CN-122011098-A - Bullfrog protein peptide and preparation method and application thereof

CN122011098ACN 122011098 ACN122011098 ACN 122011098ACN-122011098-A

Abstract

The invention belongs to the field of biotechnology, and provides a bullfrog protein peptide, a preparation method and application thereof, wherein the method comprises the steps of decoloring pretreated bullfrog offal to obtain decolored offal; stirring at normal temperature after degreasing treatment, cleaning to obtain crude protein, adding a reaction medium and protease for enzymolysis, inactivating, separating soluble peptide, and freeze-drying to obtain solid protein peptide. The invention is prepared by decolorizing, degreasing and enzymolysis of the bullfrog whole leftovers, is simple and efficient, improves the utilization of protein resources, is convenient for industrial application, can improve the oxidation resistance and the growth performance of the jewfish when being applied to the jewfish feed, improves the CAT and SOD activities of the jewfish by 40.72 percent and 51.94 percent respectively compared with a control group when being added by 1 percent, improves the weight gain rate and the specific growth rate by 11.46 percent and 4.41 percent respectively when being added by 0.5 percent, and has good application prospect.

Inventors

MA RUIJUAN
ZHANG CHUNXIAO
ZHENG MINGXIU
WANG LING
LU KANGLE
LI XUESHAN
SONG KAI

Assignees

集美大学

Dates

Publication Date: 20260512
Application Date: 20251120

Claims (10)

1. A bullfrog protein peptide is characterized in that the amino acid sequence of the protein peptide is one of Ile-Gly-Ala-Gly-Gly-Leu-Asp-Gly-Tyr, leu-Leu-Pro-Tyr-Asp-Gln-Tyr and Trp-Arg-Pro-Pro-Asn-Trp.
2. A method for preparing bullfrog protein peptide is characterized in that, a method for preparing the bullfrog protein peptide of claim 1, the method comprising: Decolorizing the pretreated bullfrog offal to obtain decolorized bullfrog offal; Adding a decoloring agent into the sheared bullfrog offal according to a first preset mass ratio, wherein the decoloring agent is sodium hydroxide solution containing 1% hydrogen peroxide, and the first preset mass ratio is that the mass ratio of the bullfrog offal to the sodium hydroxide solution is 1:3-1:5; Changing the decoloring agent according to a preset time interval until decolored bullfrog offal is obtained, wherein the preset time interval is 20-40 minutes; Degreasing the decolored bullfrog leftovers to obtain bullfrog crude proteins; adding a reaction medium and protease into the bullfrog crude protein, and carrying out enzymolysis according to preset enzymolysis time to obtain an enzymolysis product; wherein the protease comprises alkaline protease and neutral protease, and the preset enzymolysis time is 5-50 minutes; And (3) after the protease is inactivated, separating soluble bullfrog protein peptide from the zymolyte, and freeze-drying to obtain the solid bullfrog protein peptide.
3. The method for producing a bullfrog protein peptide according to claim 2, wherein the step of decoloring the pretreated bullfrog offal to obtain a decolored bullfrog offal comprises: and cleaning and shearing the bullfrog offal to a preset size, wherein the preset size is between 1 and 2 cm.
4. The method for producing a bullfrog protein peptide according to claim 2, wherein said degreasing the decolorized bullfrog offal to obtain a bullfrog crude protein comprises: Adding a degreasing agent into the decolored bullfrog offal according to a second preset mass ratio, wherein the degreasing agent is one of petroleum ether, polysorbate or lipase, and the mass ratio of the decolored bullfrog offal to the degreasing agent is 1:3-1:5; stirring the bullfrog offal and the degreasing agent according to a preset stirring time under a preset temperature condition, wherein the preset stirring time is 1-3 hours; after stirring, the bullfrog leftovers are washed to obtain bullfrog crude proteins.
5. The method for preparing bullfrog protein peptide according to claim 2, wherein the adding reaction medium and protease into the bullfrog crude protein, performing enzymolysis according to preset enzymolysis time to obtain an enzymolysis product comprises: Adding a reaction medium into the bullfrog crude protein to obtain a feed liquid, and adjusting the pH value of the feed liquid to a preset pH value, wherein the reaction medium is water, and the preset pH value is 5.5-10.5; Adding protease into the feed liquid according to a preset enzyme adding amount, wherein the preset enzyme adding amount is 100-800U; And carrying out enzymolysis according to preset enzymolysis time and preset enzymolysis temperature to obtain an enzymolysis product, wherein the protease comprises alkaline protease and neutral protease, and the preset enzymolysis time is 5-50 minutes.
6. The method for producing a bullfrog protein peptide according to claim 5, wherein the mass ratio of the bullfrog crude protein to water is 1:4 to 1:6, and the mass ratio of the alkaline protease to the neutral protease is 3:1 to 1:3.
7. The method for producing a bullfrog protein peptide according to claim 5, wherein the step of adding a reaction medium and a protease to the bullfrog crude protein, and performing enzymolysis according to a predetermined enzymolysis time to obtain an enzymolysis product, further comprises: Preprocessing according to historical experimental data to obtain a target data set, wherein the historical experimental data comprises a plurality of experimental records, each experimental record comprises an independent variable and a corresponding experimental result, the independent variable comprises pH, temperature, enzyme adding amount and enzymolysis time, and the experimental result comprises TCA-NSI and polypeptide concentration; According to the target data set, performing least square fitting by taking TCA-NSI as a response variable and simultaneously taking the independent variable and a two-factor interaction item thereof as an interpretation variable to obtain a linear regression model; performing significance test and effect quantity evaluation on the linear regression model to obtain an independent variable primary and secondary sequencing and two-factor interactive combination list; Performing secondary polynomial fitting by taking TCA-NSI as a target response according to the independent variable primary and secondary sequencing and two-factor interaction combination list to obtain a response surface model; and according to the raw material information of the current bullfrog leftovers, combining the linear regression model and the response surface model to obtain a target parameter set corresponding to the current raw materials.
8. The method for preparing bullfrog protein peptide according to claim 7, wherein the obtaining the target parameter set corresponding to the current raw material according to the raw material information of the current bullfrog offal by combining the linear regression model and the response surface model comprises: obtaining model parameters of the linear regression model and contour information of the response surface model, wherein the model parameters comprise a main effect coefficient interaction item coefficient significance test result and a fitting goodness index, and the contour information comprises a boundary of a target high response area; Ordering the main effects of the independent variables according to the significance test result, identifying significant two-factor interaction items, and obtaining a dominant factor list and an interaction constraint list; Extracting a target high response area according to the contour information and converting the target high response area into candidate ranges of respective variables; Performing characteristic extraction according to the raw material information of the current bullfrog leftovers to obtain raw material characteristic data, wherein the raw material characteristic data at least comprises tissue composition, feed liquid composition parameters and an initial pH value; Generating a process label according to the raw material characteristic data, wherein the process label comprises a compound enzyme proportioning direction label, a composition grade label and a pH deviation label; Cross screening is carried out on the value intervals of the respective variables according to the dominant factor list and the candidate range and in combination with the interaction constraint, so that the initial ranges of the respective variables are obtained; determining initial values of respective variables according to the initial range and the pH deviation label; setting the preliminary proportion of alkaline protease and neutral protease of the complex protease according to the complex enzyme proportioning direction label and the composition grade label to obtain the preliminary proportion of the complex enzyme; and according to the target high-response area, performing compliance check on the initial values of the respective variables and the preset proportion of the complex enzyme, and obtaining a target parameter set corresponding to the current raw material.
9. The method of claim 2, wherein the step of separating the soluble bullfrog protein peptide from the enzymatic hydrolysate after inactivating the protease, and freeze-drying to obtain the solid bullfrog protein peptide comprises: Inactivating the zymolyte in a boiling water bath for 5 to 15 minutes, and cooling to room temperature; Centrifuging the cooled zymolyte in a 4000-6000 rpm centrifuge for 5-15 min, separating the soluble bullfrog protein peptide from the precipitate, filtering, and taking the supernatant to obtain the soluble bullfrog protein peptide; Freezing and drying the soluble bullfrog protein peptide to obtain the solid bullfrog protein peptide.
10. The use of the bullfrog protein peptide according to claim 1, wherein the bullfrog protein peptide is used for preparing feed for aquaculture varieties.

Description

Bullfrog protein peptide and preparation method and application thereof The application is filed on day 20 and 11 of 2025, and the application is named as bullfrog protein peptide with antioxidant activity, and a preparation method and application thereof, and the application is a divisional application of the application patent application with the application number 202511708070.7. Technical Field The invention relates to the technical field of biology, in particular to a bullfrog protein peptide, a preparation method and application thereof. Background Sea bass (Lateolabrax japonicus) is used as an important variety of fresh water and sea water culture, is favored by consumers because of tender meat quality, rich nutrition and no fishy smell, and has extremely high economic value. In recent years, with the expansion of the cultivation scale, the sea bass industry is subjected to high-temperature stress (for example, the temperature of a cultivation water body in summer often exceeds 28 ℃, the metabolism rate of the sea bass is increased, the oxygen consumption is increased, the active oxygen is increased), diseases are frequently generated and the like, so that the growth rate of the sea bass is slowed down, the survival rate of the sea bass is reduced, and the development of sea bass cultivation is affected. Research shows that improving the antioxidant capacity of sea bass is a key way to alleviate the above problems, so developing an efficient, safe and low-cost antioxidant feed additive is an urgent need in the field of sea bass culture. Bullfrog (Lithobates catesbeiana) is also an important characteristic economic aquatic product, the cultivation scale is enlarged year by year, and the bullfrog is widely applied to the catering industry. However, a large amount of leftovers (such as head, feet, skin, bones and viscera) generated in the processing process of eating bullfrog are usually directly discarded, so that protein resources are wasted. The protein peptide is a small molecular peptide prepared by enzymolysis, fermentation and other methods, has the characteristics of small molecular weight, easy absorption, high bioavailability, various functional activities (such as antioxidation and immunoregulation) and the like, and has become a research hot spot in the field of feed additives. Among them, the enzymolysis method is widely used for preparing protein peptide because of mild reaction conditions, strong specificity, high safety and easy control. At present, the enzymolysis technology for the whole components of bullfrog offal in the prior art is not mature, and most researches focus on a single blood sugar reducing function, and the growth and antioxidation requirements of aquaculture animals cannot be effectively combined. For example, the Chinese patent application with publication number of CN119285752A discloses bullfrog skin peptide with DPP-IV inhibitory activity, a purification method and application thereof, wherein an enzymolysis means is adopted to extract polypeptide, then a column chromatography purification way is adopted to extract a component containing peptide segments, finally the component is evaluated and screened to obtain the purified bullfrog skin peptide with DPP-IV inhibitory activity, and then the purified bullfrog skin peptide is prepared into a medicament for reducing blood sugar. However, the method only uses bullfrog skin, the bullfrog skin which is directly peeled off in the bullfrog leftovers also comprises the bullfrog skin which is not peeled off, the method also needs to carry out screening and peeling steps of the bullfrog skin, column chromatography is also needed to carry out purification in the treatment method, the whole treatment method is relatively complex, the actual operation process time is long, and the large-scale preparation is difficult. Disclosure of Invention In view of the above, as an aspect of the present invention, an embodiment of the present invention provides a method for preparing a bullfrog protein peptide, the method comprising: Decolorizing the pretreated bullfrog offal to obtain decolorized bullfrog offal; Degreasing the decolored bullfrog leftovers to obtain bullfrog crude proteins; adding a reaction medium and protease into the bullfrog crude protein, and carrying out enzymolysis according to preset enzymolysis time to obtain an enzymolysis product; and (3) after the protease is inactivated, separating soluble bullfrog protein peptide from the zymolyte, and freeze-drying to obtain solid bullfrog protein peptide. As a second aspect of the present invention, there is provided a bullfrog protein peptide having an amino acid sequence of one of Ile-Gly-Ala-Gly-Gly-Leu-Asp-Gly-Tyr, leu-Leu-Pro-Tyr-Asp-Gln-Tyr and Trp-Arg-Pro-Pro-Asn-Trp, which is produced by the method as described in any one of the above. As a third aspect of the invention, the embodiment of the invention provides the application of the bullfrog protein peptide, which