CN115057916A - Pinctada martensii meat antioxidant polypeptide and preparation method and application thereof - Google Patents
Pinctada martensii meat antioxidant polypeptide and preparation method and application thereof Download PDFInfo
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- CN115057916A CN115057916A CN202210799601.8A CN202210799601A CN115057916A CN 115057916 A CN115057916 A CN 115057916A CN 202210799601 A CN202210799601 A CN 202210799601A CN 115057916 A CN115057916 A CN 115057916A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
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Abstract
Disclosure of the inventionAn Pinctada martensii meat antioxidant polypeptide and a preparation method and application thereof are provided. The preparation method has the advantages of simple operation, low cost and high separation efficiency, and is suitable for large-scale industrial production. The protein content of the obtained Pinctada martensii meat antioxidant polypeptide is 65-93% (dry basis), the average molecular weight is 3000-10000 Da, and the leucine content is 11-21%; oxygen Radical Absorption Capacity (ORAC) is 1000-3000 mu MTEAC/g, and has high bioavailability, remarkable antioxidant effect, and capability of remarkably increasing the Oxygen Radical Absorption Capacity (ORAC) 2 O 2 The cell survival rate of the oxidative damage of the human liver cancer cell HepG2 can be used for the development of antioxidant functional foods, and has important social and economic significance for developing the functional foods of the pinctada martensii meat with specific functions.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to pinctada martensii meat antioxidant polypeptide and a preparation method and application thereof.
Background
Pinctada martensii (Pinctada martensii), also known as Hepu nacre, is the main shellfish used in Chinese marine pearl culture. The protein nutrient solution is mainly distributed in the coastal zones of Guangxi, Guangdong, Hainan and Taiwan strait south, the meat protein content of the pinctada martensii dunker is rich, the dry weight content of crude protein is 66.71-81.21%, and the nutrient content and the free amino acid composition of the pinctada martensii dunker meat in the Chao super birch paper show that the protein nutrient value in the whole visceral organs of the pinctada martensii dunker is high, the protein nutrient solution is rich in amino acids such as G1u and Leu, can be used as a high-quality protein source for preparing functional food, and the enzymatic hydrolysate has the effects of reducing blood pressure, resisting photoaging, resisting oxidation and the like and has higher development and utilization values. However, a large amount of shellfish meat left after pearl picking causes a large amount of waste of resources due to lack of reasonable processing treatment, so that the development of the functional food of the pinctada martensii shellfish meat with specific functions can effectively improve the economic added value thereof, and has important economic and social significance.
The antioxidant peptide is a natural, safe and efficient antioxidant, and has wide application prospect in the aspects of cosmetics, health products, food, feed additives and the like. In recent years, animal antioxidant peptides, particularly marine animals, have become the focus of research. The protein of the pinctada martensii meat has high nutritive value and is a good source for preparing antioxidant peptide.
In the prior art, patent 201410040473.4 discloses a method for preparing antioxidant peptide from pinctada fucata martensii meat with antioxidant activity, wherein microwave-assisted alkaline protease is used for enzymolysis of the pinctada martensii meat, and a C18 column, a Sephadex G-25 cross-linked dextran chromatographic column and the like are used for separation and purification, so that the antioxidant peptide with strong antioxidant activity is obtained. And the animal protein is subjected to enzymolysis by using commercial enzymes such as alkaline protease, and the like, so that the method has the advantages of good enzymolysis effect, high selectivity and the like, however, after some commercial enzymes are adopted for enzymolysis, although the product has strong biological activity in vitro, the product does not show the same effect in vivo, mainly because the structure of the peptide is changed under the condition of extreme pH in the digestive absorption process of gastrointestinal tract, and the activity of the peptide is changed under the action of digestive enzyme, so that the bioavailability of the product obtained after the animal protein is subjected to enzymolysis by using the commercial enzymes cannot be ensured.
Disclosure of Invention
In view of the above, the invention discloses a Pinctada martensii meat antioxidant polypeptide and a preparation method thereof, wherein bionic biological enzymes (such as pepsin, trypsin, chymotrypsin and the like) are adopted to simulate the digestion process in vivo to carry out enzymolysis on animal proteins, so that the digestion condition of substances in vivo is reduced, the capability of the substances in exerting specific activity in vivo can be reflected more truly, and the bioactive peptides with high bioavailability are obtained
The invention aims to provide an Pinctada martensii meat antioxidant polypeptide, which has the protein content of 65-93% (dry basis), the average molecular weight of 3000-10000 Da and the leucine content of 11-21%.
Furthermore, the protein content of the Pinctada martensii meat antioxidant polypeptide is 75-93% (dry basis), the average molecular weight is 3000-10000 Da, and the leucine content is 11-21%; moreover, the pinctada martensii meat antioxidant polypeptide has good free radical scavenging capacity, and the Oxygen Radical Absorption Capacity (ORAC) is 1000-3000 mu M TEAC/g.
And after the bionic biological enzymolysis, the biological utilization rate is high, redundant free radicals in vivo can be effectively eliminated, the active oxygen balance in vivo is maintained, the harm caused by oxidative damage is reduced, and the biological enzyme has a remarkable antioxidant effect.
The invention also aims to provide a preparation method of the Pinctada martensii meat antioxidant polypeptide, wherein the antioxidant peptide is obtained by performing enzymolysis on the meat of Pinctada martensii meat through bionic biological enzymes (pepsin, trypsin and chymotrypsin), ultrafiltration, Sephadex G-25 filler separation on glucan gel and the like, and the method simulates the digestion process of substances in vivo so as to obtain the active peptide with higher bioavailability.
The purpose of the invention is realized by at least one of the following technical solutions.
The preparation method of the Pinctada martensii meat antioxidant polypeptide specifically comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shell meat, beating the shell meat into homogenate to obtain homogenate, mixing the homogenate with distilled water, adding pepsin for enzymolysis for a period of time, adding trypsin and chymotrypsin into an enzymolysis product for continuous enzymolysis for a period of time, inactivating enzyme, cooling and centrifuging, taking supernatant, and performing vacuum filtration to remove precipitates to obtain a pinctada martensii meat enzymolysis product (enzymolysis liquid);
(2) taking the enzymolysis liquid obtained in the step (1), sequentially carrying out ultrafiltration by ultrafiltration membranes with cut-off molecular weights of 10kDa, 5kDa and 3kDa respectively so as to obtain peptide liquids with molecular weights of 0-3 kDa, 3-5 kDa, 5-10 kDa and more than 10kDa respectively, and preserving at-20 ℃ after vacuum freeze drying;
(3) and (3) separating the product obtained in the step (2) by using Sephadex G-25 filler, and collecting the components according to the time sequence of peak appearance to obtain the pinctada martensii meat antioxidant polypeptide.
Preferably, the mixing ratio of the shellfish meat homogenate to the distilled water in the step (1) is 1: 1-4; the addition amount of the pepsin is 1000-4000U/g of a substrate, and the reaction conditions are as follows: the pH value is 1.0-5.0, the temperature is 20-50 ℃, and the time is 1-4 h; the addition amounts of the trypsin and the chymotrypsin are respectively 50-200U/g substrate and 10-100U/g substrate, and the reaction conditions are as follows: the pH value is 6.0-8.0, the temperature is 20-40 ℃, and the time is 1-4 h.
Further preferably, the mixing ratio of the shellfish meat homogenate and the distilled water in the step (1) is 1: 1-3; the addition amount of the pepsin is 2000-3000U/g of a substrate, and the reaction conditions are as follows: the pH value is 2.0-4.0, the temperature is 30-40 ℃, and the time is 1-3 h; the addition amounts of the trypsin and the chymotrypsin are respectively 50-100U/g substrate and 10-50U/g substrate, and the reaction conditions are as follows: the pH value is 7.0-8.0, the temperature is 30-40 ℃, and the time is 1-3 h.
Preferably, in the step (3), the specific operating conditions for separating and purifying the Sephadex G-25 filler of the Sephadex are as follows: the column volume is 100-500 mL, the sample loading concentration is 10-50 mg/mL, the sample loading volume is 2-10 mL, the mobile phase is distilled water, the flow rate is 0.5-2 mL/min, one tube is collected every 5min, and the detection wavelength is 220 nm.
When the amount of the enzyme is within the above range, the degree of proteolysis is the highest; when the enzymolysis time, the pH value and the temperature are in the ranges, the prepared sample has higher bioavailability;
moreover, when the Sephadex G-25 filler is prepared under the operation condition of separation and purification, the separation effect among the components is the best, and the yield of the component with antioxidant activity is the highest.
The invention also claims application of the Pinctada martensii meat antioxidant polypeptide in preparation of antioxidant drugs, functional foods and related daily chemical products.
Therefore, the Pinctada martensii meat antioxidant polypeptide and the preparation method and the application thereof disclosed by the invention have the following advantages and beneficial effects:
(1) the pinctada martensii is a main shellfish for pearl culture in China, and shellfish meat left after pearl collection is not reasonably processed and utilized.
(2) The invention prepares the Pinctada martensii meat antioxidant polypeptide by bionic biological enzymes (pepsin, trypsinase and chymotrypsin), the simulated substance is subjected to enzymolysis in the in-vivo digestion process, the Oxygen Radical Absorption Capacity (ORAC) activity of the polypeptide can reach 2111.95 mu M TEAC/g and is higher than the activity of glutathione, the product obtained after gastrointestinal enzymolysis still has obvious antioxidant activity, and the bioavailability of the product is as high as 76%.
(3) The pinctada martensii meat antioxidant polypeptide is prepared by enzymolysis, ultrafiltration and Sephadex G-25 filler separation, and the method is simple to operate, low in cost, high in separation efficiency and suitable for large-scale industrial production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 shows 5 fractions S1, S2, S3, S4 and S5 from comparative example 2, in which product B2a was purified by Sephadex G-25 packing.
FIG. 2 is a graph showing comparative ORAC activities of products A1-A6 and glutathione in examples 1-6.
FIG. 3 shows the results of examples 1 to 6 and comparative examples 1 to 4, in which the concentrations of A1 to A6, B1, B2a, B2B, B3a, B3B and B4 to H were 1mg/mL 2 O 2 Effect of induced survival of human liver cancer cells (HepG 2).
FIG. 4 is a graph comparing the bioavailability of the products A1-A6 of examples 1-6 and the bioavailability of the products B1, B2a, B2B, B3a, B3B, B4 of comparative examples 1-4.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings of the specification, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
Example 1
A preparation method of Pinctada martensii meat antioxidant polypeptide comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shellfish meat, beating the shellfish meat into homogenate, mixing the homogenate with distilled water at a ratio of 1:1 to obtain homogenate, adding pepsin, adding a substrate with the enzyme amount of 2000U/g, adjusting the pH value to 2.0 by using 1M HCl, oscillating the mixture in a water bath shaker at 30 ℃ for 1h, adding trypsin and chymotrypsin in a ratio of 50Ug substrate to 10U/g substrate respectively after the reaction is finished, adjusting the pH value to 6.0 by using 1MNaOH, oscillating the mixture in a water bath shaker at 30 ℃ for 1h, inactivating the enzyme in a boiling water bath for 10min after the reaction is finished, cooling the reaction system to room temperature, centrifuging the reaction system at 10000rpm for 10min, and performing vacuum suction filtration to obtain supernatant fluid of the pinctada martensii meat enzymolysis liquid.
(2) And (2) sequentially carrying out ultrafiltration on the enzymolysis liquid obtained in the step (1) by using ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa and 3kDa, taking liquid with the molecular weight of 3-5 kDa, and preserving at-20 ℃ after vacuum freeze drying.
(3) Dissolving the product obtained in the step (2) with distilled water at the concentration of 10mg/mL, filtering with a 0.22 mu m filter membrane, and separating and purifying by adopting Sephadex G-25 filler under the specific conditions that: the column volume is 100mL, the sample loading volume is 2mL, the mobile phase is distilled water, the flow rate is 0.5mL/min, one tube is collected every 5min, and the detection wavelength is 220 nm. Collecting the 4 th elution peak component according to the peak time sequence, and obtaining the product A1 after rotary evaporation and freeze-drying.
Example 2
A preparation method of Pinctada martensii meat antioxidant polypeptide comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shellfish meat, beating the shellfish meat into homogenate, mixing the homogenate with distilled water in a ratio of 1:4 to obtain homogenate, adding pepsin, adjusting the enzyme amount to be 2500U/g of a substrate, adjusting the pH to be 3.0 by using 1M HCl, oscillating the mixture for 2 hours in a water bath shaker at 37 ℃, adding trypsin and chymotrypsin according to the ratio of 75U/g of the substrate to 25U/g of the substrate after the reaction is finished, adjusting the pH to be 7.0 by using 1MNaOH, oscillating the mixture for 2 hours in the water bath shaker at 37 ℃, inactivating the enzyme for 10 minutes in a boiling water bath after the reaction is finished, cooling a reaction system to room temperature, centrifuging the mixture for 20 minutes at 10000rpm, and then carrying out vacuum suction filtration to obtain supernatant fluid of the pinctada martensii meat hydrolysate.
(2) And (2) sequentially carrying out ultrafiltration on the enzymolysis liquid obtained in the step (1) by using ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa and 3kDa, taking liquid with the molecular weight of 3-5 kDa, and preserving at-20 ℃ after vacuum freeze drying.
(3) Dissolving the product obtained in the step (2) with distilled water at the concentration of 25mg/mL, filtering with a 0.22 mu m filter membrane, and separating and purifying by adopting Sephadex G-25 filler under the specific conditions that: the column volume is 300mL, the sample loading volume is 7mL, the mobile phase is distilled water, the flow rate is 1mL/min, one tube is collected every 5min, and the detection wavelength is 220 nm. Collecting the 4 th elution peak component according to the peak time sequence, and obtaining the product A2 after rotary evaporation and freeze-drying.
Example 3
A preparation method of Pinctada martensii meat antioxidant polypeptide comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shellfish meat, beating the shellfish meat into homogenate, mixing the homogenate with distilled water in a ratio of 1:3 to obtain homogenate, adding pepsin with the enzyme amount of 3000U/g substrate, adjusting the pH to 4.0 by using 1M HCl, oscillating the homogenate for 3 hours by using a water bath shaker at 40 ℃, adding trypsin and chymotrypsin according to the ratio of 100Ug substrate to 50U/g substrate respectively after the reaction is finished, adjusting the pH to 8.0 by using 1MNaOH, oscillating the substrate for 3 hours by using a water bath shaker at 40 ℃, inactivating the enzyme for 10 minutes by using a boiling water bath after the reaction is finished, cooling a reaction system to room temperature, centrifuging the reaction system at 10000rpm for 30 minutes, and performing vacuum suction filtration to obtain supernatant to obtain the pinctada martensii meat hydrolysate.
(2) And (2) sequentially carrying out ultrafiltration on the enzymolysis liquid obtained in the step (1) by using ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa and 3kDa, taking liquid with the molecular weight range of 3-5 kDa, and preserving at-20 ℃ after vacuum freeze drying.
(3) Taking the product obtained in the step (2), dissolving the product with distilled water at the concentration of 50mg/mL, filtering the product through a 0.22 mu m filter membrane, and separating and purifying the product by adopting Sephadex G-25 filler, wherein the specific conditions are as follows: the column volume is 400mL, the sample loading volume is 10mL, the mobile phase is distilled water, the flow rate is 2mL/min, one tube is collected every 5min, and the detection wavelength is 220 nm. Collecting the 4 th elution peak component according to the peak time sequence, and obtaining the product A3 after rotary evaporation and freeze-drying.
Example 4
A preparation method of Pinctada martensii meat antioxidant polypeptide comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shellfish meat, beating the shellfish meat into homogenate, mixing the homogenate with distilled water in a ratio of 1:2 to obtain homogenate, adding pepsin with the enzyme amount of 2000U/g substrate, adjusting the pH to 3.0 by using 1M HCl, oscillating the homogenate for 2 hours in a water bath shaking table at 37 ℃, adding trypsin and chymotrypsin in a ratio of 100U/g substrate to 25U/g substrate respectively after the reaction is finished, adjusting the pH to 7.0 by using 1MNaOH, oscillating the homogenate for 2 hours in the water bath shaking table at 37 ℃, inactivating the enzyme for 10 minutes in a boiling water bath after the reaction is finished, cooling a reaction system to room temperature, centrifuging the reaction system for 20 minutes at 10000rpm, and performing vacuum suction filtration to obtain supernatant to obtain pinctada martensii meat hydrolysate.
(2) And (2) sequentially carrying out ultrafiltration on the enzymolysis liquid obtained in the step (1) by using ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa and 3kDa, taking liquid with the molecular weight of 3-5 kDa, and preserving at-20 ℃ after vacuum freeze drying.
(3) Taking the product obtained in the step (2), dissolving the product with distilled water at the concentration of 15mg/mL, filtering the product through a 0.22-micron filter head, and separating and purifying the product by adopting Sephadex G-25 filler, wherein the specific conditions are as follows: the column volume is 200mL, the sample loading volume is 10mL, the mobile phase is distilled water, the flow rate is 1.2mL/min, one tube is collected every 5min, and the detection wavelength is 220 nm. The 4 th eluting peak fractions were collected in chronological order and after rotary evaporation and lyophilization the product was recorded as a 4.
Example 5
A preparation method of Pinctada martensii meat antioxidant polypeptide comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shell meat, beating the shell meat into homogenate, mixing the homogenate with distilled water in a ratio of 1:1 to obtain homogenate, adding pepsin with an enzyme amount of 3000U/g substrate, adjusting the pH to 2.0 by using 1M HCl, oscillating the mixture for 1h in a water bath shaker at 40 ℃, adding trypsin and chymotrypsin according to a ratio of 50U/g substrate to 25U/g substrate respectively after the reaction is finished, adjusting the pH to 7.0 by using 1M NaOH, oscillating the mixture for 2h in a water bath shaker at 37 ℃, inactivating the enzyme in a boiling water bath for 10min after the reaction is finished, cooling a reaction system to room temperature, centrifuging the mixture for 20min at 10000rpm, and performing vacuum suction filtration to obtain supernatant to obtain the pinctada martensii meat hydrolysate.
(2) And (2) sequentially carrying out ultrafiltration on the enzymolysis liquid obtained in the step (1) by using ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa and 3kDa, respectively, and carrying out vacuum freeze drying on the enzymolysis liquid with the molecular weight of 3-5 kDa and then storing at-20 ℃.
(3) Dissolving the product obtained in the step (2) with distilled water at the concentration of 10mg/mL, filtering with a 0.22 mu m filter membrane, and separating and purifying by adopting Sephadex G-25 filler under the specific conditions that: the column volume is 100mL, the sample loading volume is 2mL, the mobile phase is distilled water, the flow rate is 0.5mL/min, one tube is collected every 5min, and the detection wavelength is 220 nm. Collecting the 4 th elution peak component according to the peak time sequence, and obtaining the product A5 after rotary evaporation and freeze-drying.
Example 6
A preparation method of Pinctada martensii meat antioxidant polypeptide comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shellfish meat, beating the shellfish meat into homogenate, mixing the homogenate with distilled water in a ratio of 1:2 to obtain homogenate, adding pepsin, adjusting the enzyme amount to be 2500U/g of a substrate, adjusting the pH to be 4.0 by using 1M HCl, oscillating the mixture for 3 hours in a water bath shaker at 40 ℃, adding trypsin and chymotrypsin according to the ratio of 75U/g of the substrate to 50U/g of the substrate after the reaction is finished, adjusting the pH to be 8.0 by using 1MNaOH, oscillating the mixture for 1 hour in the water bath shaker at 40 ℃, inactivating the enzyme in a boiling water bath for 10 minutes after the reaction is finished, cooling a reaction system to room temperature, centrifuging the mixture for 20 minutes at 10000rpm, and then carrying out vacuum suction filtration to obtain supernatant fluid of the pinctada martensii meat hydrolysate.
(2) And (2) sequentially carrying out ultrafiltration on the enzymolysis liquid obtained in the step (1) by using ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa and 3kDa, respectively, and carrying out vacuum freeze drying on the enzymolysis liquid with the molecular weight of 3-5 kDa and then storing at-20 ℃.
(3) Dissolving the product obtained in the step (2) with distilled water at the concentration of 10mg/mL, filtering by a 0.22-micron filter head, and separating and purifying by adopting Sephadex G-25 filler under the specific conditions that: the column volume is 300mL, the sample loading volume is 6mL, the mobile phase is distilled water, the flow rate is 0.5mL/min, one tube is collected every 5min, and the detection wavelength is 220 nm. According to the time sequence of peak, the 4 th elution peak component is collected, and after rotary evaporation and freeze drying, the product A6 is obtained.
Comparative example 1
A preparation method of a Pinctada martensii meat polypeptide comprises the following steps:
removing shells of pinctada martensii, cleaning and airing shell meat, beating the shell meat into homogenate, mixing the homogenate with distilled water in a ratio of 1:2 to obtain homogenate, adding pepsin, adding a substrate with the enzyme amount of 2000U/g, adjusting the pH value to 2.0 by using 1M HCl, oscillating the mixture for 2 hours in a water bath shaker at 37 ℃, adding trypsin and chymotrypsin according to the ratio of 100Ug substrate to 25U/g substrate respectively after the reaction is finished, adjusting the pH value to 7.0 by using 1M NaOH, oscillating the mixture for 2 hours in a water bath shaker at 37 ℃, inactivating the enzyme for 10 minutes in a boiling water bath after the reaction is finished, cooling a reaction system to room temperature, centrifuging the mixture for 10 minutes at 10000rpm, and then performing vacuum suction filtration to obtain supernatant to obtain pinctada martensi martensii meat enzymatic hydrolysate, which is named as B1 (crude enzymatic hydrolysate).
Comparative example 2
A preparation method of a Pinctada martensii meat polypeptide comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shell meat, beating the shell meat into homogenate, mixing the homogenate with distilled water in a ratio of 1:2 to obtain homogenate, adding pepsin with the enzyme amount of 2000U/g substrate, adjusting the pH to 3.0 by using 1M HCl, oscillating the mixture for 2 hours in a water bath shaker at 37 ℃, adding trypsin and chymotrypsin according to the mixture ratio of 100U/g substrate to 25U/g substrate after the reaction is finished, adjusting the pH to 7.0 by using 1M NaOH, oscillating the mixture for 2 hours in a water bath shaker at 37 ℃, inactivating the enzyme in a boiling water bath for 10 minutes after the reaction is finished, cooling a reaction system to room temperature, centrifuging the mixture for 20 minutes at 10000rpm, and performing vacuum suction filtration to obtain supernatant to obtain the pinctada martensii meat hydrolysate.
(2) And (2) sequentially carrying out ultrafiltration on the enzymolysis liquid obtained in the step (1) by using ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa and 3kDa, respectively, taking liquid with the molecular weight range of 3-5 kDa and 0-3 kDa, carrying out vacuum freeze drying, and then storing at-20 ℃, wherein the liquid is respectively marked as B2a and B2B (ultrafiltration peptides).
Comparative example 3
A preparation method of a Pinctada martensii meat polypeptide comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shellfish meat, beating the shellfish meat into homogenate, mixing the homogenate with distilled water in a ratio of 1:2 to obtain homogenate, adding pepsin with the enzyme amount of 2000U/g substrate, adjusting the pH to 2.0 by using 1M HCl, oscillating the homogenate for 2 hours in a water bath shaker at 37 ℃, adding trypsin and chymotrypsin in a ratio of 100U/g substrate to 25U/g substrate respectively after the reaction is finished, adjusting the pH to 7.0 by using 1MNaOH, oscillating the homogenate for 2 hours in a water bath shaker at 37 ℃, inactivating the enzyme for 10 minutes in a boiling water bath after the reaction is finished, cooling a reaction system to room temperature, centrifuging the reaction system at 10000rpm for 20 minutes, and performing vacuum suction filtration to obtain supernatant to obtain pinctada martensii meat hydrolysate.
(2) And (2) sequentially carrying out ultrafiltration on the enzymolysis liquid obtained in the step (1) by using ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa and 3kDa, respectively, and carrying out vacuum freeze drying on the enzymolysis liquid with the molecular weight of 3-5 kDa and then storing at-20 ℃.
(3) Dissolving the product obtained in the step (2) with distilled water at the concentration of 15mg/mL, filtering with a 0.22 mu m filter membrane, and separating and purifying by adopting Sephadex G-25 filler under the specific conditions that: the column volume is 200mL, the sample loading volume is 10mL, the mobile phase is distilled water, the flow rate is 1.2mL/min, one tube is collected every 5min, and the detection wavelength is 220 nm. According to the time sequence of the peak, the 1 st and 5 th elution peak components are collected, and are named as B3a and B3B after rotary evaporation and freeze drying.
Comparative example 4
A preparation method of a Pinctada martensii meat polypeptide comprises the following steps:
(1) removing shells of pinctada martensii, cleaning and airing shellfish meat, beating the shellfish meat into homogenate, mixing the homogenate with distilled water in a ratio of 1:1 to obtain homogenate, adding Alcalase2.4L protease into the homogenate with the enzyme addition amount of 5000U/g (adding Alcalase2.4L protease 5000U into 1g of the homogenate), oscillating the homogenate for 1.5 hours in a water bath shaker at the temperature of 58 ℃, inactivating enzyme in a boiling water bath for 10 minutes after the reaction is finished, cooling a reaction system to the room temperature, centrifuging the reaction system for 10 minutes at the rotating speed of 10000rpm, and then carrying out vacuum suction filtration to obtain supernatant fluid to obtain the pinctada martensii meat enzymolysis fluid.
(2) And (2) sequentially carrying out ultrafiltration on the enzymolysis liquid obtained in the step (1) by using ultrafiltration membranes with the molecular weight cut-off of 10kDa, 5kDa and 3kDa, respectively, and carrying out vacuum freeze drying on the enzymolysis liquid with the molecular weight of 3-5 kDa and then storing at-20 ℃.
(3) Taking the product obtained in the step (2), dissolving the product with distilled water at the concentration of 15mg/mL, filtering the product through a 0.22-micron filter head, and separating and purifying the product by adopting Sephadex G-25 filler, wherein the specific conditions are as follows: the column volume is 200mL, the sample loading volume is 10mL, the mobile phase is distilled water, the flow rate is 1.2mL/min, one tube is collected every 5min, and the detection wavelength is 220 nm. According to the time sequence of the peak, the 4 th elution peak component is collected, and after rotary evaporation and freeze drying, the product is named as B4.
Effect verification
The invention selects Pinctada martensii meat antioxidant polypeptide A1-A6 prepared in embodiment 1-6, measures the protein content, molecular weight, amino acid composition and antioxidant activity of the Pinctada martensii meat antioxidant polypeptide, compares the bioavailability and antioxidant activity of the products (A1-A6) prepared in embodiment 1-6 with the bioavailability and antioxidant activity of the products (B1, B2a, B2B, B3a, B3B and B4) prepared in comparative examples 1-4, and specifically comprises the following implementation steps:
1. determination of protein content and recovery
The protein concentration of the sample was measured by using Nanjing-constructed Total protein quantitative determination kit (BCA method) according to the procedure of the instruction, and the result was expressed in%.
2. Determination of Molecular Weight (MW)
The molecular weight of the polypeptide is determined by high performance Size Exclusion Chromatography (SEC). Standard curves were prepared with each of triglycine (GGG, MW: 189Da), oxidized glutathione (MW: 351Da), bacillase (MW: 1422.69Da), aprotinin (MW: 6511.44Da) and cytochrome C (MW: 12400 Da); respectively preparing the above different standards with ultrapure water to the concentration of 1mg/mLThe sample solution is prepared into a solution with the concentration of 8mg protein/mL by using ultrapure water, and the standard solution and the sample solution are filtered by a water-phase filter membrane with the aperture of 0.22 mu m for later use. Chromatographic conditions are as follows: a detector: shimadzu RID-10A differential detector; a chromatographic column: shimadzu TSKgel G-2000SW XL (7.8X 300 mm); mobile phase: acetonitrile: 0.1% trifluoroacetic acid (TFA) ═ 20: 80 (v/v); column temperature: 30 +/-1 ℃; flow rate: 1 mL/min; sample introduction amount: 10 mu L of the solution; detection wavelength: 220 nm. And taking the Retention Time (RT) as an abscissa and a log value (LogMW) of the molecular weight of the standard product as an ordinate, performing linear fitting on the retention time and the log of the molecular weight of the polypeptide standard product, making a standard curve, and calculating the molecular weight of the polypeptide by adopting an area normalization method to perform molecular weight distribution.
3. Amino acid analysis
The total amino acid composition in the sample is analyzed by adopting an automatic amino acid analyzer after acidolysis of the sample liquid according to the method of GB 5009.124-2016, and the tryptophan analysis is to determine the total amino acid composition in the sample liquid by adopting the automatic amino acid analyzer after alkaline hydrolysis. Amino acid detection conditions: us Waters high performance liquid chromatography, pico. tag amino acid analytical column, temperature 38 ℃, flow rate 1mL/min, detection wavelength 254 nm.
4. Oxygen Radical Absorbance Capacity (ORAC) assay
20 μ L of a sample to be tested dissolved in a 75mM pH 7.4 potassium Phosphate Buffer (PBS) was added to each well of a 96-well fluorescent plate, which was an experimental group; respectively preparing Trolox standard solutions with the concentrations of 6.25, 12.5, 25 and 50 mu M, and adding 20 mu L of the Trolox standard solutions into each well, wherein the Trolox standard solutions are used as a standard group; add 20. mu.L of LPBS per well, this is a blank set. After the addition, the well plate is incubated at 37 ℃ for 10min, then 96 mu M fluorescein sodium 200 mu L is added, after 20min presetting at 37 ℃, 120 mu M AAPH (2,2' -azobis (2-methylproprionidine) dihydrate) 20 mu L is rapidly added into each well by a multi-channel pipette to start the reaction, in addition, a control without adding AAPH is made on the basis of a blank group, the micro-well plate is placed in a fluorescence microplate reader, continuous measurement is carried out at 37 ℃ at excitation wavelength 485nm and emission wavelength 535nm, the fluorescence intensity is measured 1 time every 4.5min, and the total measurement is 150 min. And (3) drawing a standard curve by taking Trolox standard solutions with different concentrations as an abscissa and the protection area of the fluorescence attenuation curve as an ordinate, calculating the ORAC value of the sample to be detected according to the standard curve, and expressing the result by Trolox equivalent (mu MTEAC/g).
5、H 2 O 2 Protective Effect assay for induced oxidative Damage to HepG2 (human liver cancer cells) cells
Building H 2 O 2 Inducing a HepG2 (human liver cancer cell) cell oxidative stress model, researching the protective effect of enzymolysis products on cell oxidative damage, and evaluating the cell survival rate by adopting an MTT (3- (4,5) -dimethylthi ahiazo (-z-y1) -3, 5-diphenylytrazoliumromide) method, wherein the experimental method comprises the following steps: HepG2 cells at 2.5X 10 4 After being seeded in 96-well cell culture plates per well, the plates were placed at 37 ℃ and contained 5% CO 2 After the culture medium is attached to the wall for 24 hours in a DMEM high-sugar medium containing 10% fetal calf serum and 1% streptomycin double antibody, the culture medium is removed by suction, PBS is washed once, 0.1mL of complete culture medium is added into a control group and a model group, and 0.1mL of product (1.0mg/mL) obtained by different implementation cases is added into an experimental group. After 24h incubation, the medium was aspirated, 0.1mL of complete medium was added to each well of the control group, and 2000. mu. M H in complete medium was added to all groups 2 O 2 And (3) adding 0.1mL of the solution, continuously incubating for 24h, removing the culture medium by suction, adding 0.05mL of 1 XMTT solution into each hole, and incubating for 4h at 37 ℃ to reduce MTT to formazan. The culture medium is removed by suction, 0.15mL of dimethyl sulfoxide (DMSO) is added into each hole to dissolve the formazan, and the formazan is shaken uniformly by a flat shaking table. Detecting absorbance of each hole at 570nm wavelength by using a microplate reader, calculating the ratio of different concentrations to the absorbance of a control group, and judging ELD to H according to the cell survival rate value 2 O 2 Induction of the effects of HepG2 oxidatively damaging cells.
As can be seen from Table 1, the protein content of the Pinctada martensii meat antioxidant polypeptide is 65-93%, wherein the protein content of A4 is the highest, which indicates that the polypeptide product with higher purity can be obtained by the invention.
TABLE 1 protein content (dry basis) of Pinctada martensii meat antioxidant polypeptide
As can be seen from FIG. 1, when the Sephadex G-25 separation and purification of the ultrafiltration peptide is carried out by the method of the invention, 5 obvious component peaks can be obtained, and the separation effect is good.
As can be seen from FIG. 2, the ORAC activity of the products prepared by the embodiments 1-6 is between 1000-2500 μ M TEAC/g, which is higher than that of reduced glutathione, wherein the ORAC activity of A5 is strongest and higher than that of glutathione, and A4 times, which shows that the products prepared by the invention have good antioxidant effect.
TABLE 2 average molecular weight distribution of the products
As can be seen from Table 2, the molecular weight of the product obtained by the method is 3000-5000 Da, which shows that the purified small molecular peptide which is more beneficial to human body absorption can be obtained.
TABLE 3 leucine (Leu) content of the products
Leucine (Leu) is a hydrophobic amino acid required by human growth, and the antioxidant capacity of the polypeptide is proved by the literature to be related to the amino acid composition, wherein the hydrophobic amino acid can enhance the affinity of the polypeptide to lipid and improve the antioxidant capacity of the polypeptide, and as can be seen from Table 3, the leucine content of the product in the embodiment of the invention is between 11% and 21%, the tryptophan content of the product A5 is the highest, and the ORAC activity of the product is also the highest in the product, which indicates that the antioxidant activity of the polypeptide can be related to the leucine content of the polypeptide.
The results of comparing the protection effect of the products of examples 1 to 6 with the products of comparative examples 1 to 4 on the oxidative damage of cells are shown in FIG. 3. The results show that A1-A6 can reduce oxidative damage of cells and have a protective and preventive effect on oxidative stress caused by free radical attacking the cells, wherein the protective effect of A5 on the cells is strongest, and the survival rate of the cells after the A1-A6 groups are higher than that of the cells after the products in the comparative examples are treated, which shows that compared with crude peptides, the purified small molecular peptides have better antioxidant effect, and also shows that compared with other enzymolysis methods, the products with stronger protective effect on the oxidative stress of the cells can be obtained by adopting bionic biological enzyme enzymolysis.
As can be seen from FIG. 4, the bioavailability of the products A1-A6 prepared by the method is 70-76%, wherein the highest bioavailability of the product A5 can reach 76%, compared with the product B1 (bioavailability 50.34%) subjected to enzymolysis by other enzymes, the enzymolysis crude peptide (B1), the ultrafiltration crude peptide (B2a, B2B) and the purified other component peptides (B3a, B3B), the bioavailability of the products prepared by the method is greatly improved, and the products obtained by enzymolysis by the method reflect the capability of the substances to exert specific activity in vivo more truly and have higher bioavailability and bioactivity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
And the previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. The Pinctada martensii meat antioxidant polypeptide is characterized in that the protein content of the Pinctada martensii meat antioxidant polypeptide is 65-93% (dry basis), the average molecular weight is 3000-10000 Da, and the leucine content is 11-21%.
2. The Pinctada martensii meat antioxidant polypeptide as claimed in claim 1, wherein the Pinctada martensii meat antioxidant polypeptide has a protein content of 75-93% (dry basis), an average molecular weight of 3000-5000 Da and a leucine content of 11-21%.
3. The pinctada martensii meat antioxidant polypeptide as claimed in claim 1, wherein the pinctada martensii meat antioxidant polypeptide has a good free radical scavenging ability, and the oxygen radical absorption capacity is 1000-3000 μ M TEAC/g.
4. A method for preparing the pinctada martensii meat antioxidant polypeptide as claimed in any one of claims 1 to 3, comprising the steps of:
1) cleaning and airing Pinctada martensii meat, beating into homogenate, mixing with distilled water, performing enzymolysis by pepsin by adopting a bionic enzymolysis method, performing enzymolysis by mixing trypsin and chymotrypsin, inactivating enzyme, centrifuging, taking supernate, and performing vacuum filtration to obtain Pinctada martensii meat enzymolysis liquid;
2) carrying out ultrafiltration on the pinctada martensii meat enzymolysis liquid obtained in the step 1) by using ultrafiltration membranes with different molecular weights, then carrying out Sephadex G-25 filler separation on the obtained product, and collecting components to obtain the pinctada martensii meat antioxidant polypeptide.
5. The method for preparing the Pinctada martensii meat antioxidant polypeptide as claimed in claim 4, wherein in the step 1), the mixing ratio of the shellfish meat homogenate to distilled water is 1: 1-4, the addition amount of pepsin is 1000-4000U/g substrate, and the enzymolysis reaction conditions are as follows: the pH value is 1.0-5.0, the temperature is 20-50 ℃, and the time is 1-4 h.
6. The preparation method of the Pinctada martensii meat antioxidant polypeptide as claimed in claim 4 or 5, wherein the addition amount of the trypsin is 50-200U/g of substrate, the addition amount of the chymotrypsin is 10-100U/g of substrate, and the mixed enzymolysis reaction conditions are as follows: the pH value is 6.0-8.0, the temperature is 20-40 ℃, and the time is 1-4 h.
7. The method for preparing the pinctada martensii meat antioxidant polypeptide according to claim 4, wherein in the step 2), the enzymolysis solution is sequentially subjected to ultrafiltration by ultrafiltration membranes with cut-off molecular weights of 10kDa, 5kDa and 3kDa respectively, so as to obtain polypeptides with molecular weights of 0-3 kDa, 3-5 kDa, 5-10 kDa and more than 10kDa respectively.
8. The method for preparing the Pinctada martensii meat antioxidant polypeptide as claimed in claim 4 or 7, wherein the operating conditions for separating and purifying the Sephadex G-25 Sephadex filler are as follows: the column volume is 100-500 mL, the sample loading concentration is 10-50 mg/mL, the sample loading volume is 2-10 mL, the mobile phase is distilled water, and the flow rate is 0.5-2 mL/min.
9. Use of the Pinctada martensii meat antioxidant polypeptide of any one of claims 1-3 or the Pinctada martensii meat antioxidant polypeptide prepared by the method of claim 4 in the preparation of antioxidant drugs, functional foods and daily chemical products.
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