阅读说明：本技术 一种重组抗菌肽Mytichitin-A的应用 (Application of recombinant antibacterial peptide Myticitin-A ) 是由 樊振川 孙雪晴 孟德梅 高晓芳 于 2019-12-06 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种重组Mytichitin-A抗菌肽(rMytichitin-A的应用。本发明使用的是由毕赤酵母GS115高效表达的rMytichitin-A抗菌肽,具有广谱抗菌活性。所得抗菌肽具有良好的温度稳定性,耐强酸和弱碱,相对的蛋白酶稳定性；对大菱鲆和SD大鼠的红细胞具有极低的溶血性。以上特点使得rMytichitin-A抗菌肽具备在食品、畜牧和医药等领域应用的潜力。同时rMytichitin-A抗菌肽还可以广泛应用于生鲜的防腐保鲜,抑制微生物的生长,降低营养物质的流失。(The invention belongs to the technical field of biology, and particularly relates to recombinant Myticitin-A (application of the rMyticitin-A), wherein the rMyticitin-A is efficiently expressed by pichia pastoris GS115 and has broad-spectrum antibacterial activity.)

1. A recombinant antibacterial peptide Myticitin-A (rMyticitin-A) with an amino acid sequence shown in SEQ ID NO: 1 is shown.

2. The recombinant antibacterial peptide Myticitin-A of claim 1, which has a nucleotide sequence shown in SEQ ID NO: 2, respectively.

3. The preparation method according to claim 1 or 2, comprising the steps of:

fermenting and culturing the expression strain of the rMytichin-A antibacterial peptide, and performing induced expression on the expression strain by taking methanol as a unique carbon source to obtain the rMytichin-A antibacterial peptide.

4. The method for preparing the antibacterial peptide according to claim 3, wherein the expression strain is a Pichia host cell (Pichia GS 115).

5. The method for preparing the antibacterial peptide according to claim 3, wherein the method for fermentation culture comprises: culturing rMyticitin-A antibacterial peptide expression strain in BMGY culture medium at 28 deg.C and 220rpm, and shaking to OD₆₀₀When 6-8 hours are reached, the BMGY medium is replaced with BMMY medium in equal volume, shaking at 28 ℃ and 220rpm, and the BMGY medium is continuously cultured for 144 hours after every 24 hours with addition of filter-sterilized 100% methanol.

6. The method of claim 5, wherein the methanol make-up is 2% by volume.

7. The rMyticitin-A antibacterial peptide of claim 1 or 2 for use in preservation and freshness.

The technical field is as follows:

the invention belongs to the technical field of biology, and particularly relates to a recombinant Myticitin-A antibacterial peptide (rMyticitin-A) and application thereof in preservation and preservation.

Background art:

antimicrobial peptides (small polypeptides), are important components of the innate immune system in invertebrates and vertebrates. The antibacterial peptide has the characteristics of broad-spectrum antibacterial activity, small molecular weight and the like, and also has good stability, acid-base resistance and temperature change resistance, is not influenced by various proteases, and is not easy to generate drug resistance. The antibacterial peptide has stable structure and various functions. The antibacterial peptide has the effects of resisting virus, treating AIDS, inhibiting the growth of tumor cells, promoting wound healing, diminishing inflammation, sterilizing and the like, also has antioxidant activity, can effectively remove oxygen free radicals, and has great application potential. However, natural extraction has high cost and low yield, and is not suitable for industrial production. In order to meet the needs of industrial production, the antibacterial peptide gene is integrated into a pichia pastoris cell by using a genetic engineering method, exogenous protein is induced and expressed, and the production cost is reduced.

The natural antibacterial peptide Myticitin-A is obtained by separation and identification from the serum of Mytilus coruscus, consists of 55 amino acids, has the molecular weight of 6.2KDa, and can inhibit the growth of gram-negative bacteria, gram-positive bacteria and fungi, and the laboratory has published documents which show that the recombinant antibacterial peptide Myticitin-A (rMyticitin-A) is successfully transferred into Pichia pastoris GS115 by using a genetic engineering method, methanol is used as a unique carbon source, the antibacterial peptide can be induced and expressed, the yield can reach 45.5 mu g/m L, and the antibacterial activity is realized on various pathogenic bacteria.

To date, various types of antimicrobial peptides have been used in food preservation research. Due to the high-efficiency antibacterial activity, the growth of microorganisms can be effectively inhibited, the degradation of protein and the oxidation of lipid are reduced, and the sensory quality of food is kept. The antibacterial peptide can be widely applied to the fresh keeping of beverages, fruits and vegetables, dairy products, fresh meat and meat products.

The invention applies the recombinant antibacterial peptide Myticitin-A produced by the pichia pastoris GS115 to the fresh keeping of the cold fresh pork, and can effectively prolong the quality guarantee period. The invention has not been published and reported at home and abroad after retrieval, and has not been published and used at home and abroad.

The invention content is as follows:

the invention aims to provide an application of recombinant antibacterial peptide Myticitin-A in preservation and freshness, in particular an application in preservation and freshness of cold fresh meat products.

The amino acid sequence of the rMyticitin-A is shown in a sequence table SEQ ID NO: 1 is shown in the specification; the nucleotide sequence is shown as SEQ ID NO: 2, respectively.

Furthermore, the rMytichitin-A preservative solution is an rMytichitin-A aqueous solution with the concentration of 5 to 15 and 25 mu g/m L, and the shelf life of the rMytichitin-A preservative solution can be prolonged by 1 to 2 days compared with that of untreated cold fresh meat.

The preparation method comprises the following steps:

1. expression of rMyticitin-A antibacterial peptide

Carrying out fermentation culture on an expression strain of the rMytichin-A antibacterial peptide, and carrying out induction expression on the expression strain by taking methanol as a unique carbon source to obtain a rMytichin-A antibacterial peptide product;

further, the fermentation culture method comprises selecting rMyticitin-A antibacterial peptide expression strain in 1L triangular flask, shake-culturing at 28 deg.C and 220rpm in 150m L BMGY culture medium, and allowing OD to grow₆₀₀When the volume reaches 6-8, replacing BMGY medium with BMMY medium in the same volume, performing shake culture at 28 ℃ and 220rpm, supplementing 100% methanol subjected to filtration sterilization every 24h, and continuously culturing for 144 h;

furthermore, the methanol supplementation amount accounts for 2 percent of the volume of the fermentation liquor each time, the induction time is 144h, and the content of the target protein in the supernatant is determined to reach 133 mug/m L by the method of E L ISA;

2. the rMyticitin-A antibacterial peptide product has the following characteristics:

(1) the temperature stability is that the rMyticitin-A antibacterial peptide is treated for 1h at the conditions of 4 ℃, 25 ℃, 37 ℃, 60 ℃, 80 ℃ and 90 ℃ respectively and then is used for MIC experiments, the concentration of the rMyticitin-A is 10 mu g/m L, and the inhibition rates of the rMyticitin-A to staphylococcus aureus have no significant difference and are all more than 90 percent.

(2) The pH stability is that the rMyticitin-A antibacterial peptide is treated by buffer solutions with the pH values of 2, 4, 6, 8 and 10 respectively at 37 ℃ for 4h and then subjected to MIC experiments, the concentration of the rMyticitin-A is 10 mu g/m L, the inhibition rates of the rMyticitin-A antibacterial peptide on staphylococcus aureus have no significant difference and are all larger than 90 percent after the rMyticitin-A antibacterial peptide is treated by solutions with the pH values of 2, 4, 6 and 8, and the inhibition rate on staphylococcus aureus is 33 percent after the rMyticitin-A antibacterial peptide is treated by the solution with the pH value of 10.

(3) The protease stability is that pepsin, papain, proteinase K and trypsin are respectively adopted, the rMyticitin-A antibacterial peptide is treated at 37 ℃ for 2h and then is used for MIC experiments, the concentration of rMyticitin-A is 10 mu g/m L, the inhibition rates of the rMyticitin-A antibacterial peptide on staphylococcus aureus after being treated by the pepsin, the papain and the trypsin are not obviously different and are all more than 90%, and the inhibition rate after being treated by the proteinase K is 60%.

In conclusion, the antibacterial peptide rMyticitin-A used in the invention has good temperature stability and relative protease stability, and can resist strong acid and weak base.

(4) Hemolysis, namely, performing centrifugal measurement on the absorbance value of supernatant after 30min of action of rMytichitin-A antibacterial peptide with different concentrations of 10 mu L and red blood cells with 4% concentration of 90 mu L at 37 ℃, wherein the concentrations of the rMytichitin-A antibacterial peptide are 10, 20, 30, 40, 50 and 60 mu g/m L, and hemolysis does not occur on red blood cells of turbot and SD rats in the concentration range of 60 mu g/m L.

(5) The preservative and fresh-keeping effect is that fresh pork is respectively treated by 5, 15 and 25 mu g/m L rMytichitin-A, stored for 7 days at 4 ℃, the colony count and rancidity degree of the pork are detected every 24h in the period, and sensory evaluation is carried out, when the concentration of the rMytichitin-A is 25 mu g/m L, the preservative and fresh-keeping effect is relatively optimal, the colony count is lowest, the oxidative degradation degree of fat and protein is lowest, the loss of nutrient components can be inhibited, and good sensory morphology is maintained.

Has the advantages that:

(1) the rMyticitin-A antibacterial peptide used in the invention has good temperature stability and relative protease stability, and can tolerate strong acid, weak base and extremely low cell hemolysis. Therefore, the rMyticitin-A antibacterial peptide has better application prospect in the fields of food, animal husbandry, clinical medical treatment and the like.

(2) The pichia pastoris expression system used in the invention can efficiently express foreign protein, the content of rMyticitin-A antibacterial peptide in the supernatant of fermentation liquor is as high as 133 mu g/m L, and the method is favorable for future industrial production of the antibacterial peptide.

(3) The storage and preservation experimental data in the invention show that the rMyticitin-A has good preservative and preservation effects, can be widely applied to preservative and preservation of fresh pork under low-temperature storage, inhibits propagation of microorganisms, reduces loss of nutrient substances, and maintains good sensory morphology and nutritional quality of pork. Therefore, the rMyticitin-A has great potential to become a novel natural food preservative.

Description of the drawings:

FIG. 1 is a schematic diagram of the present invention;

FIG. 2 is a study of the properties of rMyticitin-A antibacterial peptide;

wherein, A: temperature stability study of rMyticitin-A;

b: rMyticitin-A pH stability study;

c: rMyticitin-A protease stability study;

FIG. 3 is a study of the bactericidal kinetics of rMyticitin-A;

FIG. 4 is a study of the hemolysis of rMyticitin-A;

wherein, A: research on hemolysis of turbot erythrocytes by rMyticitin-A, B: hemolytic study of rMyticitin-A on SD rat erythrocytes; p: 1% Triton X-100 as positive control; n: PBS was used as negative control;

FIG. 5 is the physicochemical index measurement of the preservative and fresh-keeping effect of rMyticitin-A on pork;

wherein A is the total number of colonies; b is a TBARS value; c is a TVB-N value; d is the juice loss rate; e is the pH value;

the specific implementation mode is as follows:

the invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.