Identification and partial

1707J.Food Prot.,Vol.63,No.121708HUR ETAL.FIGURE 1.Scanning electron microscopic observation of strainBH5.FIGURE 2.Production of lacticin BH5in a jar fermenter.supernatant of Kimchi onto MRS agar (Difco Laboratories,De-troit,Mich.)and incubating anaerobically at 30ЊC.Stock cultures were maintained at Ϫ70ЊC in MRS broth to which 20%(vol/vol)glycerol was added.Working cultures were propagated in MRS broth at 30ЊC for 12h before being used in experiments.Micro-coccus flavus ATCC 10240was used as the indicator strain.The strains used for the antimicrobial spectrum of activity indicator organisms were obtained from different culture collections and indicator strains were grown in appropriate media,as indicated in Tables 1and 2.Detection of antimicrobial ctis BH5was ex-amined for antimicrobial activity against indicator organisms on MRS agar plates using the modified deferred method (1).After inoculating the selected producing organism with a toothpick,ctis BH5was grown on MRS agar at 30ЊC for 24h.Five mil-liliters of soft MRS agar (0.7%agar),containing approximately 107cells of the indicator strain per overlay,was overlaid on 1.5%MRS agar plates,and after 24h of incubation at the indicator strain’s optimal growth temperature,an inhibition halo was clearly visible.The strength of the antimicrobial activity was expressed by the diameter (mm)of the halo,and results presented are means of duplicate tests.Lacticin BH5assay.The spot-on-lawn method was per-formed as follows.The soft MRS agar (0.7%agar),containing 107cells of the indicator organism per overlay,was overlaid on 1.5%MRS agar plates.When the soft agar hardened,5␮l of the bacteriocin preparation was spotted on the plate,and after about 12h of incubation at 30ЊC,an inhibition halo was clearly visible.Bacteriocin preparation was made by spotting serial twofold di-lutions of crude bacteriocin and the reciprocal of the greatest in-hibitory dilution was used to calculate the arbitrary activity units (AU)per milliliter of the original cultures.All experiments were performed in duplicate,and the results presented are the means of duplicate trials.Identification of a lacticin BH5producer.Bacteriocin-pro-ducing strain BH5was identified with Gram staining,morpholog-ical examination by scanning electron microscopy,a catalase test,and biochemical carbohydrate fermentation patterns using the API 50CHL kit (bioMe ´rieux,Marcy l’Etoile,France).Production of lacticin cticin BH5production was performed in a 5-liter jar fermenter (3.0liter working volume;Korea Fermenter Co.,Inchon,Korea)in a fermentation medium of MRS ctis BH5was inoculated (1%,vol/vol)into250ml of sterile MRS,and the seed culture (1%,vol/vol)was transferred into the jar fermenter.The temperature was controlled at 30ЊC and the pH was maintained at 6.0Ϯ0.1by the addition of 3N HCl and 3N NaOH.Agitation speed was set at 200rpm in the fermenter,and no aeration was provided.Samples were aseptically removed over a 12-h period to determine the viable cell numbers and the bacteriocin activity at different time inter-vals.Cell growth was monitored spectrophotometrically,and the bacteriocin activity of the culture broth was evaluated using the method described previously.Preparation of cell-free supernatant.Culture broth from the jar fermenter was centrifuged at 8,000ϫg for 20min at 4ЊC and the supernatant was filter-sterilized through 0.22␮m pore size of cellulose acetate (27).Partial purification of lacticin BH5.Partially purified lac-ticin BH5was obtained as follows (21).Solid ammonium sulfate was slowly added to the culture supernatant to 75%saturation at 4ЊC,with constant stirring,over a period of 5h.Slow stirring was continued for an additional 1h at 4ЊC.Precipitated proteins were pelletized by centrifugation at 12,000ϫg for 20min at 4ЊC,then resuspended in 100mM phosphate buffer (pH 7.0),and exten-sively dialyzed against 2liters of 100mM phosphate buffer (pH 7.0)for 12to 24h in dialysis tubing (molecular weight cutoff,1,000;Spectrum Medical Industries,Los Angeles,Calif.).The di-alyzed samples were stored at Ϫ70ЊC.Assay for protein concentration.The protein concentration of the partially purified lacticin BH5was determined using the method of Lowry et al.(15).Bovine serum albumin was used as the standard protein.Antimicrobial spectrum of activity.The spot-on-lawn method was used to assess the antimicrobial activity of the cell-free supernatant and the partially purified preparation of lacticin BH5against several gram-positive and gram-negative strains,in-cluding food spoilage and pathogenic organisms,a yeast,and molds.All strains were previously subcultured in appropriate growth agar medium and were propagated in liquid medium,then inoculated into soft-agar medium (0.7%agar)of the same com-position.Effects of enzymes,heat,pH,and organic solvents on bac-teriocin activity.For enzyme stability testing,partially purified lacticin BH5was treated at 37ЊC for 1h with various enzymes at a final concentration of 1mg/ml.All enzymes were dissolved in buffers as recommended by the supplier (Sigma,St.Louis,Mo.).Untreated bacteriocin plus buffers,buffers alone,and enzyme so-J.Food Prot.,Vol.63,No.12KIMCHI BACTERIOCIN1709 TABLE1.Antimicrobial spectrum of activity of partially purified lacticin BH5using the spot-on-lawn methodOrganismsCulturemedium aIncubationtemp.(ЊC)InhibitionSupernatantPartially purifiedbacteriocinGram-positive bacteriaBacillus cereus ATCC11778B.cereusBacillus pumilisBacillus subtilis ATCC6633B.subtilis IFO12113Clostridium perfringens ATCC3624b Corynebacterium xerosis NCTC9755 Enterococcus faecalis ATCC19433 Lactobacillus delbrueckii ATCC4797 Leuconostoc curvatus CA170-12 Leuconostoc mesenteroides KCCM11324 Listeria ivanovii ATCC19119Listeria monocytogenes ATCC15313 Micrococcusflavus ATCC10240 Pediococcus acidilactici KCTC1626 Staphylococcus aureus ATCC25923S.aureus KCCM32359 Staphylococcus epidermidis ATCC12228TSBNBNBTSBLBTSBTSBTSBMRSMRSMRSTSBTSBNBMRSTSBNBTSB373030373737373737252530303037373737ϩϩϩϮcϪϩϪϮϩϩϩϩϩϩϩϪϩϮϩϩϩϮϪϩϪϮϩϩϩϩϩϩϩϮϩϮGram-negative bacteriaEscherichia coli JM109E.coli KCCM32396E.coli O157:H7Pseudomonas aeruginosa ATCC15442 Pseudomonasfluorescens Pseudomonas putidaPseudomonas syringae ATCC12885 Salmonella EnteritidisSalmonella ParatyphiSalmonella TyphiSalmonella TyphimuriumShigell boydiiShigellaflexneriShigella sonneiVibrio cholerae non-O1V.cholerae O139Vibrio parahaemolyticus ATCC17802 Vibrio vulnificusYersinia enterocolitica ATCC27729LBLBTSBTSBNBNBTSBTSBTSBTSBTSBTSBTSBTSBTSBTSBTSBTSBTSB37373730303030373737373737373737373730ϪϩϪϪϩϪϪϪϪϪϪϪϪϪϮND dϪϪϮϪϩϪϪϩϪϪϪϪϪϪϪϪϪϮNDϮϮϩYeasts and moldsAspergillus oryzae KCCM11371 Aspergillus niger KCCM11239 Penicillium chrysogenum KCCM6933 Saccharomyces cerevisiae KCCM11201PDBPDBPDBYPD30303030ϪϪϪϪϪϪϪϪa NB,nutrient broth;LB,Luria broth;TSB,tryptic soy broth;MRS,lactobacilli MRS broth;PDB,potato dextrose broth;YPD,yeast extract peptone dextrose.b Incubated in anaerobic GasPak jar.c Not clear.d Not determined.lutions were used as control.To determine the effect of heat on bacteriocin activity,aliquots(500␮l)of partially purified lacticin BH5were incubated at various temperatures(40,50,60,70,80, 90,and100ЊC)for30min or autoclaved at121ЊC for15min (20).The pH stability was estimated in the partially purified lac-ticin BH5after4h of storage at4ЊC in the following buffers:50 mM citrate buffer,pH3to6;50mM phosphate buffer,pH7;50 mM Tris-HCl buffer,pH8to9(20).Partially purified bacteriocin was treated with50%organic solvents such as ethanol,methanol, acetone,toluene,isopropanol,and chloroform.Solvent-treated samples were incubated at30ЊC for1h,and residual solvents were evaporated at30ЊC for2h.In this case bacteriocin in buffer without added solvents was used as the control.The residual bac-teriocin activity was determined by the spot-on-lawn method.Re-sults are presented as the average of duplicate trials.Mode of inhibition.Cells from the log-phase of M.flavus ATCC10240were suspended in sterile100mM phosphate buffer (pH7.0).The test was carried out at30ЊC by adding1,000AU/ ml of partially purified lacticin BH5.At various times,the viable cells(CFU/ml)were determined on MRS agar plates using the standard plate counting method.J.Food Prot.,Vol.63,No.121710HUR ET AL.TABLE 2.Effect of various enzymes on partially purified lacticin BH5Enzyme treatmentResidual activity(AU/ml)ControlProtease XIV Protease IX Pepsin␣-Chymotrypsin Proteinase K ␣-Amylase Lipase 51,200051,20051,2001,60051,20051,20051,200TABLE 3.Effect of pH,heat,and organic solvents on partially purified lacticin BH5Treatment Residual activity (AU/ml)Treatment Residual activity (AU/ml)TreatmentResidual activity (AU/ml)Heat pHOrganic solvent Control 40ЊC a 50ЊC a 60ЊC a 70ЊC a 80ЊC a 90ЊC a 100ЊC a 121ЊC b102,400102,400102,400102,400102,400102,400102,40012,800Control 2.03.04.05.06.07.08.09.051,20051,20051,20051,20051,20051,20051,20051,20051,200Control Ethanol Methanol Acetone Toluene Isopropyl Chloroform51,20051,20051,20051,20051,20051,20051,200a Heat treatment for 30min.bAutoclave for 15min.Molecular weight determination by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis.To estimate the molec-ular weight of partially purified lacticin BH5,sodium dodecyl sul-fate-polyacrylamide gel electrophoresis was performed on 16%discontinuous gel.Twenty microliters of the sample and molecular weight standards were applied to the gel.Protein standards and their molecular weights were as follows:ovalbumin,43,000;car-bonic anhydrase,29,000;lactoglobulin,18,400;lysozyme,14,300;bovine trypsin inhibitor,6,200;and insulin,3,000.The sample was prepared by mixing the lacticin BH5sample and buff-er in a 1:1ratio and boiling the mixture at 100ЊC for 5min.Electrophoresis was performed in a vertical slab gel apparatus (Protean Cell II;Bio-Rad,Richmond,Calif.),with the buffer sys-tem at constant voltage (100V)for 2h.Half of the gel was stained with a Coomassie brilliant blue R-250reagent as directed by the manufacturer,while the other half was assayed for bacteriocin activity by the direct method described by Daba et al.(5).Briefly,the area of gel being assayed was fixed for 2h in 20%2-propanol and 10%acetic acid and subsequently soaked for at least 4h in sterile deionized water.It was then aseptically placed in a sterile petri dish and covered with 20ml of soft agar containing 107cells of M.flavus ATCC 10240as the indicator strain.The plate was then incubated at 30ЊC for 12h and examined for zones of inhi-bition.RESULTS AND DISCUSSIONIdentification of a lacticin BH5producer isolated from Kimchi.Strain BH5was isolated from naturally fer-mented Kimchi and identified as a bacteriocin producer.Upon dilution of lacticin BH5,the zones of inhibition on lawns of the indicator strain diminished in size without the appearance of plaques,which suggested that the inhibition was not caused by the replication of bacteriophage.The bacteriocin-producing strain BH5was identified by Gram staining,a catalase test,morphology by scanning electron microscopy,and from biochemical carbohydrate fermentation patterns using the API 50CHL rma-tion on carbohydrate utilization patterns of strain BH5co-incided with those of ctis ctis (data not shown).This isolate was gram-positive,nonmotile,cata-lase-negative,and the cells were of the coccus type (Fig.1).On the basis of these results this strain was identified as ctis,and the isolate was tentatively named as ctis cticin BH5is proposed as the tentative name of the bacteriocin produced by ctis BH5.Production of lacticin BH5.In MRS medium of pH 6.0and incubated at 30ЊC,ctis BH5produced extra-cellular inhibitory activity against M.flavus ATCC 10240(Fig.2).Production of lacticin BH5seems to follow the kinetics typical of primary metabolite cticin BH5activity reached a maximum (12,800AU/ml)after in-cubation for 5h,the early stationary phase,and was main-tained for 1h before dropping after the late stationary phase.Possible reasons for this rapid decrease in bacterio-cin activity include the formation of an inhibitor,its deg-radation by extracellular proteolytic enzymes,the binding of bacteriocin to cells,and inactivating complex formation with other extracellular products (22).Almost all the bac-teriocins of lactic acid bacteria are produced during the ex-ponential growth phase.Several lantibiotics are also syn-thesized during the exponential phase of the producer strain (7,8,26).This comparatively early bacteriocin production (5h)by ctis BH5could be exploited as an industrially useful characteristic.Partial purification of lacticin ctis BH5was cultured in a jar fermenter at 30ЊC for 6h,and the culture broth was centrifuged.Partially purified prepara-tions of lacticin BH5were made by ammonium sulfate pre-cipitation.The activity of the partially purified lacticin BH5J.Food Prot.,Vol.63,No.12KIMCHI BACTERIOCIN1711FIGURE 3.Mode of action of lacticin BH5against M.flavus ATCC 10240in phosphatebuffer.FIGURE 4.Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis of lacticin BH5after Coomassie brilliant blue staining (A)and the gel overlaid with M.flavus ATCC 10240(B)for determination of molecular ne 1,protein molecular size standards;2,cell-free supernatant;3,partially purified lacticin BH5.was 409,600AU/ml,approximately 97.6%of the lacticin BH5activity was recovered.The specific activity was in-creased 9.4-fold (data not shown).Antimicrobial spectrum of activity.To determine the antimicrobial spectrum of activity,the cell-free supernatant and partially purified lacticin BH5were tested against var-ious nonpathogenic and pathogenic bacteria,a yeast,and molds using the spot-on-lawn methods (Table 1).Lacticin BH5showed a relatively broad spectrum of activity against most of lactic acid bacteria,Staphylococcus aureus KCCM 32359,Staphylococcus epidermidis ATCC 12228,Clostridium perfringens ATCC 3624,some bacilli,M.flavus ATCC 10240,Listeria monocytogenes ATCC 15313,Listeria ivanovii ATCC 19119,Yersinia enterocol-itica ATCC 27729,Escherichia coli KCCM 32396,and Pseudomonas fluorescens using the spot-on-lawn method,when compared with other bacteriocins of lactic acid bac-teria.However,inhibitory activity was not observed against a yeast and molds.Accordingly,from its inhibitory spec-trum,lacticin BH5appeared to show similarity with lanti-biotic nisin that inhibits most gram-positive bacteria (3),rather than several bacteriocins from Lactobacillus sp.,whose activity spectrums include only strains that belong to the same genus (2,10,24).Effects of various enzymes,heat,pH,and organic solvents.As shown in Table 2,treatment with protease XIV caused a complete loss of bacteriocin activity.When the partially purified bacteriocin was treated with ␣-chymo-trypsin,the bacteriocin activity was decreased,but not com-pletely.No modification of activity was observed when lac-ticin BH5was treated with the other enzymes tested (pro-tease IX,pepsin,proteinase K,␣-amylase,and lipase).Buffers and enzyme solutions alone had no effect on the indicator strain.When the partially purified bacteriocin was treated with lipase and amylases,the bacteriocin activity was not changed.These results confirm the proteinaceous nature of the antimicrobial substance and suggest that nei-ther lipid nor carbohydrate moieties are essential for the bacteriocin activity.Lacticin BH5proved to be relatively heat stable (Table 3);partially purified lacticin BH5was stable to heat trea-ment at 90ЊC for 30min.Inhibitory activities were detectedduring treatment up to 100ЊC for 30min,however,the in-hibitory activity of partially purified lacticin BH5was in-activated after autoclaving at 121ЊC for 15min.This heat stability could be due to the formation of small globular structures and the occurrence of strongly hydrophobic re-gions,stable cross-linkage,and a high glycine content (6).This heat stability also rules out the possibility that the in-hibitory action is due to bacteriophage.In terms of food-related applications,it requires further study on the activity of lacticin BH5at refrigeration temperature,as well as sta-bility following freezing and thawing (9).Finally,partially purified bacteriocin was pH stable in the range 2.0to 9.0and it was not affected by any of the organic solvents shown in Table 3.This aspect of its sta-bility is of particular importance to the food industry.Mode of inhibition.To determine whether lacticin BH5has a bactericidal or a bacteriostatic effect,the par-tially purified lacticin BH5was added to the indicator cells suspended in phosphate buffer (pH 7.0).Lacticin BH5showed a bactericidal mode of action.A decrease in CFU per milliliter was observed after the bacteriocin was ex-posed to the indicator cells (Fig.3).However,the intrinsic nature of this inhibition has not been identified and requires further investigation.Molecular weight of lacticin BH5.Several contami-nating proteins were detected in the partially purified lac-ticin BH5(Fig.4).The 16%polyacrylamide gel,containing partially purified lacticin BH5,was cut into two vertical sections.The gel portion containing the sample and the mo-lecular weight markers was stained,while the remaining section that contained only the sample was fixed and used for the direct detection of antimicrobial activity using the method of Daba et al.(5).As shown in Figure 4,the bac-tericidal activity of lacticin BH5was associated with a band having an apparent molecular mass of 3to 3.5kDa.Thus,1712HUR ET AL.J.Food Prot.,Vol.63,No.12。