阅读说明：本技术 一种梅鱼加工副产品的抗真菌肽的制备方法 (Preparation method of antifungal peptide of meiyu processing byproduct ) 是由 陈美龄 胡嘉惠 于 2019-10-14 设计创作，主要内容包括：本发明涉及生物技术提取领域,针对现有技术的抗真菌肽抗菌活性较低及抑菌种类有限的问题,公开了一种梅鱼加工副产品的抗真菌肽制备方法,包括以下步骤：(1)、预处理；(2)、酶解；(3)、过滤得澄清液；(4)、分离纯化；(5)、多肽分级；(6)、培养观察抑菌圈。本发明提供了一种梅鱼加工副产品的抗真菌肽制备方法,该抗真菌肽具有较高的抗菌活性,且是初次从梅鱼中提取抗真菌肽；该抗真菌肽是从梅鱼的加工副产品鱼头中进行提取,实现废物再利用,成本较低；丰富了抗真菌肽的种类,能更好的对食品进行保质保鲜,助于了解利用梅鱼副产物蛋白质水解后的抗真菌肽保存食物、防止腐败和病原微生物。(The invention relates to the field of biotechnological extraction, and discloses a preparation method of antifungal peptide of a meiyu processing byproduct, aiming at the problems of low antibacterial activity and limited antibacterial types of antifungal peptide in the prior art, wherein the preparation method comprises the following steps: (1) and (4) preprocessing; (2) carrying out enzymolysis; (3) filtering to obtain clear liquid; (4) separating and purifying; (5) classifying the polypeptide; (6) and culturing and observing the bacteriostatic zone. The invention provides a preparation method of antifungal peptide of a meiyu processing byproduct, wherein the antifungal peptide has higher antibacterial activity and is extracted from the meiyu for the first time; the antifungal peptide is extracted from the fish head which is a processing byproduct of the mermaid, so that the waste is recycled, and the cost is low; the variety of the antifungal peptide is enriched, the food can be better preserved and fresh, the understanding of food preservation by the antifungal peptide after the hydrolysis of the meiyu byproduct protein is facilitated, and the putrefaction and pathogenic microorganisms are prevented.)

1. A preparation method of antifungal peptide of a meiyu processing byproduct is characterized by comprising the following preparation steps:

(1) and (3) pretreatment: cleaning and mincing the heads of the mermaid, and degreasing the heads of the mermaid in a 94-96% methanol water bath for 2-2.2h at the temperature of 48-52 ℃ and at the material-liquid ratio of 1:6-9 (m/v); after degreasing, pouring out the supernatant, cleaning the precipitate with pure water, centrifuging the degreased fish meat for 20-25min at the temperature of 0-4 ℃ and the temperature of 11900 and 12000 r/min, and collecting the precipitate;

(2) and enzymolysis: adding fish meat into enzymolysis liquid containing neutral protease for enzymolysis; wherein the concentration of the neutral protease is 0.05-0.25wt%, the mass of the enzymolysis liquid is 2-4 times of the mass of the fish, the enzymolysis temperature is 30-45 ℃, the pH value of the enzymolysis liquid is controlled to be 5-6, and the enzymolysis time is 5-5.4 h;

(3) filtering to obtain a clear solution: adding modified fruit tree activated carbon with the mass of 1-2wt% of the enzymolysis liquid into the enzymolysis liquid, and heating and decoloring the enzymolysis liquid; transferring the decolorized enzymatic hydrolysate to a plate-and-frame filter tank for filtering, and taking a clear solution;

(4) and separation and purification: bubbling the clarified liquid by air, collecting the foam liquid, and injecting water while bubbling; after the collected foam liquid is extinguished, carrying out heavy metal separation on the foam liquid; adding magnetic affinity microspheres with the mass of 1-2% of the foam liquid into the foam liquid after heavy metal separation, uniformly dispersing, standing for affinity adsorption for 50-60min, and then utilizing a magnetic field to aggregate the magnetic affinity microspheres; after separating out the magnetic affinity microspheres, eluting the magnetic affinity microspheres by using a buffer solution with the pH value of 7-7.2 to obtain an eluent, adjusting the pH value of the eluent to 6-8, extracting by using ethyl acetate, and evaporating to dryness and concentrating at 35-40 ℃ to ensure that the concentration of a concentrated solution is 25-30%;

(5) and polypeptide grading: separating the concentrated solution by using a membrane according to the molecular weight to obtain antifungal peptide with the target molecular weight;

(6) culturing and observing an antibacterial zone: the antifungal activity of the polypeptide is screened by an agar diffusion method, the peptide substance is added into a culture medium containing an Aspergillus niger standard strain, a bacteriostasis area is observed, and the bacteriostasis diameter is measured.

2. The method for preparing antifungal peptide from the prune fish processing byproduct of claim 1, wherein the modified fruit tree activated carbon of step (3) is prepared by the following steps:

for the fruit shell activated carbon, 0.5-0.8mol/L dilute sulfuric acid is used for soaking the fruit tree activated carbon for 1.8-2.2h at 60-65 ℃, a sample is filtered, 0.8-1mol/L hydrochloric acid is slowly added, the fruit tree activated carbon is soaked for 8-10h at 60-70 ℃, then the fruit tree activated carbon is washed to be neutral by deionized water, deionized water is added, the mixture is boiled for 1.8-2.2h at 98-100 ℃, then the mixture is washed for 2-3 times by the pure water and then is placed in an oven at 98-102 ℃ for drying; weighing the pretreated fruit shell activated carbon, respectively soaking the fruit shell activated carbon in 15-18% ammonia water, magnetically stirring for 1.8-2.2h, washing the fruit shell activated carbon with deionized water until the pH value reaches 6.8-7, and drying at 98-102 ℃ for 22-24 h to obtain the modified fruit shell activated carbon.

3. The method for preparing antifungal peptide derived from a byproduct of mermaid processing according to claim 1, wherein the air blowing rate in the step (4) is 3 to 4L/min, the water injection rate is 0.2 to 0.3L/min, and the temperature of the supernatant is maintained at 25 to 35 ℃.

4. The method for preparing antifungal peptide derived from a byproduct of mermaid processing according to claim 1, wherein the heating temperature in the step (3) is 70 to 80 ℃ and the decoloring time is 30 to 40 min.

5. The method for preparing the antifungal peptide derived from the byproducts of Mylopharyngodon piceus as claimed in claim 1, wherein the target molecular weight of the antifungal peptide obtained by fractionation in step (5) is 1200-3800 Da.

6. The method of claim 1, wherein the antifungal peptide is extracted from the byproduct of meiyu processing.

7. The method for preparing the antifungal peptide derived from the byproduct of mermaid processing according to claim 1, wherein the membrane of the step (5) is an ultrafiltration membrane or a nanofiltration membrane having a pore size of 10 to 50 nm.

8. The method for preparing antifungal peptide derived from a meiyu byproduct of claim 1, wherein the culturing in step (6) is performed at 36-38 ℃ for 70-72 hours.

Technical Field

The invention relates to the field of biotechnological extraction, and particularly relates to a preparation method of antifungal peptide of a meiyu processing byproduct.

Background

Food safety is an important problem in the field of public health, with the continuous improvement of the living standard of residents in China, the attention of people to the quality and safety of food is higher and higher, the food spoilage and food poisoning caused by microorganisms are the primary problems of food safety, particularly, foods with high sugar content and starch content are easily polluted by fungi, and toxic metabolites, namely mycotoxin, can be produced under certain conditions.

The antibacterial peptide is a small molecular compound, usually has 10-50 amino acids, has a very wide source, and is found in insects, mammals, amphibians, bacteria, fungi and plants; the long-term survival adaptation of marine animals living in water environment rich in various microorganisms enables the marine animals to form an effective defense function, and the antibacterial peptide is considered to be one of main components of defense systems of fishes, shrimps, shells and the like; these peptides can be produced in vitro by enzymatic or chemical hydrolysis, and in the food industry antimicrobial peptides play a positive role, which can inhibit the growth of pathogenic bacteria and fungi in food, and are a sub-group of antimicrobial peptides.

The mermaid is a small-sized marine fish with important economic value, and the mermaid is often used for preparing canned food products and generates a large amount of antifungal peptide in the processing process.

The patent number CN200910175405.8, the patent name is "an antifungal peptide and a preparation method and application thereof", the invention provides an antifungal peptide which is a polypeptide purified from a water-soluble extract of tartary buckwheat seeds and has the molecular weight of 3.909 kDa. The preparation method of the antifungal peptide comprises the steps of crushing tartary buckwheat seeds, degreasing, extracting by using buffer solution, extracting to obtain crude protein, and then carrying out heat treatment and protein chromatographic separation, namely Resource S cation exchange and Superdex peptide gel exclusion chromatography purification to obtain the antifungal peptide. The antifungal peptide has obvious inhibiting effect on the growth of white rot fungi, trichoderma viride and alternaria.

The antifungal peptide has the defects that the antifungal peptide is a plant extract, has low antibacterial activity and only has certain inhibition effect on specific strains.

Disclosure of Invention

The invention aims to overcome the problems of low antibacterial activity and limited antibacterial types of antifungal peptides in the prior art, and provides a preparation method of antifungal peptides of meiyu processing byproducts.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of antifungal peptide of a meiyu processing byproduct comprises the following preparation steps:

(1) and (3) pretreatment: cleaning and mincing the heads of the mermaid, and degreasing the heads of the mermaid in a 94-96% methanol water bath for 2-2.2h at the temperature of 48-52 ℃ and at the material-liquid ratio of 1:6-9 (m/v); after degreasing, pouring out the supernatant, cleaning the precipitate with pure water, centrifuging the degreased fish meat for 20-25min at the temperature of 0-4 ℃ and the temperature of 11900 and 12000 r/min, and collecting the precipitate;

(2) and enzymolysis: adding fish meat into enzymolysis liquid containing neutral protease for enzymolysis; wherein the concentration of the neutral protease is 0.05-0.25wt%, the mass of the enzymolysis liquid is 2-4 times of the mass of the fish, the enzymolysis temperature is 30-45 ℃, the pH value of the enzymolysis liquid is controlled to be 5-6, and the enzymolysis time is 5-5.4 h;

(3) filtering to obtain a clear solution: adding modified fruit tree activated carbon with the mass of 1-2wt% of the enzymolysis liquid into the enzymolysis liquid, and heating and decoloring the enzymolysis liquid; transferring the decolorized enzymatic hydrolysate to a plate-and-frame filter tank for filtering, and taking a clear solution;

(4) and separation and purification: bubbling the clarified liquid by air, collecting the foam liquid, and injecting water while bubbling; after the collected foam liquid is extinguished, carrying out heavy metal separation on the foam liquid; adding magnetic affinity microspheres with the mass of 1-2% of the foam liquid into the foam liquid after heavy metal separation, uniformly dispersing, standing for affinity adsorption for 50-60min, and then utilizing a magnetic field to aggregate the magnetic affinity microspheres; after separating out the magnetic affinity microspheres, eluting the magnetic affinity microspheres by using a buffer solution with the pH value of 7-7.2 to obtain an eluent, adjusting the pH value of the eluent to 6-8, extracting by using ethyl acetate, and evaporating to dryness and concentrating at 35-40 ℃ to ensure that the concentration of a concentrated solution is 25-30%;

(5) and polypeptide grading: polypeptide grading: separating the concentrated solution by using a membrane according to the molecular weight to obtain antifungal peptide with the target molecular weight;

(6) culturing and observing an antibacterial zone: the antifungal activity of the polypeptide is screened by an agar diffusion method, the peptide substance is added into a culture medium containing an Aspergillus niger standard strain, a bacteriostasis area is observed, and the bacteriostasis diameter is measured.

The antifungal peptide is extracted from the mermaid for the first time, the extraction source of the antifungal peptide is expanded, the types of the antifungal peptide are enriched, the food preservation is remarkably promoted, the antifungal peptide with high purity is extracted through multiple times of separation and purification, and the antifungal peptide with optimal activity is obtained through multiple times of separation and screening.

The mechanism of action of antifungal peptides on fungi is generally the action of antifungal peptides on the fungal cell wall, cell membrane, and organelles within the fungus. The cell wall of the fungus consists of chitin, glucan and lipid, the peptide is inserted into the protein matrix, the cell wall of the fungus is obviously subjected to internal pressure, the cell wall is damaged, and the fungus is cracked; the peptides penetrate through cell membranes and are specifically combined with targets in the cells to inhibit the synthesis of DNA, RNA and protein, so that the integrity of the cell membranes is damaged, and the aim of resisting bacteria is fulfilled.

Preferably, the preparation method of the modified fruit tree activated carbon in the step (3) comprises the following steps:

for the fruit shell activated carbon, 0.5-0.8mol/L dilute sulfuric acid is used for soaking the fruit tree activated carbon for 1.8-2.2h at 60-65 ℃, a sample is filtered, 0.8-1mol/L hydrochloric acid is slowly added, the fruit tree activated carbon is soaked for 8-10h at 60-70 ℃, then the fruit tree activated carbon is washed to be neutral by deionized water, deionized water is added, the mixture is boiled for 1.8-2.2h at 98-100 ℃, then the mixture is washed for 2-3 times by the pure water and then is placed in an oven at 98-102 ℃ for drying; weighing the pretreated fruit shell activated carbon, respectively soaking the fruit shell activated carbon in 15-18% ammonia water, magnetically stirring for 1.8-2.2h, washing the fruit shell activated carbon with deionized water until the pH value reaches 6.8-7, and drying at 98-102 ℃ for 22-24 h to obtain the modified fruit shell activated carbon.

The nutshell activated carbon is selected because the nutshell activated carbon has low polarity, so that the nutshell activated carbon is prevented from adsorbing macromolecules such as antifungal peptide, small holes and gullies on the surface of the nutshell activated carbon can be increased after modification, the adsorption capacity of the activated carbon is increased, pigments can be adsorbed, and the adsorption capacity of the activated carbon reaches a balance value after modification treatment, so that the pigments can be fully removed, and the antifungal peptide in the solution can not be absorbed.

Preferably, the air blowing rate of the step (4) is 3-4L/min, the water injection rate is 0.2-0.3L/min, and the temperature of the clear liquid is kept at 25-35 ℃.

Preferably, the heating temperature in the step (3) is 70-80 ℃, and the decoloring time is 30-40 min.

Preferably, the antifungal peptide obtained by the fractionation in the step (5) has a target molecular weight of 1200-3800 Da.

Preferably, the antifungal peptide is extracted from a byproduct of processing of mermaid.

Can realize making the best use of things, fully use the abandoned fish head again, have great promotion effect on material object bacteriostasis and corrosion prevention, and reduce the body damage caused by adding preservatives and the like.

Preferably, the membrane in the step (5) is an ultrafiltration membrane or a nanofiltration membrane with the pore diameter of 10-50 nm.

Preferably, the culture conditions in step (6) are 36-38 ℃ for 70-72 h.

Therefore, the invention has the following beneficial effects:

(1) the preparation method of the antifungal peptide of the meiyu processing byproduct is provided, the antifungal peptide has higher antibacterial activity, and the antifungal peptide is extracted from the meiyu for the first time;

(2) the antifungal peptide is extracted from the fish head which is a processing byproduct of the mermaid, so that the waste is recycled, and the cost is low;

(3) the variety of the antifungal peptide is enriched, the food can be better preserved and fresh, the understanding of the food preservation by the antifungal peptide after the hydrolysis of the meiyu byproduct protein is facilitated, and the putrefaction and pathogenic microorganisms are prevented;

(3) the preparation process has good controllability, is easy for actual large-scale industrial mass production, and the prepared product has high yield, high purity and good activity.

Drawings

FIG. 1 is a graph of the inhibition circle of the neutral protease hydrolysis to obtain the antimicrobial peptide on A.niger.

FIG. 2 is a graph of the inhibition circle of the peptide obtained by pepsin hydrolysis on Aspergillus niger.

Detailed Description

The invention is further described with reference to specific embodiments.