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Prof.Dr . Dilek Heperkan Istanbul Technical University ,

Antimicrobial activity of lactic acid bacteria on pathogens in foods Why succesful ? How succesful ?. Prof.Dr . Dilek Heperkan Istanbul Technical University , Faculty of Chemical and Metallurgical Engineering , Dept . of Food Engineering.

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Prof.Dr . Dilek Heperkan Istanbul Technical University ,

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  1. Antimicrobialactivityof lacticacidbacteria on pathogens in foodsWhysuccesful?How succesful? Prof.Dr. Dilek Heperkan Istanbul Technical University, Faculty of ChemicalandMetallurgicalEngineering, Dept. of FoodEngineering Food Technology2014 21-23 July, Las Vegas

  2. Microorganismsespeciallybacteria can be usedfor a number of beneficialpurposes. AmongthemsomearemoreprominentlikeLacticacidbacteria(LAB) and bifidobacteria. • Lacticacidbacteriahavebeenused as natural food-gradepreservativesagainsta variety of undesirablemicroorganisms. • LAB has beenusedforproduction of fermentedfoodsformanyyears. As a matter of factfermented foods existedlongbefore mankind discoveredmicroorganisms. • Duringtheiractivity in production of fermentedfoodstheirinhibitorypotential on pathogenicbacteriahavebeenrecognisedand theygainedincreasing interest in thescientificcommunity. • ThusLAB has beenused as a tool to produceantimicrobialcompounds and to controlundesirablemicroorganisms.

  3. Antibiotics; • On theotherhand, acutediarrheaduetotheloss of normal intestinalmicrobiota, drug-resistantstrainsandchronictoxicityduetowidespreaduse of antibioticsarewellknownnegativeeffects of antibiotics. Pesticides; • Therisksof toxicresidues in foods, as well as contamination of soilandwaterduetotheuse of xenobiotics in foodproductionchainareconsiderednegativelybythepublic. • Labproducing a widerange of antimicrobialmetabolitesthus, thelasttwodecadeshaveseenpronouncedadvancements in using LAB andtheirmetabolitesfornaturalfoodpreservation, as well as cropprotection, andhealthprotection. Food Technology2014 21-23 July, Las Vegas

  4. Content: • The role of lactic acid bacteria • Biopreservative agent • Probiotic potential • Antimicrobial agentWhysuccesful • Biopestiside • Chemotherapeuticagent / infectioncontrolagent • Case studies; Antimicrobial activities against • Listeria monocytogenes(themethodweusedandthe • Staphylococcus auresresultsweobtained) • Salmonella typhimurium • Industrialapplications of LABHowsuccesful Food Technology2014 21-23 July, Las Vegas

  5. LACTIC ACID BACTERIA The term lactic acid bacteria (LAB) has no strict taxonomic significance,although the LAB have been shown by serological techniques and 16Sribosomal RNA cataloguing to be phylogenetically related. They share anumber of common features: • they are Gram-positive • non-sporeformingrods or cocci • non-motile • resistant to acid and • highfermentativeability Themost are aerotolerant anaerobes which lack cytochromesand porphyrins and are therefore catalase- and oxidase-negative. Somedo take up oxygen through the mediation of flavoprotein oxidasesandthis is used to produce hydrogen peroxide and/or tore-oxidize NADHproduced during the dehydrogenation of sugars. Food Technology2014 21-23 July, Las Vegas

  6. LAB compose a heterogeneousbacterialgroup, theircommoncharacteristic is theability to ferment sugars. Food Technology2014 21-23 July, Las Vegas

  7. Lacticacidbacteria, whichconvertfermentablesugars to lacticacid and otherorganicacidsdepending on theirmetabolicpathways. • Theyarethemostimportantgroup of bacteria in fermentedfoods. Thus, duetotheacidproductionthepH of theenvironmentreducesto a pH 3.5. • However, Lactobacillusspp. plays a major role in theprocess and Leuconostoc and Pediococcus to a lesserextent. Food Technology2014 21-23 July, Las Vegas

  8. Heterofermenters produce roughly equimolar amounts of lactate,ethanol/acetate, and carbon dioxide from glucose. • Some authors also include Bifidobacterium among the lactic acid bacteria although this has less justification as they are quite distinct both phylogenetically and biochemically. • For example, hexose fermentation by bifidobacteria follows neither the EMP glycolytic pathway nor the phosphoketolase pathway but produces a mixture of acetic and lactic acids. Food Technology2014 21-23 July, Las Vegas

  9. The role of lactic acid bacteriaBiopreservativeagent • LAB arewellknown for their activity as starter cultures in the manufacture of fermented foods for a number of industries. • Lactic fermentation is one of the oldest forms of preparation and preservation of foods(Charlier et al.,2009). LAB are essential to the production of fermented products. Rawmaterialsfor HARDALIYE Food Technology2014 21-23 July, Las Vegas

  10. Cerealbased Milkbased Fishbased Fruitbased Meatbased (Adams and Moss, 2008) Food Technology2014 21-23 July, Las Vegas

  11. Healthbenefits Since the trends towards • natural (minimally processed or without additives) • high nutritional value • health-promoting • flavor richproducts Fermented food play an important role in human diet around the world due to their health benefits. Food Technology2014 21-23 July, Las Vegas

  12. Role of LAB Biopreservative Agent(Food preservation and safety) Food preservation Theirability to promote food preservation is summerizedbelow; • theycause a decrease in pH as a resultof lacticacidproduction, and additionally, • theyproduce a number of antimicrobialagentsandthusplay a role in the inhibition of pathogenic / spoilage microorganismsduringfermentationprocess in foods. • A combination of thesefactorslimitstheproliferation of undesirablemicroorganisms. • LAB thereforeundoubtedlyplay a role in promoting food safety. Food Technology2014 21-23 July, Las Vegas

  13. 2) Probiotic characteristics of lacticacidbacteria Probiotic potential is another significant character of a LAB. The most commonly used species, in probiotic preparations are Lactobacillus ssp.,Bifidobacteriumssp. andStreptococcusssp. Probiotic strains have several beneficial properties such as • Improvingintestinal tract health • producing antimicrobial substances • enhancing the immune response • reducing symptoms of lactose intolerance • enhancing the bioavailability of nutrients, and • decreasing the prevalence of allergy in susceptible individuals (Parvez et al., 2006; De Bellis et al., 2010: Mena and Aryana, 2012). Food Technology2014 21-23 July, Las Vegas

  14. Probiotic characteristics of lacticacidbacteriaBeneficial role of LAB(Charlieret al.,2009) • Thecurrentdefinition of a probiotic is a “livemicro-organismwhichwhenadministered in adequateamountsconfers a healthbenefit on thehost” (FAO/WHO report, October, 2001), whichdoes not implythatmicroorganismshaveto be orallyingested. • Thewellknownspeciesof probioticsarelactobacilliandbifidobacteria (Lb. casei, Lb. rhamnosus, Lb. acidophilus, Bf. bifidum). Food Technology2014 21-23 July, Las Vegas

  15. 3. Antimicrobialmetabolites • LAB haveantimicrobialeffectsagainstundesirablemicroorganismsthroughproducingvariousmetabolitesorthroughinhibitingthecelladhesion of pathogenicorganisms in vitro. Food Technology2014 21-23 July, Las Vegas

  16. Food Technology2014 21-23 July, Las Vegas

  17. Food Technology2014 21-23 July, Las Vegas

  18. Organicacids Organic acids have been usedas food additivesand preservatives in food industry. It is usedfor preventing food spoilage andextending the shelf life of foods. Thetype of organicaciddepends on thetype of producerspecies. Homofermentersproduce lacticacidas a single product from the fermentation of glucose.Whereasheterofermentersproduceaceticacid as well. Food Technology2014 21-23 July, Las Vegas

  19. depending on the physiologicalstatus of the organism and the physicochemical characteristics ofthe environment (Ricke, 2003). Organic acids are capable of exhibiting bacteriostaticand bactericidal properties Thereare a number of researchconducted to investigate theeffect of differentorganic acids on inhibiting the growth of pathogensin laboratory media and liquid foods. Propionic acid and acetic acid showed the strongestinhibitory effect against E. sakazakii in laboratory broth media(Backet al., 2009). Food Technology2014 21-23 July, Las Vegas

  20. How can we test antimicrobialefect of LAB againstpathogens? (Agaroverlay, discdiffusion, welldiffusion) Uploadthesupernatantcontainingmetabolites Incubation (37 C for 24 h) Measuretheinhibitedzonearoundthe sterile disc (diameter, mm or cm) Sterile disk Inoculatepathogenicbacteria Food Technology2014 21-23 July, Las Vegas

  21. Hydrogenperoxide (H2O2) • LacticacidbacteriaproduceH2O2as a lowmolecularweightantimicrobialcompound. Hydrogenperoxideshowesbacterostaticeffectagainst gram positivebacteria (including LAB) andbactericidaleffectagainst a number of gram negativebacteria in general (Yang, 2000). Mechanism • Hydrogenperoxideoxidizesthesulphidrilgroups in a moleculeandthuscausedenaturation of a variety of enzymes. • increasesthepermeability of themembranebyperoxidizingthelipids in theplasmamembrane. • has a role on production of superokside(O²-) andhydroxil (OH*) freeradicalsandcausingdamage in DNA (bactericidaleffect) (Ammor ve diğ., 2006). Food Technology2014 21-23 July, Las Vegas

  22. However, theactual role of hydrogenperoxideproducedby LAB in theinhibition of pathogens has beendifficulttodemonstrateandthus is stillcontroversial. Food Technology2014 21-23 July, Las Vegas

  23. Bacteriocinsandbacteriocin-likeinhibitorysubstances • Manyspecies of LAB producebacteriocinswithfairlylargespectraof inhibitionthustheyconsidered as promising agents for use in food preservation. There is an increasinginterest in theliteraturewhichmainlyfocusedon • Theisolationandidentification of bacteriocinproducing LAB • Theirantimicrobial / antagonisticeffectstodifferentpathogens • Characterisation of theactivemetaboliteandfinally • Possibilitytousethem as a biopreservative in foods as well as otherarea of interest. Food Technology2014 21-23 July, Las Vegas

  24. Bacteriocinsandbacteriocin-likeinhibitorysubstances • Numerous strains of LAB associated with foods produce bacteriocins, defined as proteinaceous compounds with activity against related species. • Bacteriocinsareribosomally-synthesizedpeptidesorproteinssecretedbycertainstrains of bacteria. Mechanisms • Theantagonisticactivity of LAB topathogensmayaffectthegrowth rate and/orsurvivaldepending on thetypeandtheconcentration of bacteriocin. • Mostbacteriocinskilltargetcellsbypermeabilization of thecellmembrane, andtheactivity is oftenveryspecific, since theyemployspecificreceptors on thetargetcellsurfaces(Kjos et al., 2011). Food Technology2014 21-23 July, Las Vegas

  25. Nisin(L. lactis) and pediocin(Pediococcusacidilactici) are among the most well characterisedbacteriocinsand the most frequently used in fermented products. Food Technology2014 21-23 July, Las Vegas

  26. 4. Biopestisidesenvironmentallyfriendlybiocontrolsystems • Severalspecies of lacticacidbacteriahavebeenrecognisedas producers of bioactivemetaboliteswhicharefunctionalagainst a broadspectrum of undesirablemicroorganisms. • Theyareeffectivetofungi, oomycetesandotherbacteria. • Consumersin developedcountrieshavebecome morecriticalandmorefragmentedin theirfood choices,leadingtosituationswherequality differentiationof foodproducts, has becomenecessary in ordertosatisfyconsumers. Food Technology2014 21-23 July, Las Vegas

  27. Consumer pressureagainstchemicalsin food productionand therisksof toxicresidues General publichave becomeinterested and oftencriticalwithregard to certainways of producing food- bothat thefarmlevel and at theprocessing level. As a result, discussions on organicproduction, reducetheapplication of pesticideson crops, animalwelfare, and theuse of geneticallymodified organisms (GMOs) in food production. Food Technology2014 21-23 July, Las Vegas

  28. Consumer pressure • Publicfear of usingchemicalsin food production, theperceivedrisks of toxicresidues in treatedproducts, as well as contamination of soil and water, pesticidesarelookeduponnegativelybythepublic. • Thenecessityto moveaway from traditionalchemicaltreatments,manystudieshavefocused on findingalternativebiocontrolsystems. • Ingeneral, due to thedifferentmodes of actions (i.e. antagonisticeffectsorinduction of plantdefencemechanisms),theuseof LAB as preservatives has a definitepotential(Axel et al., 2012). • Thus, theymayrepresentan interestingtoolforthedevelopment of novelconcepts in pestmanagement. Food Technology2014 21-23 July, Las Vegas

  29. 5)Antimicrobialchemotherapeuticagents • Antimicrobialchemotherapeuticagentshavebeenwidelyusedtocontrolgastrointestinalinfections. However, thewidespreaduse of antibiotics is nowbeingdiscouragedduetoproblemsincludingtheemergence of drug-resistantstrainsandchronictoxicity(Modyet al., 2003). • Inaddition, antibioticsareoftenresponsibleforacutediarrheaduetotheloss of normal intestinalmicrobiota as well as pathogenicorganisms(Van der Waaij et al., 1982). • As alternatives, lacticacidbacteriaortheirderivateshavebeenadministered. LAB havebeenusedorplanningtouseagainstpathogens not only in foodandplantprotectionindustry but also in medicalindustryas well. • Bacteriocinproducer LAB strainswhichareactiveagainst S. aureusandotherpathogenicbacteriaarealsoscreened in ordertodevelopprobioticforthehuman body (Voravuthikunchai et al., 2006). Food Technology2014 21-23 July, Las Vegas

  30. Whysuccesful ? • LAB havebeenwidelystudiedfortheirantimicrobialactivity, andseveralantibacterialandfungicidalcompoundshavebeenisolatedandcharacterisedtodate. • Amongsttheantibacterialcompounds, nisin (Lactococcuslactis) has beenusedsuccessfully as an effectivebiopreservative in somedairyproductsfordecades.Nisin is currently used commercially as a food preservative in around 50 countries. • ıtis registered as a foodpreservative E234 (No 95/2/EC 1995). • Amongotherpthogenicbacteria the growth of S. enteritidis was alsoeffectively inhibited by the presence of the(lactic acid bacteria culture condensate mixture)LCCM. • In vitro and in vivo experiments showed that the LCCM has antimicrobial effect against S. enteritidis. Ingestion of the LCCM after a meal will be helpful for preventing S. enteritidisinfection(Park et al., 2005) . Food Technology2014 21-23 July, Las Vegas

  31. Whysuccesful LAB play a crucial role in everypart of nutritionsafetysuch as • food preservation • food safetyandquality • to a lesserextent in nutrition • Majority of fermented foods is produced by the activity of lactic acid bacteria • environmentallyfriendlybiocontrolsystems (biopestiside) • Antimicrobialchemoterapeuticagent/diseasecontrol on skin infections Food Technology2014 21-23 July, Las Vegas

  32. S.aureus • Staphylococcusaureus is one of opportunisticpathogen, involvedin foodpoisoning, toxicshocksyndrome(TSS) and a widerange of infections. • Thenatural habitat of thisspeciesarethenasalcavityandthe skin of warm-bloodedanimals. Becauseof itsimportance, S. aureus is one of themoststudiedpathogen. • S.aureus is thecausativeagent of skin-releatedinfectionsandsuperficiallesionsto life-threateningsepticaemia(Charlier et al.,2009). Food Technology2014 21-23 July, Las Vegas

  33. Case studiesMaterialandMethods A total of 47 strains were isolated from 30 tulum cheese samplesobtained from east Anatolia. Thischeese is a traditional semi- softcheese. • Specieswereidentifiedby 16S rRNAusing PCR. • Antimicrobialactivities of strainsweretestedusingwelldiffusionassay • Theantimicrobialactivity of eachstrainwereconfirmedbyclassicalmicrobiologicalanalysesusingselectivemediaforeachpathogen. Food Technology2014 21-23 July, Las Vegas

  34. LAB haveantimicrobialeffectsagainstpathogenssuch as (Park et al., 2005) • EscherichiacoliO157:H7 • Vibriocholerae • Salmonellaenteritidis • SalmonellatyphimuriumATCC 14028 • Listeriamonocytogenes • StaphylococcusaureusATCC25923 • E. sakazakii • Aspergillusniger • Yeasts Food Technology2014 21-23 July, Las Vegas

  35. Table1. Thenumber of speciesisolatedfromcheese Supernatantsobtainedfrom LAB testedfortheir total antimicrobialcapasitiesafter 24 and 48hrs. Food Technology2014 21-23 July, Las Vegas

  36. Bacterialmetaboliteproduction Food Technology2014 21-23 July, Las Vegas

  37. Table 2. pH of supernatantsafterincubation Food Technology2014 21-23 July, Las Vegas

  38. Figure1. Antagonisticeffects of organicacids(non-neutralizedsupernatant) of LactobacillusbrevisagainstListeriamonocytogenes 0,27 23.1: Enterococcusfaecium

  39. Figure2. Antagonisticeffects of neutralizedsupernatant of LactobacillusbrevisagainstListeriamonocytogenes Lb. brevis 1 Lb. brevis 2 Lb. brevis 3

  40. Figure3. Antagonisticeffects of neutralizedsupernatantcontainingcatalase of LactobacillusbrevisantimicrobialsagainstListeriamonocytogenes

  41. Confirmationbyinoculatingsupernatantwithpathogen on PALCAM agarshowedcompleteinhibitonbyorganicacids but not neutralizedandneutralizedsupernatantwithcatalase. • Thustheinhibitoryeffect of L. brevisassociatedwiththe organicacid.

  42. Theresults of thisstudywere in agreementwiththeliterature theantagonisticeffects of Lb. brevisagainst; • S. aureus (Aslim B. 2005; Banerji 2011) • E. coli (Aslim B. 2005; Banerji 2011) • Y. enterocolitica (Aslim B. 2005) • Aslim B. (2005) organicacid • Banerji (2011) ise bacteriyocin-likecompounds

  43. Figure4. Antagonisticeffects of organicacids (non-neutralizedsupernatant) of Lactobacillusplantarumagainst L. monocytogenes

  44. Figure5. Antagonisticeffects of neutralizedsupernatant of Lactobacillusplantarumagainst L. monocytogenes

  45. Figure6. Antagonisticeffects of neutralizedsupernatantcontainingcatalaseof Lactobacillusplantarumagainst L. monocytogenes

  46. Theresultsof thisstudywere not inagreementwiththeliterature • Theantagonisticeffects of Lb. plantarumGonzalez et al., (2007) organicacidsonly • Neutralizedandneutralizedcontainingcatalase 3 supernatantswereeffectiveagainst L. monocytogenes

  47. StaphylococcusaureusSalmonellatyphimurium • Welldiffusion • 0, 6, 24 and 48hrs • 640nm • confirmed • After 24 and 48hrs • Staphylococcusaureus - BP agar • Salmonellatyphimurium – XLD agar

  48. Figure 7. Antagonisticeffects of organicacids (non-neutralizedsupernatant) of LactobacillusbrevisagainstStaphylococcusaureus *23.1: Enterococcusfaecium

  49. Figure 8. Antagonisticeffects of neutralizedsupernatant of LactobacillusbrevisagainstStaphylococcusaureus *23.1: Enterococcusfaecium

  50. Figure9. Antagonisticeffects of neutralizedsupernatantcontainingcatalase of LactobacillusbrevisantimicrobialsagainstS. aureus *23.1:Enterococcus faecium

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