KR100634482B1 - Method for separating ADI from Lactococcus lactis - Google Patents

Abstract

The present invention relates to an anticancer composition containing an ADI (arginine deiminase) isolated from Lactococcus lactis as an active ingredient and a method for separating ADI from Lactococcus lactis. Arginine deiminase (ADI) isolated from Lactococcus lactis according to the present invention very effectively inhibits the proliferation of cancer cell lines, particularly gastric or colorectal cancer cell lines, and therefore can be used as an anti-cancer composition, but also to remove toxicity. It can also be used as a functional food composition having an anticancer effect without the purification process. Since the composition according to the present invention needs to be formulated by separating only ADI from the cytoplasmic fraction of Lactococcus lactis, there is an advantage that the cost of production can be lowered compared to various types of anticancer agents including antibiotics.

Lactococcus lactis, cancer, arginine deiminase (ADI)

Description

Method for separating ADI from Lactococcus lactis}

Figure 1 shows the results of comparing the inhibitory activity of gastric cancer cell line growth according to the heating temperature of the cytoplasmic fraction of Lactococcus lactis S. L. lactis.

Figure 2 shows the results of comparing the inhibitory activity of gastric cancer cell line proliferation according to the pH of the cytoplasmic fraction of Lactococcus lactis S. L. lactis.

Figure 3 shows the results of comparing the gastric cancer cell line proliferation inhibitory activity and ADI activity according to the concentration of the cytoplasmic fraction of Lactococcus lactis S. L. lactis.

Figure 4 shows a photograph showing the results of the SDS-PAGE analysis of the fraction separated by the separation method of arginine deiminase (ADI) according to the present invention (lane A: marker showing the molecular weight, lane B: anion exchange chromatography performed ADI fraction obtained after performing additional gel permeation chromatography on fractions of C, lane C: ADI fraction obtained after further gel permeation chromatography on fractions of lane D, C).

Figure 5 shows the results of performing the MTT assay by treating the gastric cancer cell line SNU-1 ADI fraction of Lactococcus lactis S. L. lactis by concentration.

FIG. 6 is an electron micrograph of the apoptosis of cancer cells after adding and incubating the ADI fraction of Lactococcus lactis S. pyractis to SNU-1.

Figure 7 shows the results of performing the MTT assay by treating the cytoplasmic fraction of Lactococcus lactis S. L. lactis to the colon cancer cell line SNU-C2A by concentration.

Figure 8 shows the results of performing the MTT assay by treating the colon cancer cell line SNU-C2A ADI fraction of Lactococcus lactis S. L. lactis by concentration.

Figure 9 shows the results of the investigation of the ADI activity of the cytoplasmic fraction of various lactic acid strains (Blon: Bifidobacterium, longum , Bbre: Bifidobacterium breve , Lcas: Lactobacillus carcass Lactobacillus casei , Lpla: Lactobacillus plantarum , Sthe: Streptococcus thermophilus , Lbul: lactobacillus bulgaricpido , Bado: Bifidobacterium adolescent , Llac: Lactococcus lactis ssp.lactis ).

The present invention relates to an anticancer composition, and more particularly, to an anticancer composition containing ADI isolated from Lactococcus lactis as an active ingredient and a method for separating ADI from Lactococcus lactis.

Lactococcus lactis is a type of lactic acid bacteria. Lactic acid bacteria are microorganisms that produce lactic acid using anaerobic carbohydrates and are widely distributed in nature. Lactic acid bacteria have been applied as an important preservation means for foods in both East and West by suppressing the growth of decaying microorganisms that grow well in neutral and alkaline. Especially, it is used for processing milk, dairy products, meat products, vegetables, and various salted fish. come. Lactic acid bacteria increase the preservation of foods by reducing the pH by their various types of organic acids, because organic acids such as lactic acid and acetic acid have bacteriocide against most microorganisms.

Recently, since it has been reported that ingested lactic acid bacteria can suppress tumors in the large intestine, the importance of lactic acid bacteria in the treatment of chronic diseases associated with diet, such as cancer, has been highlighted. For example, chemically induced cancer cells in rodents were inhibited by administration of Lactobacillus casei ssp.casei (Tomita et al ., Nihon Hinyoukika Gakkai, 85: 655-663, 1994; Yokokura, Intestinal Flora and Human Health , 72-88, 1994).

In addition, it has been reported that lactic acid bacteria have a direct cytotoxic effect on cancer cells in addition to the anticancer effect by a mechanism that promotes the immune function of the host cell, the body of this activity has been reported as peptidoglycan (Fichera, GA, and Giese, G., Cancer Lett ., 85: 93-103, 1994). In addition, cell wall components of Enterococcus faecalis have been reported to have an effect of inhibiting tumor cell proliferation (Nomoto, K., Biotherapy , 1: 169-177, 1989; Park, SJ et al ., Kor. J. Appl. Microbiol.Biotechnol. 27: 8-14, 1999).

Meanwhile, the inventors of the present application reported that the peptidoglycan component in the cell wall fraction was effective in suppressing tumors in vivo, whereas the cytoplasmic fraction present in the strain was more effective than the peptidoglycan. Inhibiting was reported (Patent Registration No. 10-0411198).

In the cytoplasmic fraction of the strain showing a growth inhibitory activity against the cancer cell line, the present inventors are in the cytoplasmic fraction of Lactococcus lactis, while continuing to study which component of the cytoplasmic fraction exhibits cancer cell line growth inhibitory activity By confirming that ADI (arginine deiminase) is an active ingredient for inhibiting the proliferation of cancer cell lines, the present invention was completed.

An object of the present invention is to provide a pharmaceutical composition for anticancer containing ADI isolated from the lactic acid strain as an active ingredient.                         

Another object of the present invention is to provide a food composition containing ADI isolated from the lactic acid strain as an active ingredient.

Another object of the present invention is to provide a method for separating ADI from lactic acid strains.

In order to achieve the above object, the present invention provides an anticancer composition containing an arginine deiminase (ADI) isolated from Lactococcus lactis as an active ingredient.

In the antitumor composition of the invention, the arm is preferably preferably in the gastric cancer, wherein the Lactococcus lactis (Lactococcus lactis) are Lactococcus lactis eseueseu blood lactis (Lactococcus lactis ssp. Lactis) Do.

In order to achieve another object of the present invention, the present invention provides a food composition comprising an arginine deiminase (ADI) isolated from Lactococcus lactis ( Lactococcus lactis ).

In addition, in order to achieve another object of the present invention, the present invention is performed by chromatography from the cytoplasmic fraction of Lactococcus lactis to obtain an ADI fraction ADI from Lactococcus lactis ( Lactococcus lactis ) It provides a way to isolate (arginine deiminase).

In the ADI separation method according to the present invention, the chromatography is preferably anion exchange chromatography and gel permeation chromatography, and the cytoplasmic fraction breaks down Lactococcus lactis. Preferably, the crushed cells are obtained by centrifugation to remove the precipitate.

Hereinafter, the present invention will be described in detail.

In some strains that have previously exhibited proliferation inhibitory activity against cancer cell lines, it has been reported that peptidoglycans constituting the whole cell of the strain or the cell wall of the strain have a cancer cell line proliferation inhibitory effect. However, we have demonstrated in patent registration 10-0411198 that the cytoplasmic fraction present in the strain exhibits a much stronger cancer cell line proliferation inhibitory effect than the whole cell and peptidoglycan components of the strain.

In one embodiment of the present invention, in order to identify the active ingredient in the cytoplasmic fraction of lactic acid bacteria showing a proliferation inhibitory activity against the cancer cell line, the characteristics of the active ingredient were analyzed. As a result, when heat-treating the cytoplasmic fraction of Lactococcus lactis S. P. lactis, anticancer activity dropped sharply compared to the case without heat treatment (see FIG. 1). In addition, the cytoplasmic fraction maintained anticancer activity at pH 5-7, but the activity was reduced by 20% at the remaining pH (see Fig. 2). From the above results, it can be seen that the component having cancer cell line proliferation inhibitory activity is a kind of enzyme.

In order to identify the active ingredient suspected as an enzyme, cancer cell line proliferation inhibitory activity with increasing concentration of the cytoplasmic fraction of Lactococcus lactis SPI was compared with the activity of the enzymes contained in the cytoplasmic fraction. As a result, as the concentration of the cytoplasmic fraction of Lactococcus lactis SPI increased, the activity of ADI also increased, and it was confirmed that the activity of the ADI correlated with the inhibition rate of cancer cell line proliferation of the cytoplasmic fraction (see FIG. 3). In contrast, the activity of the remaining enzymes other than ADI did not correlate with the inhibition of cancer cell line proliferation of the cytoplasmic fraction (results not shown). From the above results, it was estimated that ADI contained in the cytoplasmic fraction of Lactococcus lactis is an active ingredient for inhibiting cancer cell line proliferation.

In order to confirm whether or not the ADI (arginine deiminase) isolated from Lactococcus lactis according to the present invention has cancer cell line proliferation inhibitory activity, the ADI fraction isolated according to the method of the present invention and the cancer cell line proliferation inhibitory activity were MTT assays were performed using cytosolic fractions of lactic acid strains known to have.

As a result, the addition of the cytoplasmic fraction of Lactococcus lactis S. p. Lactis to gastric cancer cell line did not inhibit the proliferation of gastric cancer cell line by 50% even though it was added up to 250 ug / ml (see FIG. 3). For the ADI fraction of the invention, addition of 2 ug / ml inhibited 50% of proliferation of gastric cancer cell lines (IC ₅₀ = 2 ug / ml of the ADI fraction; see FIG. 5).

Similarly, for the colorectal cancer cell line, the addition of the cytosolic fraction of Lactococcus lactis espi lactis did not inhibit the proliferation of the colorectal cancer cell line by 50% (see FIG. 7), but was isolated therefrom. The ADI fraction of the invention inhibited 50% proliferation of colorectal cancer cell lines at 5 ug / ml (IC ₅₀ = 5 ug / ml of the ADI fraction; see FIG. 8).

According to the above results, it can be seen that the cancer cell line proliferation inhibitory effect of the ADI fraction isolated therefrom is remarkably superior to the cytoplasmic fraction of Lactococcus lactis S. pyractis. Therefore, it can be seen that ADI in the cytoplasmic fraction of the known cancer cell line proliferation inhibitor Lactococcus lactis SSP lactis is an active ingredient for inhibiting the proliferation of the cancer cell line.

In another embodiment of the present invention, in order to confirm the mechanism by which the ADI fraction according to the present invention inhibits the proliferation of cancer cell lines, the ADI fractions were treated with gastric cancer cell lines and observed with an electron microscope. As a result, it was confirmed that the phenomenon of chromatin condensation, which is a part of apoptosis process, occurs in the cancer cell line (see FIG. 6). As a result, chromatin condensation occurs as a part of the process of inhibiting cancer cell line proliferation of the ADI fraction according to the present invention, and it can be confirmed that apoptosis occurs in the cancer cell line.

In another embodiment of the present invention, in order to confirm whether the cellular fraction of other lactic acid strains other than Lactococcus lactis contains ADI, the ADI activity of the various lactic acid strains was examined and compared. As a result, the cytoplasmic fraction of lactic acid strains other than Lactococcus lactis S. pyractis had little ADI activity (see Fig. 9). From the above results, it can be seen that ADI is contained in a large amount of lactococcus lactis, among lactate strains.

Accordingly, the present invention provides an anticancer composition containing an arginine deiminase (ADI) isolated from Lactococcus lactis as an active ingredient.

The cancer may include gastric cancer, colorectal cancer, leukemia, lung cancer, kidney cancer, bladder cancer, cervical cancer, breast cancer, liver cancer, or prostate cancer, but is preferably gastric cancer or colon cancer.

The ADI according to the present invention can be used separately from the lactic acid bacteria, but is preferably separated from the Lactococcus lactis, more preferably from the Lactococcus lactis S. L. lactis.

The anticancer composition according to the present invention may include an arginine deiminase (ADI) isolated from a pharmaceutically effective amount of Lactococcus lactis alone or one or more pharmaceutically acceptable carriers, excipients or diluents. Can be. As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat or prevent cancer.

A pharmaceutically effective amount of arginine deiminase (ADI) isolated from Lactococcus lactis according to the present invention is 0.1 to 100 mg / day / kg body weight, preferably 1 to 10 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the type of cancer and its severity, the age, weight, health condition, sex, route of administration and duration of treatment.

As used herein, “pharmaceutically acceptable” refers to a composition which, when administered physiologically and is human, does not normally cause an allergic reaction such as gastrointestinal disorders, dizziness or the like. Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The anticancer composition may further include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives. In addition, the compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.

The anticancer composition according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular. The dosage of the active ingredient may be appropriately selected depending on several factors such as the route of administration, the age, sex, weight of the patient, and the severity of the patient.

The anticancer composition of the present invention can be administered in parallel with a known compound having the effect of preventing or treating cancer.

In addition, ADI isolated from Lactococcus lactis according to the present invention may be added to food for the purpose of preventing or treating cancer. Accordingly, the present invention provides a food composition comprising ADI isolated from Lactococcus lactis.

Lactobacillus strains used in the present invention can be used as a food grade and can be safely ingested without a separate purification process to remove toxicity. Therefore, the ADI fraction isolated from the cytoplasmic fraction of Lactococcus lactis having cancer cell line inhibitory activity If you add it to food, you can get anti-cancer effect.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the ADI of the present invention may be prepared in the form of tea, juice, and drink for drinking, or granulated, encapsulated, and powdered.

In addition, functional foods include beverages (including alcoholic beverages), fruits and processed foods (e.g. canned fruit, canned foods, jams, marmalade, etc.), fish, meat and processed foods (e.g. ham, sausage corned beef) Breads and noodles (e.g. udon, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, syrups, dairy products (e.g. butter, cheese), edible vegetable oils, margarine, vegetable protein , Retort foods, frozen foods, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the ADI of the present invention.

In addition, in order to use the ADI isolated from the lactococcus lactis of the present invention in the form of a food additive, it may be prepared and used in powder or concentrate form.

The preferred content of ADI isolated from the Lactococcus lactis of the present invention in the food composition of the present invention is about 0.1 to 10 mg per 100 g of food.

The present invention also provides a method for isolating arginine deiminase (ADI) from Lactococcus lactis.

The method for separating ADI (arginine deiminase) from the specific strain comprises the steps of: (a) obtaining a cytoplasmic fraction by separating the cell wall from Lactococcus lactis ( Lactococcus lactis ); And (b) obtaining an ADI fraction through chromatography from the cytoplasmic fraction.

More preferably, the step of obtaining the cytoplasmic fraction of (a) is carried out by crushing the cells, centrifuging the crushed cells to remove the precipitate.

In addition, in the step of obtaining the ADI fraction of (b), it is more preferable to perform anion exchange chromatography and gel permeation chromatography one after the other.

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

However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited thereto.

Example 1 Characterization of Anticancer Active Substances in the Cytoplasmic Fraction of Lactococcus Lactis S. P. Lactis

<1-1> Determination of Protein and Sugar Content

Protein content and sugar content analysis of the cytoplasmic fraction of Lactococcus lactis S. p. Lactis were measured using known methods such as Lowry and sulfuric acid reaction method. The protein content and sugar content of the cytoplasmic fraction of Lactococcus lactis S. pyractis were about 47% and about 24%, respectively.

<1-2> Thermal Stability Analysis

The cytosolic fractions of Lactococcus lactis espi lactis were heat treated at 60 ° C, 70 ° C, 80 ° C, 90 ° C and 100 ° C for 1 hour, and then treated with SNU1, a human gastric cancer cell line, to compare the changes in anticancer activity. . The results are shown in FIG. From the above results, it was confirmed that the anti-cancer activity of the cytoplasmic fraction of the heat treated Lactococcus lactis S. L. lactis is sharply lowered compared to the case where the heat treatment was not performed.

<1-3> pH stability analysis

Cytoplasmic fractions of Lactococcus lactis espi lactis were tested for anticancer activity against SNU1 treated at pH 3, 4, 5, 6, 7, 8 and 9 for 1 hour, respectively. The results are shown in FIG. The cell line proliferation inhibitory activity of the cytoplasmic fraction of Lactococcus lactis S. p. Lactis was maintained at pH 5-7, but decreased by 20% at the remaining pH.

From the above results, it was predicted that the active ingredient of the anticancer activity in the cytoplasmic fraction of Lactococcus lactis S. p. Lactis is a kind of enzyme.

<Example 2> Comparison of cancer cell line proliferation inhibitory activity and ADI activity with increasing concentration of cytosolic fraction of Lactococcus lactis S. pyractis

The cancer cell line proliferation inhibitory activity of the cytoplasmic fraction of Lactococcus lactis espi lactis was measured in the same manner as in Example <5-1>.

In addition, the ADI activity measurement of the cytoplasmic fraction of Lactococcus lactis S. p. Lactis applied the Oginsky method, a known method using a method of measuring the amount of L-citrulline. That is, since ADI decomposes L-arginine into L-citrulline and ammonia, ADI activity can be measured by confirming the amount of L-citrulline produced. First, the cytoplasmic fraction of Lactococcus lactis espi lactis was dissolved in 10 mM arginine and 50 mM Tris (pH 7.0) to give a final volume of 1.0 ml. After incubation at 37 ° C. for 3 hours, 0.5 ml of a solution containing H ₂ SO ₄ and H ₃ PO ₄ in a ratio of 1: 3 (v / v) was added thereto to stop L-citrulline production. After the addition, diacetyl monooxime was added to check the degree of color change. One unit of ADI activity is the action of an enzyme that changes 1 mol of L-arginine to 1 mol of L-citrulline for 1 hour. Protein amount was measured using the BCA Assay Kit (Pierce Co, USA).

The results measured above are shown in FIG. 3. From the above results, the increase of ADI activity according to the concentration of the cytoplasmic fraction of Lactococcus lactis S. lac lactis and the increase of the cell proliferation inhibitory effect by the concentration of the cytoplasmic fraction of Lactococcus lactis. It has been found that there is a correlation.

Example 3 Characterization of ADI

<3-1> Optimal pH Analysis

The ADI fractions were treated for 1 hour at pH 3, 4, 5, 6, 7, 8 and 9, respectively, and then the ADI activity of the cytoplasmic fractions was measured. Measurement of ADI activity was performed in the same manner as in Example 2. As a result, the highest activity was found at pH 7.

<3-2> Optimal Buffer Analysis

Investigation of ADI activity in Tris buffer and potassium phosphate buffer used in most of the literature showed that ADI activity remained higher in Tris buffer.

Example 4 Preparation of ADI Fraction from Lactococcus Lactis S. L. Lactis

<4-1> Culture of Lactococcus lactis S. L. lactis

Lactobacillus Lactococcus lactis ssp. Lactis ( Lactococus lactis ssp. Lactis : Accession No. ATCC 7962) was inoculated in 3 l of M17 liquid medium (Difco) to which 5% glucose was added and incubated at 30 ° C. for 18 hours. After incubation, 8000 rpm, centrifuged at 4 ° C. (Vision, Korea) to recover Lactococcus lactis S. L. lactis, washed three times with distilled water and then stored by freeze drying.

<4-2> Separation of Cytoplasmic Fractions

In order to separate the cytoplasmic fraction of Lactococcus lactis espi lactis, the freeze-dried Lactococcus lactis espi lactis was suspended in distilled water at a concentration of 10 mg / ml. Suspended cells were crushed for 30 minutes using a cell crusher (Sonics and Materials Inc., Danbury, CT, USA), but the heat generated was crushed while cooling with ice. The crushed cells were centrifuged at 800 g at 5 ° C. for 30 minutes to remove cell precipitates. In order to separate the cell wall from the supernatant of the crushed cells, the supernatant cytosolic fraction was separated by ultracentrifugation (Ultracentrifuge, Hitach, Tokyo, Japan) for 30 minutes to remove the precipitate containing the cell wall.

<4-3> Isolation and Identification of ADI Fraction from Cytoplasmic Fraction

The ADI fraction was isolated and identified from the cytoplasmic fraction of Lactococcus lactis S. L. lactis isolated in Example <4-2>.

First, anion exchange chromatography was performed using a HiTrap Q sepharose Fast Flow 5ml prepacked column (Amersham Co., USA). At this time, 10 mM Tris buffer (pH 7.0; A buffer) and 10 mM Tris buffer (pH 7.0) containing 1M NaCl were used as the elution buffer. First, the cytoplasmic fraction isolated in Example <4-2> was injected while flowing A buffer, and then sufficiently washed. Then, the concentration gradient was changed from 100% A buffer to 100% B buffer for 50 minutes at an elution rate of 3 ml / min. gave. Only fractions showing ADI activity were collected according to the hourly fractions. Measurement of ADI activity was performed in the same manner as in Example 2.

Thereafter, the collected fractions showing ADI activity were subjected to gel permeation chromatography again using Sephacryl 200 16/26 column (Amersham Co., USA). 10 mM Tris buffer (pH 7.0) containing 0.15 M Nacl was used as the elution buffer, and the elution rate was 0.4 ml / min. Only fractions showing ADI activity were collected according to the hourly fractions. Measurement of ADI activity was performed in the same manner as in Example 2.

In order to confirm whether the separated fractions contained ADI, the fractions were concentrated and 12% SDS-PAGE analysis was performed, and the results are shown in FIG. 4.

As shown in C and D of Figure 4, it can be seen that the fraction separated by the chromatography of the second step contains a high concentration of ADI.

Example 5 Determination of Cancer Cell Line Proliferation Inhibition of ADI Isolated from Cell Fraction of Lactococcus Lactose S. P. Lactis

<5-1> Proliferation Inhibition of ADI on Gastric Cancer Cell Lines

As a cancer cell line, SNU-1 (KCLB 00001), a human-derived gastric cancer cell line, was used. Culture of the cancer cell line was added to 10% of FBS to RPMI-1640, penicillin (penicillin) 100 IU / ㎖, streptomycin (100 ㎍ / ㎖) using a medium, 5% CO 2, 37 ℃ The incubator was performed in Forma Scientific Co., Marjetta, OH, USA. All of the media compositions used for cell culture were GIBCO BRL (Grand Island, NY, USA).

In order to determine whether the substance showing the inhibitory effect of cancer cell line proliferation in the cytoplasmic fraction of Lactococcus lactis S. p. Lactis is the ADI fraction, the cultured SNU-1 cancer cell line was added to a 96 well plate 1 ×. 10 ⁴ cells were aliquoted, and the cytoplasmic fraction and ADI fraction isolated in Examples <4-2> and <4-3> were added at 100 µg / ml and incubated for 48 hours.

In order to measure the degree of inhibition of proliferation of cancer cells, MTT assay was performed according to a known method (Mosmann, J. Immunol. Method , 65: 55-63, 1983). Cultured cells were washed twice with phosphate buffer. 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium in 96 well plates bromide; MTT, Sigma) was added and reacted at 37 ° C. for 4 hours. The supernatant was removed by centrifuging the 96 well plate at 800 g for 25 minutes at room temperature. 0.1 ml of dimethylsulfoxide was added to the blue formazan product and reacted at 80 rpm for 30 minutes on a shaker, followed by a microplate reader (BioRad, Hercules, CA, USA). Absorbance at 570 nm was measured using. Three repeated experiments were performed according to this example, and the percentage of cell proliferation inhibition of the ADI fraction was calculated in% and is shown in FIGS. 3 and 5.

As shown in Figure 5, it can be seen that the ADI fraction inhibits cancer cell proliferation in a concentration-dependent manner. The ADI fraction of the present invention inhibited the proliferation of gastric cancer cell lines by 50% at 2 ug / ml.

As a result, it can be seen that the proliferation inhibitory effect on the gastric cancer cell line of the ADI fraction is significantly superior to the whole cytoplasmic fraction.

<5-2> Proliferation Inhibition of ADI on Colorectal Cancer Cell Lines

Cytoplasmic fraction of Lactococcus lactis S. L. lactis and ADI fraction isolated therefrom against colorectal cancer cell line SNU-C2A (KCLB0000C2A) using the method of measuring gastric cancer cell proliferation inhibition of Example <5-1> The proliferation inhibition of was measured. The results are shown in FIGS. 7 and 8. As shown in FIG. 8, the ADI fraction inhibited the proliferation of colon cancer cells in a concentration-dependent manner, and inhibited the proliferation of colon cancer cell lines by 50% at a concentration of 5 ug / ml.

As a result, it can be seen that the proliferation inhibitory effect of the ADI fraction on the colorectal cancer cell line is significantly superior to the whole cytoplasmic fraction.

<5-3> Apoptosis Confirmation

In the culture of gastric cancer cell line SNU-1, the ADI fraction was added at a concentration of 100 μg / ml, incubated for 48 hours, collected by centrifugation, and fixed with 4% paraformaldehyde. After transferring to the slides and dried at room temperature, stained with DAPI (4,6-diamidino-2-phenylindole) at a concentration of 1 ug / ㎖ for 15 minutes and observed with an electron microscope (Olympus Ix70, Japan). The results are shown in FIG. 6.

As shown in FIG. 6, when the ADI fraction was treated to cancer cell lines, it was confirmed that chromatin condensation, which is a part of apoptosis process, occurred.

Example 6 Comparison of ADI Activity by Lactic Acid Bacteria

Bifidobacterium bron gum ( Bifidobacterium, longum ), Bifidobacterium breve ( Bifidobacterium breve ) in order to determine whether a large amount of ADI is also included in the cytoplasmic fraction of other lactobacillus strains other than the Lactococcus lactis S. pyractis , Lactobacillus casei , Lactobacillus plantarum , Streptococcus thermophilus , Lactobacillus bulgaricus , Bifidobacterium The ADI activities of the cytoplasmic fractions of bifidobacterium adolescent and Lactococcus lactis ssp.lactis were investigated and compared, respectively.

The results are shown in FIG. As shown in Figure 9, it can be seen that the cytoplasmic fraction of lactic acid strains other than Lactococcus lactis S. L. lactis has little ADI activity.

As described above, the ADI component of the cytoplasmic fraction of Lactococcus lactis had a remarkably excellent proliferation inhibitory effect against cancer cell lines, especially gastric and colorectal cancer cell lines. Therefore, ADI (arginine deiminase) isolated from Lactococcus lactis according to the present invention very effectively inhibits the proliferation of cancer cell lines, especially gastric or colorectal cancer cell lines, and therefore can be used as a pharmaceutical composition for anticancer, It can also be used as a functional food composition having an anticancer effect without a separate purification process for removal. Since the composition according to the present invention needs to be formulated by separating only ADI from the cytoplasmic fraction of Lactococcus lactis, there is an advantage that the cost of production can be lowered compared to various types of anticancer agents including antibiotics.

Claims (8)

delete delete delete delete A method for separating arginine deiminase (ADI) from Lactococcus lactis comprising performing chromatographic chromatography from a cytoplasmic fraction of Lactococcus lactis to obtain an ADI fraction. 6. The method of claim 5, wherein the chromatography is anion exchange chromatography and gel permeation chromatography. 6. The method of claim 5, wherein the cytosolic fraction is obtained by disrupting Lactococcus lactis cells and centrifuging the disrupted cells to remove precipitates. The method of claim 5, wherein the Lactococcus lactis ( Lactococcus lactis ) is characterized in that Lactococcus lactis ssp .

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