KR102649548B1 - A antimicrobial peptide and a antimicrobial composition containing the same - Google Patents

Abstract

The present invention provides a peptide with antibacterial activity and an antibacterial composition containing the same as an active ingredient, specifically, Bacillus subtilis , Staphylococcus aureus , or Salmonella sp., which are harmful microorganisms and cause food poisoning. A peptide with novel antibacterial activity that is excellent in antibacterial activity and maintains antibacterial activity in a temperature and salt concentration range that can be changed during the storage and processing of marine products, an antibacterial composition containing the peptide as an active ingredient, and an effective peptide using the peptide. It relates to an antibacterial pharmaceutical composition containing the peptide as an ingredient or an antibacterial food additive containing the peptide as an active ingredient.

Description

Peptide having antibacterial activity and antibacterial composition containing the same as an active ingredient {A ANTIMICROBIAL PEPTIDE AND A ANTIMICROBIAL COMPOSITION CONTAINING THE SAME}

The present invention relates to a peptide having antibacterial activity and an antibacterial composition containing the same as an active ingredient, specifically, a novel antibacterial activity produced by a novel Halorubrum aethiopicum SAH-A6T strain (Halorubrum aethiopicum SAH-A6T), a halophilic strain. It relates to a peptide having and an antibacterial composition containing it as an active ingredient.

All living things on Earth have biological defense mechanisms that can defend or protect themselves from microorganisms such as bacteria, fungi, and viruses. One of them is a non-selective immune system using antibacterial substances such as antibacterial peptides or antibiotics. specific immunity).

The antibiotic is a substance produced by microorganisms that kills or inhibits the growth of other microorganisms. The problem of antibiotic resistance has continued since the time humans first began using antibiotics, and currently, the pace of development of new antibiotics is decreasing. We are unable to keep up with the pace of emergence of resistant bacteria. It is reported that it takes an average of 5 to 10 years for a new antibiotic to be developed and hit the market, and that 400 to 800 million dollars is spent on research and development. In addition, existing antibiotics simultaneously inhibit enzymes that regulate the host's metabolic and biosynthetic pathways, so they often cause side effects and toxicity to the human body or the administered target, and their use is limited due to resistance issues. Therefore, there is an urgent need to develop new infection treatments that can effectively control infectious diseases in the early stages, and antimicrobial peptides (AMPs) are attracting attention as a way to solve this problem.

The antimicrobial peptide (AMP) can play an important role in protecting organisms from attack by external microorganisms. In addition, antibacterial peptides actually exhibit stronger antibacterial activity against a wider range of microorganisms than existing antibiotics, and exhibit antibacterial activity through a different activation mechanism from existing antibiotics that cause antibiotic resistance, making them a new product with excellent antibacterial activity. Research on antibacterial peptides is gaining momentum.

In addition, the antibacterial peptide is harmless to the human body and has no residual properties. Unlike existing antibiotics, which are secondary metabolites, the antibacterial peptide is directly biosynthesized from a plasmid or chromosome, making it suitable for biotechnological applications through genetic manipulation. It has the advantage of being easy. In addition, since the antibacterial peptide is a protein, it is decomposed by proteolytic enzymes in the digestive system as soon as it is ingested into the human body, and does not cause problems such as side effects in the body, so it can be used as a new biopreservative for foods or fermented foods. It is expected to be very useful as a bioregulator.

Among the above antibacterial substances, antibacterial peptides, which are natural antibacterial proteins or protein-based substances produced by various types of microorganisms, include bacteriocin, halocin, and lantibiotics.

Among these, halocin is a bacteriocin-like substance derived from halophiles. There are a total of 15 halocin types (C8, H4, H1, H6, S8, SH10, A4, R1) known to date. , Sech7a, H17, KPS1, H2, H3, H5, G1), and the characteristics of 11 types (C8, H4, H1, H6, S8, SH10, A4, R1, Sech7a, H17, KPS1). This has been reported.

The mechanism of action of halocin is largely unknown, and it is known to kill target cells by destroying the H6 type Na ⁺ /H ⁺ antitransporter. Additionally, the halocin is known to be an antibacterial peptide that is stable against heat and salt.

The halocin is classified into protein halocin and micro halocin depending on its size. The halocin proteins are 30 to 40 kDa, mainly of the H1, H4, and H6 types, and the microhalosins or microcins are mostly 10 kDa or less, and include the C8, S8, A4, R1, H17, and Sech7a types. It applies.

Recently, due to increased profits and changes in consumption trends, there is a growing tendency for consumers to prefer foods with a long storage period and minimal processing. However, the antibacterial peptides used in the existing industry are mainly derived from bacteriocins and have the disadvantage of not working well at high temperatures or high salt concentrations, which limits their use in high-salt foods such as marine products, thereby overcoming the limitations of these usage conditions. There is an increasing demand for the development of antibacterial substances or natural food preservatives that can exhibit antibacterial activity even under high salinity conditions.

KR2018-0000364A KR10-1575878B KR10-1471975B

The purpose of the present invention is to solve the problems of the prior art as described above, and the present invention is to provide a novel antibacterial peptide that is separated from halophilia and has heat and salt resistance and a use thereof.

In order to achieve the above object, the present invention provides a peptide having antibacterial activity and an antibacterial composition containing the same as an active ingredient.

Specifically, the present invention may be a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof.

The peptide may have antibacterial activity against harmful microorganisms.

The harmful microorganism may be one or more species selected from the group consisting of Bacillus cereus, Staphylococcus aureus , Salmonella typhimurium, and Salmonella enteritidis .

The peptide is a novel Halorubrum etiopigum SAH-A6 strain, which is a halophilic bacterium ( Halorubrum aethiopicum SAH-A6) can be characterized as being produced.

In addition, in order to achieve the above object, the present invention provides a nucleotide encoding a peptide having the amino acid sequence of SEQ ID NO: 1.

Additionally, the present invention provides a vector containing the above nucleotide.

In addition, in order to achieve the above object, the present invention provides an antibacterial composition comprising the peptide or an acid addition salt thereof as an active ingredient.

In order to achieve the above object, the present invention also provides an antibacterial pharmaceutical composition against Bacillus cereus, Staphylococcus aureus, Typhus typhus, or Enterobacteriaceae, comprising the peptide or an acid addition salt thereof as an active ingredient.

In order to achieve the above object, the present invention also provides an antibacterial food additive against Bacillus cereus, Staphylococcus aureus, Typhus typhus, or Enterobacteriaceae, comprising the peptide or an acid addition salt thereof as an active ingredient.

The peptide having antibacterial activity of the present invention is used against harmful microorganisms, especially food poisoning-causing bacteria, such as Bacillus cereus, Staphylococcus aureus, and Salmonella strains, which are bacteria that cause food poisoning in fresh food or seafood, etc., or cause food poisoning when consumed. It has excellent antibacterial activity, maintains antibacterial activity even under high salt concentration conditions to enable application to seafood, etc., and maintains antibacterial activity even in various temperature ranges, and the antibacterial peptide, which is an active ingredient of the composition with antibacterial activity, is a proteolytic enzyme. Since it is effectively decomposed and its antibacterial activity can be easily removed from the body when the ingredient is ingested, it can be usefully used as an antibiotic that is safe for the human body.

1 shows Halorubrum , according to an embodiment of the present invention aethiopicum A genetic map that functionally analyzes the genomic genes of the SAH-A6T strain (the subsystem category distribution statistics for Halorubrum ) aethiopicum SAH-A6T).
Figure 2 shows Halorubrum , according to an embodiment of the present invention aethiopicum This is the result of comparing the amino acid sequence of halocine derived from the SAH-A6T strain and the previously reported strain.
Figures 3 and 4 confirm the antibacterial activity spectrum of HY1377 according to an embodiment of the present invention, and Figure 3 shows the antibacterial activity against each strain ( Bacillus subtillis , Staphylococcus aureus , Salmonella typhimurium , and Salmonella enteritidis ). This is a photograph, and Figure 4 shows the results of antibacterial activity according to heat treatment.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily practice it. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

The present invention relates to a peptide having antibacterial activity and an antibacterial composition containing the same as an active ingredient.

Specifically, the present invention relates to a peptide having the amino acid sequence of SEQ ID NO: 1. The present invention may be a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof.

The peptide may have antibacterial activity against harmful microorganisms.

The harmful microorganism may be one or more species selected from the group consisting of Bacillus subtilis, Staphylococcus aureus , Salmonella typhimurium , and Salmonella enteritidis .

The peptide is a novel Halorubrum etiopigum SAH-A6T strain, which is a halophilic bacterium ( Halorubrum aethiopicum SAH-A6T) can be characterized as being produced.

Additionally, in order to achieve the above object, the present invention relates to a nucleotide encoding a peptide having the amino acid sequence of SEQ ID NO: 1.

Additionally, the present invention relates to a vector containing the above nucleotide.

In addition, in order to achieve the above object, the present invention relates to an antibacterial composition comprising the peptide or its acid addition salt as an active ingredient.

In addition, in order to achieve the above object, the present invention relates to a preservative containing the peptide or its acid addition salt as an active ingredient. The preservative may be a preservative composition.

In addition, in order to achieve the above object, the present invention relates to an antibacterial pharmaceutical composition against Bacillus subtilis, Typhus typhus, or Enterobacteriaceae, comprising the peptide or an acid addition salt thereof as an active ingredient.

In addition, in order to achieve the above object, the present invention relates to an antibacterial food additive against Bacillus subtilis, murine typhus, or enteritis bacteria, comprising the peptide or an acid addition salt thereof as an active ingredient.

The inventors of the present invention proposed that existing natural antibiotics, especially antibacterial peptides, be used to increase the efficiency of prevention and preservation of contamination and spoilage of marine products or seafood, and to prevent, improve, or treat diseases that may occur during the consumption of contaminated marine products or seafood. While trying to solve the problem of losing antibacterial activity in a high-salt environment, we searched for a novel antibacterial peptide derived from halophiles that maintains antibacterial activity even at high salt concentration and high temperature. As a result, substances with antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium, and Salmonella enteritidis , which are the causative agents of enteritis, were identified, and substances with the above antibacterial activity were identified. The present invention was completed by confirming that among halocines, which belong to novel antibacterial peptides that have not previously been reported, it corresponds to micro halocines, which have excellent stability against heat and salt.

Hereinafter, the present invention will be described in more detail.

In the present invention, the term subject or laboratory animal is not particularly limited to include all animals capable of carrying out an immune response by transplantation of xenogeneic tissues or organs, preferably humans, monkeys, and cows. , may be vertebrates such as horses, sheep, pigs, goats, camels, antelopes, dogs, rabbits, mice, etc., more preferably primates, and even more preferably primates other than humans.

In the present invention, the term antibacterial composition may have the same meaning as antibiotic, which is a general term for antimicrobial agents, and may have the same meaning as antibacterial agent, disinfectant, preservative, preservative, or sterilization agent, and specifically, gram-positive bacteria, gram-negative bacteria, and fungi. (yeast and mold) refers to a substance capable of inhibiting or inhibiting the development and life functions of one or more species selected from the group consisting of yeast and mold, that is, a substance with antibacterial and antifungal effects, preferably Bacillus subtilis , It may be a substance that has antibacterial activity against B. cereus , Staphylococcus aureus , or Salmonella sp. strains.

In the present invention, culture means culturing a specific microorganism in a culture medium or culture solution, and the culture may or may not include the specific microorganism. The formulation of the culture is not limited, and for example, it may be liquid or solid. The culture medium refers to a solid or liquid containing nutrients necessary for the growth of animal cells, plant cells, or bacteria. The culture medium refers to inoculating and culturing the strain in a liquid medium. The culture medium may be one containing the strain or a culture filtrate obtained by inoculating and culturing the strain and then eliminating the strain, that is, removing the strain (cell-free).

In the present invention, a preservative is a preservative that can be used in one or more types selected from the group consisting of food, feed, cosmetics, and pharmaceuticals, and inhibits the growth of one or more types of harmful microorganisms or spoilage microorganisms selected from the group consisting of bacteria, mold, and yeast. It is an additive that preserves food, feed, and medicine by sterilizing or sterilizing it, and refers to a substance that can inhibit the growth of spoilage microorganisms or sterilize them.

In the present invention, food refers to a natural product or processed product containing one or more nutrients, and preferably means that it has undergone a certain degree of processing to become ready to eat. Examples include fruits, Vegetables, dried or cut fruits or vegetable products, fruit juice, vegetable juice, mixed juices thereof, chips, noodles, processed livestock products, processed food products, processed marine products, processed milk foods, fish products, fermented foods, pickled foods, Bean foods including tofu, jelly, grain foods, seasoned foods, microbially fermented foods, confectionery and baked goods, seasonings, processed meats, acidic beverages, licorice, herbs, etc. In the present invention, fermented foods, functional foods, and health supplements are included. and processed foods, but is not limited thereto.

The present invention relates to peptides having antibacterial activity.

The peptide having the antibacterial activity may be a peptide having the amino acid sequence of SEQ ID NO: 1 produced in a halophilic strain. The peptide having the antibacterial activity is a novel Halorubrum ethiopigum SAH-A6T strain ( Halorubrum aethiopicum It may be related to the bacteriocin produced by SAH-A6T).

In this respect, the peptide is a novel Halorubrum etiopigum SAH-A6T strain ( Halorubrum aethiopicum SAH-A6T) can be characterized as being produced.

The peptide having antibacterial activity may be a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof, preferably a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof.

The peptide may exist in isolated or substantially pure form. Additionally, the peptide may be a substance derived from a peptide having the amino acid sequence of SEQ ID NO: 1.

The acid addition salt may be a pharmaceutically acceptable acid addition salt, and the pharmaceutically acceptable acid addition salt refers to a salt that maintains the biological activity of the peptide and minimizes side effects such as unwanted toxic effects. The acid addition salt may be a pharmaceutically acceptable inorganic acid addition salt or an organic acid addition salt, and examples include hydrochloride, hydrobromide, sulfate, nitrate, acetate, benzoate, maleate, fumarate, succinate, tartrate, and citrate. , oxalate, methanesulfonate, toluenesulfonate, aspartate, or glutamate.

The peptide preferably has antibacterial activity against harmful microorganisms, specifically Gram-positive bacteria and Gram-negative bacteria, and more specifically, Bacillus sp. strains, Staphylococcus aureus strains, and Salmonella sp. Having antibacterial activity against one or more strains selected from the group consisting of strains, more specifically Bacillus subtilis , Staphylococcus aureus , Salmonella typhimurium , and Salmonella enteritidis . It may have antibacterial activity against one or more strains selected from the group consisting of. In addition, the peptide may be an antibacterial peptide that is safe for the human body, that is, halocine, as its antibacterial activity is lost by proteolytic enzymes.

In addition, the present invention may be a peptide having the amino acid sequence of SEQ ID NO: 1, preferably a nucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 1. The nucleotides may be isolated nucleotides.

Additionally, the present invention relates to a vector containing the above nucleotide. The type of the vector may vary depending on the target host and expression conditions for transformation using the vector, and as long as it contains the nucleotide, other conditions are not limited.

In addition, the present invention relates to an antibacterial composition comprising as an active ingredient a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof, preferably a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof. .

The peptide having the amino acid sequence of SEQ ID NO: 1 has antibacterial activity against Bacillus subtilis , Staphylococcus aureus , Salmonella typhimurium, and Salmonella enteritidis , specifically Bacillus subtilis. It exhibits excellent antibacterial activity against strains of the genus Salmonella, has thermal stability at 80°C to 99°C, and is stable even at a concentration of 1.5M NaCl, which corresponds to a high salt concentration, so it can be usefully used as an active ingredient in antibacterial compositions. there is.

In this respect, the peptide having the amino acid sequence of SEQ ID NO: 1 not only maintains high antibacterial activity against harmful bacteria in the temperature and salt concentration ranges belonging to the process of preserving food or cooking or storing marine products or seafood, but also maintains the antibacterial activity. Since the peptide corresponds to a proteinaceous substance decomposed by proteolytic enzymes, the antibacterial activity of the peptide is evaluated to be safe without affecting the human body that consumes food, and the peptide has a high level of resistance against harmful microorganisms. It can have a remarkably excellent effect by having both antibacterial activity and the safety required when applied to food.

The peptide is a novel Halorubrum etiopigum SAH-A6T strain, which is a halophilic bacterium ( Halorubrum aethiopicum It can be genetically produced through recombinant peptides using nucleotides isolated from the genome of (SAH-A6T), and can be separated and purified using columns, etc. after production.

The antibacterial composition may contain 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight, of the active ingredient of the peptide or acid addition salt thereof, based on the total weight of the composition, and the method and purpose of use of the antibacterial composition Accordingly, the content of the active ingredient can be appropriately adjusted.

The antibacterial composition may have antibacterial activity against harmful microorganisms that cause food poisoning, specifically, against harmful microorganisms. The antibacterial composition of the present invention can be applied directly to animals, including humans, to exhibit antibacterial activity, and can be applied to foods, cosmetics or medicines, including seafood, to inhibit or prevent spoilage of products caused by harmful microorganisms of the genus Bacillus subtilis or Salmonella. Since the antibacterial peptide, which is a substance exhibiting antibacterial activity, will be decomposed or deactivated by proteolytic enzymes, even when the antibacterial composition is used in food, cosmetics, or medicine to which the antibacterial composition is applicable, the substance exhibiting antibacterial activity It has the advantage that secondary problems do not occur.

The antibacterial composition may include carbohydrates, proteins, lipids, vitamins, and minerals in addition to the active ingredients.

The sugar may be appropriately selected depending on its purpose and purpose, and examples include honey, dextrin, sucrose, palatinose, glucose, fructose, starch syrup, sugar alcohol, sorbitol, xylitol, and maltitol. It is not particularly limited thereto. The protein may be appropriately selected depending on the purpose and purpose of use. For example, milk-derived proteins such as casein and whey protein, soy proteins, and animal-derived enzymes such as trypsin and pepsin of these proteins. and a hydrolyzate by neutrase or alkilase, but is not particularly limited thereto. The lipid may be appropriately selected depending on the purpose and purpose of use. For example, sunflower oil containing monovalent saturated fatty acids and polyunsaturated fatty acids, rapeseed oil, olive oil, safflower oil, and corn oil. , various plant-derived fats and oils such as soybean oil, palm oil, and palm oil, middle-chain fatty acids, EPA, DHA, soybean-derived phospholipids, and milk-derived phospholipids, but are not particularly limited thereto. There are no particular restrictions on the vitamins, and the minerals may be appropriately selected depending on the purpose and purpose of use. For example, potassium phosphate, potassium carbonate, potassium chloride, sodium chloride, calcium lactate, calcium gluconate, calcium pantothenate, calcium caseinate. , magnesium chloride, ferrous sulfate, and sodium bicarbonate, but is not particularly limited thereto.

Any of the sugars, proteins, lipids, vitamins, or minerals are not limited to the above specific examples and may include other ingredients as long as the antibacterial properties of the composition are maintained, and the content of each is not limited as long as the antibacterial properties are maintained. , preferably 0.1 to 15% by weight, preferably 0.5 to 10% by weight, relative to the total composition.

In addition, the present invention relates to a preservative comprising as an active ingredient a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof, preferably a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof. The preservative may be a preservative composition.

The preservative inhibits or sterilizes the growth of harmful microorganisms or spoilage microorganisms such as bacteria, mold, and yeast, and specifically Bacillus subtilis , Staphylococcus aureus , and Salmonella . typhimurium ) and enteritis ( Salmonella enteritidis ), more specifically, preventing and preserving deterioration, rancidity or decay of objects of preservation that may be infected or spoiled by harmful microorganisms of the genus Bacillus subtilis or Salmonella. It refers to a substance used to maintain the condition of an object, and may preferably be used for one or more types selected from the group consisting of food, feed, cosmetics, and medicine. The feed is not limited to its use, including livestock feed, and may be solid or liquid.

The above-mentioned harmful microorganisms or putrefactive microorganisms, specifically, one or more selected from the group consisting of Bacillus subtilis, Staphylococcus, murine typhus, and Enterobacteriaceae, and more specifically, growth inhibition and sterilizing activity against harmful microorganisms of the genus Bacillus subtilis or Salmonella. Since the bacteriocin, which is a substance showing antibacterial activity, will be decomposed or deactivated by proteolytic enzymes, the preservative will not cause secondary problems due to the substance showing antibacterial activity even when applied to food, feed, cosmetics or medicine, which are the objects of preservation for which the preservative is used. It has the advantage of not occurring.

In this respect, the preservative may be a food preservative, feed preservative, cosmetic preservative or pharmaceutical preservative. The food preservative, feed preservative, cosmetic preservative, or pharmaceutical preservative may be an additive used to prevent deterioration, decay, discoloration, and chemical change of food, feed, cosmetics, or medicine.

In that the preservative must be non-toxic and effective even in trace amounts, the invention provides a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof, preferably a peptide having the amino acid sequence of SEQ ID NO: 1 or Their acid addition salts are preferred as active ingredients of the above preservative.

The preservative may further include ingredients or substances included in conventional preservatives in addition to the effective ingredients. The preservative composition may contain 0.1 to 50 parts by weight or 0.5 to 10 parts by weight of a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof, based on 100 parts by weight of the total composition.

The preservative may further include a disinfectant, antioxidant, or antibiotic that can inhibit the growth of spoilage microorganisms or sterilize food and pharmaceuticals by inhibiting the growth of microorganisms such as bacteria, mold, and yeast.

Additionally, the preservative composition of the present invention can be used together with conventional synthetic preservatives or organic acid mixtures within the range that does not limit the effect of the present invention.

In addition, the present invention relates to Bacillus subtilis comprising as an active ingredient a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof, preferably a peptide having the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof. It relates to an antibacterial pharmaceutical composition against staphylococci, murine typhus, or enteritis bacteria.

The peptide can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation.

Specifically, the peptide can be administered in various oral or parenteral formulations. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. You can.

Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories.

Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. may be used as the non-aqueous solvent or suspension solvent. As a base for the suppository, witepsol, macrogol, tween 61, cacao, liuringe, or glycerogenatin may be used.

In addition, the peptide can be used by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and to increase stability or absorption, carbohydrates such as glucose, sucrose or dextran, and ascorbic acid ( Antioxidants such as ascorbic acid or glutathione, chelating agents, small molecular proteins or other stabilizers may be used.

The active ingredient may be included in an amount of 0.01 to 100 mg/kg or 0.1 to 10 mg/kg relative to the total composition, and may be administered once to three times a day, but this does not limit the scope of the present invention.

In the pharmaceutical composition, the peptide may be administered to the patient in a single dose in the form of a bolus or by infusion for a relatively short period of time, based on the total dose, and may be administered to the patient in multiple doses. It can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time.

The content of the active ingredient or the dosage to the patient is determined considering various factors such as the patient's age and health status as well as the route of administration and number of treatments, so the content of the active ingredient according to the purpose of the pharmaceutical composition. Alternatively, the dosage can be appropriately determined by a person skilled in the art to which the present invention pertains.

In addition, in order to achieve the above object, the present invention relates to an antibacterial food additive against Bacillus subtilis, murine typhus, or enteritis bacteria, comprising the peptide or an acid addition salt thereof as an active ingredient.

If the antibacterial food additive is used to suppress the growth of harmful microorganisms by its antibacterial power, and to exert a food preservation effect on the added food and to prevent or improve infectious diseases caused by harmful microorganisms, the type of food to which it is added There are no special restrictions. Examples of foods to which the above food additives can be added include drinks, meat, sausages, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, and various soups. , beverages, alcoholic beverages, and vitamin complexes.

The food additives can be added directly to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods.

The amount of the active ingredient included in the food additive can be appropriately determined depending on the purpose of its use. In general, the content of the active ingredient, that is, the peptide, of the antibacterial food additive may be 0.01 to 99% by weight, 0.05 to 90% by weight, or 0.1 to 50% by weight based on the total weight of the food additive.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement it. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

< Experiment example . halophilic Isolation and confirmation of strain-derived antibacterial substances>

Salt lake in Ethiopia's Afdera region, salt in Ethiopia's Danakil region, Shinan Topan salt farm and Jangpan salt farm, Gomso salt farm, salted fish (hairtail milk, bandag milk, shrimp milk, anchovy milk), which the inventor of the present invention possessed through previous research. Modified Growth Medium (MGM) containing 20% NaCl and marin broth (Difco Marin broth 2216, Fisher scientific, USA) was used, and culture conditions such as pH (pH 4, 7, 9), temperature (10 to 65°C), and salt concentration % (w/v, NaCl 0 to 30%) were used to analyze strain characteristics. and cultured, and enzyme activity (amylase, cellulase, lipase, protease, auxin production ability, and phosphorus solubilization ability) was also confirmed.

As a result of the above experiment, among halophilic strains, Halorubrum was identified as a strain capable of producing halocin-like substances. aethiopicum The SAH -A6 strain was selected, and experiments were performed based on it.

Example 1. Genome engineering-based novel halocin analysis

Halorubrum selected above aethiopicum Genomic DNA extraction from the SAH -A6 strain was performed using a standard genomic DNA isolation kit (Promega, USA). Genome sequencing was confirmed using single molecule real time (SMRT) from PacBio (Pacific Biosciences, USA). The sequences of the rRNA gene were confirmed using RNAmmer 1.2, and the tRNA gene sequences were confirmed using tRNA scan-SE 1.23.

As a result of confirming the genome sequence by the above method, Halorubrum aethiopicum The genome of the SAH -A6 strain consists of a total of 3,325,770 bp, and the GC content is 68.0%, which was confirmed to be similar to that of other Halorubrum sp. strains. As a result of functional analysis, it was found to contain antimicrobial peptides such as bacteriocin and immunity protein. has not been confirmed.

For more detailed analysis, the strain for which the genomic DNA was secured was uploaded to the RAST server (http://rast.nmpdr.org/), and the previously known halocine sequence was uploaded to NCBI (https://www. After obtaining it from (ncbi.nlm.nih.gov/), halocine sequence and genomic DNA were paired using NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Through wise comparative analysis, halocin-like sequences among hypothetical proteins were analyzed. The analysis results are shown in Figure 2.

As shown in Figure 2 above, Halorubrum aethiopicum Natrinema previously reported in some of the genomes of the SAH-A6 strain gari A region with 35% homology to a partial sequence of the Halocin C8 type, which consists of 139 amino acids (aa) produced by JCM 14663 , was identified, and the region was analyzed as a hypothetical protein, and was named HY1377.

Example 2. Halocin genome cloning

To clone and amplify HY1377, PCR was performed using the primers shown in Table 1 below. As shown in Table 1 below, PCR was performed using two types of primers.

Primer Sequence Restriction enzyme HY1377_F 5'-CCAG CATATG CAAAGCTTAGGTTTTTGTG-3' Nde I HY1377_R 5'-AATT CTCGAG TCAGATACCTGCAATGGCTAC-3' Xho I

When performing PCR, nPfu forte DNA polymerase (Enzynomics, Korea) was used to perform denaturation at 95°C for 30 seconds, primer annealing at 50°C for 40 seconds, extension at 72°C for 40 seconds repeated 30 times, and finally at 72°C for 7 minutes. Extension was performed under the following conditions.

In order to confirm the PCR product amplified through the above PCR, the size was confirmed by electrophoresis in 1% agarose, and the expected size as expected in Table 2 below (135 bp for HY1377(s), 135 bp for HY1377-2(ns)) A band matching 111 bp) was cut out and purified using a Gel extraction kit (QIAGEN, Germany).

Amino acid sequence
(4.04 kDa) MGLLVLATFVAAARIGGQRAPGADESEDTYTAITRRLSEVARTPAVWSI AFVVIALGVGALVVLSVGNFAFVEGITGSLLGVLFAVVGLLVTGFVFLGA YFGVRGRGLGNAHGVAAGSFAAGLVFLVLIAVQLLVGVIG

Mix 23.5 μl of each purified PCT product, 1.5 μl of pTOP-blunt-V2 vector (Enzynomics, Korea), and 5 μl of 6X TOP cloner buffer (1.2 M NaCl, 0.06 M MgCl₂) and react overnight at 4°C. Recombinant plasmid preparation (ligation) was performed.

Put 6 ㎕ of each recombinant plasmid (plasmid DNA) prepared above into a 1.5 ml micro centrifuge tube, and add E. coli After adding 50 ㎕ of DH-5a competent cells (Enzynomics, Korea), leave on ice for 15 minutes, heat shock at 42°C for 1 minute, leave on ice again for 1 minute, and heat at 42°C for 30 seconds. After shock, the recombinant strain was prepared by leaving it on ice for 20 minutes. 400 ㎕ of SOC medium was mixed with the above-mentioned strain and cultured at 37°C for 1 hour, then plated on LB solid medium containing a final concentration of 100 ㎍/ml Kanamycin at 37°C and cultured for 14 hours to form transformed colonies. After separating the colony, it was inoculated into 8 ml of LB liquid medium containing 100 ㎍/㎖ of kanamycin, cultured with shaking at 37°C for 14 hours, and the plasmid was extracted using a miniprep kit (QIAGEN, Germany). Macrogen (Korea) was contacted to confirm the presence of the introduced gene.

The identified plasmid was treated with restriction enzymes NdeI and XhoI to isolate the HY1377 gene, and pET-28a (Novagen, Germany), a protein expression vector containing a 6 A recombinant plasmid was prepared by combining the pET-28a vector and the HY1377 gene as described above.

10 ㎕ of the recombinant plasmid prepared above was mixed with 50 ㎕ of E. coli DH-5a competent cells (Enzynomics, Korea) and transformed as described above. Colonies identified were then submitted to Macrogen (Korea) for base sequence analysis. E. coli in the same manner as described above It was transformed into BL21(DE3) competent cells (Enzynomics, Korea). The transformed colonies were inoculated into LB liquid medium containing 100 μg/ml of kanamycin to create a 25% glycerol stock and stored at -80°C.

Example 3. Confirmation of halocin expression and activity

For protein expression of the HY1377 gene, which is inferred from the above halocin, pET-28a HY1377 transformed into E. coli BL-21(DE3) competent cells was induced until the OD ₅₅₀ was 0.6 and the final concentration of IPTG was 1 mM. After overexpression, the cells were cultured at 37°C for 5 hours and then centrifuged at 8,000 rpm and 4°C for 20 minutes. Afterwards, the settled cells were diluted with 5 ) was done. Afterwards, centrifugation was performed at 13,000 rpm and 4°C for 10 minutes, the supernatant was separated, and protein expression was confirmed.

To purify the expressed protein, 1/20 of 500 ml of binding buffer of overexpressed HY1377(s) and HY1377-2(ns) cells was suspended in the cells, and the cells were disrupted using sonication, followed by 8,000 rpm. and centrifuged for 20 minutes at 4°C to remove only the supernatant. A sample was prepared by filtering the supernatant through a 0.45 ㎛ filter (Sartorius, Hannover, Germany).

The prepared sample was subjected to Hix-tag purification using FPLC (fast protein liqid chromatography). The Ni-NTA His-tag column was a ^HisTrap HP (GE health care., USA) Ni ²⁺ column. The column was washed with sterilized distilled water and flowed with binding buffer until the column was equilibrated, and then 25 ml of the previously prepared sample was flowed through the column at a flow rate of 0.5 ml/min.

The binding buffer was equilibrated by flowing through the column at a flow rate of 0.5 ml/min, and then eluted with an elution buffer. The flow rate of the elution buffer was 0.5 ㎖/min, 500㎕ of each fraction was received, and the antibacterial activity was measured against various harmful microbial indicators shown in Table 3 below.

Harmful microorganisms (food poisoning bacteria and spoilage bacteria) Culture temperature and medium Vibrio Parahaemolyticus (KCTC 2471) 30℃ and Marin broth Vibrio alginolyticus (KCTC 2472) 37℃ and Marin broth Vibrio ordalii (ATCC 33509) 30℃ and Marin broth Salmonella typhimurium (ATCC 29630) 37℃ and Nutrint broth Salmonella enteritidis (ATCC 31194) 30℃ and Nutrint broth Bacillus subtills (KCCM 32835) 30℃ and Nutrint broth E. coli (KCTC 1924) 37℃ and L.B. Staphylococcus aureus (ATCC 25923) 37℃ and Tryptic soy broth

Antibacterial activity against the harmful microbial indicators in Table 3 was performed using the paper disc method. First, each target strain of Table 3 was spread on the entire 6mm paper disc using a sterilized cotton swab in a selective medium, and then 20 ㎕ of the elution buffer was dispensed and placed on a plate. To confirm the antibacterial activity, salt Experiments were performed with different concentrations (1.5M NaCl, Figure 4) and temperatures (treatment at 80°C and 90°C for 4 hours, Figure 5). At this time, the positive controls used were ampicillin (50 mg/ml), kanamycin (50 mg/ml), and chloramphenicol (35 mg/ml), and the negative controls were sterilized distilled water or 1.5M NaCl Lysis. It was a buffer. The results of testing the antibacterial activity with overexpressed HY1377 in the same manner as above are shown in Figures 3 to 5.

As shown in FIG. 3, among the harmful microorganisms, antibacterial activity was confirmed for Bacillus subtillis , Staphylococcus aureus , Salmonella typhimurium , and Salmonella enteritidis .

In addition, as shown in Figure 4, HY1377 showed little decrease in activity even when treated at 80°C for 4 hours, and showed little decrease in activity even when treated at 99°C for 2 hours, and heat treatment for 4 hours. In one case, it was confirmed that the size of the inhibitory ring was reduced by about 2/3, resulting in a slight decrease in activity. From the above results, it was confirmed that the thermal stability was very stable.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims are also possible. falls within the scope of rights.

<110> Industry University Cooperation Foundation Silla University <120> A ANTIMICROBIAL PEPTIDE AND A ANTIMICROBIAL COMPOSITION CONTAINING THE SAME <130> CDP20190008 <160> 1 <170>CopatentIn 2.0 <210> 1 <211> 139 <212> PRT <213> HY1377 <400> 1 Met Gly Leu Leu Val Leu Ala Thr Phe Val Ala Ala Ala Arg Ile Gly 1 5 10 15 Gly Gln Arg Ala Pro Gly Ala Asp Glu Ser Glu Asp Thr Tyr Thr Ala 20 25 30 Ile Thr Arg Arg Leu Ser Glu Val Ala Arg Thr Pro Ala Val Trp Ser 35 40 45 Ile Ala Phe Val Val Ile Ala Leu Gly Val Gly Ala Leu Val Val Leu 50 55 60 Ser Val Gly Asn Phe Ala Phe Val Glu Gly Ile Thr Gly Ser Leu Leu 65 70 75 80 Gly Val Leu Phe Ala Val Val Gly Leu Leu Val Thr Gly Phe Val Phe 85 90 95 Leu Gly Ala Tyr Phe Gly Val Arg Gly Arg Gly Leu Gly Asn Ala His 100 105 110 Gly Val Ala Ala Gly Ser Phe Ala Ala Gly Leu Val Phe Leu Val Leu 115 120 125 Ile Ala Val Gln Leu Leu Val Gly Val Ile Gly 130 135

Claims (9)

A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or an acid addition salt thereof. According to paragraph 1,
The peptide has antibacterial activity against harmful microorganisms,
The harmful microorganism is a peptide or an acid addition salt thereof, which is at least one selected from the group consisting of Bacillus cereus , Staphylococcus aureus , Salmonella typhimurium , and Salmonella enteritidis . . delete Nucleotide encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 1. A vector containing the nucleotide of claim 4. An antibacterial composition comprising the peptide of claim 1 or an acid addition salt thereof as an active ingredient. A preservative comprising the peptide of claim 1 or an acid addition salt thereof as an active ingredient. delete An antibacterial food additive against Bacillus cereus, Staphylococcus, murine typhus, or Enterobacteriaceae, comprising the peptide of claim 1 or an acid addition salt thereof as an active ingredient.