CN-121204200-B - Fish collagen peptide with high GPH content, functional food and preparation method thereof by collagenase colG

Abstract

The invention provides a fish collagen peptide with high GPH content, a functional food and a method for preparing the fish collagen peptide with high GPH content by using collagenase colG. The method comprises the steps of a) homogenizing fish scales, b) carrying out first-step enzymolysis on the homogenate by using alkaline protease, and c) carrying out second-step enzymolysis on the homogenate by using collagenase colG. The technological parameters of the first enzymolysis are that the pH is 8-10, the enzymolysis temperature is 45-55 ℃, the alkaline protease dosage is 0.5-2% of the fish scale mass, and the enzymolysis time is 0.5-3h. The technological parameters of the second enzymolysis are that the pH is 7-8, the enzymolysis temperature is 35-45 ℃, the dosage of collagenase colG is 1-2.5% of the mass of fish scales, and the enzymolysis time is 4-8h. The content of the characteristic fragment Gly-Pro-Hyp in the fish collagen peptide prepared by the method can reach 6.1 percent at most.

Inventors

• HUANG JUNYANG
• PENG GUOQING
• YANG XIONG
• XU MEI
• YU TIAN
• ZHAO DAZHOU

Assignees

• 厦门元之道生物科技有限公司

Dates

Publication Date

20260508

Application Date

20251128

Claims (6)

1. 1. A method for preparing fish collagen peptide with high GPH content by collagenase colG, which is characterized by comprising the following steps: a) Homogenizing fish scales; b) Performing first-step enzymolysis on the homogenate by using alkaline protease, wherein the dosage of the alkaline protease is 0.5-2% of the mass of fish scales, and the technological parameters of the first-step enzymolysis are that the pH is 8-10, the enzymolysis temperature is 45-55 ℃, and the enzymolysis time is 0.5-3h; c) And carrying out second-step enzymolysis on the homogenate by using collagenase colG, wherein the dosage of the collagenase colG is 1-2.5% of the mass of the fish scales, and the technological parameters of the second-step enzymolysis are that the pH is 7-8, the enzymolysis temperature is 35-45 ℃, and the enzymolysis time is 4-8h.
2. 2. The method according to claim 1, wherein the step of inactivating the alkaline protease is performed at a temperature after the first enzymatic hydrolysis step, and the step of inactivating the collagenase colG is performed at a temperature after the second enzymatic hydrolysis step.
3. 3. The method according to claim 2, wherein the inactivation parameter is that the enzyme is inactivated by heating to 90 ℃ for 30 min.
4. 4. A method according to any one of claims 1 to 3, characterized in that the method further comprises the steps of removing impurities, filtering and spray drying after step c).
5. 5. The method of claim 1, wherein the collagenase colG is expressed by a recombinant bacillus subtilis strain capable of producing clostridium histolyticum derived collagenase colG, the coding sequence of collagenase colG is shown in SEQ ID No. 1.
6. 6. The method of claim 5, wherein the strain comprises colG expression vector pP43NMK or pHT01.

Description

Fish collagen peptide with high GPH content, functional food and preparation method thereof by collagenase colG Technical Field The invention relates to a fish collagen peptide with high GPH content, a functional food and a method for preparing the fish collagen peptide with high GPH content by collagenase colG, belonging to the technical field of bioengineering and food processing. Background Collagen is composed of amino acids and its own unique triple helix structure, and this particular structure determines the value and function of protein that it cannot replace. Collagen has many applications, and its uses vary depending on the molecular weight of the desired protein. At present, collagen is widely applied as a small molecular protein, such as a cosmetic additive, a food additive and the like, but the supercoiled structure of the collagen is extremely special, and common protease is difficult to degrade the collagen, so that collagen polypeptide fragments are difficult to obtain, and researches show that bacterial collagenase from microorganism sources can be realized. The sources of the microbial collagenase are mainly bacteria, more and more collagenase-producing strains are separated and screened along with the deep research, the collagenase from different sources has large structure and property differences, and the strains capable of industrially producing the collagenase are few at present. Commercial microbial collagenases are derived from Clostridium histolyticum, and the pathogenicity of the strain limits the use of collagenases in industry, especially in the food industry. The original company BioSpecifics was to purify AUX-I and AUX-II from the fermentation supernatant by culturing Clostridium histolyticum. However, clostridium histolyticum is a pathogenic bacterium of gas gangrene, and various toxins are often mixed in the fermentation supernatant, so that the components are complex, and the food standard requirements are difficult to ensure without the strains of original research companies and the production and purification processes. The bacillus subtilis is non-pathogenic bacteria, does not produce endotoxin in the metabolic process, is acknowledged as a food-grade safe strain, has the advantages of fast growth rate, short culture period, low nutrition requirement, strong stress resistance, low culture cost, high-efficiency protein secretion capacity and the like, and is widely applied to the fields of fermentation, food, agriculture and the like. Therefore, bacillus subtilis is an excellent strain for producing collagenase, and how to industrially produce collagenase by using bacillus subtilis is the subject of study by those skilled in the art. Collagen hydrolysates are well known as dietary supplements for the treatment of skin aging, and studies have shown that oral administration of collagen hydrolysates results in elevated levels of collagen-derived peptides in the blood, particularly collagen-derived small peptides such as Gly-Pro-Hyp (GPH) and Pro-Hyp (PH), which play a role in a variety of physiological functions. Studies have shown that Hyp-containing peptides (i.e., GPH and PH) are better absorbed and reach higher plasma levels when orally administered to rats as compared to high molecular weight collagen peptide (H-CP). However, the collagen tripeptide (GTP) prepared by the conventional enzymolysis method has low Gly-Pro-Hyp (GPH) content and cannot meet the actual application requirements. Disclosure of Invention The invention provides a fish collagen peptide with high GPH content, a functional food and a method for preparing the fish collagen peptide by using collagenase colG, which can effectively solve the problems. The invention provides a method for preparing fish collagen peptide, which comprises the following steps: a) Homogenizing fish scales; b) Subjecting the homogenate to a first step of enzymatic hydrolysis using alkaline protease; c) The homogenate is subjected to a second step of enzymatic hydrolysis using collagenase colG. In some embodiments, the technological parameters of the first step of enzymolysis are that the pH is 8-10, the enzymolysis temperature is 45-55 ℃, the alkaline protease dosage is 0.5-2% of the fish scale mass, and the enzymolysis time is 0.5-3h. In some embodiments, the second step of enzymolysis has the technological parameters of pH 7-8, enzymolysis temperature 35-45 deg.c, collagenase colG in the amount of 1-2.5% of fish scale and enzymolysis time of 4-8 hr. In some embodiments, the step of inactivating the alkaline protease is included after the first step of enzymatic hydrolysis, and the step of inactivating the collagenase colG is included after the second step of enzymatic hydrolysis. In some embodiments, the inactivation parameter is heating to 90℃for 30min to inactivate the enzyme. In some embodiments, the method further comprises the steps of removing impurities, filtering, and spray drying after step c). In some embodiments, t