KR101374267B1 - Novel antibiotic peptide isolated from Nordotis discus discus and use thereof - Google Patents

Abstract

The present invention relates to antibiotic peptides having a novel amino acid sequence, and more particularly, to Nordotis of the present invention. discus The antibiotic peptides Nod-1 and Nod-2 having the amino acid sequence of SEQ ID NO: 1 or 2 isolated from discus ) are Escherichia coli ) or Staphylococcus aureus ) has excellent antibacterial activity and is stable to heat, so it is useful as an active ingredient of antibacterial and antiseptic composition, pharmaceutical composition for antimicrobial, cosmetic composition showing antimicrobial activity, health food composition for antimicrobial, pesticide preparation and feed additive for antimicrobial. Can be used.

Description

Novel antibiotic peptide isolated from Nordotis discus discus and use thereof

The present invention relates to novel antibiotic peptides and their use.

Plants and animals are known to naturally produce antimicrobial peptides [Bevins et al., Ann . Rev. Biochem ., 1990, 59 , 395-414. The antimicrobial activity of these peptides plays an important role in host defense and the innate immune system [Boman, HG, Cell, 65, 205, 1991; Boman, HG, Annu. Rev. Microbiol., 13, 61, 1995], these antimicrobial peptides are cysteine-rich β-sheet peptides, α-helical amphiphilic molecules, and proline according to amino acid sequence. It is classified as a proline-rich peptide [Mayasaki et al., Int. J. Antimicrob . Agents , 1998, 9 , 269-280]. The most common of these structures is a cysteine residue-free molecule that forms an amphiphilic alpha helical, and cecropin found in insects is a representative antibiotic peptide structure.

In addition, in addition to the peptide produced in vivo, the bacteriocin protein is also known to perform a function as an antibiotic that is secreted to compete with the surrounding homologous or heterologous bacteria to kill harmful bacteria. These bacteriocins are classified into three classes according to their structure: lantibiotics, nonlantibiotics, and signal peptides (Cintas et al., J. Bad . , 1998, 180 , 1988-1994]. Peptides or bacteriocins, which are produced in vivo and perform antibiotic functions, can solve problems such as antibiotic resistance and antibiotic shock caused by antibiotics caused by conventional antibiotics. Can be.

Therefore, efforts to replace existing antimicrobials with natural antimicrobials have been steadily progressing. Here, the natural antimicrobial agent is an antimicrobial agent selected from natural materials or microorganisms to maximize biochemical or physiological differences, selectively control viruses, bacteria, yeasts, molds, etc., and harm the human body or minimize toxicity. . Currently reported natural antimicrobial agents include henokithiol in cypress, megnonole in magnolia, and grapefruit seed extract DF-100, but the scale currently used is very small. Currently, natural herbal extracts have been reported as safe natural antimicrobial agents that inhibit cell proliferation mechanisms, have a high bactericidal effect against microorganisms, are nontoxic as plant natural bactericidal disinfectants, and are not corrosive. However, plants currently used as natural antimicrobial agents are limited in their use as natural antimicrobial agents due to their cultivation scale is small or many are difficult to cultivate in Korea as foreign species.

Abalone is a mollusk of the abalone family of primitive abalone. Generally, more than 100 varieties of abalone are known around the world.The Korean abalone, Haliotis discus hannai , and turbulent black abalone, Haliotis discus), the end of rollover (N. gigantea), Siebold abalone (H. Sieboldii), ohbunjagi (Sulculus diversicolor aquatilis), Sack ohbunjagi (H. diversicolor supertexta ), round abalone ( Nordotis) discus discus ) and the like. Abalone is widely distributed according to species from 4 to 20 m in depth. Most of the wild products in Korea are abalone, and abalone and five-molecule abalone are caught in Jeju, and most of the farming is abalone. The abalone lives for about 12 years and lives mainly in tropical and temperate seas, where the water temperature is 10-23 ° C. It lives on shallow reefs or small rocks. Abalone has abundant protein and vitamins, so it has excellent effects on skin care, tonic, postpartum cooking, weak constitution, and is widely used for food as well as medicinal use.

Therefore, the inventors of the present invention abalone ( Nordotis) discus discus ) as starting material, in particular abalone ( Nordotis) discus extract, heat, isolate and purify from discus ) to obtain a novel heat stable peptide, the heat stable peptide is Escherichia coli ) or Staphylococcus The present invention was completed by elucidating that it can be usefully used as a natural antibiotic since it shows excellent antibacterial activity against aureus ).

It is an object of the present invention to provide a novel antibiotic peptide derived from abalone.

Still another object of the present invention is to provide a method for preparing the antibiotic peptide.

Another object of the present invention to provide an antimicrobial composition using the antibiotic peptide.

Another object of the present invention to provide an antiseptic composition using the antibiotic peptide.

In order to achieve the above object, the present invention provides an antibiotic peptide having the amino acid sequence of SEQ ID NO: 1.

In addition,

1) crushing the abalone and then extracting with water, alcohol or an aqueous alcohol solution;

2) recovering the supernatant fraction of the extract by centrifuging the extract of step 1);

3) recovering the supernatant fraction by heating the supernatant fraction of step 2) and then centrifuging; And

4) It provides a method for producing an antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2, comprising the step of separating and purifying the fraction of step 3).

The present invention also provides an antimicrobial and antiseptic composition comprising the antibiotic peptide as an active ingredient.

 The present invention also provides an antimicrobial pharmaceutical composition comprising the antibiotic peptide as an active ingredient.

The present invention also provides an antimicrobial cosmetic composition comprising the antibiotic peptide as an active ingredient.

The present invention also provides an antimicrobial health food composition comprising the antibiotic peptide as an active ingredient.

In addition, the present invention provides a pesticide preparation comprising the antibiotic peptide as an active ingredient.

The present invention also provides a food preservative comprising the antibiotic peptide as an active ingredient.

In addition, the present invention provides a cosmetic preservative comprising the antibiotic peptide as an active ingredient.

The present invention also provides a pharmaceutical preservative comprising the antibiotic peptide as an active ingredient.

In addition, the present invention provides an antimicrobial feed additive comprising the antibiotic peptide as an active ingredient.

The antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 of the present invention is Escherichia coli ) or Staphylococcus aureus ) shows excellent antimicrobial activity and is stable to heat, antimicrobial and antiseptic composition, antimicrobial pharmaceutical composition, antimicrobial cosmetic composition, antimicrobial health food composition, pesticide preparation, food preservative, cosmetic preservative, It can be usefully used as an active ingredient of pharmaceutical preservatives and antimicrobial feed additives.

FIG. 1 is overturned (Nordotis discus discus ) is a diagram illustrating a series of steps to extract the peptide-containing extract from.
Figure 2 shows the abalone extract Escherichia coli ( Esherichia coli ) and Staphylococcus aureus ) shows the results of measuring the antimicrobial activity.
3 is a diagram showing the results of separating two single peptides using C18 HPLC from the abalone extract of 10 kDa or less obtained through ultrafiltration.
Figure 4 is a diagram showing the results confirmed by the peptide isolated using HPLC using SDS-PAGE (Sodoum Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis).
FIG. 5 shows Nod-1 and Nod-2, respectively, in Escherichia coli ) and Staphylococcus aureus ) shows the results of measuring the antimicrobial activity.
6 is a diagram showing the results of confirming the amino acid sequence of the isolated peptide.
Figure 7 is a diagram showing the result of confirming the molecular weight of the peptide isolated using Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS).

Hereinafter, the present invention will be described in detail.

The present invention provides antibiotic peptides having the amino acid sequence of SEQ ID NO: 1 or 2.

The antibiotic peptide is overturned ( Nordotis) discus discus ), but is not limited thereto.

The abalone may be used without limitation, those grown specifically, commercially available.

Preferably, the antibiotic peptide has thermal stability at 60 ° C to 80 ° C, and more preferably at 70 ° C, but is not limited thereto.

Specifically, the antibiotic peptide has a molecular weight of 10 kDa or less, more preferably 5 kDa to 8 kDa, and most preferably 6.1 kDa, but not limited thereto.

The antibiotic peptide has antimicrobial activity, specifically Escherichia coli coli) or Staphylococcus aureus (S taphylococcus aureus ) preferably have antimicrobial activity, but is not limited thereto.

In order to extract antibiotic peptides, the inventors used Nordotis discus discus ) was extracted with a solvent to obtain an extract. The extract was centrifuged to recover the supernatant fraction of the extract, the supernatant fraction was heated and centrifuged to recover the supernatant fraction again, and the supernatant fraction was separated and purified to obtain an antibiotic peptide.

At this time, the separation and purification is first separated the extract of 10 kDa or less through ultra-filtration of the supernatant fraction, and the extract obtained from the above step C ₁₈ -column (5μm, 300Å, 4.6 × 250 mm, Vydac, Hesperia, CA) Isolated peptide was obtained using Reverse Phase HPLC (Shimadzu, Japan). The isolated peptides were named Nod-1 and Nod-2 peptides, respectively. The isolated peptide was confirmed to have a molecular weight of about 6.1 kDa using SDS-PAGE (see FIGS. 4A and B). The isolated peptide is Escherichia coli ), Staphylococcus aureus ) was confirmed to have antimicrobial activity. Staphylococcus All peptides isolated against aureus were excellent in antimicrobial activity, Escherichia coli ), it was confirmed that Nod-2 had better antimicrobial activity than Nod-1 (see A and B of FIG. 5).

In order to confirm the amino acid sequence and molecular weight of the isolated peptide, the inventors confirmed using an amino acid analyzer and MALDI-MS (Matrix Assisted Lasr Desorption Ionizaion-Mass Spectrometry), where Nod-1 has the amino acid sequence of SEQ ID NO: 1. It is confirmed that the molecular weight is 6130.9 Da (see FIG. 7A), the Nod-2 has the amino acid sequence of SEQ ID NO: 2 (see FIG. 6), and the molecular weight is 6116.7 Da (see FIG. 7B).

Thus, the novel peptide of the present invention is Escherichia coli ) or Staphylococcus It can be seen that the antibiotic peptide exhibits excellent antimicrobial activity against aureus ) and has thermal stability at 60 ° C to 80 ° C.

In addition,

1) Overture (Nordotis discus milling discus ), followed by extraction with water, alcohol or an aqueous solution of alcohol;

2) recovering the supernatant fraction of the extract by centrifuging the extract of step 1);

3) recovering the supernatant fraction by heating the supernatant fraction of step 2) and then centrifuging; And

4) It provides a method for producing an antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 comprising the step of separating and purifying the supernatant fraction of step 3).

In the above production method, abalone of step 1) can be used without limitation, specifically grown and commercially available.

In the above production method, the alcohol of step 1) is preferably methanol or ethanol, the aqueous solution is more preferably 30% to 80% (v / v) aqueous solution, 50% (v / v) aqueous solution Most preferred but not limited thereto.

In the manufacturing method, the heating of step 4) is preferably heated to 60 ℃ to 80 ℃, more preferably 70 ℃ but is not limited thereto.

In the above production method, the separation of step 4) is preferably, but not limited to, separating the material having a size of 10 kDa or less with an ultra-fine filter.

In the above production method, the purification of step 4) is preferably using reverse phase HPLC, but is not limited thereto.

The present invention also provides an antimicrobial or antiseptic composition comprising an antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.

The antimicrobial or antiseptic composition is specifically Escherichia coli ( Esherichia coli ) and Staphylococcus aureus ) preferably have an antimicrobial activity, but is not limited thereto.

The antiseptic composition includes food preservatives, cosmetic preservatives or pharmaceutical preservatives. The food preservatives, cosmetic preservatives and pharmaceutical preservatives are additives used to prevent the deterioration, decay, discoloration and chemical changes of foods or drugs, including fungicides and antioxidants, and inhibit the growth of microorganisms such as bacteria, fungi and yeasts. Functional antibiotics such as inhibiting or sterilizing the growth of decay microorganisms in food and pharmaceuticals are included. Ideal conditions for such preservative compositions should be nontoxic and effective in trace amounts.

The antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 of the present invention is Escherichia coli ) or Staphylococcus aureus ) shows excellent antimicrobial activity and has thermal stability at 60 ℃ to 80 ℃, it can be usefully used as an antibacterial and antiseptic composition.

The present invention also provides an antimicrobial pharmaceutical composition comprising an antibiotic peptide having an amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.

The antimicrobial pharmaceutical composition is specifically Escherichia coli ( Esherichia coli ) or Staphylococcus aureus (S taphylococcus aureus ) to have an antimicrobial activity, but is not limited thereto.

The antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 of the present invention is Escherichia coli ) or Staphylococcus aureus ) shows excellent antimicrobial activity and has a thermal stability at 60 ℃ to 80 ℃, it can be usefully used as an active ingredient of the pharmaceutical composition for antibacterial.

The antibiotic peptide of the present invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

In other words, the antibiotic peptide of the present invention can be administered in various parenteral formulations, and when formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao paper, Liu ling, glycerogelatin and the like can be used.

 In addition, the antibiotic peptide of the present invention can be used in admixture with a number of carriers (pharmaceutically acceptable) such as physiological saline or organic solvent, carbohydrates such as glucose, sucrose or dextran, to increase the stability or absorption Antioxidants such as ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers may be used as medicaments.

The effective dose of the antibiotic peptide of the present invention is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and may be administered once to three times a day.

In the pharmaceutical composition of the present invention, the total effective amount of the novel peptide of the present invention may be administered to the patient in a single dose by bolus form or by infusion for a relatively short period of time. Multiple doses may be administered by a fractionated treatment protocol with long term administration. Since the concentration of the effective dose of the patient is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient, in view of this point, Lt; RTI ID = 0.0 > of < / RTI > novel peptide as a pharmaceutical composition.

The present invention also provides an antimicrobial cosmetic composition comprising an antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.

The antimicrobial cosmetic composition is specifically Escherichia coli ( Esherichia coli ) or Staphylococcus aureus (S taphylococcus aureus ) to have an antimicrobial activity, but is not limited thereto.

The antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 of the present invention is Escherichia coli ) or Staphylococcus aureus ) shows excellent antimicrobial activity, and has a thermal stability at 60 ℃ to 80 ℃, it can be usefully used as an active ingredient of the cosmetic composition for antibacterial.

The cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the antibiotic peptide, and includes, for example, conventional auxiliaries such as sulfates, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

In the cosmetic composition of the present invention, the peptide of the present invention may be added to the cosmetic composition usually contained in an amount of 0.1 to 50% by weight, preferably 1 to 10% by weight.

The cosmetic composition of the present invention can be prepared into any of the formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion (skin), nutrition lotion (milk lotion), nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder .

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanta, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide are used as carrier components. Can be.

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, and microcrystals are used as carrier components. Soluble cellulose, aluminum metahydroxy, bentonite, agar or tragacanta and the like can be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component is selected from aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

The present invention also provides an antimicrobial health food composition comprising an antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.

The antibacterial health food composition is specifically Escherichia coli ( Esherichia coli) or Staphylococcus aureus (S taphylococcus aureus ) preferably have an antimicrobial activity, but is not limited thereto.

The antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 of the present invention is Escherichia coli ) or Staphylococcus aureus ) shows an excellent antimicrobial activity, and has a thermal stability at 60 ℃ to 80 ℃, it can be usefully used as an active ingredient of the health food composition for antibacterial.

The kind of the food composition is not particularly limited. Examples of the foods to which the above substances can be added include dairy products including dairy products such as drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The antibiotic peptide of the present invention may be added to a food product as it is, or used with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). In general, the amount of the peptide in the dietary supplement may be added at 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The antimicrobial health food composition of the present invention is not particularly limited to other ingredients except the fermented product as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the antibiotic peptides of the present invention are various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the antibiotic peptides of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not so critical but the antibiotic peptides of the present invention are generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight.

The present invention also provides a pesticide preparation, food preservatives, cosmetic preservatives, pharmaceutical preservatives or antimicrobial feed additive comprising an antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 as an active ingredient.

Antibiotic peptide having the amino acid sequence of SEQ ID NO: 1 or 2 of the present invention is E. scherichia coli ) or Staphylococcus aureus ) has excellent antimicrobial activity and thermal stability, can be useful as an active ingredient of pesticides, food preservatives, cosmetic preservatives, pharmaceutical preservatives or antimicrobial feed additives.

Hereinafter, the present invention will be described in detail with reference to examples and preparation examples.

However, the following Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Preparation Examples.

< Example  1> abalone( Nordotis discus discus Extract from

Overture (Nordotis discus discus ) was frozen through liquid nitrogen and pulverized with a grinder, and the powder was added well to 50% methanol for 1 hour at room temperature and mixed well. After centrifugation for 20 minutes at 12,000 rpm to recover the supernatant fraction of the extract. The supernatant is heated at 70 ° C. and centrifuged again at 12,000 rpm for 20 minutes to recover a thermally stable supernatant fraction. Ultrafiltration gave an extract of 10 kDa or less.

< Example  2> Confirm the antimicrobial activity of Abalone extract

In order to measure the antimicrobial activity of the abalone extract obtained from the <Example 1> was experimented as follows.

Specifically, the number of bacteria is mixed with 50 μl diluted in sodium phosphate medium and 50 μl of abalone extract (100 μg) so that 5 × 10 ⁵ cells per 1 ml, and in the negative control only by dispensing the medium instead of peptide in a 37 ℃ incubator Incubated for 4 hours. Then, in order to directly check the antimicrobial activity by diluting and spreading it in agar medium and incubated for 18 hours in 37 ℃ incubator.

As a result, Escherichia coli ), the antimicrobial activity of E. coli was observed in the negative control group E. coli was observed in the negative control (Fig. 2 A), E. coli was not observed after the abalone extract treatment (A2 in Figure 2) It was confirmed.

In addition, Staphylococcus aureus ), as a result of measuring the antimicrobial activity, Staphylococcus aureus was observed in the negative control group (Fig. 2 B), while yellow Staphylococcus aureus was not observed after the abalone extract treatment (B2 in Figure 2) yellow of the abalone extract Antimicrobial activity against staphylococci was confirmed.

< Example  3> Of peptide  detach

In order to separate the peptide from the abalone extract obtained from the <Example 1> was carried out as follows.

Specifically, abalone extract C _{18 -} was purified using a column (5μm, 300 Å, 4.6 x 250 mm, Vydac, Hesperia, CA) reversed phase HPLC (Reverse Phase HPLC) (Shimadzu , Japan). At this time, the solution was eluted by a gradient (linear gradient, 10-60%, 1 ml / min, 50 minutes) of the buffer solution (acetoanitrile B with 0.1% TFA) (FIG. 3).

As a result, the abalone extract was purified to isolate a single peptide. Two peaks (peaks) were identified (A of FIG. 3), and the front peak (B of FIG. 3) and the rear peak (C of FIG. 3) were identified, respectively. Each of the isolated peptides named the front peak Nod-1 and the back peak the Nod-2 peptide.

< Example  3> Two single Of peptide  Detach Confirm

In order to confirm the separation of the two single peptides obtained in Example 2 was carried out as follows.

Specifically, Tricine SDS-PAGE (Tricine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) was performed. At this time, two single peptides and standard protein were loaded on 16.5% tricin acrylamide gel. After electrophoresis, the gel was fixed with 5% glutaraldehyde, stained with coomassie brilliant blue G-250, and destained with destaining buffer (10% acetic acid) to confirm the band of the peptide. .

As a result, the separation of the peptide of the front peak (A of FIG. 4) and the rear peak (B of FIG. 4) of <Example 2> was confirmed.

< Example  4> two single Of peptide  Antimicrobial activity measurement

In order to measure the antimicrobial activity of a single peptide obtained from the <Example 3> was experimented as follows.

Specifically, the number of bacteria was mixed 50 μl of bacteria diluted with sodium phosphate medium and 50 μl of peptide (25 μg) so that 5 × 10 ⁵ cells per ml, and in the negative control only the medium instead of peptide was dispensed in 4 ℃ 37 Incubated for hours. Then, in order to directly check the antimicrobial activity by diluting and spreading it in agar medium and incubated for 18 hours in 37 ℃ incubator.

As a result, Escherichia coli ), the E. coli was observed in the negative control group (A1 in FIG. 5), whereas the antimicrobial activity was observed after Nod-1 peptide treatment (A2 in FIG. 5), and after the Nod-2 peptide treatment. Antimicrobial activity was observed (A3 of Figure 5).

In addition, Staphylococcus aureus ), the antimicrobial activity was observed in the negative control group (Fig. 5 B1), while the antibacterial activity was observed after the Nod-1 peptide treatment (Fig. 5 B2), after the Nod-2 peptide treatment Antimicrobial activity was observed.

In summary, both Staphylococcus aureus and Nod-1 and Nod-2 were superior in antimicrobial activity, but E. coli was able to confirm that Nod-2 had better antimicrobial activity than Nod-1.

The preparation examples for the composition of the present invention are illustrated below.

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

<1-1> Sanje  Produce

20 mg of peptide of the present invention

Lactose 20 mg

After mixing the above components, the mixture was packed in an airtight container to prepare a powder.

<1-2> Preparation of tablets

10 mg of peptide of the present invention

Corn starch 100 mg

100 mg of milk

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

10 mg of peptide of the present invention

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

<1-4> Liquid  Produce

20 mg of peptide of the present invention

10 g of isomerized sugar

5 g of mannitol

Purified water

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

<1-5> Preparation of Injection

10 μg / ml peptide of the invention

Dilute hydrochloric acid BP until pH 7.6

Sodium chloride up to 1 ml for injection

The peptide of the present invention was dissolved in a suitable volume of main sodium chloride BP, and the pH of the resulting solution was adjusted to pH 7.6 using dilute hydrochloric acid BP, and the volume was adjusted using a main volume of sodium chloride BP and mixed well. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.

<1-6> Preparation of the ring

1 g of the peptide of the present invention

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-7> Preparation of Granules

150 mg of the peptide of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> manufacture of cosmetics

<2-1> flexible cosmetics ( skin )

In order to produce antimicrobial softening longevity comprising the peptide of the present invention, it can be formulated according to the production method in the general cosmetic field by combining as shown in the following [Table 1].

ingredient Content (% by weight) Peptides of the Invention 0.1 to 30% 1,3-butylene glycol 3.0 glycerin 5.0 Polyoxyethylene (60) hardening castor oil 0.2 ethanol 8.0 Citric acid 0.02 Sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water To 100

<2-2> Nourishing lotion (lotion)

In order to produce an antimicrobial nutrient for longevity comprising the peptide of the present invention, it may be formulated according to the preparation method in the conventional cosmetic field by combining as described in the following [Table 2].

ingredient Content (% by weight) Peptides of the Invention 0.1 to 30% 1,3-butylene glycol 8.0 glycerin 5.0 Squalane 10.0 Polyoxyethylene sorbitan monooleate 2.0 Flux oil 0.1 to 30% 1,3-butylene glycol 3.0 glycerin 5.0 Polyoxyethylene (60) hardening castor oil 0.2 ethanol 8.0 Citric acid 0.02 Sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water To 100

<2-3> essence

In order to produce an antimicrobial essence comprising the peptide of the present invention, it may be blended as described in the following [Table 3] and prepared according to a conventional manufacturing method in the cosmetic field.

ingredient Content (% by weight) Peptides of the Invention 0.1 to 30% Sitosterol 1.7 Polyglyceryl 2-oleate 1.5 Ceramide 0.7 Setares-4 1.2 cholesterol 1.5 Dicetyl phosphate 0.4 Concentrated glycerin 5.0 Carboxyvinyl polymer 0.2 Xanthan gum 0.2 antiseptic a very small amount Spices a very small amount Purified water To 100

<2-4> face wash ( Cleansing Foam )

In order to produce an antibacterial face wash (cleansing foam) containing the peptide of the present invention, it may be blended as described in the following [Table 4] and manufactured according to a manufacturing method in a conventional cosmetic field.

ingredient Content (% by weight) Peptides of the Invention 0.1 to 30% N-acyl glutamate 20.0 glycerin 10.0 PEG-400 15.0 Propylene glycol 10.0 POE (15) oleyl alcohol ether 3.0 Laurin derivative 2.0 Methyl paraben 0.2 EDTA-4Na 0.03 Spices 0.2 Purified water To 100

<2-5> Nutrition Cream

To prepare an antimicrobial nutrition cream comprising the peptide of the present invention, it is prepared according to the manufacturing method in the conventional cosmetic field as described in Table 5 below.

Compounding ingredient Content (% by weight) Peptides of the Invention 0.1 to 30% vaseline 7.0 Liquid paraffin 10.0 Wax 2.0 Polysorbate 60 2.5 Sorbitan sesquioleate 1.5 Squalane 3.0 Propylene glycol 6.0 glycerin 4.0 Triethanolamine 0.5 Xanthan gum 0.5 Tocophenylacetate 0.1 Incense, preservative a very small amount Purified water To 100

<2-6> massage cream

To prepare an antibacterial massage cream containing the peptide of the present invention, it is prepared according to the preparation method in the general cosmetic field as described in Table 6 below.

Compounding ingredient Content (% by weight) Peptides of the Invention 0.1 to 30% Propylene glycol 6.0 glycerin 4.0 Triethanolamine 0.5 Wax 2.0 Tocophenylacetate 0.1 Polysorbate 60 3.0 Sorbitan sesquioleate 2.5 Cetearyl alcohol 2.0 Liquid paraffin 30.0 Xanthan gum 0.5 Incense, preservative a very small amount Purified water To 100

<2-7> pack

To prepare an antimicrobial pack containing the peptide of the present invention, it is prepared according to the preparation method in the general cosmetic field as described in Table 7 below.

Compounding ingredient Content (% by weight) Peptides of the Invention 0.1 to 30% Propylene glycol 2.0 glycerin 4.0 Polyvinyl alcohol 10.0 ethanol 7.0 Fiji-40 Hydrogenated Castor Oil 0.8 Triethanolamine 0.3 Incense, preservative a very small amount Purified water To 100

< Manufacturing example  3> Manufacturing of food

Foods containing the peptide of the present invention were prepared as follows.

<3-1> Production of flour food

0.5 to 5.0 parts by weight of the peptide of the present invention was added to the flour, and using this mixture to prepare bread, cakes, cookies, crackers and noodles to prepare an antibacterial health food.

<3-2> dairy products ( dairy products )

5 to 10 parts by weight of the peptide of the present invention was added to milk, and the milk was used to prepare various dairy products such as butter and ice cream.

<3-3> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The peptide of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a sprayer and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the dry powder of the peptide of the present invention prepared above were formulated in the following ratio.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Peptide (3 parts by weight) of the present invention,

(0.5 parts by weight), and

Rhubarb (0.5 parts by weight).

<3-4> Preparation of Health Supplements

100 mg of the peptide of the present invention

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

< Manufacturing example  4> Health drink  Produce

       100 mg of the peptide of the present invention

       Citric acid 100 mg

       100 mg of oligosaccharide

       Plum concentrate 2 mg

       100 mg taurine

       Purified water was added to 500 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the resulting solution was filtered to obtain a sterilized 1 liter container, sealed sterilized, It is used in the production of the health beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

The present invention is not limited to the above-described embodiments and preparation examples, and can be variously modified and changed by those skilled in the art, and can be applied to cosmetics of various uses including other color cosmetics. It can be used in the manufacture of a medicament, that is, an ointment that can be applied thinly to the human body depending on its efficacy, which is included in the spirit and scope of the present invention as defined in the appended claims.

Attach an electronic file to a sequence list

Claims (10)

Antibiotic peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2.
The method of claim 1, wherein the antibiotic peptide is Nordotis discus antibiotic peptides, characterized in that separate from the discus).
The antibiotic peptide of claim 1, wherein the antibiotic peptide has thermal stability.
The method of claim 1, wherein the antibiotic peptide is Escherichia coli ) or Staphylococcus aureus ) antibiotic peptide characterized in that it has an antimicrobial activity.
1) milling abalone ( Nordotis discus discus ) and extracting with water, alcohol or aqueous alcohol solution;
2) recovering the supernatant fraction of the extract by centrifuging the extract of step 1);
3) recovering the supernatant fraction by heating the supernatant fraction of step 2) and then centrifuging; And
4) A method of preparing an antibiotic peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, comprising the step of separating and purifying the supernatant fraction of step 3).
6. The method of claim 5, wherein the alcohol of step 1) is an antibiotic peptide, characterized in that methanol or ethanol.
The method of claim 5, wherein the separation of step 4) is characterized in that the production of antibiotic peptides, characterized in that to obtain a protein of less than 10 kDa through ultrafiltration.
delete Antimicrobial pharmaceutical composition against Escherichia coli or Staphylococcus aureus comprising the antibiotic peptide of claim 1 as an active ingredient.
Antimicrobial cosmetic composition for Escherichia coli or Staphylococcus aureus comprising the antibiotic peptide of claim 1 as an active ingredient.

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