799

Journalof Food Protection, Vol. 65, No. 5, 2002, Pages 799– 805 Copyright Q,InternationalAssociation for FoodProtection

Behavior of Listeria monocytogenes and Staphylococcusaureus inY ogurtFermented witha Bacteriocin-ProducingThermophilic Starter

NOREDDINE BENKERROUM, 1* HAFIDA OUBEL, 2 AND LAMIAE BEN MIMOUN 1

1InstitutAgronomique et Ve´te´rinaireHassan II, De´partementdes Sciences Alimentaireset Nutritionnelles,BP: 60202,Instituts, 10101-Rabat, Morocco;and 2Universite´MohamedV, Faculte´des Sciences, Laboratoirede Microbiologieet deBiologie Mole ´culaire,Avenue Ibn Batouta, BP:

1014,Rabat, Morocco Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/5/799/1673166/0362-028x-65_5_799.pdf by guest on 27 September 2021

MS01-352:Received 26September 2001/ Accepted 17December 2001

ABSTRACT

Streptococcussalivarius subsp. thermophilus Bproducinga bacteriocinactive against Listeriamonocytogenes ATCC 7644 and Staphylococcusaureus SAD 30was isolated from bakery yeast. The bacteriocin was partially puriŽ ed by an ad- sorption/desorptiontechnique, and its spectrum of action was compared to that of a neutralizedcell-free supernatant (CFS). Althoughthe CFS inhibiteda numberof gram-positiveand -negative bacteria of healthand spoilage signiŽ cance, the spectrum ofactionof thepartially puriŽ ed bacteriocinwas limited to gram-positivebacteria. L.monocytogenes wasthe most sensitive toboth preparation s.The bacteriocin -producingstreptococ calstrain was used in combination with a Bac 2 Lactobacillus delbrueckii subsp. bulgaricus CYstrainisolated from commercial yogurt to assess the effectivene ssof the resulting ther- mophilicstarter in controlling L.monocytogenes and S. aureus inyogurt during fermentatio nandstorage at refrigeratio n (ca. 78C)orabuse (ca. 22 8C)temperature.Yogurtsamples were contaminate dwith L.monocytogenes or S. aureus to the approximatelevels of 10 3 and 106 CFU/mlof milk, respectivel y.The results showed that in situ bacteriocin production wasmore active against L.monocytogenes thanagainst S. aureus invitro and in contaminate dsamples.While L. mono- cytogenes leveledoff below the detectable limit in a 1-mlsample of yogurt within 24 h ofprocessing , S. aureus survived in Bac1 and Bac2 samplesduring 10 days of storage at room temperature (ca. 22 8C).Useof a Bac 1 starterresulted in a 5-dayextension of the shelf life.

Dairyproducts have been associated with foodborne talstress including heat, acidity, and reduced water activity diseases.Recurrent cases of listeriosis (11, 27) and staph- (13). Suchresistance may even increase in stressful con- ylococcalintoxications (11) dueto the consumption of con- ditions,leading to unusual acid (14, 26) or heat (19, 23) taminatedmilk and milk derivatives have been reported tolerance. throughoutthe world whenever accurate records were avail- Mostof therecent strategies for controllingthe growth ablefor audit. ofundesirable microorganisms in foods tend to apply the Arecentsurvey revealed that Listeriamonocytogenes ‘‘hurdleconcept,’ ’ advocatingthe combination of morethan and Staphylococcusaureus were involvedin 22 and 15%, onebarrier having different modes and spectra of action. respectively,of recorded foodborne illnesses caused by Theaim of this approach is to increase the potential of dairyproducts in eightdeveloped countries (11). According eliminatinga broaderrange of pathogens and spoilage mi- tothesame report, S. aureus was responsiblefor morethan croorganismsand thus to enhance the safety and keeping 85%of the dairy-borne diseases in France. Moreover , L. qualityof foods. Bacteriocins or bacteriocin-producin g monocytogenes was responsiblefor ca.25% of the esti- strainshave often been suggested as partof thehurdle com- matedfoodborne disease-related deaths in the United States binationto reduce the prevalence of L.monocytogenes and (27). Therefore,a tremendouseffort has been made during S. aureus inyogurt and other food products (22,35, 37). thelast two decades to control these pathogens in foods. Nisinhas Ž rst beensuggested because of its generally Inthis regard, yogurt received the least attention recognizedas safe statusin many countries and because amongdairy products. Its highacidity and milk pasteuri- ofits efŽ cacy in removing the pathogens from dairyprod- zationwere oftenthought to be effective barriers against ucts (6, 10). Nonetheless,the bacteriocin has been shown theproduct’ s beingsubjected to L.monocytogenes and S. toinhibit the thermophilic strains used in yogurt starters aureus contamination.However ,itis now well established (21) andhence may seriously impede the fermentation. thatboth pathogens can occur in yogurt as a resultof their Otherbacteriocins of anti- Listeria classIIa havealso been survivalduring processing or postcontamination (4, 8, 29, suggested (22), butthey still need to be legally approved 31).L. monocytogenes isnaturallyresistant to environmen- for suchuse. Furthermore, thermophilic starter cultures commonlyused in dairy fermentations were shownto be *Authorfor correspondence. T el: 212(0) 37 77 17 58/ 59;Fax: 212 (0) particularlysensitive to the class I lantibioticsnisin A and 3777 81 35; E-mail: [email protected]. Z,aswell as to other class II bacteriocins (28). Therefore, 800 BENKERROUM ETAL. J.FoodProt., Vol. 65, No. 5

TABLE 1. Sensitivityof selected gram-positive and -negative bacteria to the cell-free supernatant (CFS) or to the partially puriŽ ed bacteriocin(PPB) of Streptococcussalivarius subsp. thermophilus

Extentof inhibition b

Indicatorbacteria Origina CFS PPB

Gram-positivebacteria Listeriamonocytogenes 7644 ATCC 1111 1111 L.monocytogenes2 CHU 1111 1111 Staphylococcusaureus SAD 30 IAVHASSAN II 11 11 Micrococcus avus NCIB8166 OSU 2 ND Bacillus sp. TE1 Ourcollection 1 ND Streptococcusfaecalis SF1 IAVHassanII 11 ND

Enterococcusfaecium A2 Ourcollection 2 ND Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/5/799/1673166/0362-028x-65_5_799.pdf by guest on 27 September 2021 S.salivarius subsp. thermophilus B This study 2 2 Lactobacillusdelbrueckii subsp.bulgaricus CY This study 2 2 E. faecius SPH1 Ourcollection 2 2 S.thermophilus SPH2 Ourcollection 2 2 Lactococcuslactis subsp. lactis M Ourcollection 2 2 L. lactis S Ourcollection 2 2 L. lactis H Ourcollection 2 2 Gram-negativebacteria Salmonella TyphimuriumSTP1 IAVHassanII 1 2 Salmonella Typhi ST20 IAVHassanII 11 2 Salmonella Hadar SH4 IAVHassanII 1 2 Pseudomonasputida PPOSU OSU 11 2 Pseudomonasaeruginosa PAOSU OSU 11 2 Escherichiacoli EC4 Ourcollection 1 2 Enterobacter sp. EI3 HMI MV 1 2 Proteusmirabilis HM10 HMI MV 1 2 Escherichiacoli V517 OSU 2 2 Klebsiellapneumoniae E30PIP135 HMI MV 11 2 Enterobactercloacae B703 HMI MV 11 2 Shigella sp. SI1 Ourcollection 11 2 a ATCC, AmericanType Culture Collection; OSU, OregonState University; HMI MV,HoˆpitalMilitaire d’ InstructionMohamed V , Rabat;CHU, CentreHospitalier Universitaire Mohamed V ,Rabat;IA VHassanII, InstitutAgronomique et Ve ´te´rinaireHasssan II, Rabat. b 1111,diameterof the inhibition zone (ø ) . 20 mm; 111, 16 , ø , 20 mm; 11, 12 , ø , 16 mm; 1, ø , 12 mm; 2, no inhibition;ND, notdetermined. Data are averages of three determinations, each one in duplicate. thesearch for newbacteriocin-producin gstrainsnormally 2208Cinappropriatebroths containing 25% glycerol. Before use, usedin thermophilic starters that can efŽ ciently enhance lacticacid bacteria (LAB) werepropagated in deMan Rogosa thesafety and extend the shelf life of dairyproducts seems Sharpe(MRS) broth (12), andthe other microorganisms were worthwhile. propagatedin tryptic soy broth (Biokar ,France). Theobjective of the present study was toisolate and Acommerciallyophilized yogurt starter (Redi-set, Hansen characterizea bacteriocin-producingstrainof Streptococcus Laboratories,Inc., Milwaukee, Wis.) was used for control pur- poses.Before use, this starter culture was activated in sterile non- salivarius subsp. thermophilus andto evaluate its potential fatdry milk according to the manufacturer’ s recommendationsand L. mon- toprotect yogurt from thesurvival and growth of testedfor inhibitory activity against L.monocytogenes ATCC ocytogenes and S. aureus duringprocessing and storage at 7644by the spot test (33) toconŽ rm its suitability as a control refrigeration(ca. 7 8C)orabuse (ca. 22 8C)temperature. (Bac2). MATERIALS AND METHODS ConŽrmation of the bacteriocinogenic nature of the in- Bacterialstrains and media. S.salivarius subsp. thermo- hibitorysubstance produced by S.thermophilus B. To exclude philus B,abacteriocin-producingstrain (the ‘ ‘producer’’), was theeffect of organic acids, the pH of an overnight culture of the isolatedin this study by the method described previously (5). It producerstrain in MRS brothfortiŽ ed with 2.5% yeast extract wasidentiŽ ed by morphological and physiological tests (9). A wasadjusted to pH 6.0with 3 MNaOH solutionand assayed for nonbacteriocin-producing Lactobacillusdelbrueckii subsp. bul- inhibitoryactivity against L.monocytogenes ATCC 7644with the garicus strainCY wasisolated from commercial yogurt and iden- welldiffusion test (36). Sensitivitytests for catalase, pronase E, tiŽed according to Kandler and W eiss (20). trypsin,and a-chymotrypsinwere performed to exclude the effect Bacteriaused in the present study (T able1) were stored at ofhydrogenperoxide (catalase treatment) and to conŽrm the pro- J.FoodProt., Vol. 65, No. 5 EFFECTOF BAC 1 STARTER ON L.MONOCYTOGENES AND S. AUREUS IN YOGURT 801 teinaceousnature (protease treatment) of the inhibitory substance Spectrumof action of the partially puriŽ ed bacteriocin. produced by S.thermophilus B.Allenzymes were obtained from Theactivity of theCFS orthepartially puriŽ ed bacteriocin (PPB) SigmaChemical Company (St. Louis, Mo.), except for catalase, againstdifferent gram-positive and -negative pathogenic or spoil- whichwas purchased from Serva (Heidelberg, Germany). Buffer agebacteria (T able1) was tested by the well diffusion test (36). preparationsand enzyme treatments were conducted as described previously (5). Yogurttrials. Nonfatdry milk was added to raw milk to adjustfat and dr ymattercontents to approximately 17 and 150 Stabilityto pH and heat. Twoseriesof cell-freesupernatant g/liter,respectively.After a thoroughmixing, milk was pasteur- (CFS) sampleswere prepared. In the Ž rstseries, aliquots of the izedin a waterbath at 80 8Cfor30 min, and then cooled to 40 8C crudeCFS sampleswere made and exposed to heat treatments of inice water. Pasteurized milk was divided into two batches of 6 increasingseverity. In the second, the pH wasadjusted to different literseach, one of which was inoculated with S.thermophilus B valuesranging from 2.0 to 11.0 with concentrated HCl or10 M and L.bulgaricus CY(1.5%each). The other (control) was in- NaOH solutionsand was heat treated (121 8Cfor15 min). The oculatedwith the commercial Bac 2 starter(Redi-set, Hansen). Eachbatch was further divided into two parts. The Ž rstpart was supernatantsof both series were tested for inhibitory activity Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/5/799/1673166/0362-028x-65_5_799.pdf by guest on 27 September 2021 against L.monocytogenes ATCC 7644by thewell diffusion assay. contaminatedwith an overnight culture of L.monocytogenes Nonheat-treatedCFS samplesof each series were tested simulta- ATCC 7644 or S. aureus SAD 30(0.03 or 3.0 ml) to achieve an 3 6 neouslyto serve as controls. approximateinocula of 10 and 10 CFU/mlof milk,respectively. Thesecond part was not contaminated and hence served as a PartialpuriŽ cation of the bacteriocin produced by S. negativecontrol. Inoculated milk was then dispensed into plastic thermophilus B. ThepH-dependent adsorption/ desorptiontech- cupscontaining 100 ml, incubated at 40 8Cuntilcoagulation, and niquedescribed by Y anget al. (39) wasperformed with some thentransferred to the refrigerator (ca. 7 8C).At 24h, half of the adjustmentsto partially purify the bacteriocin. yogurtcups of each trial were withdrawn from the refrigerator to BeforepuriŽ cation, the effect of thepH on the adsorption of bestored at ambient temperature (ca. 22 8C). thebacteriocin to thecell wall of theproducer organism was stud- Enumerationof microorganisms and pH measurement. ied. S.thermophilus Bwasgrown overnight in 500 ml of MRS Microbialenumerations were performed at regular intervals dur- brothfortiŽ ed with 2.5% yeast extract at 30 8C.Theculture was ingprocessing and storage until alteration was perceivable. LAB thenheated at 70 8Cfor25 min to kill producer cells and to in- wereenumerated on MRS agar (12) after24 to48hofincubation activateany proteolytic enzymes that may have been present. Ar- at 408C, L.monocytogenes wasenumerated on Al-zoreki-Sandine bitraryunits, deŽ ned as the reciprocal of the highest dilution ListeriaMedium (3) after48 to 72 h ofincubation at 37 8C, and showinga distinctzone of inhibition of the indicator strain, were S. aureus wasenumerated on Chapman agar (Oxoid, Hampshire, determined(AU ).Theheated culture was then aseptically dis- T UK) afterincubation at 37 8Cfor24 to48 h. ThepH wasmeasured pensedin sterile test tubes (10 ml each) and adjusted to different everyhour until coagulation, and then at 24h, witha JenwaypH pHvalues ranging from 1.5 to 8.0 by adding 1 MHCl or3 M meter(Model 3310, Felsted, Dunmow, Essex, UK). NaOH solutions.T ubeswere kept in the refrigerator for 30 min topromote desorption of the bacteriocin from the producer cell Statisticalanalysis. Experimentswere repeated three times wall.Each suspension was then adjusted to pH 6.0,and cells were for L. monocytogenes andtwice for S. aureus. Alldeterminations removedby centrifugationin a microfuge(235C; Fisher ScientiŽ c, weredone in duplicate. Analysis of variance ( a 5 0.05%) and Fairlawn,N.J.) at maximumspeed for 10min.Bacteriocin activity Student’s t testwere performed for comparison of means. wasassayed in the supernatant (AU S).Thefollowing terms were usedto determinethe percentage of adsorbedbacteriocin: AU T 5 RESULTS thetotal bacteriocin activity (AU/ ml)determined in the culture; Detectionof an anti- Listeria bacteriocin-producing AU 5 theunbound bacteriocin activity (AU/ ml)determined in S strain of S.thermophilus. Amongseveral bacteriocin-pro- theCFS. Theamount of bacteriocin adsorbed to the cell wall couldbe calculated by the formula: Adsorbed Activity (%) 5 ducingstrains previously isolated from differentMoroccan products,one isolated from bakeryyeast and identiŽ ed as [(AUT 2 AUS)/AUT] 3 100. Oncethe adsorption proŽ le of thebacteriocin to the producer S.thermophilus was inhibitoryto L.monocytogenes, to S. cellwall as a functionof the pH was known, the puriŽ cation aureus, andto somegram-negative bacteria (T able1). Such proceededas described by Y anget al. (39). A culture of S. ther- resultssuggest that this strain has a goodpotential to en- mophilus Bwasprepared by inoculation of MRS broth(1 liter) hancethe safety and keeping quality of yogurt. Therefore, andovernight incubation at 37 8C.ThepH of the culture was ad- furtherstudy focused on the characterization of the inhib- justedto 6.0 with 3 MNaOH. Theculture was then heated at itorysubstance it produced and on the assessment of its 708Cfor25 min, and cells were harvested by centrifugation at inhibitoryperformance against these speciŽ c pathogensin 14,000 3 g for15 min. The cell pellets were washed twice with yogurtduring processing and storage at refrigeration (ca. phosphatebuffer (5 mM, pH6.0) and resuspended in 50 ml of 78C)orambient (ca. 22 8C)temperature. NaClsolution (100 mM) atpH 1.5. The cell suspension was Theinhibitory substance produced by S.thermophilus stirredwith a stirbar for 1 hat7 8C,andcells were removed by Bpossessesthe main criteria of a bacteriocin.It retained centrifugationat 14,000 3 g for20 min. The supernatant was itsactivity at pH 6.0, resisted catalase, was hydrolyzedby exhaustivelydialyzed and concentrated at 7 8Cina dialysismem- brane(500 molecular weight cutoff) against NaCl solution (100 proteolyticenzymes (T able2), and was bactericidalto sen- mM, pH1.5). Preliminary assays showed that a dialysismem- sitivestrains (data not shown). Unlike the most frequently branewith an 8,000-molecular weight cut-off limit did not retain producedbacteriocins by LAB, it seems to have a broad thebacteriocin. Preparations were then freeze dried and stored at spectrumof action that extends to gram-negative organ- 78C.Ineach step of the puriŽ cation procedure, arbitrary units isms.It is worth mentioning, however ,thatthe inhibition weredetermined to estimate bacteriocin yields and losses. zonesof sensitive gram-negative bacteria were usually 802 BENKERROUM ETAL. J.FoodProt., Vol. 65, No. 5

TABLE 2. Sensitivityof Listeriamonocytogenes ATCC 7644 to TABLE 3. Combinedeffect of heattreatment (121 8C,15min)and thecell-free supernatant of Streptococcussalivarius subsp. ther- pHon the stability of the bacteriocin produced by Streptococcus mophilus Bexposedto various enzymes and treatments a salivarius subsp. thermophilus Ba

Diameter ofinhibition zone (mm) b Meandiameter ofthe inhibition zone (mm) b

Control pH Control(nonheated) Heated (121 8C, 15 min) (untreated Treatment Treated sample sample) 2.0 19 Ac (62.6)d 13 B (62) 4.0 16 A (62) 14 A (61.7) Enzymes 6.0 18 A (62) 17 A (62.6) Catalase 19.5 Ac (61.3)d 19.7 A (61.5) 8.0 16 A (61) 8 B (60) Pronase E NIe 19.3 (60.6) 10.0 9 (61.7) NI Trypsin NI 19.7 (60.6) 11.0 NIe NI

a-Chymotrypsin NI 19.0 (61.7) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/5/799/1673166/0362-028x-65_5_799.pdf by guest on 27 September 2021 a Pepsin 9.3 B (61.2) 18.0 A (62) Listeriamonocytogenes ATCC 7644was used as the indicator strainfor sensitivity testing by the well diffusion assay (36). Heat b Meansfrom three experiments. V aluesinclude the diameter of 608Cpendant30 min thewell (7 mm). 808Cpendant30 min 19.3 A (62.1) 19.0 A (61.7) c Meansin arowwith the same letter are not signiŽcantly different 1008Cpendant30 min 20.3 A (62.1) 19.5 A (61.3) (a 5 0.05%). Autoclaving(121 8C, 19.0 A (63.5) 19.3 A (62.1) d Standarddeviation. 15 min) 20.3 A (61.5) 19.7 A (61.2) e NI,no inhibition. a Listeriamonocytogenes ATCC 7644was used as the indicator strainfor sensitivity testing by the well diffusion assay (36). ofPPB from theCFS, which could be considered good b Meansfrom three experiments. V aluesinclude the diameter of performanceif a highpurity level was notrequired. thewell (7 mm). Yogurtmanufacture. Evolutionof the pH of yogurt c Meanswith the same letter are not signiŽ cantly different ( a 5 0.05%). sampleswas monitoredduring fermentation and storage at d Standarddeviation. refrigeration(ca. 7 8C)orambient (22 8C)temperature.The e NI,no inhibition. pHdecreased in standard yogurt fermentation. No signiŽ - cantdifference ( P . 0.05)was observedbetween Bac 1 and Bac2 samplesduring fermentation and storage. At ambient 2 smallerand less clear than those observed with gram-pos- temperature,the Bac sampleswere alteredby the growth itivebacteria. Listeria strainswere themost sensitive. ofyeasts and molds on the surface at day 15, while in the Bac1 samples,such alteration marks were perceivable5 Stabilityof bacteriocin to pH andheat. Stabilityof days later. thebacteriocin produced by S.thermophilus Bwas assessed byexposingthe crude CFS todifferentheat treatments. The Effect ofin situ bacteriocinproduction on L. mon- activityof the crude CFS was notaffected ( P . 0.05) by ocytogenes. Thein situ effect of bacteriocinproduction by anyof the heat treatments tested (T able2). Therefore, the combinedeffect of pHandheat was studiedby autoclaving theCFS adjustedto different pH values ranging from 2.0 to11.0. The results showed that such resistance was pH- dependent.Although the bacteriocin could withstand auto- clavingat acidic pH values (maximum resistance between pH4.0and 6.0), it was readilydestroyed at pHvalues less acidicthan 8.0 by thesame treatment. In fact, the bacterio- cinwas unstablein alkaline conditions even without heat- ing,as evidenced by the drastic reduction in its inhibitory activityat pH 8.0 and its complete loss of activity at pH valuesless acidic than 10.0 (T able3). Extractionand partial puriŽ cation of bacteriocin. Adsorptionof the bacteriocin onto the producer cell wall was dependenton the pH of the medium. Approximately 100%of thebacteriocin was attachedto thecells at pH 6.0. Conversely,virtually all of theactivity produced was found inthe supernatant at pH 1.5(Fig. 1). Recovered activity in theCFS was reducedat a pHless acidic than 6.0. There- fore,the respective pH valuesof 6.0and 1.5 were usedfor FIGURE 1. Percentageof bacteriocin adsorbed onto the producer theadsorption and desorption steps in the puriŽ cation pro- cells of Streptococcussalivarius subsp. thermophilus B as a func- cedure.Such pH adjustmentallowed the recovery of 96.2% tionof the pH. J.FoodProt., Vol. 65, No. 5 EFFECTOF BAC 1 STARTER ON L.MONOCYTOGENES AND S. AUREUS IN YOGURT 803 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/5/799/1673166/0362-028x-65_5_799.pdf by guest on 27 September 2021

FIGURE 3. Growthof lactic acid bacteria ( —m—, —M—) and FIGURE 2. Growthof lactic acid bacteria ( —m—, —M—), Lis- Staphylococcusaureus SAD 30 (—v—, —V—)inyogurt fer- teriamonocytogenes (—v—, —V—), and Staphylococcusau- mentedwith Bac 1 (—m—, —v—) or Bac2 (—M—, —V—) reus (— —, — —)duringfermentation of yogurt with Bac 1 thermophilicstarter during storage at refrigeration temperature (—m—, —v—, — —) or Bac2 (—M—, —V—, — —) ther- (ca. 78C). mophilicstarter.

pathogensurvived for 10days of storage at refrigeration 1 2 S.thermophilus onthe growth and survival of L. monocy- temperature(7 8C) in Bac and Bac samples(Fig. 3). togenes inyogurt made from milkcontaminated with ca. DISCUSSION 103 CFU/ml of Listeria was studiedduring fermentation andstorage. The growth of LAB was monitoredsimulta- Thestrain of S.thermophilus Bisolatedin this work neously.The regular increase in numbers of LAB during producesa bacteriocinthat resists autoclaving under acidic fermentation(Fig. 2), along with the steady increase in conditions(T able3). In that respect, it differs from ther- acidity,indicates the normal processing of yogurtfermented mophilin347 (38) andthermophilin T (2), whichare in- eitherwith the Bac 1 or Bac2 starterculture. In both cases, activatedin these conditions. The main physicochemical countsof LAB were ca.10 8 CFU/mlat8hoffermentation characteristicsof the bacteriocin produced by S. thermo- (Fig.2), and no signiŽ cant difference ( P . 0.05)was ob- philus Bareshared by most class IIa bacteriocins.It is a servedin their count during processing. thermostablesmall peptide that passes through a dialysis L.monocytogenes fellbelow the detectable level in 1 membraneof an8,000-molecular weight cut-off limit, it has ml in the Bac1 samplesbetween 8 and24 h, whereas in anarrowspectrum of action (PPB), andit is strongly in- the Bac2 samples,only a 1-logunit reduction in the Listeria hibitory to L.monocytogenes. Furthercharacterization at countswas observedat 24 h (Fig.2). Moreover ,thepath- themolecular level is as yet needed to determine whether ogensurvived in the Bac 2 samplesthroughout the storage ornotit isanovelbacteriocin and to conŽrm itsrelatedness periodat both storage temperatures. tothe bacteriocins of class IIa. NeutralizedCFS samplesof the producer strain inhib- Effect ofinsitu bacteriocinproduction on S. aureus itedgram-positive as well as some gram-negative bacteria in yogurt. Thebehavior of S. aureus SAD 30in yogurt (Table1). Although it is well known that bacteriocins of fermentedwith Bac 1 or Bac2 starterwas studiedin the LABhavea narrowspectrum of actionessentially directed samemanner as for L.monocytogenes ATCC7600,except towardgram-positive species related to the producer organ- thata higherinitial inoculum (10 6 CFU/ml) was used. ism,some exceptions have been reported to inhibit gram- Staphylococci areindeed usually encountered in milk and negativebacteria also (7, 24, 32). Infact, all of these au- milkderivatives in elevated numbers. Furthermore, their thorsreported on the inhibitory activity of the spent broth minimuminfectious dose is higher than that of Listeria, for orthe spot-on-lawn rather than the puriŽ ed bacteriocin or whichmany countries apply the status of zero tolerance in PPB.In the present study, spectra of actionof theCFS and a25-g(or ml) sample.The results of Figure 2 showthat thePPB were compared.The results showed that the PPB numbers of S. aureus increasedduring the Ž rst 2hby0.9 didnot inhibit the gram-negative bacteria sensitive to the and1.5 logarithmic units in Bac 1 and Bac2 samples,re- CFS(Table1). The spent broth may contain a mixtureof spectively.Afterward, asharpdecrease in CFUs was ob- constituentsderived from bacterialmetabolism or from the servedin both types of yogurt samples. At 24h, Staphy- culturemedium, which would act synergistically with the lococcus countswere reducedto 6.6 3 102 and 9.3 3 103 bacteriocinto extend its spectrum of action to gram-nega- CFU/mlin Bac 1 and Bac2 samples,respectively. Overall, tivebacteria. Different substances and ecological factors insitubacteriocin production by S.thermophilus B appears havebeen demonstrated to interfere with the integrity of togivemoderate protection against S. aureus SAD 30 com- thelipopolysaccharide layer and to sensitize gram-negative pared to L.monocytogenes ATCC7644,since the former bacteriato bacteriocins (15, 34). Also, S.thermophilus B 804 BENKERROUM ETAL. J.FoodProt., Vol. 65, No. 5 mayproduce more than one bacteriocin having different andits incidence on human health make it worthwhile to spectraof action, as has been shown for otherLAB (30). investigateits behavior in yogurt fermented with a bacte- Theputative bacteriocin speciŽ cally inhibiting gram-nega- riocin-producingstarter .Inthis regard, the present study tivebacteria would not be recovered by the puriŽ cation showedthat, although reported to bemoresensitive to acid- procedureused. In this regard, Gomez et al. (17) also re- ity than L.monocytogenes(4), itsurvived yogurt process- portedon a strainof the Streptococcus genus,which lost ingand 10 daysof storageat refrigerationtemperature (Fig. inhibitoryactivity against gram-negative bacteria upon par- 3).A signiŽcant increase in numbers of Staphylococci was tialpuriŽ cation. evennoted in the Ž rst 2hoffermentation(Fig. 2). Similar Applicationof the adsorption/ desorptionmethod for behavior of S. aureus hasbeen reported previously in yo- thepuriŽ cation of the bacteriocin produced by S. thermo- gurt (4) andDomiati cheese (1). Extrinsicand intrinsic philus Ballowed96.2% recovery of anti- Listeria activity. growthparameters during the Ž rst hoursof fermentationare Infact, such a levelof recoveryis usual when this method encouragingto the growth of this organism. Neither the is applied (39). Someauthors even reported yields of re- aciditynor the bacteriocin was producedin sufŽ cient Downloaded from http://meridian.allenpress.com/jfp/article-pdf/65/5/799/1673166/0362-028x-65_5_799.pdf by guest on 27 September 2021 coveredactivity exceeding 100% (18). Althoughthis pro- amountsto exert the inhibitory effect on thepathogen. The ceduretypically does not provide a highdegree of purity, pHwas stillca. 6.0, and bacteriocin is not produced in a ithas proven useful for apartialpuriŽ cation and for bac- detectableamount during the early hours of fermentation. teriocinconcentration as the Ž rst stepin any puriŽ cation Moreover,milkobviously provides an adequate culture me- strategy.Partial puriŽ cation of thebacteriocin produced by diumfrom thenutritional standpoint, and the temperature S.thermophilus Bwas carriedout essentially to conŽ rm isnot a limitingfactor . inhibitoryaction against gram-negative bacteria. Restricted Thebacteriocin produced by S.thermophilus B was inhibitoryactivity of the PPB comparedto the crude CFS lessactive in removing S. aureus than Listeria from yogurt. suggeststhat in foodsystems, in situproduction of thebac- Nonetheless,accelerated decreases in Staphylococci num- teriocinwould inhibit a broaderrange of undesirable mi- bersduring processing in Bac 1 comparedto Bac 2 samples croorganismsthan the puriŽ ed bacteriocin. Therefore, use (Fig.2) suggest that bacteriocin production inhibits, to ofthe producer strain in the starter culture appears to be someextent, the growth of S. aureus inyogurt. The high moreadvantageous to food preservation and thus has a numberof inoculum used to contaminate yogurt with S. greaterpotential to enhance the safety and extend the shelf aureus SAD 30certainlyaccounts for thepersistence of the lifeof the food product. Nevertheless, the producer strain pathogenin yogurt samples. It is welldocumented that the shouldnormally be a partof the starter culture and, under quantityof the initial inoculum has an effect on the inhi- theseconditions, be able to produce sufŽ cient bacteriocin bitionof pathogensin foods (5, 22, 35). Inaddition, in vitro inthe particular food product. In fact, a studyon the dy- testsshow that S. aureus isless sensitive than L. monocy- namicsof bacteriocin production by the strain in reconsti- togenes tothe bacteriocin produced by S.thermophilus B tutedmilk showed that activity was producedin a detect- (Table 1). ableamount at 5 hofincubation and reached ca. 550 AU/ ACKNOWLEDGMENTS mlat 24 h (unpublisheddata). Preliminary in situ experi- mentsneed to be carried out when the bacterium is to be Thisresearch has beensupported partially by the ministry for re- usedfor suchpurposes. search, highereducation, and culture (grant PARS AGRO 025). 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