阅读说明：本技术 一种具有抗炎活性的鲨鱼复合短肽及制备方法与应用 (Shark composite short peptide with anti-inflammatory activity, preparation method and application ) 是由 王悦 沈东炎 陈清西 于 2020-07-10 设计创作，主要内容包括：一种具有抗炎活性的鲨鱼复合短肽及制备方法与应用,涉及海洋生物技术领域。制备方法：将新鲜或冻存解冻后去骨的鲨鱼,剪成小块,用醇类溶液去除脂肪,反复漂洗至无味,沥干；用纯水浴浸烫处理鲨鱼块；冷却至室温后进行匀浆处理,用蛋白酶酶解得到酶解液,加热终止反应；将酶解液进行粗过滤,离心处理,取上清液过滤,将滤液除去溶剂,灭菌干燥,即得鲨鱼复合短肽。所述鲨鱼复合短肽具有抗炎活性,可作为配料在制备功能性食品和特殊医学用途配方食品中应用。具有显著的抗炎保肝护肾等生物学活性。简化以往处理原材料的复杂工序,获得了抗炎功效良好的优质复合短肽,达到可工业规模化,成本效益化生产的目的。(A shark composite short peptide with anti-inflammatory activity, a preparation method and an application thereof relate to the technical field of marine organisms. The preparation method comprises the following steps: cutting fresh or frozen and thawed shark with bone removed into small pieces, removing fat with alcohol solution, repeatedly rinsing until no smell, and draining; scalding shark in pure water bath; cooling to room temperature, homogenizing, performing enzymolysis with protease to obtain enzymolysis solution, and heating to terminate the reaction; and (3) roughly filtering the enzymolysis liquid, centrifuging, taking supernate, filtering, removing the solvent from the filtrate, sterilizing and drying to obtain the shark composite short peptide. The shark composite short peptide has anti-inflammatory activity and can be used as an ingredient to prepare functional foods and formula foods with special medical application. Has obvious biological activities of resisting inflammation, protecting liver and kidney, and the like. The complex process of processing the raw materials in the past is simplified, the high-quality composite short peptide with good anti-inflammatory effect is obtained, and the purposes of industrial scale and cost-effective production can be achieved.)

1. A preparation method of shark complex short peptide with anti-inflammatory activity is characterized by comprising the following steps:

1) cutting fresh or frozen and thawed shark with bone removed into small pieces, removing fat with alcohol solution, repeatedly rinsing until no smell, and draining;

2) scalding the shark meat mass obtained in step 1) with a pure water bath;

3) cooling to room temperature, homogenizing, performing enzymolysis with protease to obtain enzymolysis solution, and heating to terminate the reaction;

4) and (3) carrying out coarse filtration and centrifugal treatment on the enzymatic hydrolysate, taking supernatant, filtering, removing the solvent from the filtrate, sterilizing and drying to obtain the shark composite short peptide.

2. As claimed in claim 1A preparation method of shark composite short peptide with anti-inflammatory activity is characterized in that in the step 1), the shark raw material is fish skin or fish meat of shark, and the shark can be one of griseus shark, striped bamboo shark or big blue shark; the small pieces can be cut to about 1cm³Small pieces of (a).

3. The process for producing a shark complex oligopeptide having anti-inflammatory activity according to claim 1, wherein in the step 1), the alcohol solution is at least one selected from the group consisting of an aqueous ethanol solution, an aqueous isopropanol solution and n-butanol; the concentration of the alcohol solution can be 10-20 wt%.

4. The method for preparing a shark complex oligopeptide with anti-inflammatory activity according to claim 1, wherein in the step 1), the specific method for removing fat comprises the following steps: soaking the mixture for 10 hours by using alcohol solution with 10-20 times of volume/weight, and changing the solution once at intervals of 8-12 hours; the usage amount of the alcohol solution is 10 times of the volume of the wet weight of the shark blocks in terms of l/kg; the repeated rinsing can be carried out by pouring 150-mesh stainless steel screen mesh and repeatedly rinsing with water; the water can adopt secondary reverse osmosis pure water, and the ultrapure water is clean and free of impurities and has a better desalting effect for the preparation of the shark composite short peptide.

5. The method for preparing a shark complex oligopeptide with anti-inflammatory activity according to claim 1, wherein in the step 2), the shark mass obtained in the step 1) is subjected to scalding treatment by a pure water bath, the amount of the pure water is 10-15 times of the wet weight of the shark mass in terms of l/kg, the temperature of the scalding treatment can be 90-95 ℃, the temperature is raised under a set normal pressure, the stirring speed is set to 30rpm, stirring is performed for 5min clockwise, and stirring is performed for 5min counterclockwise, and the treatment time is 20-30 min.

6. The method for preparing a shark complex oligopeptide with anti-inflammatory activity according to claim 1, wherein in the step 3), the protease is at least one selected from the group consisting of acid protease, subtilisin, collagenase, ficin, papain and bromelain, and the protease is preferably papain.

7. The method for preparing a shark complex oligopeptide with anti-inflammatory activity according to claim 1, wherein in the step 3), the protease is added in an amount of 1-6% by weight, preferably 3 ± 0.5% by weight, based on the wet weight of the shark tissue; the conditions of the protease enzymolysis can be as follows: the pH value is 5.5 +/-5.5, the temperature is 53 +/-2.0 ℃, and the enzymolysis time is 3.5 +/-0.5 h; the specific conditions for the heating termination reaction may be: heating to 90-95 deg.C, and maintaining for 10-20 min to terminate the reaction.

8. The method for producing a shark complex oligopeptide with anti-inflammatory activity according to claim 1, wherein in the step 4), the coarse filtration is performed by using a 300 mesh screen and a cotton gauze; the centrifugation speed can be 8000-10000 rpm, and the centrifugation time can be 10-20 min; the drying can be freeze drying or centrifugal spray drying.

9. The shark complex oligopeptide as claimed in any one of claims 1 to 8, which is prepared by the method for preparing the shark complex oligopeptide with anti-inflammatory activity.

10. The shark complex oligopeptide with anti-inflammatory activity as claimed in any one of claims 1 to 8, which is prepared by the method, and the shark complex oligopeptide is used as an ingredient in the preparation of functional foods and formula foods for special medical use.

Technical Field

The invention relates to the technical field of marine organisms, in particular to a shark composite short peptide with anti-inflammatory activity, a preparation method and application thereof.

Background

The abundant proteins in marine species hold up valuable bioactive peptide resources. In recent years, researchers extract mixed polypeptide from various organisms to treat inflammation, and the mixed polypeptide is used for replacing the traditional whole organism. Research shows that the polypeptide of pilose antler can produce anti-inflammatory action on NIH3T3 cells and is the result of combined action of various peptides (high cold, Wanghao sky, bovine daohui, etc.. the extraction and function research of polypeptide protein of pilose antler [ J ]. Jilin Chinese medicine, 2018,38(7): 825-828-). Shark cartilage in the sea contains immune active factors, and shark chondroitin extracted from the sea also proves to have certain anti-inflammatory activity (Reineckia, Pengwulin, Mali. shark cartilage active substance's current research [ J ]. Navy medical journal, 2007, 28(3): 279-281). According to traditional Chinese medicine, shark meat has the effects of sweet taste, appetizing and eating, tonifying spleen and qi, resisting fatigue and the like, but whether the shark meat has an anti-inflammatory effect is rarely reported. In the traditional processing process of sharks, shark fins, livers and cartilages with high commercial values are mainly obtained, and the skin and the fish meat are not effectively utilized, so that resource waste is caused.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a shark composite short peptide with anti-inflammatory activity, a preparation method and application thereof in order to fully develop and utilize marine biological resources.

The preparation method of the shark complex short peptide with anti-inflammatory activity comprises the following steps:

1) cutting fresh or frozen and thawed shark with bone removed into small pieces, removing fat with alcohol solution, repeatedly rinsing until no smell, and draining;

2) scalding the shark meat mass obtained in step 1) with a pure water bath;

3) cooling to room temperature, homogenizing, performing enzymolysis with protease to obtain enzymolysis solution, and heating to terminate the reaction;

4) and (3) carrying out coarse filtration and centrifugal treatment on the enzymatic hydrolysate, taking supernatant, filtering, removing the solvent from the filtrate, sterilizing and drying to obtain the shark composite short peptide.

In step 1)The shark is selected from one of Mustelus griseus, Chilosellum platinosum or Prionia lappa; the small pieces can be cut to about 1cm³Small pieces of (2); the alcohol solution can be at least one selected from ethanol water solution, isopropanol water solution, n-butanol, etc.; the concentration of the alcohol solution can be 10-20 wt%; the specific method for removing fat may be: soaking the mixture for 10 hours by using alcohol solution with 10-20 times of volume/weight, and changing the solution once at intervals of 8-12 hours; preferably, the amount of the alcohol solution is 10 times of the wet weight of the shark blocks in volume in terms of l/kg; the repeated rinsing can be carried out by pouring 150-mesh stainless steel screen mesh and repeatedly rinsing with water; the water can adopt secondary reverse osmosis pure water, and the ultrapure water is clean and free of impurities and has a better desalting effect for the preparation of the shark composite short peptide.

In the step 2), the shark blocks obtained in the step 1) are subjected to scalding treatment by a pure water bath, wherein the consumption of the pure water is 10-15 times of the wet weight of the shark blocks in terms of l/kg, the scalding treatment temperature can be 90-95 ℃, the temperature is increased at a set normal pressure, the stirring speed is set to be 30rpm, the stirring is carried out for 5min in a clockwise mode, the stirring is carried out for 5min in an anticlockwise mode, and the treatment time is 20-30 min; the pure water bath scalding treatment enables the shark tissue protein to be heated uniformly and denatured, and is beneficial to the subsequent enzymolysis reaction.

In step 3), the protease may be selected from at least one of acid protease, subtilisin, collagenase, ficin, papain, bromelain, etc., preferably papain; the adding amount of the protease is 1-6% of the wet weight of the shark tissue, and is preferably 3 +/-0.5%; the conditions of the protease enzymolysis can be as follows: the pH value is 5.5 +/-1.5, the temperature is 53 +/-2.0 ℃, and the enzymolysis time is 3.5 +/-0.5 h; the specific conditions for the heating termination reaction may be: heating to 90-95 deg.C, and maintaining for 10-20 min to terminate the reaction.

In the step 4), the coarse filtration can adopt a 300-mesh screen and cotton gauze for filtration; the centrifugation speed can be 8000-10000 rpm, and the centrifugation time can be 10-20 min; the drying can be freeze drying or centrifugal spray drying.

The shark composite short peptide has anti-inflammatory activity and can be used as an ingredient for preparing functional foods and formula foods with special medical application.

The shark composite short peptide has obvious biological activities of resisting inflammation, protecting liver and kidney and the like through in vitro and in vivo tests.

The sharkskin and the shark meat contain rich protein, are treated by a modern enzymolysis technical method through process technology improvement and mild reaction process conditions, obtain high-quality composite short peptide with nutritive value and bioactivity, and further improve the added value and the effective utilization of biological resources.

The preparation method of the shark composite short peptide with anti-inflammatory activity simplifies the prior complex process for processing raw materials, obtains the high-quality composite short peptide with good anti-inflammatory effect, and achieves the purposes of industrial scale and cost-effective production.

The shark composite short peptide with anti-inflammatory activity has rich types of short peptides and obvious comprehensive anti-inflammatory effect, and expands the research and application of the composite short peptide in functional foods and medical formula foods.

Experimental research shows that the shark composite short peptide prepared by the method can moderately promote the activity of macrophage RAW264.7, and the activity of the shark composite short peptide is increased and is in positive correlation with time and concentration in a certain range, which indicates that the shark composite short peptide can promote the anti-inflammatory capability of an organism by promoting the phagocytosis of the macrophage.

Further animal model research shows that in a mouse anti-inflammatory experiment, after the shark composite short peptide prepared by the application is perfused into a mouse for 15 days and is injected with bacterial Lipopolysaccharide (LPS) to induce acute inflammation, the shark composite short peptide prepared by the application can obviously inhibit the acute inflammatory rise of the liver function index of the mouse; the acute inflammatory rise of creatinine in renal function indexes is remarkably inhibited; the compound has the anti-inflammatory effect which is equivalent to that of the classic anti-inflammatory drug aspirin in combination, and has no obvious side effect. Research results show that the shark composite short peptide prepared by the application has good anti-inflammatory activity, avoids tissue and organ function damage caused by inflammation, is beneficial to conditioning an immune system, and is expected to be developed into a novel anti-inflammatory functional food ingredient.

The invention aims to fully develop and utilize marine biological resources, uses shark skin and fish meat as raw materials to extract a shark composite short peptide with anti-inflammatory activity, and has biological activities of resisting inflammation, protecting liver and kidney and the like besides higher protein nutritive value through cytology and animal experiment tests, so that the shark composite short peptide is expected to be further developed into a novel anti-inflammatory functional food ingredient.

Drawings

FIG. 1 is a bar graph showing the results of promoting the activity of macrophage RAW264.7 in vitro by using the shark compound oligopeptide prepared according to the embodiment of the invention.

FIG. 2 is a bar chart of the detection result of alleviating acute liver and kidney function damage of mice caused by bacterial endotoxin with shark complex oligopeptide prepared in the embodiment of the invention.

Detailed Description

The following examples will further illustrate the present invention with reference to the accompanying drawings.