KR102542359B1 - An Antimicrobial Composition Comprising Immunized Tenebrio molitor Larvae Extract and Menufacturing Method thereof - Google Patents

Abstract

The present invention relates to an antibacterial composition that is environmentally friendly and has excellent antibacterial activity isolated from immunized brown mealworm larvae and a method for producing the same, which is excellent in inhibiting the growth of harmful microorganisms including spoilage bacteria and fungi that spoil food and deteriorate quality. It has excellent stability against heat, pH and salinity.

Description

Antimicrobial Composition Comprising Immunized Tenebrio molitor Larvae Extract and Menufacturing Method thereof}

The present invention relates to an antibacterial composition isolated from immunized brown mealworm larvae, which is environmentally friendly and has excellent antibacterial activity, and a method for preparing the same.

Preservation of food is a problem that has been studied for a very long time. In human history, as the first method developed to preserve food, methods such as drying, smoking, or salting have been used. Then, in 1810, the canning method was developed, followed by the development of temperature-controlled refrigerators and freezers, which greatly improved food preservation. In addition, as a method for preserving food, food preservatives that are directly added to food have been developed. Food preservatives include preservatives that inhibit the growth of spoilage bacteria and antifungal agents that inhibit the growth of mold. Any of them acts on microorganisms in food to show a preservation effect, but this means that they are toxic to some extent, so the law is strictly regulating the target food and usage amount.

However, most processed foods such as bread, meat products, confectionery, canned food, instant noodles, and cup noodles contain preservatives, flavoring agents, coloring agents, preservatives, and chemical seasonings. These synthetic additives are a factor that harms the health of both humans and livestock. The reason is that when chemical additives are ingested into the body, 50-80% is discharged through the respiratory or excretory organs, but most of the rest accumulates in the body and causes allergies, diabetes, poor growth due to calcium, osteoporosis, osteomalacia, asthma, hunchback, and insomnia. This is because it not only causes various diseases such as heart palpitations, neurosis, joint pain, arteriosclerosis, and high blood pressure, but also causes cancer.

Since chemical preservatives cause many problems related to human safety, it is preferable to use natural preservatives obtained by extracting from natural organisms in consideration of human safety. So far, as natural preservatives that inhibit microbial contamination and growth, hinokitiol, a cypress extract, megnonol, a magnolia extract, and DF-100, a grapefruit seed extract, have been developed, but their scale is 17 It is only 1% and is not meeting the demand.

Therefore, there is an urgent need to develop a food preservative having excellent human body stability, antiseptic and antiseptic effects, and excellent economic feasibility at the same time as solving the above problems.

Republic of Korea Patent Publication No. 10-2020-0084258

Therefore, the present invention has been made in view of the above problems, and an object of the present invention is to provide an antibacterial composition having excellent antibacterial effect against harmful microorganisms and excellent stability against heat, pH and salinity, and a food preservative containing the same. is to provide

Another object of the present invention is to provide a manufacturing method for mass production of an antimicrobial composition.

In order to achieve the above object, the present invention provides an antibacterial composition comprising an extract of brown mealworm larvae immunized by Lactobacillus plantarum as an active ingredient.

According to one embodiment of the present invention, the extract may be extracted with 10 to 30% (v / v) weak acid of immunized brown mealworm larvae powder.

In order to achieve the above object, the present invention provides a food preservative comprising the antimicrobial composition.

In order to achieve the above other object, the present invention provides a method for preparing an antimicrobial composition comprising the following steps.

a) injecting Lactobacillus plantarum into the body cavity of brown mealworm larvae;

b) inducing an immune response by leaving the brown mealworm larvae injected with Lactobacillus plantarum for 10 to 15 hours; and

c) obtaining an extract from the immunized brown mealworm larvae using a weak acid solution.

According to one embodiment of the present invention, in the step of c) extraction, the immunized brown mealworm larva powder may be immersed in a 10 to 30% (v/v) weak acid solution for 30 to 60 hours.

According to various embodiments of the present invention, it has an excellent growth inhibitory effect against harmful microorganisms including spoilage bacteria and fungi that spoil food and deteriorate quality, and exhibits excellent stability against heat, pH and salinity.

Therefore, the antibacterial composition of the present invention is prepared using edible lactic acid bacteria and larvae, so that it can be used as a food preservative having excellent antiseptic or antiseptic effects while being stable to the human body.

1 is a view schematically showing a process for preparing an antibacterial composition of the present invention.
Figure 2 shows that Lactobacillus plantarum was injected into brown mealworm larvae, and according to various immune induction periods (6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours), atacin (attacin) ) (a), defencin (b), tenenesin (c) and choleoptericin (coleoptericin) (d) is a graph measuring the expression levels.
Figure 3a is an antibacterial activity analysis result for the immunized brown mealworm larvae extract (distilled water) prepared by various extraction methods.
Figure 3b is the antibacterial activity analysis results for the immunized brown mealworm larval extract (ethanol) prepared by various extraction methods.
Figure 3c is the antibacterial activity analysis results for the immunized brown mealworm larvae extract (20% (v / v) acetic acid solution) prepared by various extraction methods.
4 is an analysis result of the antibacterial activity of the immunized brown gourd larvae extract prepared in Example 1 against Escherichia coli, Bacillus cereus , which is a food poisoning bacterium, and Staphylococcus aureus .
5 is an analysis result of the antibacterial activity of the immunized brown mealworm larvae extract prepared in Example 1 against Lactobacillus acidophilus .
6 is an analysis result of the antibacterial activity of the immunized brown mealworm larvae extract prepared in Example 1 against Aspergillus flavus and Aspergillus parasiticus .
7 is a result of analyzing the antimicrobial activity of the immunized brown gourd larvae extract prepared in Example 1 against Aspergillus oryzae .

In the following, various aspects and various embodiments of the present invention will be described in more detail.

According to one aspect of the present invention, it relates to an antibacterial composition comprising an extract of brown mealworm larvae immunized with lactic acid bacteria, more preferably an extract of brown mealworm larvae immunized by Lactobacillus plantarum It relates to an antibacterial composition comprising as an active ingredient.

In the present invention, brown mealworm ( Tenebrio molitor ) refers to insects belonging to the mealworm family, and the brown mealworm has a life cycle of eggs, larvae, pupae, and adults, and the antibacterial composition of the present invention uses brown mealworm larvae.

The brown mealworm larvae ( Tenebrio molitor Larvae) is one of the widely known edible insects, also called 'mealworm' or 'gosoae', and is permitted to be used as a food ingredient in many countries around the world, including Korea. .

In the present invention, "active ingredient" means a component that exhibits the desired activity alone or can exhibit activity in combination with a carrier having no activity itself.

In the present invention, "immunization" may refer to a process of imparting the ability to increase an immune response (including cellular immunity such as antibodies or effector CTLs) to a target antigen or epitope. Such immunization can increase a subject's immune response to an antigen by providing an active immune response.

The immunization does not mean complete immunity, but means an immune response that is relatively greater than the baseline. For example, if a cellular or humoral immune response to the target antigen is increased or generated by the immunization, it can be evaluated as being immunized against the target antigen.

The immunization is not particularly limited as long as it is a method of inducing an immune response by introducing an antigen to a specific subject, and is, for example, standard intramuscular or intradermal injection, transdermal particle delivery, inhalation, topical, oral, intranasal or mucosal administration. It can be directly introduced into the subject by

Specifically, the brown mealworm larvae may be immunized by introducing a lactic acid bacteria such as a Lactobacillus spp. strain, and preferably, the lactic acid bacteria may be Lactobacillus plantarum .

The Lactobacillus plantarum may be Lactobacillus plantarum KACC 15357, and the specific scientific name may be Lactobacillus plantarum subsp. plantarum. there is. The Lactobacillus plantarum is injected into the body cavity of brown mealworm larvae at a concentration of 1×10 ⁸ CFU/ml to 1×10 ¹² CFU/ml to induce immunization of brown mealworm larvae. When the inoculation concentration of Lactobacillus plantarum is less than the above range, there is a problem that a sufficient immune response cannot be induced from brown mealworm larvae, and when it exceeds the above range, the growth of brown mealworm larvae is adversely affected. can affect

After injecting lactic acid bacteria into the brown mealworm larvae, the immunization induction period is preferably performed for 10 to 15 hours. If the immunization induction period is less than 10 hours or exceeds 15 hours, antibacterial peptides are not sufficiently produced through the immunization reaction. There may be problems with not being able to.

The extract may be extracted with a 10 to 30% (v / v) weak acid solution of immunized brown gourd larvae powder, but when extracted with a solvent other than a 10 to 30% (v / v) weak acid solution, it does not show antibacterial activity. there is a problem i can't Preferably, the weak acid solution is not particularly limited as long as it is a weak acid solution used as an extraction solvent, but may be an acetic acid solution most preferably.

In order to obtain the best antibacterial activity of the extract, the immunized brown mealworm larvae powder is immersed in 10 to 30% (v / v) acetic acid aqueous solution for 30 to 60 hours, 35 to 55 hours, and 40 to 50 hours It may be extracted through In the present invention, the aqueous acetic acid solution was used by diluting glacial acetic acid.

The extract may contain one or more antimicrobial peptides selected from the group consisting of attacin, defencin, tenenecin and coleoptericin. In particular, the present invention It was confirmed that the extract contained all of atacin, defencin, tenecin, and coleoptericin.

In the present invention, "antibacterial" means the ability to resist germs, and means all mechanisms made to defend against the action of microorganisms such as bacteria, molds, yeasts, and the like. Such an antibacterial effect may include a preservative effect that inhibits the growth of putrefying bacteria and an antimicrobial effect that inhibits the growth of mold.

The microorganisms refer to microscopically small organisms or units that are not easily identified with the naked eye, and include bacteria, molds, yeasts, etc. that cause phenomena or diseases harmful to health in other organisms such as humans or mammals can include

The microorganisms may include spoilage bacteria as well as fungi, and in particular, Escherichia coli, Bacillus cereus , Staphylococcus aureus , which are harmful microorganisms that influence food stability in the fermentation process of soy sauce, Any one or more bacteria selected from the group consisting of Aspergillus flavus and Aspergillus parasiticus that produce aflatoxin may be included.

In this respect, the antibacterial peptides expressed are different depending on the type of larvae and the strain to be injected, and the types of useful ingredients are different depending on the extraction method and conditions. Of course, since the effect (target strain of antibacterial activity) also varies depending on the useful component, it is most preferable to satisfy all of the above conditions in order to have the antibacterial effect of the present invention.

Since the extract of the present invention exhibits excellent antibacterial activity against harmful microorganisms in food, it can bring about a stable food production effect without toxicity, so it can be usefully used industrially. In addition, since the extract has thermal stability at 40 to 100 ° C. and stability at high pH and salt concentration, it can be usefully used as an antibacterial composition.

In the above aspect, since the extract can maintain high antibacterial activity against harmful microorganisms in the range of temperature, pH and salt concentration belonging to the preservation of food or fermentation or storage of fermented soybeans, it can be applied to food with high antibacterial activity against harmful microorganisms. It can have remarkably excellent effects by having all the stability required at the time of application.

The antibacterial composition may include 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight, of the extract active ingredient of the brown mealworm larvae immunized by the Lactobacillus plantarum , based on the total weight of the composition, , It can be appropriately adjusted according to the method of use and purpose of use of the antibacterial composition.

The antibacterial composition has antibacterial activity against harmful microorganisms, specifically harmful microorganisms, can be applied directly to animals, including humans, to exhibit antibacterial activity, and can be applied to foods, cosmetics or medicines including seafood, It is possible to inhibit or prevent spoilage of products caused by harmful microorganisms of the genus Pergillus or Streptococcus, and since the antimicrobial peptide, which is a substance exhibiting the antibacterial activity, will be decomposed or deactivated by a protease, the antimicrobial composition is Even when the composition is used for applicable foods, cosmetics, or medicines, there is an advantage in that secondary problems are not caused by the substance exhibiting the antibacterial activity.

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

The saccharide may be selected according to the purpose and purpose of use, and may be, for example, honey, dextrin, sucrose, palatinose, glucose, fructose, starch syrup, sugar alcohol, sorbitol, xylitol, maltitol, It is not particularly limited thereto. The protein may be selected according to the purpose and purpose of use, and for example, milk-derived proteins such as casein and whey protein, soybean proteins, and animal-derived enzymes such as trypsin and pepsin of these proteins And it may be 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, and for example, monovalent saturated fatty acids and polyunsaturated fatty acids, including sunflower oil, rapeseed oil, olive oil, safflower oil, and corn oil. , soybean oil, palm oil, and various plant-derived oils such as palm oil, middle-chain fatty acids, EPA, DHA, soybean-derived phospholipids, and milk-derived phospholipids, but are not particularly limited thereto. The vitamins are not particularly limited, 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, or sodium bicarbonate, but is not particularly limited thereto.

For any of the carbohydrates, proteins, lipids, vitamins or minerals, other components may be included without being limited to the above specific examples as long as the antibacterial properties of the composition are maintained, and each content is not limited as long as the antimicrobial properties are maintained. , Preferably it may be 0.1 to 15% by weight, preferably 0.5 to 10% by weight relative to the total composition.

The antibacterial composition contains an extract of brown mealworm larvae immunized by Lactobacillus plantarum as an active ingredient, and may also be used as a cosmetic composition, food composition, or pharmaceutical composition, specifically food and cosmetics. , It can be applied by adding to antibacterial objects such as external skin preparations, pharmaceuticals and quasi-drugs, and also immersing machines or instruments such as tableware, kitchen equipment, beauty equipment, and medical equipment in the composition of the present invention or its dilution, These machines can apply the composition of the present invention or a dilution thereof in a spray manner to the surface of the appliance.

When the antibacterial composition is used as a food composition, it may include all types of food compositions such as functional food, nutritional supplements, health food, and food additives. Food compositions of this type can be prepared according to conventional methods known in the art. Since the food composition contains the extract of brown mealworm larvae immunized by Lactobacillus plantarum as an active ingredient, the growth of harmful microorganisms is effectively inhibited, and the preservation of food can be remarkably improved.

The food composition may contain food additives, and the food additives, unless otherwise specified, conformity according to the standards and standards for the item in accordance with the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety. can judge

Items described in the food additives code include, for example, ketones, glycine, potassium citrate, chemical compounds such as nicotinic acid and cinnamic acid, natural additives such as persimmon pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, sodium L-glutamate preparations, and noodles. and mixed preparations such as added alkali agent, preservative preparation, and tar color preparation.

In addition, the food composition may be prepared and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions, and pills.

In addition, when the antibacterial composition is used as a pharmaceutical composition, the pharmaceutical composition contains the extract of brown mealworm larvae immunized by Lactobacillus plantarum as an active ingredient, so that harmful microorganisms, especially Escherichia coli, Bacillus cereus ( Bacillus cereus ), Staphylococcus aureus ( Staphylococcus aureus ), Aspergillus flavus ( Aspergillus flavus ) and It has excellent inhibitory activity against Aspergillus parasiticus and the like. The harmful microorganism is a microorganism related to food poisoning, and since it has an antibacterial effect against it, it can be used as a pharmaceutical composition and included in medicines for preventing, improving, and treating microbial infectious diseases that can be caused by harmful microorganisms. there is.

In addition, the antibacterial is as described above, and thus may be included in the pharmaceutical composition for the purpose of antibacterial, Escherichia coli, Bacillus cereus , Staphylococcus aureus , Asper Aspergillus flavus and Aspergillus parasiticus ( Aspergillus parasiticus ) may have a preventive or therapeutic effect on microbial infectious diseases related to food poisoning (food poisoning).

The pharmaceutical composition may further include carriers, excipients and diluents used in the preparation of conventional pharmaceutical compositions.

The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, but is not limited thereto.

In addition, the composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, It can be prepared by mixing lactose or gelatin. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

As the liquid formulation for oral administration, suspensions, internal solutions, emulsions, syrups, etc. may be used, and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to simple diluents and liquid paraffin.

Sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories may be used for the preparations for parenteral administration. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspending agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, and glycerogeratin may be used.

The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type, severity, activity of the drug, and drug It may be determined according to factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is preferable to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

In addition, when the antibacterial composition is used as a feed composition, the feed composition is Lactobacillus plantarum (Lactobacillus plantarum) as an active ingredient, so harmful microorganisms, especially Escherichia coli (Escherichia coli),Bacillus cereus (Bacillus cereus), Staphylococcus aureus (Staphylococcus aureus), Aspergillus flavus (Aspergillus flavus)and Aspergillus parasiticus (Aspergillus parasiticus)Proliferation of harmful microorganisms on the back can be effectively inhibited, and preservation can be remarkably improved.

The feed is a material that supplies organic or inorganic nutrients necessary for the growth of livestock. The animal ingests organic matter as a nutrient, and is a creature having a differentiated digestive, embryonic, and respiratory tract, and specifically, may be an echinoderm, crustacean, mollusk, fish, amphibian, reptile, bird, or mammal, but is limited thereto It is not.

The feed composition may further include grain, vegetable protein feed, animal protein feed, sugar or dairy products. Specifically, the grain may be ground or crushed wheat, oats, barley, corn and rice, the vegetable protein feed may be rape, soybean and sunflower as main components, and the animal protein feed may be blood meal, meat meal, bone meal and fish Powder, and the sugar or dairy product may be a dry ingredient consisting of various powdered milk and whey powder.

The feed composition may further include components such as nutritional supplements, digestion and absorption enhancers, growth promoters, or disease preventive agents.

In addition, the feed composition may be made of a hard granular or granular material depending on the degree of grinding of the ingredients, and may be supplied as a mesh or formed into a desired separated shape for further processing and packaging. In addition, the composition may be pelletized, expanded, or extruded for storage, and may be dried and distributed for ease of storage.

In addition, the antibacterial composition of the present invention can also be used as a food preservative. In particular, since all components are edible raw materials with low irritation, it can also be used as a preservative for food preservation for humans, companion animals and livestock. The preservative may be a preservative composition.

The food preservative inhibits or sterilizes the growth of harmful microorganisms such as bacteria, molds and yeasts, prevents deterioration, rancidity or decay of preservation objects that may be infected or decayed by harmful microorganisms, and maintains the state of preservation objects means the material used. Preferably, it may be used for at least one selected from the group consisting of food, feed, cosmetics, and pharmaceuticals, as well as food.

In this respect, the preservative may be a food preservative, a feed preservative, a cosmetic preservative or a 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 or feed, cosmetics or medicine.

In addition to the above components, other materials may be added to the composition of the present invention as long as the antibacterial activity of the composition of the present invention is not lowered. At this time, the material added is preferably a material that can be used for food or food additives.

The composition of the present invention may include a solvent for dissolving or dispersing the above components. At this time, the solvent is preferably alcohol, water, or a mixture thereof. In addition, the composition of the present invention may be formulated in various forms that are pharmaceutically, food, or cosmetically acceptable.

The present invention also provides a method for preventing or sterilizing the antibacterial object from microbial contamination by combining it with an antimicrobial object based on the functional effect of the component used as the active ingredient of the composition of the present invention as described above. At this time, it is preferable to apply the concentration of the two components at the concentration suggested above.

In the method of the present invention, when mixing the extract of brown mealworm larvae immunized by Lactobacillus plantarum with an antibacterial object, it is preferable to use a mixer or homogenizer to uniformly distribute the antibacterial object do.

The food preservative of the present invention inhibits the propagation of harmful microorganisms by its antibacterial activity, and if the food preservative is used to exhibit the effect of preserving food and preventing or improving infectious diseases caused by harmful microorganisms, the type of food to be added is There are no particular restrictions. Examples of foods to which the food preservative may be added include drinks, meat, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, and various soups , sodas, alcoholic beverages, and vitamin complexes.

The food preservative may be added to food as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.

The addition amount of the active ingredient included in the food preservative may be appropriately determined depending on the purpose of use. In general, the active ingredient content of the food preservative may be 0.001 to 99.99% by weight, preferably 0.1 to 90% by weight, based on the total weight of the food preservative.

Another aspect of the present invention relates to a method for preparing an antimicrobial composition comprising the following steps.

a) injecting Lactobacillus plantarum into the body cavity of brown mealworm larvae;

b) inducing an immune response by leaving the brown mealworm larvae injected with Lactobacillus plantarum for 10 to 15 hours; and

c) obtaining an extract from the immunized brown mealworm larvae using a weak acid solution.

Referring to Figure 1 will be described in detail the manufacturing method of the antibacterial composition. First, a) by injecting Lactobacillus plantarum into the body cavity of brown mealworm larvae, an immune response of brown mealworm larvae can be induced. The Lactobacillus plantarum may be Lactobacillus plantarum KACC 15357, and the specific scientific name may be Lactobacillus plantarum subsp. plantarum. there is. The Lactobacillus plantarum is injected into the body cavity of brown mealworm larvae at a concentration of 1×10 ⁸ CFU/ml to 1×10 ¹² CFU/ml to induce immunization of brown mealworm larvae. If the inoculation concentration of Lactobacillus plantarum is less than the above range, there is a problem that sufficient immune response cannot be induced from brown mealworm larvae, and if it exceeds the above range, it adversely affects the growth of brown mealworm larvae. can affect

Next, b) the Lactobacillus plantarum injected brown mealworm larvae are left for 10 to 15 hours to induce an immune response. At this time, the immune reaction time is preferably performed for 10 to 15 hours. If the immune reaction time is less than 10 hours or exceeds 15 hours, a problem may occur in that antibacterial peptides are not sufficiently produced through the immunization reaction.

Between steps b) and c), b') rapid freezing with liquid nitrogen may be further included, through which attacin, defencin, An extract containing a large amount of useful components such as any one or more antimicrobial peptides selected from the group consisting of tenecin and coleoptericin can be prepared.

Finally, c) an extract is obtained from the immunized brown mealworm larvae using a weak acid solution. The weak acid solution is not particularly limited as long as it is a weak acid solution usable for extraction, but may preferably be an acetic acid solution.

Preferably, the extraction step c) may be to mix a 10 to 30% (v/v) weak acid solution with the immunized brown mealworm larvae powder and soak for 30 to 60 hours. Specifically, it may be extracted by adding a 10 to 30% (v / v) weak acid solution in a 1 to 10-fold volume ratio based on the immunized brown mealworm larvae powder. When extracted with a solvent other than a 10 to 30% (v/v) weak acid solution, there is a problem in not exhibiting antibacterial activity.

The extraction method may be cold extraction, ultrasonic extraction, or heat extraction, and is not particularly limited thereto, but is preferably immersed and extracted for 30 to 60 hours, 35 to 55 hours, and 40 to 50 hours.

In order to obtain the best antibacterial activity of the extract, it may be extracted through a process of immersing the immunized brown mealworm larva powder in 10 to 30% (v / v) acetic acid aqueous solution for 40 to 50 hours.

After the step c), d) centrifuging the extract obtained from the step c), recovering the supernatant, filtering and concentrating the extract may be further included. That is, impurities can be removed by purifying the extract, and the purification process is not particularly limited as long as it is a conventional method, but may preferably be a filtration process through a filter. The concentration is not particularly limited as long as it is a conventional concentration method, but may be used after concentrating through centrifugation and then drying. The drying may be performed through a drying process such as freezing or spraying.

Hereinafter, the present invention will be described in more detail through examples and the like, but the scope and contents of the present invention are reduced or limited by the examples below and cannot be interpreted. In addition, based on the disclosure of the present invention including the following examples, it is clear that a person skilled in the art can easily practice the present invention for which no experimental results are specifically presented, and the patents to which such variations and modifications are attached. It goes without saying that it falls within the scope of the claims.

In addition, the experimental results presented below are only representative experimental results of the above examples and comparative examples, and each effect of various embodiments of the present invention that is not explicitly presented below will be described in detail in the corresponding section.

Example 1. Immunized brown mealworm larvae extract

Brown mealworm larvae ( Tenebrio molitor larvae, TML) (species larvae) were injected into the body cavity of Lactobacillus plantarum ( Lactobacillus plantarum ) at a concentration of 1×10 ¹⁰ CFU, and immunoreacted for 12 hours. The brown mealworm larvae in which the immune response was induced were rapidly frozen using liquid nitrogen and then pulverized. The pulverized immunized brown mealworm larvae powder was immersed in 20% (v/v) glacial acetic acid for 48 hours, and then centrifuged at 4,500 rpm and 4° C. for 30 minutes. Only the supernatant was recovered, filtered with a 0.45 um syringe filter, concentrated in a vacuum concentrator centrifuge for 9 to 24 hours, diluted in 20% (v / v) acetic acid solution (glacial acetic acid), and immunized brown mealworm larvae An extract was prepared.

Experimental Example 1. Optimal induction conditions for overexpression of various antibacterial peptides in the body of brown gourd larvae

Brown mealworm larvae ( Tenebrio molitor larvae, TML) are edible insects registered in the Food Code. Before preparing the antibacterial extract from the brown gourd larvae, the maximum expression time of various antimicrobial peptides (AMPs) was determined.

First, Lactobacillus plantarum 1 × 10 ¹⁰ CFU concentration was injected into the body cavity of brown mealworm larvae, and various immune response times (6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours) bred with After extracting the body fluids from the brown mealworm larvae whose immune response was induced at various times, four antibacterial agents consisting of attacin, defencin, tenenecin, and coleoptericin were extracted. The transcriptome expression pattern of the peptide was confirmed through qRT-PCR.

For the qRT-PCR analysis, total mRNA was extracted from the body fluids of larvae for each immune induction period, and qRT-PCR analysis was performed with a gene-specific primer set (Table 1) as follows, attasin and defensin for each immune induction period The expression levels of defencin, tenecin, and coleoptericin were measured. Specifically, it was performed using a Rotor-Gene-Q (Qiagen, USA) equipped with a 72-well rotor. All samples were mixed with 5 μl of SYBR GreenTM master mix (Elpis-biotech, Korea), 3 μl of cDNA and 2.5 μM of gene-specific primer set (Table 1), and RT-PCR was performed as follows; 40 cycles of 10 sec at 94 °C, 10 sec at 62 °C and 10 sec at 72 °C were repeated.

division Sequence (5‘-3’) attacin forward SEQ ID NO: 1: 5'-GAAACGAAATGGAAGGTGGA-3' reverse SEQ ID NO: 2: 5'-TGCTTCGGCAGACAATACAG-3' defencin forward SEQ ID NO: 3: 5'-AAATCGAACAAGGCCAAACAC-3' reverse SEQ ID NO: 4: 5'-GCAAATGCAGACCCTCTTTC-3' tenecin forward SEQ ID NO: 5: 5'-CAGCTGAAGAAATCGAACAAGG-3' reverse SEQ ID NO: 6: 5'-CAGACCCTCTTTCCGTTACAGT-3' coleoptericin forward SEQ ID NO: 7: 5'-GGACAGAATGGTGGATGGTC-3' reverse SEQ ID NO: 8: 5'-CTCCAACATTCCAGGTAGGC-3'

Figure 2 shows that Lactobacillus plantarum was injected into brown mealworm larvae, and according to various immune induction periods (6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours), atacin (attacin) ) (a), defencin (b), tenenesin (c) and choleoptericin (coleoptericin) (d) is a graph measuring the expression levels.

As shown in Figure 2, it was expressed at a high level from 12 hours after injection and then gradually decreased. Through this, it was confirmed that the antimicrobial peptides were expressed for 10 to 30 hours, and the expression of all antimicrobial peptides was maximized at 10 to 15 hours. In particular, since the expression was highest when the immunity induction period was 12 hours, the immunity induction period was set to 12 hours in subsequent experiments.

Experimental Example 2. Optimal extraction conditions from immunized brown gourd larvae

Brown mealworm larvae ( Tenebrio molitor larvae, TML) were injected into the body cavity of Lactobacillus plantarum ( Lactobacillus plantarum ) at a concentration of 1×10 ¹⁰ CFU, and immunoreacted for 12 hours. The brown gourd larvae in which the immune response was induced were prepared in various solvents and various methods to obtain extracts, and then used as samples for measuring antibacterial activity.

With three extraction solvents: distilled water (D.W), ethanol (ethanol, EtOH), and 20% (v/v) acetic acid solution (glacial acetic acid, 20% (v/v) AcOH), boil for 20 minutes or (Boil), soaking for 48 hours (Soak), or ultrasonic disruption (Sonication) were combined to prepare immunized brown mealworm larvae extracts under a total of nine extraction conditions.

The antibacterial efficacy of each extract was confirmed through inhibition zone assay. The strain used in the antibacterial activity test was Escherichia coli , which is classified as a harmful bacteria in the production of soy sauce in the Food Code. E. coli was grown under optimal culture conditions and then cultured in an optimal liquid medium such as Luria-Bertani broth for about 8 hours to adjust the turbidity of the culture medium to OD 600 = 1.25, and then the inhibition zone assay was performed. 0.1 μl of the culture medium of E. coli was evenly spread on an optimal medium such as nutrient agar medium, and the concentration of the extract was 500 μg/disc or 1000 μg/disc. A sterilized paper disc (Whatman chromatography paper, 6 mm diameter) was used after absorbing and drying the sample under aseptic conditions. The antibacterial activity of the extract was measured, and the experiment was repeated three times. As a negative control group, only the corresponding extraction solvent was used.

Figure 3a is an antibacterial activity analysis result for immunized brown mealworm larvae extract (distilled water) prepared by various extraction methods, and Figure 3b is an antibacterial activity analysis result for immunized brown mealworm larvae extract (ethanol) prepared by various extraction methods 3c is the result of analyzing the antibacterial activity of the immunized brown gourd larvae extract (20% (v / v) acetic acid solution) prepared by various extraction methods.

As shown in FIG. 3, both the immunized brown mealworm larvae extract (distilled water) and the immunized brown mealworm larvae extract (ethanol) did not show antibacterial activity regardless of the extraction method. On the other hand, it was confirmed that the immunized brown gourd larvae extract (20% (v / v) acetic acid solution) increased the antibacterial activity in a concentration-dependent manner. When the extract was prepared using a 20% (v / v) acetic acid solution from the immunized brown mealworm larvae, it was confirmed that immersion for 48 hours showed the clearest zone, the immunized brown mealworm larvae extract of the present invention It can be seen that the one prepared by immersing for 48 hours using a 20% (v/v) acetic acid solution is most preferable.

Experimental Example 3. Analysis of antibacterial activity of immunized brown gourd larvae extract

It was analyzed whether the immunized brown mealworm larvae extract of Example 1 prepared under optimized conditions through Experimental Example 1 and Experimental Example 2 had selective antibacterial activity against beneficial and harmful bacteria.

The strains used in the antibacterial activity test were Escherichia coli , which is classified as harmful bacteria in the production of soy sauce in the food code, Bacillus cereus , and Staphylococcus aureus , which are food poisoning bacteria. As in Experimental Example 2, the antibacterial activity was analyzed by inhibition zone assay. A 20% (v/v) acetic acid solution from the negative control group was used, and the concentration of the immunized brown mealworm larvae extract of Example 1 was 500 μg/disc or 1000 μg/disc.

As the beneficial bacteria , Lactobacillus acidophilus, a lactic acid bacterium inhabiting the human intestine, was used. The antibacterial activity thereof was assayed by a radial diffusion assay (RDA). For RDA analysis, pre-cultured Lactobacillus acidophilus in sterilized RDA underlay gel (9 mM sodium phosphate, 1 mM sodium citrate, pH7.4, 1% low electroendosmosis agarose, 0.03% TSB) ( Macfarland turvidity No 0.5) was mixed and poured into a 100 mm square plate and hardened. A hole with a diameter of 3.5 mm was made in the underlay gel, and the immunized brown mealworm larvae extract of Example 1 was put into each hole at a concentration of 500 μg / 10 μl or 1000 μg / 10 μl, and the antibacterial peptide was spread at 37 ° C. for 3 hours. After incubation, an overlay gel (6% TSB, 1% low electroendosmosis agarose) for RDA was poured over it, solidified, and incubated at 37 °C for 18 hours. Then, the size of the clear zone was measured to compare the antibacterial activity. A 20% (v/v) acetic acid solution from the negative control was used.

4 is an analysis result of the antibacterial activity of the immunized brown gourd larvae extract prepared in Example 1 against Escherichia coli, Bacillus cereus , which is a food poisoning bacterium, and Staphylococcus aureus .

As shown in FIG. 4, it was confirmed that the immunized brown gourd larvae extract prepared in Example 1 had superior inhibitory activity against harmful bacteria than the 20% (v/v) acetic acid solution used as a negative control. The immunized brown mealworm larvae extract prepared in Example 1 gradually increased its antibacterial activity against harmful bacteria in a concentration-dependent manner.

5 is an analysis result of the antibacterial activity of the immunized brown mealworm larvae extract prepared in Example 1 against Lactobacillus acidophilus .

As shown in FIG. 5, it was confirmed that the immunized brown mealworm larvae extract of Example 1 did not have inhibitory activity (antibacterial activity) against beneficial bacteria.

Experimental Example 4. Analysis of antibacterial activity against harmful fungi and beneficial fungi of immunized brown gourd larvae extract

Experiments on antibacterial activity against two harmful fungi ( Aspergillus flavus and Aspergillus parasiticus ) that produce aflatoxins among fungi and Aspergillus oryzae , a beneficial fungus used in meju production, using the immunized brown mealworm larvae extract of Example 1 In the same manner as in Example 2, inhibition zone assay was performed. As a negative control group, a 20% (v/v) acetic acid solution was used.

6 is an analysis result of the antibacterial activity of the immunized brown gourd larvae extract prepared in Example 1 against Aspergillus flavus and Aspergillus parasiticus , and FIG. ( Aspergillus oryzae ) Antibacterial activity analysis result of the immunized brown mealworm larvae extract prepared in Example 1.

As shown in FIGS. 6 and 7, the immunized brown mealworm larvae extract prepared in Example 1 was confirmed to have antibacterial activity against two harmful fungi and one beneficial fungus that produce aflatoxin. That is, it can be seen that the immunized brown mealworm larvae extract prepared in Example 1 has excellent antibacterial activity against fungi.

Experimental Example 5. Minimum inhibitory concentration assay of immunized brown gourd larvae extract

Using the immunized brown mealworm larvae extract prepared in Example 1, Escherichia coli , food poisoning bacteria Bacillus cereus , and Staphylococcus aureus , which are classified as harmful bacteria in the production of soy sauce in the food code, The minimum inhibitory activity (minimal inhibitory concentration, MIC) was measured.

After shaking each strain in a liquid medium at 37 °C and 150 rpm for 18 hours, 2% of the culture medium was added to a new liquid medium, and secondary culture was performed under the same conditions to a concentration of 4×10 ⁶ cfu/ml. did After dispensing 90 μl of bacterial culture medium (1×10 ⁵ cfu/ml) into each well of a 96-well microplate, the immunized brown mealworm larvae extract of Example 1 was added in various concentrations to each well in stages. Thereafter, after incubation at 37 ° C. for 18 hours, absorbance was measured using a spectrophotometer (600 nm) to measure MIC, which is shown in Table 2.

Name of harmful bacteria extract concentration 80 µg 90 µg 100 µg E. coli 1.152 0.208 0.044 B. cereus 0.646 0.267 0.042 S. aureus 1.103 0.164 0.041

As shown in Table 2, the minimum inhibitory activity of the immunized brown mealworm larvae extract of Example 1 against three harmful bacteria was confirmed to be 1×10 ⁵ CFU/100 μg.

Experimental Example 6. Stability analysis of immunized brown gourd larvae extract

1) Thermal stability analysis

The antibacterial activity of the immunized brown mealworm larvae extract of Example 1 was measured according to the temperature. Specifically, the change in the minimum inhibitory activity concentration of the immunized brown mealworm larvae extract of Example 1 according to the heating temperature and time was measured in the same manner as in Experimental Example 5. The heating temperature and time conditions and the results thereof are shown in Table 3.

heating conditions extract concentration 80 µg 90 µg 100 µg 40 ℃, 36 hours 1.148 0.195 0.051 40 ℃, 48 hours 1.195 0.210 0.043 40 ℃, 72 hours 1.145 0.187 0.041 100 ℃, 5 sec 1.200 0.174 0.038 100 ℃, 10 sec 1.317 0.147 0.038 100 ℃, 15 sec 1.166 0.121 0.042

As shown in Table 3, it was confirmed that no change was observed in the minimum inhibitory activity concentration of the immunized brown mealworm larvae extract of Example 1 according to the heating temperature and time. That is, it can be seen that the antibacterial activity of the immunized brown mealworm larvae extract of Example 1 is stably maintained for a long time at 40 ° C or higher.

2) pH stability analysis

The antibacterial activity of the immunized brown mealworm larvae extract of Example 1 was measured according to pH conditions. Since the immunized brown mealworm larvae extract of the present invention can be used in soy sauce, it was investigated whether it stably shows activity within the pH range of commercially available red pepper paste and soy sauce. Since the pH of gochujang is 5.04 ± 0.07 and the pH of soy sauce is 4.74 ± 0.09, the pH conditions were set to pH 4 to 6 based on this.

pH extract concentration 80 µg 90 µg 100 µg 4 1.138 0.217 0.040 5 1.097 0.225 0.041 6 1.126 0.237 0.036

As shown in Table 4, it can be seen that the immunized brown mealworm larvae extract of Example 1 has stability to pH because the minimum inhibitory activity concentration does not change with pH change.

3) Salinity stability analysis

The antibacterial activity of the immunized brown mealworm larvae extract of Example 1 was measured under various salt concentration conditions. Since the immunized brown mealworm larvae extract of the present invention can be used in soy sauce, it was investigated whether it stably shows activity in the salt range of commercially available red pepper paste and soy sauce. As a result of measuring the salinity of gochujang and soy sauce by the Mohr method, the salinity of gochujang was 6.96 ± 0.30% and that of soy sauce was 19.33 ± 1.68%. were set at 20% and 25%.

salt(%) extract concentration 80 µg 90 µg 100 µg 5 4 1.138 0.217 7.5 5 1.097 0.225 10 6 1.126 0.237 15 0.710 0.244 0.042 20 0.823 0.243 0.041 25 0.748 0.242 0.042

As shown in Table 5, it can be seen that the immunized brown mealworm larvae extract of Example 1 has stability against high salt concentration conditions because the minimum inhibitory activity concentration does not change with various salt concentration changes.

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Claims (4)

An antibacterial composition containing an immunized brown gourd larvae extract as an active ingredient,
The immunized brown mealworm larvae extract is treated with Lactobacillus plantarum at a concentration of 1 × 10 ⁸ CFU / ml to 1 × 10 ¹² CFU / ml in the body of the brown mealworm larvae, and for 10 to 15 hours After immunization, it is characterized by rapidly freezing with liquid nitrogen and extracting the pulverized powder with a 20% (v / v) aqueous acetic acid solution,
An antibacterial composition characterized in that it has a stable antibacterial activity under high temperature, high salinity and high pH conditions. delete a) injecting Lactobacillus plantarum into the body cavity of brown mealworm larvae at a concentration of 1×10 ⁸ CFU/ml to 1×10 ¹² CFU/ml;
b) inducing an immune response by leaving the brown mealworm larvae injected with Lactobacillus plantarum for 10 to 15 hours; and
c) rapidly freezing the immunized brown mealworm larvae obtained in step b) with liquid nitrogen and immersing in a 20% (v/v) acetic acid aqueous solution to obtain an extract from the immunized brown mealworm larvae; A method for producing an antimicrobial composition having stable antibacterial activity under high salinity and high pH conditions. delete

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