KR100654232B1 - THERMOSTABLE PEPTIDE ISOLATED FROM SOLANUM TUBEROSUM L cv. GOLDEN VALLEY AND FUNCTIONAL RESOURCES USE OF THE SAME - Google Patents

Abstract

The present invention relates to a thermostable peptide represented by SEQ ID NO: 1 extracted from potato of the Golden Valley variety ( Solanum tuberosum L cv. Golden Valley).

Peptides isolated from the potato ( Solanum tuberosum L cv. Golden Valley) of the present invention is a composition for the preparation of ensuring safety against cytotoxicity, it has an antimicrobial activity against Candida albicans strains as an active ingredient Containing antimicrobial agents; Or it is useful as a protease inhibitor containing it as an active ingredient having an inhibitory activity on the protease selected from chymotrypsin, trypsin or papain.

Potatoes, Antibiotic Peptides, Cytotoxicity, Pesticides, Pharmaceuticals, Golden Valley, Valley Potatoes

Description

Thermostable peptides isolated from potatoes of the Golden Valley varieties and their use as functional ingredients containing the peptides as active ingredients {THERMOSTABLE PEPTIDE ISOLATED FROM SOLANUM TUBEROSUM L cv. GOLDEN VALLEY AND FUNCTIONAL RESOURCES USE OF THE SAME}             

1 is a photograph showing a cut section of potato ( Solanum tuberosum L cv. Golden Valley) of the Golden Valley variety of the present invention,

Figure 2 is a result showing the molecular weight of the peptide isolated from the potato of the Golden Valley variety of the present invention,

Figure 3 is a result of measuring the degree of cell destruction (Hemolytic) for human red blood cells peptides isolated from the potato of the Golden Valley variety of the present invention,

Figure 4 is a photograph showing the antifungal activity of the peptide isolated from the potato of the Golden Valley varieties of the present invention, Candida albicans strain, A: negative control group, B: experimental group,

5a, 5b and 5c is a result of the thermal stability of the peptide isolated from the potato of the Golden Valley variety of the present invention,

Figure 6a is a result showing the activity of the peptide isolated from the potato of the Golden Valley variety of the present invention as a protease inhibitor against chymotrypsin,

Figure 6b is a result showing the activity of the peptide isolated from the potato of the Golden Valley variety of the present invention as a protease inhibitor against trypsin,

Figure 6c is a result showing the activity of the peptide isolated from the potato of the Golden Valley variety of the present invention as a protease inhibitor against papain.

The present invention relates to a peptide isolated from potato of the Golden Valley variety ( Solanum tuberosum L cv. Golden Valley) and to the use of the peptide. More specifically, among potato varieties, potato of the Golden Valley variety ( Solanum tuberosum L cv. Peptides extracted from Golden Valley) and the use of the peptide as an active ingredient and functional material, the activity of the peptide is proved by measuring its activity.

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 sequences. 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 antimicrobial 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 subclasses of lantibiotics, nonlantibiotics and signal peptides, depending on their structure [Cintas et al., J. Bad. , 1998 , 180 , 1988-1994. Peptides or bacteriocins that perform antibiotic functions while being produced in vivo can solve problems such as antibiotic resistance and antibiotic shock caused by antibiotics caused by conventional antibiotics made of chemicals.

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.

Recently, natural herb extracts have been reported as a safe natural antimicrobial agent that inhibits cell proliferation mechanisms, has a high bactericidal effect against microorganisms, is nontoxic as a plant natural bactericidal disinfectant, and is harmless to humans because of its small cultivation scale, Many of them are difficult to use as natural antimicrobial agents due to the problems that are difficult to cultivate in Korea as foreign species.

As one of the folk remedies, dried powdered potato peels and sprinkled in the bathroom or in a damp place do not cause bugs, and when heated in hot water, the potatoes are ground or sliced and applied to the affected area without scarring. It has been handed down. Therefore, it is said that potatoes play a role as a natural antimicrobial agent. Since these potatoes have a large growing area and a large number of them, it is easy to secure materials and lower the raw material unit cost. It can provide a smooth advantage at the same time.

As a result of our efforts to develop a variety of potato gene sources, the inventors have developed new edible potatoes with the advantages of conventionally imported varieties Sumi and Atlantic, as well as varying the color of the flesh of the potatoes so that they can be applied according to various uses. It has developed over 100 varieties of potato potatoes that have improved the quality, and has been filed and registered with the National Seed Service for breed protection.

Therefore, the inventors of the present invention as a starting material of the potato varieties of natural plant material, in particular, from the potato varieties, extracts, separation and purification from the Golden Valley varieties of potatoes ( Solanum tuberosum L cv. Golden Valley) to obtain an active fraction, The present invention was completed by measuring anti-fungal activity and peptides on hemolytic activity in human erythrocytes, making them safe for cytotoxicity and functioning as natural antibacterial agents.

It is an object of the present invention to provide a novel peptide represented by SEQ ID NO: 1 isolated from potato of the Golden Valley variety ( Solanum tuberosum L cv. Golden Valley).

Another object of the present invention is to provide a use of a peptide isolated from the potato of the Golden Valley varieties ( Solanum tuberosum L cv. Golden Valley) as a functional material.

In order to achieve the above object, the present invention is Golden Potato varieties ( Solanum tuberosum L cv. Golden Provided is a peptide represented by SEQ ID NO: 1 isolated from the valley.

The peptide is a peptide having thermal stability at a temperature of 70 ~ 120 ℃.

In addition, the peptide of the present invention ensures safety against cytotoxicity, and provides a composition for preparations containing the same as an active ingredient.

In other words, the peptide of the present invention is an antibacterial activity against Candida albicans strain, a causative agent of vaginal germs, Tricosforum Bayzellai strain, a causative agent of dandruff, Tricophyton rubrum, a causative agent of athlete's foot, and Listeria strain, a food poisoning causative bacterium. Since the present invention provides an antimicrobial agent containing the peptide as an active ingredient.

In addition, since the peptide of the present invention has an inhibitory activity on proteolytic enzymes selected from the group consisting of chymotrypsin, trypsin and papain, it provides a use as a protease inhibitor containing the peptide as an active ingredient.

Hereinafter, the present invention will be described in detail with reference to the drawings.

The present invention provides a peptide extracted from potato of the Golden Valley variety ( Solanum tuberosum L cv. Golden Valley), and represented by SEQ ID NO: 1 in the Sequence Listing.

1 is a photograph showing a cut section of potato ( Solanum tuberosum L cv. Golden Valley) of the Golden Valley variety of the present invention. Golden Valley is a low calorie potato species that can be used as a dietary diet because it has a calorie of 90.7 kcal / 100g, which is equivalent to 41% of the normal potato species. It is very small, but when steamed or cooked, it is soft and tastes good for children and toddlers. In addition, the content of vitamin C in Golden Valley is 30.9 mg / 100 g, which is much higher than Sumi (8.6 mg) and Daesa (9.2 mg), so if one 150-160 g of Golden Valley is eaten, all adults need 50 mg of vitamin C. There are benefits to ingest.

The peptide was ground to the powder of the tuber of the Solanum tuberosum L cv.Golden Valley of the Golden Valley varieties using liquid nitrogen to break the cell wall of the potato, and the powder was 50 mM Tris-HCl, 10 mM EDTA, 150 Extracting the protein using a buffer consisting of mM NaCl, 1% DMSO and 0.1% β-mercaptoethanol to prepare an extract, centrifuging the extract to recover the supernatant fraction of the extract, and the fraction was C ^18. Obtained after separation and purification by dialysis under conditions of HPLC chromatography and ammonium bicarbonate solution.

As a result of measuring the molecular weight of the peptide obtained by using a mass spectrometer, it shows 5,579 Da ( FIG. 2 ).

Figure 3 is a result of measuring the degree of cellular destruction (Hemolytic) of the peptides isolated from the potato of the Golden Valley varieties of the present invention, human peptides, when treated with a known peptide melittin, the peptide concentration of 5 μM While erythrocyte cells were destroyed 100% under the conditions of, the destruction of erythrocyte cells was not found even when the same concentration conditions were observed.

Therefore, in the case of containing the peptide isolated from the potato of the Golden Valley variety of the present invention, since no human red blood cell destruction is observed, it provides a composition for the preparation for cytotoxic safety containing it as an active ingredient.

Figure 4 is a photograph showing the antifungal activity of the peptide isolated from the potato of the Golden Valley varieties of the present invention, Candida albicans strain, A: negative control group, B: experimental group. The peptide of the present invention exhibits strong antifungal activity against Candida albicans strain, which is a causative agent of vaginal bacterium in women.

5a to 5c is a result of the thermal stability of the peptide isolated from the potato of the Golden Valley variety of the present invention. Figure 5a is a negative control, Figure 5b is carried out at 70 ℃ and Figure 5c conditions 120 ℃, by confirming that there is no change in the antimicrobial activity of the peptide against the Candida albicans strain, from the potato of the Golden Valley varieties of the present invention The isolated peptide has thermal stability at a temperature of 70-120 ° C.

In addition, the peptide isolated from the potato of the Golden Valley variety of the present invention, as a result of performing the function as a proteinase inhibitor (Protease inhibitor), chymotrypsin ( Figure 6a ), trypsin ( Figure 6b ) and papain ( Figure 6c ) In addition, at a peptide concentration of 5 µg / ml or more, the activity of the protease can be confirmed to be suppressed from as little as 40% to as much as 100%. Therefore, the peptide isolated from the potato of the Golden Valley variety of the present invention is useful as a protease inhibitor containing it as an active ingredient. Protease inhibitors can inhibit or treat diseases caused by the mechanism of action of protease, that is, effective for the soreness of skin caused by weak and fragile skin or pressure sores, and has recently attracted attention as an alternative to the AIDS treatment.

In addition, since the peptide isolated from the potato of the Golden Valley variety is secured in cytotoxicity, it can be applied to functional cosmetics, functional food ingredients or food preservatives, pharmaceutical preservatives and pesticides safe for humans.

Hereinafter, the present invention will be described in detail by way of examples.

The following examples are merely illustrative of the present invention, but the content of the present invention is not limited to the following examples.

Example 1 Protein Extraction Step from Potatoes

Potatoes of Golden Valley varieties ( Solanum tuberosum L cv. Golden Valley) were pulverized into powder using a mortar and pestle in the presence of liquid nitrogen, and the powder was then extracted with protein extraction buffer ( 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 150 mM NaCl, 1% DMSO, 0.1% β-mercaptoethanol were mixed well and the protein was extracted using a centrifuge. At this time, the protein extraction method of the present invention was carried out by modifying a known method [Ana Segura et al, Mol Plant Microbe Interact ., 12 (1): 16-23, 1999 ]. Thereafter, the extracted extract was centrifuged at 6,000 × g for 30 minutes, and then only the supernatant was taken and 50 mM ammonium was prepared using a Spectra / Por 7 (MWCO: 3,500) membrane (Spectrum Laboratories, Inc.). Purification was performed by dialysis at 4 ° C. with bicarbonate buffer (pH 8.0).

Experimental Example 1 Protein Electrophoresis

The extract obtained in the above step was subjected to SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) according to the Laemry method [Laemmli UK, Nature , 27: 680-685, 1972 ] using a slab gel. At this time, the extract and the standard protein were loaded on 15% acrylamide gel, and the molecular weight of the protein was confirmed using the same.

-락트알부민(Bovine Milk

-Lactalbumin) 14.2 kDa, 소 허파로부터 추출된 아프로티닌(Bovine Lung Aprotinin ) 6.5 kDa을 이용하였다. After electrophoresis, the gel was stained with coomassie brilliant blue R-250, and destained with destaining buffer (methanol: glacial acetic acid: distilled water = 30:10:60) to confirm the band of the protein. At this time, the molecular weight standard protein as low molecular weight region bovine serum albumin (Bovine Serum Albumin) 66 kDa, chicken egg Ovalbumin extracted from chicken egg (Chicken Egg Ovalbumin) 45 kDa, glyceraldehyde-3-phosphate di extracted from rabbit muscle Labine Muscle Glyceraldehyde-3-phosphate Dehydrogenase 36 kDa, Bovine Erythrocytes Carbonic Anhydrase 29 kDa, Bovine Pancreas Trypsinogen 24 kDa ), Soybean Trypsin Inhibitor 20 kDa) extracted from soybean, extracted from milk

Lactalbumin (Bovine Milk)

-Lactalbumin) 14.2 kDa, Bovine Lung Aprotinin 6.5 kDa extracted from bovine lung was used.

Experimental Example 2 Reversed Phase HPLC

particle; 12.0±1.0 nm pore; Shimadzu) 역상 HPLC(Reverse Phase HPLC)를 이용하여 정제하였다. 이때, 완충용액(0.1% TFA가 포함된 아세토아니트릴 B)의 기울기(linear gradient, 5∼65%, 2%/min, 30 min)에 의해 용출한 후, 피크의 분획을 모아 항진균 활성을 측정 하였다.Peptides isolated from potatoes of the Golden Valley varieties obtained from the above step were prepared using Shim-pack VP-ODS, C ¹⁸ column (4.6 × 250 mm; 4.6 ± 0.3).

particle; 12.0 ± 1.0 nm pore; Shimadzu) was purified using Reverse Phase HPLC. At this time, after eluting with a gradient (linear gradient, 5 to 65%, 2% / min, 30 min) of the buffer solution (acetonitrile B containing 0.1% TFA), the peak fractions were collected to measure antifungal activity. It was.

Experimental Example 3 Confirmation of Molecular Weight and Amino Acid Sequence and Molecular Weight

Peptides isolated from potato of the Golden Valley variety obtained from the above step was dissolved in 6N-HCl and then hydrolyzed and dried at 110 ° C. for 22 hours, and the amino acid sequence was confirmed using an amino acid analyzer. The amino acid sequence of the peptide isolated from the potato of the Golden Valley variety is described as SEQ ID NO: 1 in the Sequence Listing.

In addition, by measuring the molecular weight of the peptide using a mass spectrometer (company name; Shimadzu, Kratos, AXIMA-CFR), it was confirmed that the peptide having a molecular weight of 5,579 Da (Fig. 2).

Experimental Example 4 Measurement of Erythrocyte Hemolytic Activity

Peptides isolated from the potato of the Golden Valley variety were carried out as follows to determine the cytotoxicity through human erythrocyte hemolytic activity.

Human erythrocytes were washed three times with PBS (phosphate-buffered saline: 35 mM phosphate buffer / 0.15 M NaCl mixed solution, pH 7.0) buffer, and 8.0% red blood cell solution diluted with PBS buffer solution. 100 μl of each 96-well microtiter plate was loaded, and then 100 μl of the peptide solution isolated from the potato extract of the Golden Valley variety obtained from the above step was incubated at 37 ° C. for 1 hour. The absorbance at 414 nm was then measured. In addition, the same peptide was performed in the same manner as melittin (melittin) and compared.

At this time, the value when treated with 0.1% Triton X-100 was calculated as 100% hemolysis, and the hemolysis rate of the peptide, that is, the degree of cell destruction (hemolysis) was calculated by the following Equation 1 . In the following, A is the absorbance of 141 nm in the peptide solution, B is the absorbance of 141 nm in 0.1% Triton X-100, and C shows the absorbance of 141 nm in the PBS solution.

Figure 3 is a result of measuring the degree of cellular destruction (Hemolytic) for human red blood cells peptides isolated from the potato of the Golden Valley varieties of the present invention. In the comparative example treated with melittin, a known peptide, the hemolysis rate of the cells was 35% even at a peptide concentration of 0.31 μM, and 100% of the red blood cells were destroyed at the peptide concentration of 5 μM.

On the other hand, the peptide isolated from the potato of the Golden Valley varieties of the present invention showed that red blood cells were not destroyed at all, even if the peptide concentration of 5 μM or more, it was confirmed that the safety in cytotoxicity.

Experimental Example 5 Antifungal Activity Measurement

In order to measure the antifungal activity of the peptide isolated from the potato of the Golden Valley varieties obtained from the above step was carried out as follows.

At this time, in order to measure the antifungal activity, the pathogenic fungus Candida albicans was used as a strain. Fungal water was diluted to 2 × 10 ³ cells per well in an Agar-hole, and 100 μl of YPD medium containing fungi was dispensed on the plate, and then only Candida albicans was dispensed into the negative control (B). Experimental group (A) was inoculated with a peptide isolated from the Candida albicans fungi and the potato extract of the Golden Valley variety obtained from the above step and incubated for 24 hours in a 30 ℃ incubator.

Figure 4 is the result of measuring the antifungal activity of the peptide isolated from the potato of the Golden Valley variety of the invention. More specifically, A is a negative control group that dispensed only Candida albicans strain, B is an experimental group incubated with Candida albicans strain and peptide isolated from the potato of the Golden Valley variety of the present invention. As a result of observing the growth of the cells with the negative control group (A) and the experimental group (B), it was observed that the growth of the strain in the experimental group (B), that is, from the potato of the Golden Valley variety of the present invention The antifungal activity of the isolated peptide was confirmed.

In addition, in order to measure the stability to heat in the experiment using the Candida albicans strain peptide isolated from the potato of the Golden Valley varieties of the present invention, Figure 5a is a negative control, Figure 5b is 70 ℃ and Figure 5c at 120 ℃ conditions After the peptide was heat treated for 20 minutes or more, the Candida albicans strain was observed after treatment for one day. At this time, the peptide of the present invention at the temperature condition of 70 ~ 120 ℃ did not change the antimicrobial activity against Candida albicans strain.

Experimental Example 6 Antifungal Activity Measurement 2

In order to measure the antifungal activity, except for using Tricosforum Bayzelae as a strain, the same as in Experimental Example 5, the antifungal activity was confirmed.

Experimental Example 7 Antifungal Activity Measurement 3

In order to measure the antifungal activity, except for using the athlete's foot trichophyton rubrum as a strain, it was carried out in the same manner as in Experimental Example 5, the antifungal activity was confirmed.

Experimental Example 8 Antifungal Activity Measurement 4

In order to measure the antifungal activity, except for using the food poisoning-inducing bacteria Listeria as a strain, it was carried out in the same manner as in Experimental Example 5, the antifungal activity was confirmed.

Experimental Example 9 Activity as a Protease Inhibitor Measure

In addition, the protease inhibitor activity of the peptide isolated from the potato of the Golden Valley variety was measured.

Proteolytic enzymes include chymotrypsin, which is made with trypsin in the interest of vertebrates, trypsin, a protease secreted from the liquid of interest, and papain, a protease in papaya juice. The activity of the protease was observed for various concentrations of peptide (µg / ml).

Figure 6a is a result showing the activity of the peptide isolated from the potato of the Golden Valley variety of the present invention as a protease inhibitor against chymotrypsin, the inhibition of protease activity as the peptide concentration increases. In other words, when the peptide was not contained, the absorbance intensity was about 2100 while the initial absorbance intensity was reduced to 50% when the peptide concentration was 5 ㎍ / ml, and when the peptide concentration was above 10 ㎍ / ml, In contrast, more than 90% of the protease inhibitory activity was confirmed.

Figure 6b is a result of the peptide isolated from the potato of the Golden Valley varieties of the present invention showing the activity as a protease inhibitor against trypsin, the activity of the protease was inhibited as the peptide concentration increases. More specifically, when the peptide was not contained, the absorbance intensity of about 800 was shown, whereas when the peptide concentration was 5 µg / ml, the inhibitory effect of the proteolytic enzyme of about 75% was observed as the absorbance intensity 200. In addition, when the peptide concentration is 10 ㎍ / ㎖ or more, by confirming the result that the absorbance intensity is lowered to the value of less than 30, the peptide derived from the Golden Valley varieties of the present invention as a strong protease inhibitor against trypsin, a protease at low concentration Confirmed by function.

Figure 6c is a result of the peptide isolated from the potato of the Golden Valley variety of the present invention showing the activity as a protease inhibitor for papain, when the peptide does not contain, the absorbance intensity of about 800, while the peptide concentration As the increased the activity of the protease was inhibited, more preferably, at a peptide concentration of 20 ㎍ / ㎖, it was confirmed that the activity inhibitory effect of the proteolytic enzyme of at least 80%.

From the above results, it was confirmed that the peptide isolated from the potato of the Golden Valley variety of the present invention has an excellent function of inhibiting the activity of protease, and is more preferable as a protease inhibitor of chymotrypsin or trypsin.

As described above, the present invention

First, a peptide represented by SEQ ID NO: 1 was extracted, isolated, and purified from tubers of Golden Valley varieties Potato ( Solanum tuberosum L cv. Golden Valley),

Secondly, to provide a formulation composition for ensuring the safety against cytotoxic containing the peptide of the present invention,

Third, there was provided an antimicrobial agent containing the peptide of the present invention as an active ingredient,

Fourth, to provide a protease inhibitor containing the peptide of the present invention as an active ingredient,

Fifth, it is suitable as a pesticides, food preservatives, cosmetics, and pharmaceutical preservatives that are safe for the human body by using the formulation composition for ensuring safety against cytotoxicity of the present invention.

Although the present invention has been described in detail only with respect to the embodiments described, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical spirit of the present invention, and such modifications and variations belong to the appended claims. .

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Claims (9)

delete delete delete delete delete delete delete Potatoes of the Golden Valley variety ( Solanum tuberosum L cv. Golden Valley) and an antimicrobial composition, characterized in that it contains a peptide represented by SEQ ID NO: 1 secured thermal stability at a temperature of 70 ~ 120 ℃ as an active ingredient. The method of claim 8, The antimicrobial composition is an antimicrobial composition, characterized in that it has an antimicrobial activity against any strain selected from the group consisting of Candida albicans strain, Tricos forum Bezellai strain, Tricophyton rubrum strain and Listeria strain.

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