Isolation of bacteriocin nisin producing Lactococcus lactis from dairy ...

J. Acad. Indus. Res. Vol. 1(10) March 2013 627RESEARCH ARTICLEISSN: 2278-5213Isolation of bacteriocin nisin producing Lactococcus lactis from dairy productsP.K. Nagalakshmi * , R. Sumathi, K. Kanimozhi and T. SivakumarDept. of Microbiology, Kanchi Shri Krishna College of Arts and Science, Kilambi, Kancheepuram-631551, TN, Indianagsmicro@yahoo.co.in*; +91 9840558346; 9865206694______________________________________________________________________________________________AbstractMost inhibitory substances produced by lactic acid bacteria are safe and effective natural inhibitors ofpathogenic and food spoilage bacteria in various foods. In this study, bacteriocin nisin producing Lactococcuslactis was isolated from fermented milk and cheese. The test isolate was grown in MRS medium underoptimized conditions. Bacteriocin activity of the test isolate was determined by agar well diffusion assayagainst Escherichia coli, Staphylococcus aureus and Bacillus sp. The optimum temperature of L. lactis inMRS medium was 37C and the highest viable cell count of L. lactis at 37C reached about 8.54 log cfu/mL in8 h and 8.10 log cfu/mL in 14 h. Bacteriocin nisin production was maximized when L. lactis was grown incontrolled pH fermentation medium of 6.5. Bacteriocin supernatant (CBS) of L. lactis was heat stable, as theinhibition activity remained constant after heating at 80C for 20 min. The results revealed that E. coli wasleast sensitive to crude bacteriocin supernatant of L. lactis as compared to other indicator strains.Keywords: Lactic acid bacteria, bacteriocin nisin, Lactococcus lactis, Staphylococcus aureus, Bacillus sp.IntroductionLactic acid bacteria (LAB) have been used for centuriesin the fermentation of foods, not only for flavour andtexture, but also due to their ability to prevent the growthof pathogenic microorganisms. They are also responsiblefor the fermentative processing and preservation of manyfood products including dairy, meat, vegetables andbakery products (McAuliffe et al., 2001). Preservation offermented foods is due primarily to the conversion ofsugars in organic acids with a concomitant lowering ofthe pH and removal of large amounts of carbohydratesas nutrient sources. These effects extend the shelf lifeand safety of the final product (Delves-Broughton, 1990).Later, it has been identified that the preservation effectresults from the antimicrobial action of bacteriocins aswell as metabolites, such as lactic acid and hydrogenperoxide, produced by LAB. Bacteriocins are ribosomallysynthesized, extracellularly released bioactive peptide orpeptide complexes which have a bactericidal orbacteriostatic effect on other species. In all cases, theproducer cell exhibits specific immunity to the action ofits own bacteriocins. Bacteriocin-producing strains canbe readily identified in a deferred antagonism assay, inwhich colonies of the putative producer are overlaid witha bacterial lawn of a sensitive strain (Deegan et al.,2006).Nisin is the most extensively characterized bacteriocin ofantimicrobial proteins produced by LAB (Jack et al.,1991). Nisin is secreted by Lactococcus lactis has beenused as a food preservative for more than 50 years andno significant bacterial resistance against nisin has beenreported. Lactococcus lactis is a microbe classifiedinformally as lactic acid bacteria because it ferments milksugar (lactose) to lactic acid.Lactococci are typically spherical or ovoid cells, about1.2 µm/1.5 µm, occurring in pairs and short chains. Theyare gram-positive, non-motile and do not form spores.Lactococci are found associated with plant material,mainly grasses, from which they are easily inoculatedinto milk. Hence, they are found normally in milk and maybe a natural cause of souring (Hansen and Liu, 1990).Although nisin has been approved as a food preservativeby Food and Drug Administration (FDA), the low rateproduction by the producer strains has restricted itsapplication in the fermentation systems. Therefore, theisolation and characterization of new strains having highproduction ability has been the main subject in thisrespect. The first nisin producers were isolated andidentified in fermented milk products, but since, then theyhave been isolated from various dairy products (Mitraet al., 2005), traditional fermented vegetables (Olasupoet al., 1999), fermented meat products (Choi et al.,2000), river water (Zendo et al., 2003) and human milk(Beasley and Saris, 2004). In view of the above facts,this study was initiated to isolate bacteriocin nisinproducing Lactococcus lactis from dairy products alongwith characterization and optimization of nisin producingorganisms.Materials and methodsIsolation of lactic acid bacteria: Fermented milk wasdirectly submitted to isolation of lactic acid bacterialstrains, while cheese is serially diluted in saline solutionand then plated on MRS agar. The plates were incubatedaerobically at 37C for 48 h and then several colonieswere picked randomly for identification of lactic acidbacteria.©Youth Education and Research Trust (YERT) Nagalakshmi et al., 2013

J. Acad. Indus. Res. Vol. 1(10) March 2013 628The isolated colonies were further subcultured andtransferred to MRS slants incubated at 37C for 48 h andpreserved at 4C for further work.Morphological and biochemical tests: The isolates weregrouped as lactic acid bacteria after examining theirgram reaction, cell and colony morphology, catalasereaction and gas production from glucose fermentation.The test isolate LAB, Lactococcus lactis was used forfurther optimization process.Effect of heat treatment on bateriocin production:Aliquots (5 mL) of sterile CBS were heated at 70, 80, 90and 100C for 20 min in a thermostatically controlledwater bath and at 121C for 20 min in an autoclave. Thetreated samples were tested for antibacterial activityagainst the indicator bacterial strains. Untreated cultureconcentrated served as a positive control.Plate 1. Growth of L. lactis on MRS media.Optimum temperature for growth of L. lactis: Lactococcuslactis culture was inoculated in 100 mL MRS broth at100 rpm in a shaking incubator at different temperaturesranging from 30, 35, 37 and 40C. Samples were takenevery hour for the first 12 h and then at 14, 16, 20 and24 h for enumeration of LAB in MRS agar and incubatedat 37C for 24-36 h.Controlled pH for growth of L. lactis: Lactococcus lactisstrain was grown in MRS broth supplemented with2% yeast extract, 0.2% glucose and 0.2% meat extractincubated at 37C for 24 h. The pH of fermentation wasmaintained constantly at 6, 6.5 and 7 by addition of1 M NaOH and uncontrolled-pH fermentation was alsoinvestigated. Samples were taken every hour for the first12 h and then were taken at 14, 16, 20 and 24 h.Preparation of crude bacteriocin supernatant (CBS):Overnight cultures of L. lactis were inoculated to 25 mLMRS broth incubated at optimum temperature for12-16 h to reach stationary phase. The cultures wereprepared for CBS by centrifugation at 12,000 x g for20 min at 4C for determining bacteriocin activity by agarwell diffusion assay. The inhibition activity was reportedas diameter (mm) of a clear inhibition zone.Bacteriocin assay method by agar well diffusion:Bacteriocin screening was performed by well diffusionmethod. The target strains used in this study wereE. coli, S. aureus and Bacillus sp. and they weresubcultured in tryptone soy broth (TSB) for 24 h foractivation prior to antimicrobial activity. The antimicrobialactivities of the isolates were quantified by agar welldiffusion method. An isolated colony was selected fromMRS agar plate culture and the growth is transferred intoa tube containing sterile MRS broth (5 mL), incubated at35C for 24 h. To get the culture filtrate, a 24 h culturewas centrifuged at 10,000 rpm for 20 min and heattreatment process was done. An actively growing testorganism in TSB of 24 h culture were dipped with asterile cotton swab and overlaid on MHA plates. Wells ofapproximately 6 mm were bored in the agar medium.The well was filled with culture filtrates. After 12-24 h ofincubation, each plate was examined. The diameter ofthe zones of complete inhibition was examined, includingthe diameter of the well.Table 1. Physiological and biochemical characteristics of L. lactis.Colony morphologyRaised and circularCell morphologyCocciCell arrangementPairs and short chainsGram reactionPositiveSpore formationNegativeCatalase activityNegativeGlucose fermentationPositiveSalt tolerance testNaCl at 2%PositiveNaCl at 4%PositiveNaCl at 6.5%PositiveResultsA total of 120 colonies from the fermented food sampleswere examined, of which 12 colonies were recorded aspositive, producing inhibition zones on agar media.Of these colonies, only one strain No. 12 stably secretedinhibitory substances into culture broth. This bacteriocinproducing strain was gram positive, catalase-negative,coccus, non-motile and produced no gas from glucosewas potentially identified as Lactococcus lactis (Plate 1and Table 1). Optimum temperature of L. lactis growth inMRS broth was found to be 37C in 8 h (Table 2).When the pH of fermentation of L. lactis was constantlyheld at 6.0, 6.5, 7.0 and uncontrolled, at 37 o C then theviable cell counts of L. lactis and absorbance at 600 nmwere determined. Viable cell counts of L. lactis andabsorbance of fermented media during 24 h wasobserved. The maximum of absorbance (in term ofabsorbance reading at 600 nm) and bacterial growthwere observed at 8 h at pH of 6.5 (Table 3). Antimicrobialactivity of standard antibiotics was tested against E. coli,S. aureus and Bacillus sp. and degree of inhibitiontowards test organisms were given in Table 4.©Youth Education and Research Trust (YERT) Nagalakshmi et al., 2013

J. Acad. Indus. Res. Vol. 1(10) March 2013 629Table 2. Growth of L. lactis in MRS broth at different temperatures.TemperatureGrowth (OD at 600 nm)0 h 2 h 4 h 6 h 8 h 10 h 12 h 14 h 16 h 18 h 20 h 22 h 24 h30C 6.51 6.58 7.01 7.21 7.62 8.12 8.26 8.02 7.98 7.72 7.50 6.50 5.7135C 6.81 7.00 7.50 8.00 8.12 8.35 7.90 7.61 7.35 7.10 6.95 6.85 6.5037C 6.53 6.80 7.20 8.01 8.54 8.50 8.31 7.80 7.35 7.20 6.98 6.50 6.0040C 6.50 6.67 6.98 7.15 7.20 7.65 7.91 8.10 7.65 7.10 6.80 6.09 5.50Table 3. Growth of L. lactis in MRS broth at different pHs.pHGrowth (OD at 600 nm)0 h 2 h 4 h 6 h 8 h 10 h 12 h 14 h 16 h 18 h 20 h 22 h 24 h6.0 0.0 0.0 0.20 1.25 2.53 2.49 2.30 2.25 2.20 2.18 2.15 2.14 2.136.5 0.0 0.0 0.60 2.50 2.68 2.40 2.28 2.24 2.19 2.17 2.15 2.15 2.157.0 0.0 0.0 0.75 2.25 2.43 2.25 2.15 2.14 2.13 2.10 2.09 2.08 2.00Control 0.0 0.0 0.11 0.25 0.50 0.95 1.10 1.15 1.19 1.20 1.22 1.25 1.29In this study, maximum zone of inhibition was observedin ciprofloxin (31 mm) and minimum inhibitory of 11 mmobserved in streptomycin. The effect of heat treatment onbacteriocin activity was determined using the indicatorbacteria. The inhibitory substance produced by L. lactiswas considered to be nisin and heat stable (Table 5 and6).DiscussionLactic acid bacteria isolated from fermented milkproducts includes Lactobacillus, Lactococcus,Pediococcus and Streptococcus. All LAB were subjectedto inhibitory activity test using agar well diffusion method.Girum et al. (2005) observed varying degree of inhibitionon various food borne pathogens by the culture filtrate ofLAB. In this study, essentially it is noted that bacteriocinhas a lytic bactericidal mode of action. There is aphysiological link between temperature, growth and nisinconcentration. This may be due to the direct effect oftemperature on microbial growth. Optimum temperatureof L. lactis in MRS broth could be considered at 37C.The highest viable cell count of L. lactis at 37C reached8.54 log cfu/mL in 8 h, whereas incubation at 30, 35 and40C, the highest viable cell counts reached8.26 log cfu/mL in 11 h, 8.35 (9 h) and 8.10 log cfu/mL in14 h, respectively. It was obvious that the growth ofL. lactis at 37C was better than at other temperatures.The optimum temperature at 37C falls in line with theprevious reports by Matsusaki et al. (1998) andBenkerroum et al. (2000). Crude bacteriocin supernatantproduced by L. lactis in controlled pH fermentationrevealed that bacteriocin production could be maximizedby maintaining the pH of growth media at 6.5. The effectof pH on bacteriocin production has been welldocumented where control of pH during growth results inhigher bacteriocin titers (Yang and Ray, 1994; Franzet al., 1997). Therefore, fermentation of bacteriocinproducing bacteria at controlled pH of 6.5 wasconsidered as more suitable than at pH 6 and 7.Decreasing of bacteriocin activities was observed at 11,14, 12 and 12 h for the fermented media at controlled pHof 6, 6.5, 7 and uncontrolled pH.Table 4. Antibiogram results of test strains withtraditional antibiotics.AntibioticsE. coli S. aureus Bacillus sp.(mm) (mm) (mm)Amikacin (30 µg) 20 23 25Gentamycin (10 µg) 21 20 21Streptomycin (10 µg) 20 20 11Ciprofloxin (5 µg) 31 25 24Table 5. Effect of heat treatment on bacteriocin activity byagar well diffusion method.Test organismBacteriocinHeat treatmentactivity(⁰C)(mm)70 2080 16E. coli90 12100 10121 08Control 2070 2380 21S. aureus90 18100 15121 10Control 2370 2580 23Bacillus sp.90 18100 15121 10Control 25Decrease in bacteriocin activity was observed during thelate of fermentation for both controlled and uncontrolledpH media. O’Keeffe and Hill (1999) described thisphenomenon due to the protein degradation, proteinaggregation and complex formation. Crude bacteriocinsupernatant produced by L. lactis was considered to beheat stable, as the inhibition activity remained constantafter heating at 80C for 20 min, but decreased in activitywas observed when heating at 90C from 10 to 20 min.Heating at 100C for 20 min, the activity decreased downthereafter, when CBS was autoclaved at 121 o C for20 min and its activity remained slightly.©Youth Education and Research Trust (YERT) Nagalakshmi et al., 2013

J. Acad. Indus. Res. Vol. 1(10) March 2013 630This indicated that crude bacteriocin produced byL. lactis was heat stable at 100C. The hypothesis of thisstudy was to demonstrate that the optimum temperaturefor cell growth should be the optimum for bacteriocinproduction as well. Therefore, the bacteriocin productionwas conducted at 37C. E. coli was least sensitive toinhibitory substance produced by LAB as compared toother indicator strain. The resistance of gram-negativebacteria is attributed to the particular nature of their cellenvelop, the mechanism of action described forbacteriocin. This could be due to fact that these isolatesare nisin producers (Soomro et al., 2002) and theprimary target of nisin antimicrobial action is the cellmembranes. Nisin has a inhibitory effect against a widevariety of gram-positive food borne pathogens andspoilage microorganisms (Rodriguez, 1996). In thisstudy, wider inhibition zone were observed by theLactococcus on the test strains. Similarly, Kellyet al. (1996) and Hernandez et al. (2005) reported thatseveral Lactococci produced nisin like activity andshowed a broad inhibitory spectrum against the indicatorstrains tested.ConclusionThe culture filtrates from L. lactis isolated from dairyproducts exhibited antimicrobial activity against threeindicator test strains. The test isolate was grown in MRSmedium under optimized conditions. Bacteriocin activityof the test isolate was determined by agar well diffusionassay against Escherichia coli, Staphylococcus aureusand Bacillus sp. The optimum temperature of L. lactis inMRS medium was 37C. Highest viable cell count ofL. lactis at 37C reached about 8.54 log cfu/mL in 8 h.Bacteriocin nisin production was maximized whenL. lactis was grown in controlled pH fermentation mediumof 6.5. Bacteriocin supernatant of L. lactis was heatstable, as the inhibition activity remained constant afterheating at 80C for 20 min. 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Biochem. 67: 1616-1619.©Youth Education and Research Trust (YERT) Nagalakshmi et al., 2013