydrolysis, so that the solubility of the pork protein is enhanced, and the hydrolysis effect of hydrolase on the pork protein is improved;
2. the pork protein is glycosylated by selecting the alginate jelly oligosaccharide, so that the hydrophilic property of the pork protein is enhanced, and the solubility of the pork protein is improved conveniently.
Detailed Description
Examples
Example 1: a method for preparing polypeptide of hydrolyzed pork protein of porket comprises the following steps:
preparation of alginate jelly oligosaccharide: preparing alginate jelly into 1% alginate jelly solution, adjusting pH to 7.0, adding alginate jelly lyase 8U/mL into the alginate jelly solution, boiling in water bath at 40 deg.C for 5 hr, centrifuging, and collecting supernatant; adjusting the pH value of the supernatant to 2.85, centrifuging and collecting a centrifugate; evaporating and concentrating the centrifugate to obtain a concentrated solution, adding 90% ethanol into the concentrated solution, shaking for 20min, centrifuging, collecting precipitate, and freeze-drying to obtain trehalose oligosaccharide.
Preparation of glycosylated pork protein: weighing pork protein, and dissolving the pork protein in a phosphoric acid buffer solution with the pH value of 7.0, wherein the concentration of the pork protein is 8%; continuously adding 0.25g/g of pork alginate-derived oligosaccharides into the mixed solution, uniformly stirring, placing in a 75 ℃ constant-temperature water bath condition for glycosylation reaction, reacting for 5h, taking out a sample, and freeze-drying to obtain the glycosylated pork protein.
Hydrolysis of pork protein: dissolving glycosylated pork protein in water, and adjusting the feed-liquid ratio to be 1: 20, adjusting the pH value to 7.0, continuously adding papain with the mass of 1.5 percent of the glycosylated pork protein, carrying out enzymolysis for 2.5h at 50 ℃, boiling for 10min after the enzymolysis is finished, centrifuging and collecting the supernatant to obtain the enzymolysis liquid.
And (3) polypeptide purification: separating the enzymolysis liquid by an ultrafiltration membrane with the molecular weight cutoff of 5ku, wherein the sample loading amount is 25mL, the nitrogen pressure is 0.02Mpa, and collecting the cutoff liquid.
Example 2: example 2 differs from example 1 in that the glycosylated pork protein is dissolved in water, and the feed-to-liquid ratio is adjusted to 1: 20, adjusting the pH value to 8.0, continuously adding trypsin accounting for 1.5 percent of the mass of the glycosylated pork protein, carrying out enzymolysis for 2.5h at 37 ℃, boiling for 10min after the enzymolysis is finished, centrifuging and collecting supernatant to obtain enzymolysis liquid.
Example 3: example 3 differs from example 1 in that the glycosylated pork protein is dissolved in water, and the feed-to-liquid ratio is adjusted to 1: 20, adjusting the pH value to 7.0, continuously adding neutral protease accounting for 1.5 percent of the mass of the glycosylated pork protein, carrying out enzymolysis for 2.5h at the temperature of 45 ℃, boiling for 10min after the enzymolysis is finished, centrifuging and collecting supernatant to obtain enzymolysis liquid.
Example 4: example 4 differs from example 1 in that the addition amount of alginate oligosaccharides was 0.2g/g pork during the preparation of glycosylated pork protein.
Example 5: example 5 differs from example 1 in that the addition amount of alginate oligosaccharides was 0.3g/g pork during the preparation of glycosylated pork protein.
Example 6: a method for preparing polypeptide of hydrolyzed pork protein of porket comprises the following steps:
preparation of alginate jelly oligosaccharide: preparing alginate jelly into 1% alginate jelly solution, adjusting pH to 7.0, adding alginate jelly lyase of 5U/mL into the alginate jelly solution, boiling in water bath at 35 deg.C for 4 hr, centrifuging, and collecting supernatant; adjusting the pH value of the supernatant to 2.85, centrifuging and collecting a centrifugate; evaporating and concentrating the centrifugate to obtain a concentrated solution, adding 90% ethanol into the concentrated solution, shaking for 20min, centrifuging, collecting precipitate, and freeze-drying to obtain trehalose oligosaccharide.
Preparation of glycosylated pork protein: weighing pork protein, and dissolving the pork protein in a phosphoric acid buffer solution with the pH value of 7.0, wherein the concentration of the pork protein is 8%; continuously adding 0.25g/g of pork alginate-derived oligosaccharides into the mixed solution, uniformly stirring, placing in a constant-temperature water bath condition of 70 ℃ for glycosylation reaction, reacting for 4h, taking out a sample, and freeze-drying to obtain the glycosylated pork protein.
Hydrolysis of pork protein: dissolving glycosylated pork protein in water, and adjusting the feed-liquid ratio to be 1: 20, adjusting the pH value to 7.0, continuously adding papain with the mass of 1.5 percent of the glycosylated pork protein, carrying out enzymolysis for 2.5h at 50 ℃, boiling for 10min after the enzymolysis is finished, centrifuging and collecting the supernatant to obtain the enzymolysis liquid.
And (3) polypeptide purification: separating the enzymolysis liquid by an ultrafiltration membrane with the molecular weight cutoff of 5ku, wherein the sample loading amount is 25mL, the nitrogen pressure is 0.02Mpa, and collecting the cutoff liquid.
Example 7: a method for preparing polypeptide of hydrolyzed pork protein of porket comprises the following steps:
preparation of alginate jelly oligosaccharide: preparing alginate jelly into 1% alginate jelly solution, adjusting pH to 7.0, adding 10U/mL alginate jelly lyase into the alginate jelly solution, boiling in 45 deg.C water bath for 6 hr, centrifuging, and collecting supernatant; adjusting the pH value of the supernatant to 2.85, centrifuging and collecting a centrifugate; evaporating and concentrating the centrifugate to obtain a concentrated solution, adding 90% ethanol into the concentrated solution, shaking for 20min, centrifuging, collecting precipitate, and freeze-drying to obtain trehalose oligosaccharide.
Preparation of glycosylated pork protein: weighing pork protein, and dissolving the pork protein in a phosphoric acid buffer solution with the pH value of 7.0, wherein the concentration of the pork protein is 8%; continuously adding 0.3g/g of pork alginate-derived oligosaccharides into the mixed solution, uniformly stirring, placing in a constant-temperature water bath condition of 80 ℃ for glycosylation reaction, reacting for 6h, taking out a sample, and freeze-drying to obtain the glycosylated pork protein.
Hydrolysis of pork protein: dissolving glycosylated pork protein in water, and adjusting the feed-liquid ratio to be 1: 20, adjusting the pH value to 7.0, continuously adding papain with the mass of 2% of the glycosylated pork protein, carrying out enzymolysis for 3h at 50 ℃, boiling for 10min after the enzymolysis is finished, and centrifuging and collecting the supernatant to obtain an enzymolysis liquid.
And (3) polypeptide purification: separating the enzymolysis liquid by an ultrafiltration membrane with the molecular weight cutoff of 5ku, wherein the sample loading amount is 25mL, the nitrogen pressure is 0.02Mpa, and collecting the cutoff liquid.
Comparative example
Comparative example 1: the difference between comparative example 1 and example 1 is that the addition amount of alginate oligosaccharides was 0.1g/g pork during the preparation of glycosylated pork protein.
Comparative example 2: the difference between comparative example 2 and example 1 is that the addition amount of alginate oligosaccharides was 0.4g/g pork during the preparation of glycosylated pork protein.
Comparative example 3: the difference between comparative example 3 and example 1 is that pork protein was hydrolyzed by adding it directly to papain without glycosylation.
Performance test
The degree of hydrolysis of the pork protein in examples 1 to 7 and comparative examples 1 to 3, respectively, was determined, the degree of hydrolysis = (moles of free amino nitrogen/moles of total nitrogen) × 100%, the moles of total nitrogen were determined according to GB5009.5-2010, and the moles of free amino nitrogen were determined by formaldehyde titration, with the results shown in table 1.
The weight of the dry matter of the retentate obtained after freeze-drying in examples 1 to 7 and comparative examples 1 to 3 was respectively weighed, and the results are shown in Table 1.
TABLE 1
| Measurement items | Degree of hydrolysis% | Mass fraction% | Measurement items | Degree of hydrolysis% | Mass fraction% |
| Example 1 | 17.34 | 79.39 | Comparative example 1 | 17.04 | 79.36 |
| Example 2 | 18.92 | 75.27 | Comparative example 2 | 17.49 | 79.42 |
| Example 3 | 15.27 | 68.44 | Comparative example 3 | 12.31 | 75.63 |
| Example 4 | 17.48 | 79.38 | |||
| Example 5 | 17.51 | 79.41 | |||
| Example 6 | 17.30 | 79.37 | |||
| Example 7 | 17.38 | 79.40 |
From the data in table 1 for the various groups of examples and comparative examples it can be seen that: among the three hydrolases, trypsin has the highest hydrolysis degree on pork protein; along with the increase of the addition amount of the alginate oligosaccharide, the glycosylation degree of the pork protein is higher, and the solubility of the pork protein is improved, so that the hydrolysis degree of the papain to the pork protein is improved; when the addition amount of the alginate oligosaccharide is lower than the range defined in the embodiment, the hydrolysis degree of the papain to the pork protein is reduced, and when the addition amount of the alginate oligosaccharide is lower than the range defined in the embodiment, the hydrolysis degree of the papain to the pork protein is not greatly changed; when the pork protein is directly added into the papain for hydrolysis without glycosylation, the hydrolysis degree of the papain to the pork protein is obviously reduced.
After the papain hydrolyzes pork protein, the ratio of the components is the highest when the components are less than 5 ku; with the increase of the addition amount of the alginate jelly oligosaccharide, the ratio of the components less than 5ku in the total components does not change greatly; when the addition amount of the alginate oligosaccharide is more or less than the range defined in the examples, the ratio of the component less than 5ku in the total component does not change greatly; when pork protein is directly added to papain for hydrolysis without glycosylation, the proportion of components less than 5ku in the total components is reduced.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (5)
1. A method for preparing polypeptide of hydrolyzed pork protein of suckling pigs is characterized in that: the method comprises the following steps:
preparation of glycosylated pork protein: dispersing pork protein in a phosphoric acid buffer solution with the pH value of 7.0, and uniformly stirring to obtain a pork protein dispersion liquid with the mass fraction of 8%; adding alginate-derived oligosaccharide into the pork protein dispersion liquid for glycosylation, and freeze-drying after reaction to obtain glycosylated pork protein;
hydrolysis of pork protein: dissolving the glycosylated pork protein in water, and adjusting the feed-liquid ratio to be 1: 20 obtaining a dissolved solution; adding hydrolase with the mass being 1-2% of that of the glycosylated pork protein into the dissolved solution for enzymolysis reaction, boiling for 10min after enzymolysis for 2-3h, centrifuging and collecting supernatant to obtain an enzymolysis solution;
in the process of hydrolyzing the pork protein, the hydrolase is trypsin, the reaction pH is 8.0, and the reaction temperature is 37 ℃.
2. The method for preparing polypeptide of hydrolyzed pork protein of porket according to claim 1, wherein: in the preparation process of the glycosylated pork protein, the addition amount of the alginate oligosaccharide is 0.2-0.3g/g pork, the reaction temperature is 70-80 ℃, and the reaction time is 4-6 h.
3. The method for preparing polypeptide of hydrolyzed pork protein of porket according to claim 1, wherein: the preparation method of the alginate jelly oligosaccharide comprises the following steps: preparing seaweed gel into a seaweed gel solution with the mass fraction of 1%, adjusting the pH to 7.0, adding seaweed gel lyase into the seaweed gel solution for hydrolysis reaction, boiling after hydrolysis is finished, centrifuging and collecting supernatant; adjusting the pH value of the supernatant to 2.85, centrifuging and collecting a centrifugate; evaporating and concentrating the centrifugate to obtain a concentrated solution, adding 90% ethanol into the concentrated solution, centrifuging after the reaction is finished, collecting the precipitate, and freeze-drying to obtain the trehalose oligosaccharide.
4. The method for preparing polypeptide of hydrolyzed pork protein of porket according to claim 3, wherein: in the enzymolysis process of the seaweed gel, the addition amount of the seaweed gel lyase is 5-10U/mL, the reaction temperature is 35-45 ℃, and the reaction time is 4-6 h.
5. The method for preparing polypeptide of hydrolyzed pork protein of porket according to claim 1, wherein: separating the enzymolysis liquid by an ultrafiltration membrane with the molecular weight cutoff of 5ku, wherein the sample loading amount is 25mL, the nitrogen pressure is 0.02Mpa, and collecting the cutoff liquid.
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