INTRODUCTION

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Lactic acid bacteria have been widely used for the fermentation of many fermented products, such as cheese, yogurt, yakult, buttermilk, sour cream, sauerkraut, sausages, silage, and pickles (22). They are well known to possess a variety of beneficial functions for humans, including antimicrobial (7, 22), antitumor (1, 11, 12), and antimutagenic (11, 18) activities, as well as effects on modulating the immune system (8, 20), lowering cholesterol levels (27), and reducing lactose intolerance in the host (2, 23).

Currently, one of the most important factors in human longevity is the control of tumors. Therefore, studies on the beneficial effects of lactic acid bacteria have been largely focused on their antitumor effects. A variety of lactic acid bacteria isolated from fermented milk products have been previously reported as displaying antitumor and anticarcinogenic activities in experimental mice (1, 11, 12) as well as antimutagenic activity on Salmonella enterica serovar Typhimurium (11, 18). Three molecules possessing antitumor effects, glycopeptide, polysaccharide, and phosphopolysaccharide, were purified and characterized from lactic bacteria, but none of the antimutagenic substances has been purified thus far (3, 16, 17).

Lactobacillus plantarum, a lactic acid bacterium participating in the fermentation of fermented milk products as well as fermented vegetables, is also well known to exhibit antitumor activity against mouse fibrosarcoma and ascite tumors (26). However, little is known about the antimutagenic effects of L. plantarum because its antimutagenic activity has not been studied as intensively as its antitumor activities. Concerning the antimutagenic activity of lactic acid bacteria, Hosono et al. (10) were the first to report that milk fermented with Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis subsp. lactis, or Enterococcus faecalis exhibited antimutagenic activity. The lactic acid bacteria that display antimutagenic activity on S. enterica serovar Typhimurium cells now include L. delbrueckii subsp. bulgaricus, Lactobacillus helveticus, L. lactis subsp. lactis, and Streptococcus thermophilus (18). All these strains originate mainly from fermented-milk products.

In a previous study, we isolated L. plantarum KLAB21 from kimchi, a Korean traditional fermented vegetable, that produces antimutagenic compounds (19, 21). It was also demonstrated that the bacteria possesses a high antimutagenic activity against various mutagens, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline-1-oxide, 4-nitro-O-phenylenediamine, and aflatoxin B1, on S. enterica serovar Typhimurium His reversion as well as Bacillus subtilis spore rec assays (19). The majority of the activity by L. plantarum KLAB21 cells was exhibited in the culture supernatant fraction, thereby suggesting that its antimutagenic substance is of an extracellular type (21). In this study, the antimutagenic substances were purified from a culture supernatant of L. plantarum KLAB21 and found to be composed of three different glycoproteins.

In this study, the antimutagenic substances were purified from a culture supernatant of L. plantarum KLAB21 cells and found to be composed of three different glycoproteins.

	MATERIALS AND METHODS

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Strains and media. L. plantarum KLAB21 has been previously described in detail (19, 21). S. enterica serovar Typhimurium TA100 (hisG46 rfa uvrB) was used for the antimutagenic test using the preincubation method (14, 29). The MRS broth was used for the production of antimutagenic substances by L. plantarum KLAB21 (5). S. enterica serovar Typhimurium TA100 cells were grown in nutrient broth (Difco, Detroit, Mich.). The minimal glucose agar media used for the counting of His⁺ revertants of S. enterica serovar Typhimurium have been previously described in detail (14, 19, 21).

Preparation of bacterial culture supernatant. L. plantarum KLAB21 cells were grown in MRS media at 37°C for 36 h with shaking at a speed of 150 rpm for the production of antimutagenic substances. After the culture broth was centrifuged at 25,000 × g for 30 min, the supernatant was removed and stored at 20°C for further experiments.

Purification of antimutagenic substances. The antimutagenic substances were purified from the culture supernatant by using four steps; ammonium sulfate fractionation, anion-exchange chromatography, and Sephadex G200 and Sepharose CL-6B gel filtrations. For the first step, solid ammonium sulfate was added to the culture supernatant to yield 70% saturation. The mixture was incubated at 4°C for 8 h and centrifuged at 25,000 × g for 30 min to collect the precipitants. The precipitants were resuspended in 50 mM phosphate buffer (pH 7.0) and dialyzed against the same buffer for 24 h. The dialyzed solution was then applied to a DEAE-cellulose column (2.0 by 40 cm) equilibrated with buffer of the same composition. After washing the column with the same buffer, antimutagenic substances were eluted with a linear gradient of 0 to 0.5 M NaCl at a flow rate of 40 ml/h to collect 5 ml of each fraction. The fractions containing antimutagenic activity were concentrated using an ultrafiltration kit with an Amicon YM10 membrane (Millipore Co., Waltham, Mass.). The concentrated fraction was then applied to a Sephadex G200 column (1.8 by 80 cm) equilibrated with the same buffer. Antimutagenic substances were eluted at a flow rate of 6 ml/h to collect 5 ml of each fraction. The fractions containing antimutagenic activity were concentrated using the ultrafiltration kit and applied to a Sepharose CL-6B column (1.8 by 80 cm) equilibrated with the same buffer. Antimutagenic substances were eluted at a flow rate of 25 ml/h to collect 5 ml of each fraction. The active fractions were pooled together and used as purified antimutagenic fractions for their characterization. All the purification steps were carried out at 4°C.

Mutagenic and antimutagenic tests. MNNG (Sigma Co., St. Louis, Mo.) was dissolved in distilled water and used as the mutagen at a final concentration of 5 µg per plate for the mutagenic and antimutagenic tests of the fractions, using S. enterica serovar Typhimurium TA100 cells as previously described (14, 29). For the antimutagenic activity test, 100 µl of each fraction being tested, 50 µl of mutagen solution, 100 µl of an overnight culture of S. enterica serovar Typhimurium cells, and 0.5 ml of a 0.2 M sodium phosphate buffer (pH 7.0) were mixed in glass cap tubes. The mixture was then preincubated at 37°C for 30 min with agitation in a shaking incubator. Following the incubation, 3 ml of a molten top agar solution containing histidine and biotin was added and the resulting mixtures were plated on a minimal glucose agar medium. After the plates were incubated at 37°C for 2 days in the dark, the number of His⁺ revertants per plate was counted. The antimutagenic activity was expressed as the percentage inhibition of mutagenesis as follows: percent antimutagenic activity = 100% × [(A  B)/(A  C)], where A is the number of His⁺ revertants induced by a mutagen in the absence of a sample, B is the number of His⁺ revertants induced by a mutagen in the presence of a sample, and C is the number of spontaneous His⁺ revertants in the absence of a mutagen. For the mutagenic test of the purified fractions, 50 µl of distilled water was used instead of the mutagen solution and all the other procedures were essentially the same as those described above. All the data represent the average of at least three trials that were performed in triplicate.

Polyacrylamide gel electrophoresis. The purity of the purified antimutagenic substances was determined by the native gel electrophoresis in a 7.5% polyacrylamide gel with 0.08 M Tris0.3 M diethyl barbituric acid buffer (pH 7.0) by the method described by Schagger and van Jagow (24). After electrophoresis, protein staining was carried out with Coomassie brilliant blue R-250 by a general method (9, 28). To test for glycoprotein, each gel was subjected to staining for carbohydrate using fuchsin solution by the method described by Zacharius et al. (30).

Analytical methods. Molecular weights of the purified antimutagenic substances were determined by gel filtration through a Sepharose CL-6B column. The elution patterns were calibrated using the following proteins: bovine carbonic anhydrase (29.0 kDa), horse heart cytochrome c (12.4 kDa), and bovine lung aprotinin (6.5 kDa). Protein amounts were determined at 595 nm by the method described by Bradford (4), using bovine serum albumin as a standard. Total sugar contents were measured at 470 nm by the phenol-H₂SO₄ method (6) and calculated as the glucose amount.

	RESULTS

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Purification of antimutagenic substances of L. plantarum KLAB21. In order to purify antimutagenic substances of L. plantarum KLAB21, culture supernatant was prepared by centrifugation of the bacterial culture broth in MRS media and concentrated with ammonium sulfate precipitation. After dialysis against 50 mM phosphate buffer (pH 7.0), the concentrated solution was subjected to DEAE-cellulose column chromatography (Fig. 1A). When the bound substances were eluted with a linear gradient of 0 to 0.5 M NaCl, the antimutagenic substances were separated into two fractions: one in fractions 17 to 36 and the other in fractions 100 to 119. The antimutagenic substances in fractions 17 to 36 and 100 to 119 were pooled together and designated the R1 and R2 fractions, respectively.

View larger version (45K): [in this window] [in a new window]   FIG. 1.   Purification of the antimutagenic substances produced by L. plantarum KLAB21. (A) DEAE-cellulose ion-exchange chromatogram of antimutagenic substances. The culture supernatant was applied to a DEAE-cellulose column (2.0 by 40 cm) that was equilibrated with a buffer of the same composition. After washing the column with the same buffer, antimutagenic substances were eluted at a flow rate of 40 ml/h with a linear gradient of 0 to 0.5 M NaCl to collect 5 ml of each fraction. The first and second peaks were designated the R1 and R2 fractions, respectively, and used for further purification. (B) Gel filtration chromatogram of the R1 antimutagenic fraction on a Sephadex G-200 column. The R1 fraction (80 ml) obtained from DEAE-cellulose ion-exchange chromatography was concentrated to 5 ml by using an ultrafiltration kit (Millipore Co.) with a YM10 membrane and applied to a Sephadex G200 column equilibrated with 50 mM phosphate buffer (pH 7.0). Antimutagenic substances were eluted with a buffer of the same composition at a flow rate of 6 ml/h to collect 5 ml of each fraction. The antimutagenic fractions were divided into two peaks again, designated R1-1 and R1-2 fractions. (C to E) Gel filtration chromatograms of the antimutagenic fractions R1-1 (C), R1-2 (D), and R2 (E) on a Sepharose CL-6B column. Each fraction was concentrated to 5 ml using the ultrafiltration kit and applied to a Sepharose CL-6B column equilibrated with 50 mM phosphate buffer (pH 7.0). Antimutagenic substances were eluted with a buffer of the same composition at a flow rate of 25 ml/h to collect 5 ml of each fraction. During all the purification steps, antimutagenic activities () of the fractions were assayed against MNNG, and their protein amounts () were determined using Coomassie brilliant blue G-250 and using bovine serum albumin as a standard.

Gel electrophoresis of purified antimutagenic fractions. When each purified fraction was resolved in a 7.5% polyacrylamide gel and stained with Coomassie brilliant blue R-250 for protein staining, all three fractions showed a single band (Fig. 2). The gel was also stained with fuchsin solution for the test of glycoproteins. All three fractions were successfully stained with fuchsin, suggesting that the purified antimutagenic substances are glycoproteins.

View larger version (30K): [in this window] [in a new window]   FIG. 2.   Polyacrylamide gel electrophoresis of purified antimutagenic fractions of L. plantarum KLAB21 cells. The culture supernatants (lanes C) and the purified antimutagenic fractions (lanes P) were resolved on a native 7.5% polyacrylamide gel. After the electrophoresis, the gels were stained with a Coomassie brilliant blue R-250 solution (CBB) for protein staining or a fuchsin sulfate solution for sugar staining.

Activities of purified antimutagenic fractions. Preliminary experiments showed that up to 100 µl of each fraction were neither toxic nor mutagenic to S. enterica serovar Typhimurium TA100 cells (data not shown). The purified fractions were tested for the antimutagenic activity of the strain against MNNG by using various amounts of up to 100 µl (Table 1). When 20 µl of the purified fractions was used, the highest antimutagenic activity was obtained in each fraction. When an amount greater than 20 µl was used for the activity assay, the antimutagenic activity decreased somewhat. The reason for this is not yet understood. Among the three fractions, the R1-1 fraction showed the highest activity, which was 81.0%. In addition, the antimutagenic activities of the R1-2 and R2 fractions were 72.1 and 60.0%, respectively.

Characteristics of purified antimutagenic fractions. Molecular masses of the three purified fractions were determined using Sepharose CL-6B gel filtration chromatography. They were estimated to be 16 kDa (R1-1), 11 kDa (R1-2), and 14 kDa (R2) (data not shown).

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Thin-layer chromatogram of the purified antimutagenic fractions of L. plantarum KLAB21 for the identification of their sugar compositions. After being treated at 110°C for 24 h in the presence of 6 N HCl, each purified fraction was dried on an evaporator. The dried fractions were dissolved in H2O and applied to a silica gel plate for development with an n-propanol-ethanol-water (7:2:1) mixture for 5 h. After development, the plate was stained with 0.2% orcinol solution in a methanol-sulfuric acid (9:1) mixture and the Rf values of the fractions R1-1 (lane C), R1-2 (lane D), and R2 (lane E) were compared with those of standard glucose (lane A) and galactose (lane B).

	DISCUSSION

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L. plantarum KLAB21 isolated from the Korean fermented vegetable kimchi has been shown to produce antimutagenic substances against MNNG (19, 21). The antimutagenic substances were purified from the culture supernatant by using DEAE-cellulose anion-exchange column chromatography followed by Sephadex G200 and Sepharose CL-6B gel filtrations (Fig. 1). The antimutagenic substances were separated into three different fractions, all of which contained glycoproteins (Fig. 1 and 2). Although their electrophoretic patterns were similar (Fig. 2), their molecular sizes as well as sugar and amino acid compositions were different from each other (Tables 2 and 3; Fig. 3). Therefore, it was proposed that at least three different glycoproteins participate in the antimutagenic activity of L. plantarum KLAB21.

Although it has been well-established that various lactic acid bacterial strains originating from fermented milk, such as L. delbrueckii subsp. bulgaricus, L. helveticus, L. lactis subsp. lactis, and Streptococcus thermophilus, possess antimutagenic activities, there have been no studies on the purification of their antimutagenic substances thus far (18). However, some of the components demonstrating antitumor activity have already been purified and identified. They can be divided into three different groups, depending on the strain: glycopeptides, polysaccharides, or phosphopolysaccharides. The glycopeptide was purified from L. delbrueckii subsp. bulgaricus cells (3), the polysaccharide was from L. kefiranofaciens cells (16), and the phosphopolysaccharide was from L. lactis subsp. cremoris cells (17). The antitumor glycopeptide was reported to be composed of N-acetylglucosamine, N-acetyl muramic acid, and five different amino acids, suggesting that it is a component of the bacterial cell wall (3). The sugar compositions of the purified antimutagenic fractions in this study were found to be glucose or galactose (Fig. 3). In addition, the antimutagenic glycoproteins were purified from the culture supernatant because they have been proposed to be extracellular types (19). Therefore, the antimutagenic glycoproteins purified in this study were thought to have different features from the antitumor glycopeptide purified from L. delbrueckii subsp. bulgaricus cells (3).

Nowadays, the effect of glycosylation on the biological activities of glycoproteins is of increasing interest largely due to their potential therapeutic use. However, it is not yet known whether the glycosylation in these purified antimutagenic fractions plays a key role in their activities. The purified fractions are now under study for antitumor activity using several cell lines in order to ascertain that they possess antitumor activities as well.