CN105132510A - Method for preparing biological active peptide by conducting hydrolysis on snail slime through enzymatic method - Google Patents

Abstract

The invention discloses a method for preparing biologically active peptides by enzymatically hydrolyzing snail mucus. The method is as follows: collecting snail mucus, adjusting the pH value of the snail mucus to the optimum pH value of the protease used, adding protease, heating and stirring, and carrying out enzymolysis, and after the enzymolysis is completed, carry out enzymatic treatment to obtain the enzymolysis liquid; The obtained enzymolysis solution is decolorized and filtered, and the obtained filtrate is concentrated and dried to obtain the bioactive peptide product. The invention adopts the enzymatic hydrolysis method to hydrolyze the snail mucus, and the proteolysis degree is high, which can reach 22.06%. The product yield is high, 103g of snail mucus can be collected per 1000g of fresh snail raw materials, and 1.67g of active peptide products can be obtained. The molecular weight of bioactive peptide products is 98% distributed below 1000Da, the molecular weight is small and evenly distributed, which is more conducive to human body absorption. The reaction conditions are mild, and no environmental pollution and no toxic and harmful substances are produced.

Description

A method for preparing bioactive peptides by enzymatically hydrolyzing snail mucus

technical field

The invention relates to the technical field of protein hydrolysis, in particular to a method for preparing biologically active peptides by enzymatically hydrolyzing snail mucus.

Background technique

Snail mucus is a slimy liquid secreted by the snail's foot glands, which helps the snail protect the gastropod muscles from being damaged when they rub against the ground during crawling; it can also allow the snail to adsorb on the rock surface when it is at rest, forming a temporary closed structure. Snail mucus has a high density and is not easy to evaporate, thereby slowing down the loss of water in the body. Therefore, snail mucus can reduce water loss and moisturize the skin. The water content in snail mucus is 95%-99%, the protein content is 5.0-5.1mg/g, and the sugar content is 0.3-0.6mg/g, which is the most important active substance in snail mucus. In addition, it also contains a large number of active substances such as glycoprotein, allantoin, glycolic acid, collagen, chondroitin, vitamins, small molecule antioxidant substances and other essential materials for making cosmetics. For example, protein can increase skin nutrition, make the skin more delicate and smooth, and also has a strong antioxidant effect: collagen, an important connective tissue component of the skin, together with elastin to build a complete skin, has the effect of maintaining moisture; elasticity Protein can maintain skin tissue. When the age increases, the skin loses its elasticity and produces wrinkles. Proper supplementation of elastin can prevent wrinkles from appearing early and reduce the damage of ultraviolet rays to the skin. Allantoin in mucus can effectively repair scars, stimulate cell regeneration, and is a skin softener and antioxidant. In addition, snail mucus is also a natural antibiotic, which can kill certain bacteria on the skin, eliminate skin spots and acne. In addition to being used in cosmetics, snail mucus has remarkable effects in healing burnt skin and treating large scarred skin.

However, due to the large molecular weight, complex structure and high viscosity of the protein in snail mucus, the absorption rate of the human body is not high. Therefore, the protein in the mucus can be hydrolyzed into bioactive peptides with a smaller molecular weight, which can enhance the skin's absorption of the protein in the snail mucus and expand the application range of the snail mucus.

Bioactive peptides refer to peptide compounds that are beneficial to the life activities of organisms or have physiological functions. Compared with protein, active peptide has good physical and chemical properties, such as good solubility and hydration, low viscosity, high bioavailability, easier absorption than amino acids, and has the effect of regulating human physiological functions. It is the original protein and its constituent amino acids do not have. Therefore, it is widely used in food, health products and cosmetics.

In recent years, various bioactive peptides have been extracted and separated from food proteins such as milk protein, soybean protein, corn protein, fish and shellfish protein, and collagen in various countries around the world. Some of them have been industrialized as health food and medicine, and have achieved huge economic benefits. Active peptide foods, health products and cosmetics have been launched in Japan, the United States, and Europe, and Japan is at the forefront of the world in the production of active peptides. However, the research on active peptides started relatively late in my country, and the products are rare. Therefore, making full use of my country's rich protein resources to develop peptide products with good biological activity has a very broad market and application prospects.

At present, the application and development of snail products are mostly concentrated in the fields of food and cosmetics. There are more studies on polysaccharides, helicase, secretion extraction, etc., but there is no research on the hydrolysis of proteins in snail mucus. Bioactive peptides prepared by hydrolysis of proteins in snail mucus have an amino acid composition and ratio more similar to human amino acids than products of plant origin. In terms of production process, the traditional method mostly uses acid method to hydrolyze animal and vegetable protein, the production process is difficult to control, resulting in a certain degree of damage to amino acids; and acid and high temperature conditions often lead to carcinogenic substances in the hydrolyzed solution, especially the content of chloropropanol Exceeding the standard greatly limits the use of the product. The invention adopts enzymatic method to hydrolyze snail mucus to prepare biologically active peptides, and has the advantages of mild reaction conditions, complete preservation of active ingredients of raw materials, safe products, no generation of toxic and harmful by-products, no destruction or racemization of amino acids, and no environmental pollution.

Contents of the invention

The purpose of the present invention is to provide a method for enzymatically hydrolyzing snail mucus to prepare biologically active peptides, to fully develop and utilize my country's abundant high-quality protein resources, to release biologically active peptide products through in vitro protease hydrolysis technology, and to play the role of original protein. have physiological functions.

The technical scheme that the present invention adopts for realizing the above object is as follows:

A method for enzymatically hydrolyzing snail mucus to prepare biologically active peptides, the method comprising: collecting snail mucus, adjusting the pH value of the snail mucus to the optimum pH value of the protease used, adding protease, heating and stirring, performing enzymolysis, and enzymolysis ends Afterwards, an enzymatic hydrolysis solution is obtained by inactivating the enzyme; the above-mentioned enzymatic hydrolysis solution is decolorized and filtered, and the obtained filtrate is concentrated and dried to obtain the biologically active peptide product. Specifically include the following steps:

(1) Mucus collection: Scrape the mucus from the snail's foot into the container with a medicine spoon, rinse the residual mucus from the snail's foot with deionized water, and combine the liquid.

(2) Enzymolysis: adjust the pH value of the snail mucus to the optimum pH value of the enzyme used, add the enzyme, heat and stir for enzymolysis. After the enzymatic hydrolysis, the hydrolyzate was boiled for 5-10 minutes to inactivate the enzyme.

(3) Decolorization: add activated carbon to the enzymolysis solution, stir to decolorize.

(4) Filtration: use filter aid diatomite to pre-coat the filter medium, and filter the decolorized enzymolysis solution to obtain a colorless and transparent liquid.

(5) Concentration: The filtered liquid is concentrated to remove most of the water.

(6) Drying: the concentrated viscous liquid is dried to obtain a powdery bioactive peptide product.

The snail mucus described in step (1) is preferably the mucus produced by white jade snails.

The enzyme described in step (2) is a protease, which can be one or a composite enzyme of acid protease, alkaline protease, neutral protease, papain, pancreatin, flavor protease, bromelain and the like. The optimum pH value of the enzyme is: acid protease 3.0-5.0, alkaline protease 8.0-11.5, neutral protease 6.5-7.5, papain 5.0-7.0, trypsin 7.0-8.5, flavor protease 6.5-7.5, pineapple Protease 6.0-8.0.

The amount of the enzyme added in step (2) is 0.1-3% (w/v) of the volume of the snail mucus, that is, the percentage of the enzyme mass to the volume of the snail mucus is 0.1-3%, preferably 1-2%.

The enzymolysis time in step (2) is 0.5-4h, preferably 2-3h; the enzymolysis temperature is 40-60°C, preferably 50°C.

The amount of activated carbon added in step (3) is 0.5-2w/v% of the liquid volume, preferably 1%; the decolorization temperature is room temperature-50°C, preferably room temperature; the decolorization time is 0.5-3h, preferably 1h.

The filtration described in step (4) can adopt methods such as vacuum suction filtration, plate and frame filtration, centrifugation, and filter aids such as diatomite are used during filtration, and the usage amount of diatomite is 0.5-2w/v% of the liquid volume, 1% is preferred.

The concentration described in step (5) can be but not limited to vacuum concentration.

The drying described in step (6) can be freeze-dried or spray-dried.

Compared with the prior art, the beneficial effects of the present invention are as follows:

(1) The present invention uses an enzymatic hydrolysis method to hydrolyze the snail mucus, and the degree of proteolysis is high, up to 22.06%. The degree of proteolysis refers to the degree or percentage of peptide bonds being cleaved during the process of protein hydrolysis, that is, 22.06% of the peptide bonds are cleaved into small molecular peptides in this process.

(2) The product yield is high, 103g of snail mucus can be collected per 1000g of fresh snail raw materials, and 1.67g of active peptide products can be obtained.

(3) The present invention obtains the best parameter conditions of the enzymatic hydrolysis process through experiments, so that 98% of the molecular weight of the obtained bioactive peptide product is distributed below 1000 Da, and the molecular weight is small and evenly distributed, which is more conducive to human body absorption.

(4) The reaction conditions are mild, and no environmental pollution and toxic and harmful substances are produced.

Detailed ways

The technical solution of the present invention is illustrated below through specific examples. The raw materials and reagents used in the present invention are all commercially available.

Example 1

Scrape the snail foot mucus into the Erlenmeyer flask with a medicine spoon, wash the snail foot with a small amount of deionized water, combine the liquid to obtain the snail mucus, and freeze it in a -20°C refrigerator for later use. Take 20mL of snail mucus, adjust the pH value of the solution to 7.0, add 1% (ie 0.2g) neutral protease, stir evenly, place in a water bath shaker at 55°C for 2 hours, boil for 5 minutes to kill the enzyme, add 1 % (w/v, ie 0.2g) activated carbon was decolorized at room temperature for 1 h, pre-coated with 1% (w/v, ie 0.2g) diatomaceous earth as a filter medium, and suction filtered to obtain a clear colorless transparent liquid. The filtrate was concentrated by rotary evaporation to remove most of the water, and then dried with a freeze dryer to obtain a white powder bioactive peptide product. The degree of hydrolysis of the snail protein in the process is 22.06%; 1000g of snail raw material (wet basis) can produce 1.67g of bioactive peptide product (dry basis); the molecular weight of the bioactive peptide in the product is less than 1000Da.

Example 2

Take 20mL of the prepared snail mucus, adjust the pH value of the solution to 8.5, add 1.5% (ie 0.3g) alkaline protease, stir evenly, place in a water bath shaker at 50°C for 3 hours, and boil for 5 minutes to inactivate the enzyme. The solution was decolorized by adding 0.5% (w/v, 0.1g) of activated carbon at 50°C for 2 hours, pre-coated with 1.5% (w/v, 0.3g) of diatomaceous earth, and suction filtered to obtain a clear colorless Transparent liquid. The filtrate was concentrated by rotary evaporation to remove most of the water, and then dried with a freeze dryer to obtain a white powder bioactive peptide product. The degree of hydrolysis of the snail protein in the process is 21.86%; 1000g of snail raw material (wet basis) can produce 1.58g of bioactive peptide product (dry basis); the molecular weight of the bioactive peptide in the product is less than 1000Da.

Example 3

Take 20mL of the prepared snail mucus, adjust the pH value of the solution to 4.0, add 2% (ie 0.4g) acid protease, stir evenly, place in a water bath shaker at 50°C for 1 hour of enzymatic hydrolysis, boil for 5 minutes to inactivate the enzyme, and enzymolyze Add 1.5% (w/v, ie 0.3g) of activated carbon to the solution for decolorization at 40°C for 1 hour, pre-coat the filter medium with 1% (w/v, ie 0.2g) diatomaceous earth, and filter with suction to obtain a clear colorless and transparent liquid. The filtrate was concentrated by rotary evaporation to remove most of the water, and then dried with a freeze dryer to obtain a white powder bioactive peptide product. The degree of hydrolysis of the snail protein in this process is 19.37%; 1000g of snail raw material (wet basis) can produce 1.44g of bioactive peptide product (dry basis); the molecular weight of the bioactive peptide in the product is less than 1000Da.

The above are only some preferred embodiments of the present invention, and the present invention is not limited to the content of the embodiments. For those skilled in the art, various changes and modifications can be made within the concept scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims (10)

1. A method for enzymatically hydrolyzing snail mucus to prepare biologically active peptides, the method comprising the steps of: Step 1, collecting the snail mucus, adjusting the pH value of the snail mucus to the optimum pH value of the protease used, adding protease, heating and stirring, performing enzymatic hydrolysis, and after the enzymatic hydrolysis, performing an enzymatic treatment to obtain an enzymatic hydrolyzate; Step 2, decolorize and filter the enzymolysis solution obtained above, and concentrate and dry the obtained filtrate to obtain the biologically active peptide product. 2. A method for preparing bioactive peptides by enzymatically hydrolyzing snail mucus according to claim 1, characterized in that: said snail mucus is mucus produced by white jade snails. 3. a kind of enzymatic hydrolysis snail mucus preparation method for biologically active peptide as claimed in claim 1 is characterized in that: described protease is selected from acid protease, alkaline protease, neutral protease, papain, trypsin, local flavor One or more of protease and bromelain. 4. a kind of enzymatic hydrolysis snail mucus preparation method for bioactive peptide as claimed in claim 3 is characterized in that, the optimum pH value of described protease is: acid protease 3.0-5.0, alkaline protease 8.0-11.5, Neutral protease 6.5-7.5, papain 5.0-7.0, trypsin 7.0-8.5, flavor protease 6.5-7.5, bromelain 6.0-8.0. 5. A method for enzymatically hydrolyzing snail mucus to prepare bioactive peptides as claimed in claim 1, characterized in that: the amount of protease added in the step 1 is 0.1-3% (w/v) of the volume of snail mucus . 6. A method for enzymatically hydrolyzing snail mucus to prepare bioactive peptides as claimed in claim 1, characterized in that: in step 1, the enzymatic hydrolysis temperature of protease is 40-60°C, and the enzymatic hydrolysis time is 0.5-4h . 7. A method for enzymatically hydrolyzing snail mucus to prepare biologically active peptides as claimed in claim 1, characterized in that the enzymatic treatment method in step 1 is: boiling the enzymatic solution for 5-10 minutes. 8. A method for preparing bioactive peptides by enzymatically hydrolyzing snail mucus according to any one of claims 1-7, characterized in that: the filtration in the step 2 adopts centrifugation or/and suction filtration, during suction filtration Add diatomaceous earth filter aid. 9. A method for preparing bioactive peptides by enzymatic hydrolysis of snail mucus according to any one of claims 1-7, characterized in that, the filtrate decolorization method in the step 2 is: add activated carbon for decolorization, the addition of activated carbon The amount is 0.5-2% (w/v) of the liquid volume, the decolorization temperature is 15-50°C, and the decolorization time is 0.5-3h. 10. A method for preparing bioactive peptides by enzymatically hydrolyzing snail mucus according to any one of claims 1-7, characterized in that: the concentration in the step 2 adopts vacuum concentration, and the drying adopts freeze-drying or spray-drying .