CN110499352B - Preparation method of oxhide gelatin polypeptide with antioxidant activity - Google Patents
Preparation method of oxhide gelatin polypeptide with antioxidant activity Download PDFInfo
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- CN110499352B CN110499352B CN201910895398.2A CN201910895398A CN110499352B CN 110499352 B CN110499352 B CN 110499352B CN 201910895398 A CN201910895398 A CN 201910895398A CN 110499352 B CN110499352 B CN 110499352B
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 38
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 28
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 27
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- 238000002360 preparation method Methods 0.000 title claims abstract description 12
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- 238000000034 method Methods 0.000 claims abstract description 20
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 17
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 17
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- 239000000243 solution Substances 0.000 description 15
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- 102000004169 proteins and genes Human genes 0.000 description 5
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- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
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- GJCOSYZMQJWQCA-UHFFFAOYSA-N 9H-xanthene Chemical compound C1=CC=C2CC3=CC=CC=C3OC2=C1 GJCOSYZMQJWQCA-UHFFFAOYSA-N 0.000 description 1
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- RBRPTFMVULVGIC-ZTIIIDENSA-N Xanthatin Chemical compound C1C=C(\C=C\C(C)=O)[C@@H](C)C[C@@H]2OC(=O)C(=C)[C@H]21 RBRPTFMVULVGIC-ZTIIIDENSA-N 0.000 description 1
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- QDLAGTHXVHQKRE-UHFFFAOYSA-N lichenxanthone Natural products COC1=CC(O)=C2C(=O)C3=C(C)C=C(OC)C=C3OC2=C1 QDLAGTHXVHQKRE-UHFFFAOYSA-N 0.000 description 1
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- 230000008736 traumatic injury Effects 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
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- RBRPTFMVULVGIC-UHFFFAOYSA-N xanthatin Natural products C1C=C(C=CC(C)=O)C(C)CC2OC(=O)C(=C)C21 RBRPTFMVULVGIC-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a preparation method of oxhide gelatin polypeptide with antioxidant activity, which comprises the following steps: adding gelatinum oxhide into PBS buffer (pH7.4), and heating to room temperatureMelting the oxhide gelatin, and adding (NH)4)2SO4Standing, centrifuging, and removing upper oily floating substances to obtain lower liquid; adding pancreatin into the lower layer liquid for enzymolysis; the obtained enzymolysis liquid is poured into a 10KD ultrafiltration tube for centrifugation, and the permeation liquid positioned at the lower layer of the ultrafiltration tube is gelatin polypeptide. The oxhide gelatin polypeptide prepared by the method has good oxidation resistance.
Description
Technical Field
The present invention belongs to a preparation method of gelatin polypeptide.
Background
The oxhide gelatin is prepared from skin of cattle of family Bovidae by decocting, and has effects of nourishing yin, moistening dryness, stopping bleeding, and relieving swelling, and can be used for treating consumptive disease, consumptive lung disease, cough, hemoptysis, hematemesis, metrorrhagia, traumatic injury, carbuncle, swelling, scald, etc.
In recent years, the problems of raw material shortage, quality reduction and cost increase of the donkey-hide gelatin occur, the price of the yellow gelatin is low, the raw materials are easy to obtain, and the literature mentions that the yellow gelatin has similar or alternative efficacy with the donkey-hide gelatin. In addition, the comparison between the yellow gelatin and the donkey-hide gelatin shows that the total content of amino acid in the yellow gelatin is higher than that of the donkey-hide gelatin, so that the preparation of the low peptide by using the yellow gelatin as a raw material is a good choice. However, a method for producing a oxhide gelatin polypeptide is still lacking.
A method for preparing colla Corii Asini low peptide with publication number CN 106831938A comprises (1) dissolving colla Corii Asini to obtain colla Corii Asini water solution with set concentration; (2) acid-adding high-pressure hydrolysis: mixing the donkey-hide gelatin solution obtained in the step (1) with oxalic acid, adjusting the pH value to 1.5-2.5, and performing high-pressure acidic hydrolysis; (3) and (4) adjusting the pH value: adjusting the pH value of the product after high-pressure hydrolysis to 3.5-4.5; (4) decoloring and removing impurities: adding activated carbon for decolorization and impurity removal, and then removing the activated carbon to obtain the donkey-hide gelatin micromolecule low peptide. The method adopts organic acid to prepare acidic environment, and oxalic acid is weak acid, and can sufficiently break peptide bonds between donkey-hide gelatin proteins and decompose the donkey-hide gelatin proteins into small molecular low peptides; the high pressure cooking method is used to quickly denature proteins and break peptide bonds. The method combining the two is favorable for obtaining the small molecular low peptide with smaller molecular weight and uniformity. The small molecular weight low peptide can be well absorbed and utilized by human body.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of oxhide gelatin polypeptide with strong oxidation resistance.
In order to solve the technical problems, the invention provides a preparation method of oxhide gelatin polypeptide with antioxidant activity, which comprises the following steps:
1) and pretreatment:
firstly, adding the yellow gelatin into a PBS buffer solution (pH7.4) according to the material-liquid ratio of 1 g/20-60 ml, and heating until the yellow gelatin is melted (stewed) to form a mixed system;
the mixed system is cooled to room temperature and then (NH) is added4)2SO4Stirring to (NH)4)2SO4Completely dissolved of said (NH)4)2SO4The dosage ratio of the buffer solution to PBS buffer solution (pH7.4) is 0.8-1.2 g/10ml, the mixture is placed still (30 +/-10) min and then put into a centrifuge to be centrifuged (10 +/-2) min at 3500 +/-500 rpm, and oily floating substances on the upper layer are removed to obtain liquid on the lower layer;
note: the purpose of standing is to fully combine ammonium sulfate and gelatinum oxhide, so that protein is separated from impurities such as lipid; after centrifugation, the mixture is layered, the upper layer of floating oily substances are removed, and the lower layer of liquid is reserved for further enzymolysis.
2) And enzymolysis:
adding pancreatin into the lower layer liquid until the content is 2500 + -250U/g, mixing, and performing enzymolysis at 37 + -0.5 deg.C for 4 h;
3) and purifying:
pouring the enzymolysis liquid obtained in the step 2) into a 10KD ultrafiltration tube, and putting the ultrafiltration tube into a centrifuge to centrifuge at (4000 +/-500) rpm for (20 +/-2) min;
the permeate at the lower layer of the ultrafiltration tube is gelatin polypeptide which is low peptide with the molecular weight below 10 KD.
The improvement of the preparation method of the oxhide gelatin polypeptide with antioxidant activity of the invention comprises the following steps: in the step 2), after the enzymolysis time is up, heating to inactivate the enzyme (heating to 80 ℃ and keeping for 15min), and cooling (cooling to room temperature) to obtain the enzymolysis liquid.
As a further improvement of the preparation method of the oxhide gelatin polypeptide with antioxidant activity of the present invention: the heating temperature in the step 1) is (60 +/-5) DEG C.
As a further improvement of the preparation method of the oxhide gelatin polypeptide with antioxidant activity of the present invention: in the step 1), the feed-liquid ratio of the oxhide gelatin to the PBS buffer solution (pH7.4) is 1g/60ml, (NH)4)2SO4The amount ratio of the buffer solution to PBS buffer solution (pH7.4) was 1g/10 ml.
In the invention process, the following preparation process screening is carried out:
1. pretreatment before enzymolysis
5 tubes were collected, and 0.2g of xanthene gelatin and 4mL of PBS buffer (pH7.4) were added thereto, respectively, and the mixture was stewed at 60 ℃. After cooling to room temperature, 0.4g, 0.8g, 1.2g, 1.6g and 2.0g of ammonium sulfate were added and stirred until dissolved, and the mixture was allowed to stand for 30min before observation (see FIG. 1). The ammonium sulfate has the capability of removing protein, and the enzymolysis efficiency can be effectively improved after a certain amount of ammonium sulfate is added to remove impurities such as lipids and the like. As can be seen from FIG. 1, after more than 10% ammonium sulfate was added, a certain amount of precipitate was formed in the solution, indicating that an excess of ammonium sulfate resulted in precipitation of proteins and adversely affecting the enzymatic yield. Therefore, 10% ammonium sulfate was selected as the ammonium sulfate purification concentration. FIG. 1 shows the phenomenon after 30min of standing with ammonium sulfate, which is 50%, 40%, 30%, 20%, 10% from left to right. 10% represents the ratio of ammonium sulfate to PBS buffer (g/ml), and so on.
Then, 5 tubes after standing for 30min were placed at 3500rpm, centrifuged for 10min, and the delamination was observed. Centrifuging 10% of the test tube, and separating into two layers, namely liquid and oily floating substances on the upper surface of the liquid; and 20% -50% of 4 test tubes are separated into liquid and sediment below the liquid after centrifugation.
TABLE 1 protein content in the liquid and sediment/oil floes after centrifugation with addition of ammonium sulfate at different concentrations
2. Time of enzymolysis
Mixing gelatinum oxhide and PBS at a ratio of 1:20(g/ml), stewing at 60 deg.C, cooling, adding 10% ammonium sulfate, dissolving, standing for 30min, centrifuging at 3500rpm for 10min, and removing upper layer oil (upper oily floating substance). Adding 10000U/g pancreatin, and placing in water bath at 37 ℃ for enzymolysis. 3mL of the enzymolysis liquid is put into a 10KD ultrafiltration tube at different time, the centrifugation is carried out for 20min at 4000rpm, and the hydrolysis degree of the lower layer of the permeation liquid is measured by a Coomassie brilliant blue method. As shown in FIG. 2, the yield of the polypeptide with less than 10KD obtained by enzymolysis for 4h is the highest.
3. Amount of enzyme added
Taking 9 test tubes, wherein each test tube is as follows: mixing xanthan gum and PBS buffer (pH7.4) at a ratio of 1:20(g/ml) and stewing in water bath at 60 deg.C. Cooling, adding 10% ammonium sulfate, dissolving, standing for 30min, centrifuging at 3500rpm for 10min, and removing upper layer oil. Adding pancreatin 0, 2500, 5000, 7500, 10000, 20000, 30000, 40000 and 50000U/g respectively, and placing in water bath at 37 deg.C for enzymolysis. Putting the enzymolysis solution for 4h into 10KD ultrafilter tube, centrifuging at 4000rpm for 20min, and measuring hydrolysis degree of the lower layer of the permeate by Coomassie brilliant blue method. When the amount of the pancreatin added is 2500U/g, the yield of the polypeptide below 10KD is the highest (figure 3).
4. Ratio of material to liquid
Taking 5 test tubes, putting the gelatin and PBS (Ph7.4) into the PBS according to the material-liquid ratio (g/ml) of 1:10, 1:20, 1:40, 1:60 and 1:80, mixing, and stewing in water bath at 60 ℃. Cooling, adding 10% ammonium sulfate, dissolving, standing for 30min, centrifuging at 3500rpm for 10min, and removing upper layer oil. Adding 10000U/g pancreatin, and placing in water bath at 37 ℃ for enzymolysis. Putting the enzymolysis solution for 4h into 10KD ultrafilter tube, centrifuging at 4000rpm for 20min, and measuring hydrolysis degree of the lower layer of the permeate by Coomassie brilliant blue method. The yellow gelatin and PBS (pH7.4) are subjected to enzymolysis at a ratio of 1:60(g/ml), and the yield of the polypeptide with the concentration of less than 10KD is the highest (figure 4).
In the invention, the process of removing impurities by ammonium sulfate is added, so that the grease and the protein can be further separated, and the enzymolysis efficiency is increased. Also, it is important to set the concentration of ammonium sulfate, which is directly related to the concentration of protein in the liquid obtained by centrifugation.
The invention uses single pancreatin without repeatedly adjusting pH, so that the process is simpler and more convenient, and the efficiency is improved.
The yellow gelatin has antifatigue, antiaging and antioxidant effects. The antioxidant activity of the low peptide was examined in the present invention, and it was found that the antioxidant activity of the low peptide having a molecular weight of 10kD or less in the case of a concentration of 0.316mg/mL is equivalent to that of 0.552mg/mL of ascorbic acid.
In conclusion, the method firstly removes the impurities such as grease contained in the yellow gelatin, thereby improving the enzymolysis efficiency in the subsequent enzymolysis process, and obtaining the low peptide with the required molecular weight by enzymolysis according to the requirement. The antioxidant capacity of the enzymolytic oligopeptide is determined to have good antioxidant capacity, and an OH free radical scavenging experiment with ascorbic acid as a positive control shows that the antioxidant capacity of the oligopeptide of 0.316mg/mL is equivalent to that of the ascorbic acid of 0.552 mg/mL.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the phenomenon of FIG. 1 after 30min of standing with ammonium sulfate (50%, 40%, 30%, 20%, 10% from left to right);
FIG. 2 is a graph showing the comparison of the polypeptide yields for different enzymatic hydrolysis times;
FIG. 3 is a graph comparing the effect of enzyme addition on enzymatic hydrolysis;
FIG. 4 effect of feed-solution ratio on enzymatic hydrolysis;
FIG. 5 is a standard curve of antioxidant capacity.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1, a method of making a gelatin polypeptide, comprising the steps of:
1) and pretreatment
Firstly, adding 0.4g of oxhide gelatin into 8ml of PBS buffer solution (pH7.4) according to the material-liquid ratio of 1g/20ml, and putting into a water bath kettle at 60 ℃ to melt (stew) the oxhide gelatin to form a mixed system;
after cooling to room temperature, 0.8g of (NH) was added4)2SO4Stirring to (NH)4)2SO4Completely dissolved, i.e., (NH)4)2SO4Mixing with PBS buffer (pH7.4) at a ratio of 1g/10ml, standing for 30min, and addingCentrifuging at 3500rpm for 10min in a centrifuge; after centrifugation, the oily floating substances in the upper layer are removed, and the liquid in the lower layer is reserved for further enzymolysis.
2) And enzymolysis:
adding pancreatin to the lower layer liquid until the active concentration is 2500U/g, mixing well, and placing in 37 deg.C water bath for enzymolysis. Taking out after enzymolysis for 4h, putting into 80 deg.C water bath, heating for 15min to completely inactivate enzyme, and cooling for use.
3) And purifying:
pouring the enzymolysis liquid obtained in the step 2) into a 10KD ultrafiltration tube, and putting the ultrafiltration tube into a centrifuge for 20min at 4000 rpm. After the centrifugation is finished, the permeate at the lower layer of the ultrafiltration tube is gelatin polypeptide which is low peptide with the molecular weight below 10 KD; the yield thereof was found to be 36%.
Example 2, the ratio of the feed liquid in the step 1) of the example 1 is changed from 1g/20ml to 1g/60ml, namely, the step 1) is as follows:
firstly, adding 0.4g of oxhide gelatin into 24ml of PBS buffer solution (pH7.4) according to the material-liquid ratio of 1g/60ml, and putting the oxhide gelatin into a water bath kettle at the temperature of 60 ℃ to melt (stew) the oxhide gelatin to form a mixed system;
after cooling to room temperature, 2.4g of (NH) were added4)2SO4Stirring to (NH)4)2SO4Completely dissolved, i.e., (NH)4)2SO4Mixing with PBS buffer solution (pH7.4) at a ratio of 1g/10ml, standing for 30min, and centrifuging at 3500rpm for 10min in a centrifuge; after centrifugation, the oily floating substances in the upper layer are removed, and the liquid in the lower layer is reserved for further enzymolysis.
The rest is equivalent to embodiment 1.
The yield obtained is about 50%.
The experimental method for activity evaluation adopted by the invention is as follows (which is a conventional experimental method):
determination of OH radical scavenging rate for evaluation of antioxidant capacity: preparing 0.15 mmol/o-diazaphenanthrene solution and 0.75 mmol/o-FeSO4·7H2O solution, 0.01% (v/v) H2O2Solution, 20mg/mL ascorbic acid solution.
OH free radical clearance is calculated from the formula:
clearance (%) ═ aSample (A)-AInjury of the skin)/(AWithout damage-AInjury of the skin)；
AInjury of the skinThe method of (3) comprises: mixing 1.0mL of o-diazaphenanthrene solution with 2.0mL of PBS and 1.0mL of distilled water, and sequentially mixing with FeSO4·7H2O solution 1.0mL, H2O2And mixing 1.0mL of the solution, uniformly mixing, and placing in a water bath at 37 ℃ for reacting for 60min to obtain a mixed solution. 4mL of distilled water was mixed with 2mL of PBS, and the mixture was placed in a 37 ℃ water bath for 60min as a reference. The absorbance of the mixture was measured at 536 nm. Measured data is AInjury of the skin。
AWithout damageThe method of (3) comprises: in the last step H2O2The solution was replaced with distilled water. The reference is the same. Absorbance A at 536nmWithout damage。
Drawing a standard curve: 5 tubes were taken, 10, 20, 30, 40 and 50. mu.L ascorbic acid, respectively, and made up to 1.0ml with water. Substitution AInjury of the skinDistilled water in the method of (1). The reference is the same. The measurement data were plotted as an ascorbic acid standard curve. The results of the standard curve are shown in FIG. 5. The standard curve formula is that y is 0.129x + 0.0669.
The method for measuring the antioxidant capacity of the sample comprises the following steps: taking a proper amount of enzymolysis ultrafiltrate (namely the oxhide gelatin polypeptide obtained in the embodiment 1 of the invention), and adding water to supplement 1.0mL to obtain a solution to be detected. Substitution of the test solution AInjury of the skinThe distilled water in the method of (1) is ASample (A). As a reference, 1.0mL of the test solution was mixed with 2.0mL of PBS, and then mixed with 3.0mL of distilled water, and the same treatment was carried out (ultraviolet absorbance at 536 nm). The measured data are recorded as ASample (A)。
ASample (A)＝0.138、AInjury of the skin＝0.092、AIs not damaged0.260, carry-in clearance (%) (a)Sample (A)-AInjury of the skin)/(AWithout damage-AInjury of the skin) The clearance rate is 27.38%.
A is to beSample (A)The substitution y 0.129X +0.0669, 0.138, gives X0.552.
That is, the sample clearance was calculated to be 27.38%, i.e., a 0.316mg/mL solution of oxhide gelatin polypeptide was equivalent to the antioxidant capacity of 0.552mg/mL ascorbic acid.
In addition, the clearance of the gelatin polypeptides obtained in example 2 was not significantly different from the clearance of the gelatin polypeptides obtained in example 1 (P > 0.05).
Comparative example 1 (0.8 g of NH in example 1 was omitted4)2SO4The rest is equivalent to example 1.
The yield of the obtained xanthatin polypeptide is about 20%.
Comparative example 2, with reference to the procedure described in CN 106831938A, starting from gelatine and finally purified according to step 3) of the present invention using a 10KD ultrafiltration tube, gave a yield of only about 0.05%.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (2)
1. The preparation method of the oxhide gelatin polypeptide with antioxidant activity is characterized by comprising the following steps:
1) and pretreatment:
firstly, adding the yellow gelatin into PBS buffer solution with the pH value of 7.4 according to the material-liquid ratio of 1g/60ml, and heating until the yellow gelatin is melted to form a mixed system, wherein the heating temperature is 60 +/-5 ℃;
the mixed system is cooled to room temperature and then (NH) is added4)2SO4Stirring to (NH)4)2SO4Dissolving of (NH) described4)2SO4The dosage ratio of the buffer solution to PBS buffer solution with pH7.4 is 1g/10ml, standing for 30 +/-10 min, centrifuging in a centrifuge at 3500 +/-500 rpm for 10 +/-2 min, and removing upper oily floating substances to obtain lower liquid;
2) and enzymolysis:
adding pancreatin into the lower layer liquid until the concentration is 2500 plus or minus 250U/g, mixing uniformly, and performing enzymolysis at 37 plus or minus 0.5 ℃ for 4 h;
3) and purifying:
pouring the enzymolysis liquid obtained in the step 2) into a 10KD ultrafiltration tube, and putting the ultrafiltration tube into a centrifuge to centrifuge for 20 +/-2 min at 4000 +/-500 rpm;
the permeate liquid at the lower layer of the ultrafiltration tube is gelatin polypeptide.
2. The method for preparing oxhide gelatin polypeptide with antioxidant activity as claimed in claim 1, wherein: in the step 2), after the enzymolysis time is up, heating to inactivate the enzyme, and cooling to obtain an enzymolysis liquid.
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