阅读说明：本技术 一种小黄鱼骨胶原抗菌肽及其应用 (small yellow croaker ossein antibacterial peptide and application thereof ) 是由 叶常青 于 2019-08-26 设计创作，主要内容包括：本发明提供一种小黄鱼骨胶原抗菌肽及其应用,属于生物学技术领域,抗菌肽的氨基酸序列为HECEDCYSGCRNSCQH,分子量为1.87Da,可以抑制食品上的食品腐败微生物的生长,防止或抑制了被该微生物引起的所述食品的腐败变质,为实际无毒级,具有副作用或危险低的优势,安全性高；本发明抗菌肽可用于制备制备食品防腐剂,防止或抑制微生物引起的食品腐败变质；本发明抗菌肽与香豆素的合用具有较强的协同作用,可以增强抗菌活性,降低抗菌肽的使用量。(the invention provides a little yellow croaker ossein antibacterial peptide and application thereof, belonging to the technical field of biology, wherein the amino acid sequence of the antibacterial peptide is HECEDCYSGCRNSCQH, the molecular weight is 1.87Da, the antibacterial peptide can inhibit the growth of food spoilage microorganisms on food, prevent or inhibit the spoilage of the food caused by the microorganisms, is actually non-toxic, has the advantages of low side effect or risk and high safety; the antibacterial peptide can be used for preparing food preservatives to prevent or inhibit food spoilage caused by microorganisms; the combination of the antibacterial peptide and the coumarin has stronger synergistic effect, can enhance the antibacterial activity and reduce the using amount of the antibacterial peptide.)

1. the goldfish ossein antibacterial peptide is characterized in that: the amino acid sequence is shown in SEQ ID NO. 1.

2. the antibacterial peptide of the bone collagen of the small yellow croaker as claimed in claim 1, wherein: the molecular weight of the antibacterial peptide is 1.87 Da.

3. Use of the parvus fulvus collagen antibacterial peptide according to claim 1 or 2 in the preparation of a food preservative.

4. Use according to claim 3, characterized in that: the food preservative prevents or inhibits food spoilage by microorganisms.

5. Use according to claim 4, characterized in that: the microorganism is a bacterium or a fungus.

6. Use according to claim 4, characterized in that: the bacteria are of a food spoilage species selected from the group consisting of pseudomonas or bacillus.

7. use according to claim 4 or 6, characterized in that: the fungus is a food spoilage fungus selected from the following genera: alternaria alternata, aspergillus, fusarium, botrytis cinerea, anthrax and yeast.

8. Use according to claim 3, characterized in that: the food product is derived from, provided by, or is, a fruit, nut, vegetable, seed, sugar, dairy product, meat, fish, or bread.

9. Use according to claim 3, characterized in that: the preservative further comprises coumarin.

10. A method for preventing or inhibiting spoilage of a food product by a microorganism, comprising: comprising administering to a food product in need thereof an effective amount of a preservative as claimed in any one of claims 3 to 9.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a goldfish ossein antibacterial peptide and application thereof.

Background

Collagen is a tissue structural protein in animals and accounts for about 25% of the total protein content of animals. Collagen is the most prominent fibrous protein in the extracellular matrix, is the main fibrous component of connective tissue and interstitial tissue of the body, and is also an important component of the cytoskeleton. Collagen is widely present in skin, tendon, bone, ligament and other parenchymal organs, so that the tissue and organs have certain structure and mechanical properties, and the collagen can protect the organism and support the organs. The collagen polypeptide is an enzymolysis product of collagen, the molecular weight is much lower than that of the collagen, and the peptide chain is broken and can be directly absorbed and utilized by a human body. At present, a large number of studies have shown that collagen polypeptide, a hydrolysate obtained by hydrolyzing animal bones with protease, has various bioactive functions, such as lowering blood pressure, resisting oxidation, preventing and treating arthritis, resisting aging, and the like. The antibacterial peptides (ABP) are a general name of peptides with antibacterial activity, not only have broad-spectrum antibacterial activity, but also have the effects of resisting cancers, viruses, parasites, promoting wound healing and the like, and have great medicinal development value. The antibacterial peptide extracted from natural living organism is mostly composed of 12-100 amino acid residues, and has typical amphiphilic structure with molecular mass of about 4-10 kDa. In recent years, many researchers have studied antibacterial peptides in order to develop the antibacterial peptides into novel food preservatives, antibacterial and anticancer drugs, and the like.

China is a large country for aquatic product production and consumption, and the total quantity of aquatic product production of China is the first world in continuous years according to statistics. However, the aquatic product of China is not equal to the aquatic product strong country, the aquatic product structure of China is unreasonable, and the processing level still stays at the primary stage. Mainly shows that the amount of primary processed products is large, the industrialization degree is low, the amount of deep processed products is small, the proportion of processed products is low, the technical content is low, and novel technology is urgently needed to be improved. At present, the processing rate of aquatic products in China is only far lower than that of developed countries in fishery; and the waste utilization level of the aquatic product processing industry in China is low, and a large amount of aquatic product leftovers generated every year cannot be effectively processed and utilized, so that the method becomes a key point influencing the economic benefit of the aquatic industry. Taking the production and processing of the small yellow croakers as an example, China contains rich small yellow croaker resources, and a large amount of fishbone waste is not effectively utilized in the processing and production process every year. In the face of such huge bone resources, if the bone resources are not utilized or are not utilized reasonably, the problem of environmental pollution is inevitably brought about. The production of the collagen polypeptide is a process of changing waste into valuable, although the raw materials are low, the price of the finished product is very high, the large-batch production can drive the development of aquaculture industry, the income of fishermen is increased, the economic development is greatly promoted, the environmental pollution is reduced, and the method has important significance for sustainable development.

Disclosure of Invention

The invention aims to provide a zonula-source antibacterial peptide additive which has strong antibacterial activity, can inhibit the growth of food spoilage microorganisms on food and prevent or inhibit the food from spoilage.

The technical scheme adopted by the invention for realizing the purpose is as follows:

An antibacterial peptide of bone collagen of little yellow croaker, the amino acid sequence of which is shown in SEQ ID NO. 1. The antimicrobial peptide can inhibit the growth of food spoilage microorganisms on food, and prevent or inhibit food spoilage caused by the microorganisms. And the maximum oral tolerance of the antibacterial peptide to mice is more than 15g/kg, which is an actual nontoxic grade.

Preferably, the antimicrobial peptide has a molecular weight of 1.87 Da. The isoelectric point pI in silico is 5.39.

The invention also aims to provide application of the goldfish ossein antibacterial peptide in preparation of food preservatives.

the little yellow croaker ossein antibacterial peptide has the advantages of low side effect or risk and high safety, and can be decomposed into amino acid by digestive enzyme through oral administration, and further absorbed as nutrient substance. Therefore, the antimicrobial peptide can be used for preparing food preservatives having antifungal activity which are free from problems of occurrence of drug-resistant bacteria or side effects and are extremely high in safety.

preferably, the food preservative prevents or inhibits spoilage of the food by microorganisms.

preferably, the microorganism is a bacterium or a fungus.

Preferably, the bacteria are of a food spoilage species selected from the group consisting of pseudomonas or bacillus.

Preferably, the fungus is a food spoilage species of fungus selected from the following genera: alternaria alternata, aspergillus, fusarium, botrytis cinerea, anthrax and yeast. Further preferably, the fungus is a food spoilage species fungus selected from the genera kluyveromyces or zygosaccharomyces.

Preferably, the food product is derived from, provided with, or is, a fruit, nut, vegetable, seed, sugar, dairy, meat, fish, or bread.

Preferably, the preservative further comprises coumarin. The combination of the antibacterial peptide and the coumarin has stronger synergistic effect, can enhance the antibacterial activity and reduce the using amount of the antibacterial peptide. In addition, the use of this combination may reduce the use of other preservative techniques, such as heating, to preserve the flavor of the food itself; the use of this combination may also reduce the use of other chemical preservatives, thereby reducing their side effects.

Another object of the present invention is to provide a method for preventing or inhibiting food spoilage by microorganisms, the method comprising: comprising administering to a food product in need thereof an effective amount of the above-described preservative.

Compared with the prior art, the invention has the beneficial effects that: the amino acid sequence of the antibacterial peptide is HECEDCYSGCRNSCQH, so that the antibacterial peptide can inhibit the growth of food spoilage microorganisms on food, prevent or inhibit the food spoilage caused by the microorganisms, is actually nontoxic, has the advantages of low side effect or risk and high safety; the antibacterial peptide can be used for preparing food preservatives to prevent or inhibit food spoilage caused by microorganisms; the combination of the antibacterial peptide and the coumarin has stronger synergistic effect, can enhance the antibacterial activity and reduce the using amount of the antibacterial peptide.

The invention adopts the technical scheme to provide the goldfish ossein antibacterial peptide and the application thereof, overcomes the defects of the prior art, and has reasonable design and convenient operation.

Drawings

FIG. 1 is a gel chromatography in example 1 of the present invention;

FIG. 2 shows the bacteriostatic effect of each component peak in the gel chromatogram in example 1 of the present invention;

FIG. 3 is an ion exchange chromatogram of example 1 of the present invention;

FIG. 4 shows the bacteriostatic effect of each component peak in the ion exchange chromatogram in example 1 of the present invention;

FIG. 5 is a reversed-phase high performance liquid chromatogram in example 1 of the present invention;

FIG. 6 shows the bacteriostatic effects of antibacterial peptides and coumarin in test example 2 of the present invention.

Detailed Description

The preservation of food by the polypeptide of the invention can occur during the storage, transportation, handling, processing or display of the food.

the following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

one embodiment of the invention is a yellow croaker ossein antibacterial peptide, the amino acid sequence of which is shown in SEQ ID NO. 1. The antimicrobial peptide can inhibit the growth of food spoilage microorganisms on food, and prevent or inhibit spoilage of said food by said microorganisms.

In one embodiment, the antimicrobial peptide has a molecular weight of 1.87 Da. The isoelectric point pI in silico is 5.39.

One embodiment of the invention is a method for preparing a yellow croaker ossein antibacterial peptide, which comprises the following steps:

Step 1: trimming, cleaning and crushing the small yellow fish bones into fish bone particles with the diameter of 1-20 mm;

step 2: adding phosphate buffer solution (containing vanillin 0.01-0.05 μ M) with pH of 8.0-9.0 into fishbone particles at solid-to-liquid ratio of 1:1-2g/mL, grinding with tissue homogenizer, and steaming in a sealed container at 40-60 deg.C for 15-30 min; in the processing process of aquatic products, the heating degree of the raw materials in the high-temperature cooking process can seriously affect the quality of a final product, but the cooking temperature and time are not easy to control, so that the protein denaturation is insufficient or excessive, and the enzymolysis effect is affected. The step can change the structure of the bone collagen of the little yellow croaker at a lower cooking temperature, so that more enzyme cutting sites are exposed out of collagen molecules, the combination of protease and protein is facilitated, and the antibacterial peptide with the amino acid sequence of HECEDCYSGCRNSCQH is obtained, and the enzymolysis reaction rate and the yield of the antibacterial peptide can be increased by the treatment of the step;

Step 2: adding alkaline protease and flavourzyme into the zonula fish obtained in the step 1, wherein the enzyme base ratio of the alkaline protease is 400-600U/g, the enzyme base ratio of the flavourzyme is 300-500U/g, carrying out enzymolysis for 4-6h at the temperature of 50-60 ℃, and then inactivating the enzyme to obtain an enzymolysis solution;

And step 3: and (3) adding the enzymolysis liquid obtained in the step (2) into a centrifugal ultrafiltration tube with the molecular weight cutoff of 3kDa, centrifuging for 20-30min at the rotating speed of 3000-5000r/min to obtain a part of crude product with the molecular weight cutoff of less than 3kDa, and then purifying by adopting gel chromatography separation, ion exchange chromatography separation and reversed-phase high performance liquid chromatography to obtain the antibacterial peptide additive.

One embodiment of the invention is application of the goldfish ossein antibacterial peptide in preparation of food preservatives.

Food spoilage by microorganisms refers to any change in the food, e.g., taste, odor, or appearance (e.g., shape, color, texture, hardness), by a microorganism, thereby reducing its nutritional and/or commercial value. The little yellow croaker ossein antibacterial peptide has the advantages of low side effect or risk and high safety, and can be decomposed into amino acid by digestive enzyme through oral administration, and further absorbed as nutrient substance. Therefore, the antimicrobial peptide can be used for preparing food preservatives having antifungal activity which are free from problems of occurrence of drug-resistant bacteria or side effects and are extremely high in safety.

in one embodiment, the concentration of antimicrobial peptide in the preservative is 0.01% to 2% by mass.

in one embodiment, the food preservative prevents or inhibits microbial spoilage of food.

in one embodiment, the microorganism is a bacterium or a fungus.

in one embodiment, the bacteria are of a food spoilage species selected from pseudomonas or bacillus.

In one embodiment, the fungus is a food spoilage species fungus selected from the following genera: alternaria alternata, aspergillus, fusarium, botrytis cinerea, anthrax and yeast. Further preferably, the fungus is a food spoilage species fungus selected from the genera kluyveromyces or zygosaccharomyces.

In one embodiment, the food product is derived from, provided to, or is, a fruit, nut, vegetable, seed, sugar, dairy, meat, fish, or bread. Fruit-derived food products include fruit wines and juices. Plants providing seeds include cereals (e.g., corn, wheat, barley, sorghum, millet, rice, oats, and rye) and legumes (e.g., soybeans, peas, and lentils). The sugar, preferably sucrose, may be derived from sugar beet or sugar cane. Dairy products include milk, cream, cheese and yoghurt. Liquid or pasty foods include soups, sauces, pickles, mayonnaises, salad dressings and other salad dressings, preserves, syrups and baby foods. The meat and/or fish food product may be treated or untreated and may be cooked or uncooked.

In one embodiment, the preservative further comprises coumarin. Coumarins are lactones formed by dehydration of cis-o-hydroxycinnamic acid, and have biological activities such as anti-tumor, anti-virus, anti-oxidation, antimicrobial, anticoagulant and the like, and are generally classified into 4 types: simple coumarins (such as daphnelactone, osthole, etc.), furocoumarins (such as xanthotoxol, decursinol, etc.), pyranocoumarins (such as decursin, decursinol, etc.), and other coumarins (such as swollenin A, coumestrol, etc.). The combination of the antibacterial peptide and the coumarin has stronger synergistic effect, can enhance the antibacterial activity and reduce the using amount of the antibacterial peptide. In addition, the use of this combination may reduce the use of other preservative techniques, such as heating, to preserve the flavor of the food itself; the use of this combination may also reduce the use of other chemical preservatives, thereby reducing their side effects.

In one embodiment, the molar ratio of antimicrobial peptide to coumarin is from 1:0.05 to 3. The antibacterial peptide and the coumarin in the molar ratio range have the strongest synergistic effect.

One embodiment of the present invention is a method of preventing or inhibiting spoilage of a food product by a microorganism, characterized by: comprising administering to a food product in need thereof an effective amount of the above-described preservative.

The invention is further illustrated by the following examples. It is to be understood that the examples are for illustrative purposes only and are not intended to limit the scope and spirit of the present invention.