京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19046号 / 農博第2124号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4928(農学部図書室) / 31997 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 加納 健司, 教授 植田 充美 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Die Verbraucherakzeptanz von fermentierten Milchprodukten wie Joghurt oder Frischkäse ist von ihrer Textur abhängig. Zur Einstellung der produktspezifischen Textur ohne Zusatz von spezifischen Milchproteinen zur Basismilch oder milchfremden Hydrokolloiden werden in der Milchindustrie spezielle Starterkulturen eingesetzt, die während der Fermentation Exopolysaccharide (EPS) bilden. Diese EPS werden in der Regel als freie EPS ins Serum abgegeben oder verbleiben als kapsuläre EPS an der Bakterienzelle. Freie EPS lassen sich weiter anhand ihres Effektes im Produkt in fadenziehend und nicht-fadenziehend unterteilen. Aufgrund der hohen Biodiversität der EPS sowie des komplexen Zusammenhanges zwischen EPS-Eigenschaften, Substratzusammensetzung und Verfahrensbedingungen existieren jedoch widersprüchliche Ergebnisse zu ihrer Funktionalität im Produkt. Ziel dieser Arbeit war es, die wichtigsten Einflussfaktoren durch Einsatz verschiedener EPS von Lactococcus lactis und Streptococcus thermophilus in definierten Modellsystemen für stichfeste, gerührte und konzentrierte fermentierte Milchprodukte zu identifizieren und die Textureigenschaften mit den strukturellen und makromolekularen Eigenschaften der EPS in Verbindung zu bringen. Neunundzwanzig Einzelstämme von verschiedenen Starterkulturherstellern wurden auf Basis von der Fadenlänge als Indikator für den Fadenzug und der EPS-Menge in sechs Cluster eingeteilt. Aus jedem Cluster wurden Vertreter zur Herstellung von Modellsystemen genutzt, die Textur und Synärese der Produkte instrumentell erfasst und statistisch bewertet, um Einflussfaktoren auf die Eigenschaften der Modellprodukte zu identifizieren. Stichfester Modelljoghurt mit fadenziehenden EPS wies eine höhere Steifigkeit auf als jener mit nicht-fadenziehenden EPS, welche nicht mit der EPS-Menge oder der Fermentationszeit korrelierte. Unabhängig von der Art der freien EPS war beim Vorhandensein von kapsulären EPS die Gelsteifigkeit zusätzlich erhöht. Für gerührten Modelljoghurt ergab eine Komponentenanalyse zur EPS-Menge, zur Säuerungskinetik und zu Produkteigenschaften, dass fadenziehende EPS texturrelevante Eigenschaften wie Partikelgröße und Scherviskosität signifikant beeinflussen. Dies wurde auf die höhere intrinsische Viskosität für fadenziehende EPS im Vergleich zu nicht-fadenziehenden EPS zurückgeführt. Experimente mit einem Dehnrheometer ergaben, dass die Dehnviskosität stärker als die Scherviskosität mit der Fadenlänge zunahm. Ein Grund dafür sind intensivere Wechselwirkungen von fadenziehenden EPS mit Proteinen oder untereinander, was anhand höherer Relaxationszeiten in Dehnung bei höherer Fadenlänge geschlussfolgert wurde. Fadenziehende EPS erhöhten zudem die Fließgrenze von Modellfrischkäse, was hauptsächlich auf EPS-Wechselwirkungen und nicht auf die EPS-Konzentration oder Partikelgröße zurückgeführt wurde. Eine niedrigere Synärese und höhere Steifigkeit wurde entweder beim Vorhandensein von kapsulären EPS oder freien EPS beobachtet, die in Experimenten mit dynamischer Wasserdampfsorption eine höhere Feuchtebeladung aufwiesen. Fadenziehende EPS und kapsuläre EPS besaßen auch im Doppelrahmfrischkäse nach moderater Bruchhomogenisierung (0,05 MPa oder 15 MPa) eine hohe Funktionalität, was mit der höheren intrinsischen Viskosität dieser EPS korrelierte. Die Bruchhomogenisierung bei einem Druck von 30 MPa führte jedoch zu einer niedrigeren Funktionalität aufgrund der scherinduzierten Reduzierung der molekularen Masse der EPS und Umlagerungen in der Mikrostruktur der Käse. Die Ergebnisse zeigen, dass die Technofunktionalität nicht von der Konzentration der in situ produzierten EPS abhängt. Schlüsselfaktoren sind insbesondere die EPS-Lokalisierung (frei oder kapsuläre), intensivere Wechselwirkungen der fadenziehenden EPS mit Proteinen oder untereinander und ein hohes Wasserbindungsvermögen der EPS. Jedoch sollte der Energieeintrag bei der postfermentativen Verarbeitung so niedrig wie möglich sein, um die Funktionalität der fadenziehenden EPS und kapsulären EPS weitestgehend zu erhalten. Dieses Wissen ist für die Auswahl von EPS-bildenden Starterkulturen für den industriellen Maßstab entscheidend, um die gewünschten Produkteigenschaften zu erhalten.

La sicurezza e la qualità degli alimenti sono tutt’ora un problema critico per i paesi in via di sviluppo. Le diete a basso contenuto di acido folico, per esempio, possono causare gravi problemi di salute, soprattutto nei bambini. Gravi disturbi legati al tubo neurale (DTN) nei neonati possono derivare infatti da madri che hanno insufficiente apporto di acido folico (400-600 g / giorno) durante il periodo di gravidanza. Inoltre, se non adeguatamente protetti o trattati, I prodotti alimentari possono essere vettori di funghi e batteri patogeni rappresentando una fonte potenziale di malattie per l’uomo e una perdita economica per le industrie agro-alimentari. Nella seguente tesi si è quindi quindi studiato il ruolo di batteri lattici selezionati (LAB) in grado di aumentare il valore nutrizionale del latte attraverso la produzione di acido folico durante il processo di fermentazione. Inoltre, ci si è concentrati sul loro uso come "bio-conservanti" contro funghi e batteri, attraverso la sintesi di composti antimicrobici (batteriocine) in grado di inibire la crescita di funghi filamentosi e/o batteri patogeni. / The safety and quality of food are still a critical issue in developing countries. Diets with a low content of folic acid, for example, may cause serious health problems, especially in children. Severe disorders related to neural tube (NTD) in infants may arise from mothers having inadequate intakes of folic acid (400-600 g/dia) during the mother pregnancy period. Moreover foods, when not properly protected or treated, can be vectors of pathogenic fungi and bacteria thereby representing a potential source of human diseases and an economical loss for the food industry. In the following thesis we have therefore investigated the role of selected lactic acid bacteria (LAB) in increasing the nutritional value of milk through the production of folic acid during the fermentation process. In addition, we focused on their use as “bio-preservatives” against fungal and bacterial spoilage, through the synthesis of antimicrobial compounds (bacteriocins) able to inhibit the growth of filamentous fungi and /or pathogenic bacteria.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The equine gastro-intestinal tract (GIT) is a relatively unexplored niche concerning the presence of natural microbiota. Studies have shown that disruption of the microbial population naturally present in the GIT leads to the onset of several forms of gastro-intestinal disorders. To maintain a balanced microbiota, probiotic bacteria need to be administered at specific levels. Beneficial microorganisms assist with digestion of the feed, absorption of nutrients from the GIT, strengthens the immune system and improves the animal‟s growth. Various combinations of lactic acid bacteria (LAB) have been administered to horses, but have failed to benefit the host in any of the latter criteria. The screening for alternative strains with probiotic properties is thus necessary. Two strains (Lactobacillus equigenerosi Le1 and Lactobacillus reuteri Lr1) were originally isolated from horse faeces. Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, both bacteriocin-producing strains, were isolated from sorghum beer and soy beans, respectively. All four strains survived growth at acidic conditions (pH 3) and the presence of 0.5%, 1.0% and 1.5% (w/v) bile salts. L. reuteri Lr1 was the most resistant to these conditions. All strains adhered to buccal (cheek) epithelium cells sampled from horses. L. equigenerosi Le1 and E. mundtii ST4SA, however, invaded the cells, but without visible signs of disrupting the cells. None of the strains contained genes encoding adhesion to collagen (Ace), resistance to vancomycin A, B and C, or, production of aggregation substance (AS), cytolysin (Cyl) and, non-cytolysin (β hemolysin III), suggesting that they are non-virulent. Of all strains, L. equigenerosi Le1 competed the best with Clostridium sp. C6 for adherence to epithelial cells. L. equigenerosi Le1 and L. reuteri Lr1, showed the highest level of co-aggregation with Clostridium sp. C6. When the four strains were administered to horses over a period of 10 days, L. reuteri Lr1 was retained the longest (8 days) in the GIT. The numbers of viable cells of Clostridium spp. and Salmonella spp. remained constant during administration of the four strains. Blood analyses showed no negative effects from administering the strains. Total white blood cell counts remained unchanged. However, a small but tentative increase in neutrophil and eosinophil cell numbers has been recorded, suggesting that the LAB may have elicited a mild, transient, intolerance reaction. The glucose, lactate and urea levels decreased during administration with the four LAB strains. / AFRIKAANSE OPSOMMING: Die spysverteringstelsel (SVS) van die perd is 'n relatief onbekende nis wat die voorkoms van natuurlike mikrobiota betref. Studies het getoon dat versteuring van die natuurlike mikrobiese populasie in die SVS aanleiding kan gee tot die ontwikkeling van menige vorms van gastro-intestinale ongesteldhede. Om 'n gebalanseerde mikrobiota te verseker, moet probiotiese bakterieë teen 'n spesifieke vlak toegedien word. Voordelige mikroorganismes bevorder vertering en absorpsie van nutriënte vanaf die SVS, versterk die immuunsisteem en bevorder die groei van die dier. Verskeie kombinasies van melksuurbakterieë is reeds aan perde toegedien, maar sonder ooglopende voordele vir die dier. Die soeke na alternatiewe stamme met probiotiese eienskappe is dus noodsaaklik. Twee melksuurbakterieë (Lactobacillus equigenerosi Le1 en Lactobacillus reuteri Lr1) is oorspronklik uit perdemis geïsoleer. Lactobacillus plantarum 423 en Enterococcus mundtii ST4SA, beide bakteriosienproduserende stamme, is afsonderlik van sorghumbier en sojabone geïsoleer. Al vier spesies groei by lae pH (pH 3) en in die teenwoordigheid van 0.5%, 1.0% en 1.5% (m/v) galsoute. L. reuteri Lr1 is die mees bestand onder hierdie toestande. Al vier stamme het aan wang epiteelselle van perde geheg. L. equigenerosi Le1 en E. mundtii ST4SA het egter die epiteelselle binnegedring, maar sonder opsigtelike vernietiging van die selle. Nie een van die stamme besit gene wat kodeer vir aanhegting aan kollageen (Ace), bestandheid teen vankomisien A, B en C, of produksie van, sel-aggregasie (AS), sitolisien (Cyl) en nie-sitolisien (β-hemolisien III), wat daarop dui dat hulle nie-virulent is. Van al die stamme het L. equigenerosi Le1 die beste met Clostridium sp. C6 vir aanhegting aan epiteelselle gekompeteer. L. equigenerosi Le1 en L. reuteri Lr1, het die beste vlak van ko-aggregasie met Clostridium sp. C6 getoon. Met die toediening van 'n kombinasie van die vier stamme aan die perde oor 'n periode van 10 dae, het L. reuteri Lr1 die langste retensie (8 dae) in die SVS getoon. Die aantal lewende selle van Clostridium spp. en Salmonella spp. het konstant gebly tydens toediening van die vier stamme. Toediening van die vier stamme het geen negatiewe effek getoon met resultate verkry van bloed analises nie. Die totale witbloed seltellings het onveranderd gebly. 'n Klein, maar tentatiewe, toename in neutrofiel- en eosinofiel selgetalle is waargeneem, wat daarop dui dat die melksuurbakterieë 'n geringe allergiese reaksie teweeggebring het. Die glukose, laktaat en ureum vlakke het gedaal tydens die toediening van die vier melksuurbakterie stamme.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: During winemaking, a number of biochemical changes occur as a result of the metabolic activity of wine lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). The latter process, which occurs mostly after alcoholic fermentation by wine yeasts, involves the conversion of L-malate to L-lactate and CO2, thus resulting to wine acidity reduction, microbiological stabilization and alterations of wine organoleptic quality. Although Oenococcus oeni is predominantly the most preferred species suitable for carrying out MLF in wine owing to its desirable oenological properties, Lactobacillus plantarum has also been considered as a potential candidate for MLF induction. Other species in the genera of Lactobacillus and Pediococcus are often associated with wine spoilage. These microorganisms induce wine spoilage by producing off-flavours derived from their metabolic activity. It is therefore of paramount importance to understand the mechanism by which wine microbiota cause spoilage. The purpose of this study was to investigate the presence of genes encoding enzymes of oenological relevance in wine-associated LAB strains. In order to achieve this, different sets of specific primers were designed and employed for a wide-scale genetic screening of wine LAB isolates for the presence of genes encoding enzymes involved in various metabolic pathways, such as citrate metabolism, amino acid metabolism, hydrolysis of glycosides, degradation of phenolic acids as well as proteolysis and peptidolysis. PCR detection results showed that the majority of the tested strains possessed most of the genes tested for. It was also noted that, among the O. oeni strains tested for the presence of the pad gene encoding a phenolic acid decarboxylase, only two strains possessed this gene. None of the O. oeni strains has previously been shown to possess the pad gene, and this study was the first to report on the presence of this gene in O. oeni strains. In an attempt to genetically characterize this putative gene, DNA fragments from the two positive O. oeni strains were sequenced. The newly determined sequences were compared to other closely related species. Surprisingly, no match was found when these sequences were compared to the published genomes of three O. oeni strains (PSU-1, ATCC BAA-1163 and AWRI B429). This reinforced a speculation that the pad gene in these two strains might have been acquired via the horizontal gene transfer. In addition, it remains to be further determined if the presence of this gene translates to volatile phenol production in wine. In this study, a novel strain isolated from South African grape and wine samples was also identified and characterized. The identification of this strain was performed through the 16S rDNA sequence analysis, which indicated that this strain belongs to Lactobacillus florum (99.9% sequence identity). A novel PCR assay using a species-specific primer for the rapid detection and identification of Lb. florum strains was also established. For further characterization, this strain was also investigated for the presence of genes encoding enzymes of oenological relevance. PCR detection results indicated that the Lb. florum strain also possess some of the genes tested for. In addition to genetic screening of wine LAB isolates for the presence of different genes, this study was also aimed at evaluating the regulation of the mleA gene encoding malate decarboxylase in three oenological strains of O. oeni. The regulation of this gene was tested in a synthetic wine medium under various conditions of pH and ethanol. From the expression analysis, it was observed that the mleA gene expression was negatively affected by high ethanol content in the medium. On the other hand, low pH of the medium seemed to favour the expression of this gene as the mleA gene expression was more pronounced at pH 3.2 than at pH 3.8. The findings from this study have shed more light on the distribution of a wide array of enzyme-encoding genes in LAB strains associated with winemaking. However, it remains unknown if the enzymes encoded by these genes are functional under oenological conditions, given that wine is such a hostile environment encompassing a multitude of unfavourable conditions for the enzymes to work on. Evaluating the expression of these genes will also help give more insights on the regulation of the genes under winemaking conditions. / AFRIKAANSE OPSOMMING: Gedurende wynmaak, sal 'n aantal biochemiese veranderinge plaasvind as gevolg van die metaboliese aktiwiteit van wyn melksuurbakterieë (MSB) wat betrokke is by appelmelksuurgisting (AMG). Die laasgenoemde proses, wat meestal na alkoholiese fermentasie deur wyngiste plaasvind, behels die omskepping van L-malaat na L-laktaat en CO2, om sodoende die wyn se suur te verminder, mikrobiologiese stabiliteit en verandering van wyn organoleptiese kwaliteit. Alhoewel Oenococcus oeni hoofsaaklik die mees gewenste spesies is wat geskik is vir die uitvoering van AMG in wyn weens sy geskikte wynkundige eienskappe, Lactobacillus plantarum word ook beskou as 'n potensiële kandidaat vir AMG induksie. Ander spesies in die genera Lactobacillus en Pediococcus word dikwels geassosieer met wynbederf. Hierdie mikro-organismes veroorsaak wynbederf deur die produksie van wangeure as gevolg van hul metaboliese aktiwiteite. Dit is dus van kardinale belang dat die meganisme van die wynbederf verstaan word. Die doel van hierdie studie was om die teenwoordigheid van koderend ensieme gene van wynkundige belang in wynverwante MSB stamme te ondersoek. Ten einde dit te bereik, was verskillende stelle van spesifieke peilers ontwerp en toegepas vir 'n groot skaal se genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van ensiemkoderende gene betrokke by verskeie metaboliese paaie, soos sitraat metabolisme, aminosuur metabolisme, hidrolise van glikosiede, agteruitgang van fenoliese sure sowel as proteolise en peptidolise. PKR opsporings resultate het getoon dat die meerderheid van die stamme getoets, die meeste van die gene getoets voor besit. Dit is ook opgemerk dat, onder die O. oeni stamme getoets vir die teenwoordigheid van die pad geen, slegs twee stamme hierdie geen besit. Geen O. oeni stamme het voorheen gewys dat hul die pad geen besit, en hierdie studie was die eerste bewys oor die teenwoordigheid van hierdie geen in O. oeni stamme. In 'n poging om die geen geneties te karakteriseer, is DNA-fragmente van die twee positiewe O. oeni stamme se sekwens volgorde bepaal. Die DNA volgorde is vergelyk met ander nouverwante spesies. Verrassend, was geen passende DNA volgorde gevind met die gepubliseerde genome van drie O. oeni stamme (PSU-1, ATCC BAA-1163 en AWRI B429) nie. Dit versterk die spekulasie dat die pad geen in hierdie twee stamme via die horisontale geen-oordrag verkry is. Verder moet dit nog bepaal word of die teenwoordigheid van hierdie geen lei na vlugtige fenol produksie in wyn. In hierdie studie, is ongeïdentifiseerde stam geïsoleerd van Suid-Afrikaanse druiwe en wyn monsters ook geïdentifiseer en karakteriseer. Die identifisering van hierdie stam is uitgevoer deur middel van die 16S rDNA volgorde analise, wat aangedui het dat hierdie stam behoort aan Lactobacillus florum (99.9% volgorde identiteit). PKR toetse met behulp van die spesie-spesifieke peiler vir die vinnige opsporing en identifikasie van Lb. florum stamme is ook ontwikkel. Vir verdere karakterisering, was hierdie stam ook ondersoek vir die teenwoordigheid van koderende ensiem gene van wynkundige belang. PKR opsporings resultate het aangedui dat die Lb. florum stam ook oor 'n paar van die gene getoets voor besit. Bykomend tot genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van verskillende gene, het die studie ook die evaluering van die regulering van die mleA geen, kodering malaatdekarboksilase in drie wyn stamme van O. oeni. Die regulering van hierdie geen was getoets in die sintetiese wynmedium onder verskillende pH en etanol kondisies. Van die uitdrukkingsresultate, is daar waargeneem dat die mleA geenuitdrukking is negatief geraak deur hoë etanol-inhoud in die medium. Aan die ander kant, in die lae pH medium was die uitdrukking van hierdie geen bevoordeel by pH 3.2 as by pH 3.8. Die bevindinge van hierdie studie het meer lig gewerp op die verspreiding van die wye verskeidenheid van ensiem-koderende gene in MSB stamme wat verband hou met wynmaak. Dit bly egter steeds onbekend of die ensieme gekodeer deur hierdie gene funksioneel is onder wynkondisies, gegewe dat wyn so 'n vyandige omgewing is menigte ongunstige toestande vir die werking van ensieme. Evaluering van die uitdrukking van hierdie gene sal ook help om meer insigte gee oor die regulering van die gene onder wynmaak toestande.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The quality of wine is influenced and determined by various factors, one of which includes the process of malolactic fermentation (MLF). MLF plays an integral role in the flavour and sensory profile of most red wines as well as some white wines like Chardonnay. This process is conducted by lactic acid bacteria (LAB), specifically of the genera Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Of these, Oenococcus oeni is best adapted to survive in the harsh wine environment. MLF is defined as the conversion of L-malic acid to L-lactic acid and carbon dioxide. The conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid results in a decrease in acidity and an increase in pH, to give a softer mouthfeel and more favourable flavour profile. A further reason for conducting MLF in wine includes the improvement of microbial stability due to the removal of malic acid as a possible substrate for microorganisms. Recently, research focus has shifted to the ability of MLF and LAB to alter the aroma profile of wine via the production and/or modification of certain aroma compounds. In order for wine LAB to conduct MLF, they need to be able to survive the harsh and challenging wine environment. Conditions in South African wines are particularly challenging due to the long, hot ripening seasons resulting in high sugar concentrations which give high ethanol concentrations. Some LAB also struggle to adapt to an environment with high pH and low malic acid concentrations. These factors, combined with the use of sulphur dioxide, cause LAB to struggle in conducting and completing successful MLF. Many of the commercial starter cultures that are currently available contain LAB that have not been isolated from South African wine and are therefore not optimal for use under these challenging wine conditions. Oenococcus oeni is also the single LAB culture present in all commercially available starter cultures. The overriding goal of this study was to create a MLF starter culture containing a mixture of LAB cultures, namely O. oeni and Lactobacillus plantarum, which can successfully convert malic acid to lactic acid, ensure microbial stability, but also make a positive contribution to the wine aroma profile. Lactobacillus plantarum has previously been considered for possible use in a commercial starter culture. The LAB isolates used in this study were selected from the Institute for Wine Biotechnology culture collection as well as isolated from spontaneous MLF. The first objective was to characterise these LAB strains for important traits and for possible use as a MLF starter culture. A total of 23 strains were identified as O. oeni and 19 strains as Lb. plantarum. The identified strains were screened in a synthetic wine medium for their ability to convert malic acid to lactic acid. Based on the LAB strain performance in the synthetic wine medium, seven strains of both O. oeni and Lb. plantarum were selected. These 14 strains were screened for the presence of genes encoding for enzymes responsible for biogenic amine production and were found to contain none of the genes associated with the formation of histamine, tyramine or putrescine. The LAB strains were genetically screened for enzymes associated with aroma modification by LAB during MLF. The enzymes of interest that were screened for included β-glucosidase, esterase, protease and phenolic acid decarboxylase (PAD). The Lb. plantarum strains were found to possess more diverse enzymatic profiles related to aroma than O. oeni. The biggest differences were observed for the presence of β-glucosidase and PAD. The second objective was to perform small-scale fermentations with the individual LAB isolates. The individual isolates were evaluated in Pinotage and based on these results; three strains of each O. oeni and Lb. plantarum were selected for evaluation in mixed culture fermentations. The mixed cultures were evaluated in Pinotage, Shiraz and Cabernet Sauvignon in the 2008 vintage. As a third objective, the wines were also analytically and sensorially evaluated to investigate the changes in the aroma profile that could be attributed to the presence of the mixed LAB isolates. Based on the fermentation data as well as data pertaining to the aroma modification, three mixed cultures were selected for evaluation in the 2009 vintage in Pinotage, Cabernet Sauvignon and Chardonnay. The mixed cultures were able to successfully complete MLF in fermentation periods comparable to that of a commercial culture used as control. The different LAB cultures had distinct and diverse effects on the wine aroma profile. The O. oeni strain played a larger role in the ester concentration present after MLF, while the Lb. plantarum strain had a larger effect on the higher alcohol and volatile fatty acid concentration upon completion of MLF. The results generated by this novel study clearly indicate the potential of a mixed LAB starter culture for conducting MLF. The mixed cultures successfully completed MLF and made a positive contribution to the wine aroma profile. / AFRIKAANSE OPSOMMING: Die kwaliteit van wyn word beïnvloed en bepaal deur verskeie faktore en wynbereidings prosesse, wat die proses van appelmelksuurgisting (AMG) insluit. AMG speel ’n integrale rol in die sensoriese profiel van meeste rooiwyne, sowel as sommige witwyne soos Chardonnay. AMG word gedefinieër as die omskakeling van L-appelsuur na L-melksuur en koolstofdioksied. Hierdie omskakeling kan toegeskryf word aan die teenwoordigheid van melksuurbakterieë (MSB), spesifiek spesies van die genera Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Vanuit hierdie wyn MSB, is Oenococcus oeni die spesies wat die beste aanpas en oorleef onder stresvolle wyn kondisies. Die omskakeling van appelsuur, ’n dikarboksielsuur, na melksuur, ’n monokarboksielsuur, lei tot ‘n vermindering in suurheid en ’n verhoging in pH. Hierdie vermindering in suurheid gee ’n sagter en meer geronde mondgevoel aan die wyn en dra by tot ‘n meer aangename geurprofiel. ’n Verdere rede vir AMG in wyn is om mikrobiese stabiliteit te verseker deurdat appelsuur verwyder word as ’n moontlike koolstof substraat vir mikroörganismes. Onlangs het navorsing begin fokus op AMG en die vermoë van MSB om die aroma profiel van wyn te beïnvloed deur die produksie/modifisering van sekere aroma komponente. Vir MSB om AMG te kan deurvoer, moet hulle kan oorleef in die stresvolle wynomgewing. Wyntoestande in Suid-Afrika is veral uitdagend vir die oorlewing van mikroörganismes as gevolg van lang, warm somers wat lei tot ’n matriks met ’n hoë suikerkonsentrasie en wyn met ’n hoë etanolkonsentrasie. ‘n Omgewing met ‘n hoë pH en lae appelsuur konsentrasie, kan ook bydrae tot stresvolle kondisies vir MSB. Hierdie parameters, tesame met die gebruik van swaweldioksied, maak dit moeilik vir MSB om AMG te inisieer en te voltooi. Sommige van die kommersiële aanvangskulture wat tans beskikbaar is, bevat nie MSB wat onder Suid-Afrikaanse wyntoestande geïsoleer is nie en daarom is dit nie altyd optimaal vir gebruik nie. Oenococcus oeni is ook die enkele MSB kultuur wat in alle kommersiële kulture gebruik word. Die hoofdoelwit van hierdie studie was om ’n potensiële kommersiële aanvangskultuur te ontwikkel wat ‘n mengsel van MSB bevat. Hierdie aanvangskultuur moet AMG suksesvol kan voltooi, mikrobiologiese stabiliteit bevorder en steeds die wynaroma positief kan beïnvloed. Bakterierasse van O. oeni en Lb. plantarum is geselekteer vir gebruik in hierdie studie. Lactobacillus plantarum het reeds in vorige studies potensiaal getoon as ‘n moontlike aanvangskultuur. Die MSB isolate vir hierdie studie is geselekteer uit die Instituut vir Wynbiotegnologie se kultuurversameling en geïsoleer uit spontane AMG fermentasies. Die eerste doelwit was om hierdie MSB isolate te karakteriseer vir belangrike eienskappe en die moontlike gebruik as ’n kommersiële AMG aanvangskultuur. ‘n Totaal van 23 O. oeni en 19 Lb. plantarum isolate is geïdentifiseer. Hierdie isolate is in ’n sintetiese wynmedium geëvalueer vir hul vermoë om appelsuur na melksuur om te skakel. Op grond van hul reaksie in die sintetiese wynmedium, is sewe isolate van elk van die O. oeni en Lb. plantarum geselekteer. Hierdie 14 isolate is ondersoek vir die teenwoordigheid van die gene wat kodeer vir biogeenamien produksie en daar is gevind dat geen van die isolate enige van die biogeenamien gene wat ondersoek is, naamlik histamien, tiramien en putresien besit nie. Die MSB isolate is geneties ondersoek vir die teenwoordigheid van dié gene wat kodeer vir ensieme wat die aromaprofiel tydens AMG beïnvloed. Dié ensieme sluit β-glukosidase, esterase, protease, fenoliese suurdekarboksilase en sitraatliase in. Daar is gevind dat die Lb. plantarum isolate meer diverse ensiemprofiele as O. oeni besit. Die grootste verskille in die ensiemprofiele kan toegeskryf word aan die teenwoordigheid van β-glukosidase en fenoliese suurdekarboksilase. Die tweede doelwit was om kleinskaalse AMG fermentasies met die individuele MSB isolate uit te voer. Die individuele isolate is in Pinotage geëvalueer. Volgens hierdie resultate is drie isolate van elk van die O. oeni en Lb. plantarum geselekteer om in gemengde kulture getoets te word. Die gemengde kulture is in Pinotage, Shiraz en Cabernet Sauvignon in 2008 geëvalueer. As ’n derde doelwit is hierdie wyne ook analities en sensories geëvalueer om die veranderinge in die aromaprofiele as gevolg van die teenwoordigheid van die MSB te ondersoek. Op grond van die fermentasiedata, sowel as die data oor die aromaveranderinge, is drie gemengde kulture geselekteer vir evaluering in Pinotage, Cabernet Sauvignon en Chardonnay in 2009. Die gemengde kulture kon AMG suksesvol voltooi met fermentasietempo’s wat vergelykbaar was met dié van ‘n kommersiële AMG kultuur wat as kontrole gebruik is. Die verskillende MSB kulture het spesifieke en uiteenlopende uitwerkings op die wynaroma gehad. Die O. oeni isolaat in die gemengde kultuur blyk ‘n belangriker rol te speel in die esterkonsentrasie na AMG, terwyl die Lb. plantarum isolaat ’n groter effek het op die hoër alkohol en vlugtige vetsuurinhoud na AMG. Die resultate wat deur hierdie unieke studie gegenereer is, gee ’n aanduiding van die potensiaal van ’n gemengde MSB aanvangskultuur vir AMG. Die gemengde kulture kon AMG suksesvol voltooi en ‘n positiewe bydrae tot die aromaprofiel van die wyn lewer.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The tolerance of Enterococcus mundtii ST4SA to stressful gastro-intestinal conditions in humans and animals is vital to its success as a probiotic. The need for new effective probiotics with stronger inhibitory (bacteriocin) activity has arisen due to the increasing number of antibiotic resistant pathogens. Enterococci are used in the fermentation of sausages and olives, cheese making and as probiotics. Their role as opportunistic pathogens in humans makes them a controversial probiotic (Moreno et al., 2005). Enterococci occur naturally in the gastro-intestinal tract which renders them intrinsic acid and bile resistance characteristics. E. mundtii ST4SA produces a 3950 Da broad-spectrum antibacterial peptide active against Gram-positive and Gram-negative bacteria, and viruses. The bacteria include Enterococcus faecalis, Streptococcus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae and Staphylococcus aureus. E. mundtii ST4SA inactivates the herpes simplex viruses HSV-1 (strain F) and HSV-2 (strain G), a measles virus (strain MV/BRAZIL/001/91, an attenuated strain of MV), and a polio virus (PV3, strain Sabin). This study focuses on the genetic stability of E. mundtii ST4SA genes when exposed to stress factors in the human and animal gastrointestinal tract. Based on results obtained by real-time PCR, the expression of genes encoding bacST4SA, RecA, GroES and 23S rRNA by E. mundtii ST4SA were not affected when the cells were exposed to acid, bile and pancreatic juice. This suggests that these genes of E. mundtii ST4SA will remain stable in the intestine. This could indicate that other genes of E. mundtii ST4SA could remain stable in the host. Further studies on the stability of genes encoding antibiotic resistance and virulence factors should be conducted to determine their stability and expression in the host in stress conditions. Concluded from this study, E. mundtii ST4SA is an excellent probiotic strain. / AFRIKAANSE OPSOMMING: Enterococcus mundtii ST4SA se weerstandsvermoë teen stresvolle gastrointestinale kondisies is essensieel vir die sukses van hierdie organisme as ‘n probiotikum. Die aanvraag vir nuwe, meer effektiewe probiotika met sterker inhibitoriese (bakteriosien) aktiwiteit is as gevolg van die toename in antibiotikum weerstandbiedende patogene. Enterococci word algemeen gebruik as probiotika, sowel as in die fermentasie van worse, olywe en kaas. Hulle rol as oppertunistiese patogene in mense veroorsaak kontroversie as gevolg van hul toenemende gebruik as probiotika. Enterococci is deel van die natuurlike mikroflora in die gastrointestinale weg van mense en diere. Dit verleen aan hierdie spesies ‘n natuurlike weerstandsvermoë teen maagsure, galsoute en pankreatiese afskeidings. E. mundtii ST4SA produseer ‘n 3950 Da wye spektrum anti-bakteriese peptied, aktief teen Gram positiewe en Gram negatiewe bakterieë sowel as virusse. Hierdie bakterieë sluit Enterococcus faecalis, Streptococcus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae en Staphylococcus aureus in. E. mundtii ST4SA inaktiveer die herpes simpleks virus HSV-1 en HSV-2, ‘n masels virus (MV/BRAZIL/001/91), en ‘n polio virus (PV3, stam Sabin). Hierdie studie fokus op die genetiese stabiliteit van E. mundtii ST4SA gene, wanneer hulle blootgestel word aan stress faktore in die mens en dier gastrointestinale weg. “Intydse” PKR data gebasseer op die uitdrukking van die bacST4SA, RecA, GroES en 23S rRNA gene in stresvolle kondisies dui aan dat E. mundtii ST4SA nie geaffekteer word wanneer die sel blootgestel word aan suur, gal en pankreatiese vloeistowwe nie. Hierdie resultate dui aan dat hierdie gene van E. mundtii ST4SA stabiel sal bly in die intestinale weg van die mens en dier. Dit kan aandui dat ander gene van E. mundtii ST4SA soos die wat kodeer vir virulensie faktore en antibiotikum se weerstandsvermoë stabiel mag bly in die gasheer. Verdere studies wat fokus op die stabiliteit van gene wat kodeer vir antibiotikum weerstandbiedendheid en virulensie faktore moet uitgevoer word om hulle stabiliteit en uitdrukking in die gasheer te bepaal. Bevindings van hierdie studie dui aan dat E. mundtii ST4SA goeie potensiaal het as ‘n probiotikum.

Listeria monocytogenes causa a listeriose, uma doença zoonótica grave que causa infecções do sistema nervoso central (meningite, encefalite e meningoencefalite), bacteremia primária e septicemia. A doença apresenta baixa morbidade e alta mortalidade e acomete, principalmente, grupos de risco, como mulheres grávidas, neonatos, indivíduos imunocomprometidos e idosos. L. monocytogenes tem sido encontrada com freqüência em alimentos in natura e/ou processados, como queijos e outros produtos lácteos. Esse estudo objetivou isolar bactérias lácticas a partir de leite de cabra, capazes de produzir peptídeos antimicrobianos (bacteriocinas), identificar estas cepas, caracterizar as bacteriocinas produzidas e avaliar o seu potencial de aplicação no controle da multiplicação de L. monocytogenes em queijo de cabra durante armazenamento a 8-10°C. Trabalhando-se com leite de cabra cru, foi possível isolar seis cepas produtoras de bacteriocinas (DF2Mi, DF3Mi, DF4Mi, DF5Mi, DF6Mi e DF60Mi). Através de testes fenotípicos apropriados e sequenciamento do 16S rRNA, essas cepas foram identificadas como Lactococcus lactis subsp. lactis (DF2Mi, DF3Mi, DF4Mi e DF5Mi), Leuconostoc lactis (DF6Mi) e Lactobacillus paracasei subsp. paracasei (DF60Mi). A caracterização físico-química e biológica das bacteriocinas produzidas pelas cepas DF4Mi, DF6Mi e DF60Mi indicou que eram resistentes ao calor e extremos de pH, mas apresentavam características diferentes em relação ao espectro de ação, sensibilidade a agentes químicos, adsorção à células-alvo e lise das células de L. monocytogenes. O efeito do pH, temperatura e composição do meio de cultura na produção das bacteriocinas foi também cepa-dependente. A cepa DF4Mi apresentou melhor atividade antimicrobiana e foi selecionada para o estudo de inibição de L. monocytogenes em queijos. Para isso, foram preparados lotes de queijo frescal, feito com leite de cabra pasteurizado adicionado e não adicionado da cepa DF4Mi (106 UFC/mL), experimentalmente contaminado com L. monocytogenes (103 UFC/g), além dos controles positivo (queijo adicionado de nisina 12,5 mg/Kg) e negativo (leite adicionado de uma cepa de L. lactis subsp. lactis não bacteriocinogênica). Observou-se que a cepa DF4Mi apresentou efeito bacteriostático no queijo, sendo capaz de inibir a multiplicação do patógeno durante o armazenamento a 8-10°C por 10 dias. No entanto, inibição semelhante foi obtida nos queijos com a bactéria lática não bacteriocinogênica, indicando que a inibição não pode ser creditada às bacteriocinas. Nos queijos-controle com nisina, foi observada uma redução de 2 log na contagem de L. monocytogenes após 10 dias a 8-10°C. Já nos queijos preparados sem nisina e sem nenhuma bactéria lática, as contagens de L. monocytogenes atingiram contagens elevadas (106 UFC/g) após 10 dias de armazenamento a 8-10°C. / Listeria monocytogenes causes listeriosis, a serious zoonotic disease that causes infections in the central nervous system (meningitis, encephalitis and meningoencephalitis), bacteremia and septicemia. The disease presents low morbidity and high mortality and affects mainly those in the risk group, such as pregnant women, neonates, immunocompromised individuals and the elderly. L. monocytogenes has been frequently detected in in natura and processed foods. Like cheeses and other dairy products. This study aimed to isolate lactic acid bacteria capable of producing antimicrobial compounds from goat milk, identify the isolates, characterize the bacteriocins and evaluate their potential application in controlling the growth of L. monocytogenes in goat cheese during storage at 8-10°C. Six bacteriocinogenic strains (DF2Mi, DF3Mi, DF4Mi, DF5Mi, DF6Mi e DF60Mi) were successfully isolated from raw goat milk. Using appropriate phenotypic tests and 16S rRNA sequencing, these strains were identified as Lactococcus lactis subsp. lactis (DF2Mi, DF3Mi, DF4Mi e DF5Mi), Leuconostoc lactis (DF6Mi) and Lactobacillus paracasei subsp. paracasei (DF60Mi). The physico-chemical and biological characterization of the bacteriocins produced by the strains DF4Mi, DF6Mi e DF60Mi indicated that they were resistant to heat and pH extremes, but presented different spectrum of activity, sensitivity to chemicals, adsorption to target cells and lysis of L. monocytogenes. The effect of pH, temperature and culture media composition in bacteriocin production was also strain-dependent. The strain DF4Mi presented the best antimicrobial activity and was selected for the studies on inhibition of L. monocytogenes in cheese. Frescal cheese was manufactured with pasteurized goat milk added of a culture of DF4Mi (106 CFU/mL), and experimentally contaminated with L. monocytogenes (103 CFU/g). Control cheeses were also prepared: those added of 12.5 mg/Kg nisin (positive control) and those added of a non-bacteriocinogenic L. lactis subsp. lactis strain. The strain presented a bacteriostatic effect, controlling the growth of the pathogen for 10 days at 8-10°C. However, a similar effect was observed in the cheeses prepared with the non-bacteriocinogenic strain, indicating that the inhibition cannot be credited to bacteriocins. In the cheeses containing nisin, a 2 log reduction in the counts of L. monocytogenes was achieved after 10 days at 8-10°C. In the cheeses with no added nisin or lactic acid bacteria, the counts of L. monocytogenes after 10 days at 8-10°C were high (106 CFU/g).

Após a constatação da escassez de estudos realizados com vegetais crus na busca por novas estirpes de bactérias láticas (BAL) produtoras de bacteriocinas e diante do potencial tecnológico da aplicação destas cepas tanto como agentes de conservação em alimento, bem como cultura probiótica em alimentos funcionais, este estudo objetivou isolar e identificar cepas de bactérias láticas potencialmente bacteriocinogênicas de amostras de rúcula obtidas no comércio local de São Paulo, SP - Brasil, identificar e caracterizar as bacteriocinas produzidas pelos isolados e avaliar o potencial probiótico dos isolados testando sua sobrevivência no modelo dinâmico do trato gastrointestinal TNO gastro-Intestinal Model - TIM-1 disponível no TNO (The Netherlands Organization for Applied Scientific Research) divisão Quality of Life (Zeist, Holanda). A produção de bacteriocinas neste modelo também foi avaliada, comparando-se com L. sakei 2a, também produtora de bacteriocinas e ainda avaliou-se a interferência na viabilidade de E. faecium LMA1. A cepa Lactococcus lactis subsp. lactis MK02R de rúcula produziu uma bacteriocina sensível à enzimas proteolíticas, termoestável e não influenciada pelo pH, sendo capaz de inibir Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii e Listeria Monocytogenes de diferentes grupos sorológicos. Os ensaios genéticos utilizando primers Nisf e Nisr confirmaram que a bacteriocina MK02R é uma nisina, apresentando uma alteração dos aminoácidos no peptídeo líder em relação às nisinas A, Z, Q, F e U, porém com a estrutura do peptídeo maduro idêntica ao da nisina F. Estes resultados foram confirmados por espectrometria de massas de amostras purificadas por HPLC. L. lactis MK02R resistiu à passagem no modelo dinâmico TIM-1, apresentando uma alta capacidade de sobreviver nas condições simuladas do trato gastrointestinal humano. Entretanto, não foi capaz de causar a redução no número de E. faecium LMA1. Em contrapartida, L. sakei 2a, mesmo apresentando uma sobrevivência menor, foi capaz de causar uma redução de 70% na população de E. faecium LMA1 no ambiente simulado do TGI. Não foi detectada atividade residual da ação antimicrobiana das bacteriocinas produzidas por L. lactis MK02R ou L. sakei 2a após a passagem pelo modelo dinâmico TIM-1. Estes resultados evidenciam a possível aplicação de L. lactis MK02R como um agente de controle biológico na conservação de alimentos e também como uma cultura potencialmente probiótica. / Given the scarcity of studies performed with raw vegetables addressing the search for new bacteriocinogenic strains of lactic acid bacteria (LAB) and considering the technological application of these strains as food preservatives and probiotic cultures in functional foods, this study was aimed at isolation and identification of bacteriocinogenic LAB strains from samples of rocket salad obtained in the local market of São Paulo, SP - Brazil, subsequent characterization of the bacteriocins produced by these LABs and evaluation of their probiotic potential by testing their survival in the dynamic gastrointestinal model TNO gastro- Intestinal-Model - TIM-1, available at the TNO (Netherlands Organization for Applied Scientific Research) Quality of Life division (Zeist, Netherlands). The studies in the TIM-1 model were also done with another bacteriocinogenic strain L. sakei 2a for comparison, evaluating their interference on the viability of E. faecium LMA1. The bacteriocin produced by strain Lactococcus lactis subsp. lactis MK02R isolated from rocket salad was sensitive to proteolytic enzymes, heat-stable and not influenced by the pH. The bacteriocin inhibited the growth of Enterococcus faecium, Lactobacillus sakei, Listeria innocua, Lactobacillus delbrueckii the primers Nisf and Nisr indicated that the bacteriocin produced by the strain MK02R is a nisin, with a change in the amino acid sequence of the leader peptide when compared to nisin A, Z, Q, U and F, but with the structure of the mature peptide homologous to that of nisin F. These results were confirmed by mass spectrometry of purified samples obtained by HPLC. L. lactis MK02R withstood the test in the dynamic model TIM-1, presenting capability to survive in the simulated conditions of the human gastrointestinal tract. However, the strain was not able to cause a reduction in the number of E. faecium LMA1. On the other hand, L. sakei 2a, even presenting lower survival, was able to cause 70% reduction in the population of E. faecium LMA1 in the gut simulated environment. No residual antimicrobial activity of bacteriocin produced by L. lactis MK02R or L. sakei 2a was detected after the transit through the dynamic model TIM-1. These results demonstrate the possible application of L. lactis MK02R both as a biocontrol agent in food preservation and as a potentially probiotic culture.

Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria (LABs), which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. However, when this bacteriocin has not been completely characterized regarding its amino acid and the nucleotide sequences of the corresponding gene, the qualified term bacteriocin-like inhibitory substance (BLIS) is recommended. In order to increase the antimicrobial activity of bacteriocins, the ability of probiotics LABs, such as Pediococcus pentosaceus, to ferment different carbon and nitrogen sources has been studied. For the development of an improved culture medium, carbon and nitrogen sources must be considered as nutrients responsible for cell growth and bacteriocin production. The best condition, after 48 h of cultivation, for growth (3.420 g/L) and for BLIS production by Pediococcus pentosaceus ATCC 43200 was in Man, Rogosa and Sharp (MRS) culture medium supplemented with 1.5% peptone, initial pH 6.0 and under the following culture conditions: anaerobiosis, 30oC and agitation of 200 rpm. Compared with control (MRS without supplement), the growth of Pediococcus was significantly lower (1.995 g/L) as well as it reduced significantly its generation time from 2.05 h (control) to 1.28 h (MRS supplemented), a reduction of approximately 62.5%. Moreover, addiction of peptone to MRS medium promoted reduction of 4 h to the Pediococcus exponential phase onset. Regarding BLIS antimicrobial activity, addition of nitrogen source to MRS medium was also quite significant. Through the agar diffusion method, BLIS showed inhibition halos between 12.50 and 19.50 mm against LABs strains (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 and Carnobacterium piscicola CECT 4020). Against Listeria strains (Listeria innocua NCTC 111288 and Listeria seeligeri NCTC 11289), their antimicrobial activity was better detected in liquid medium assay, evaluating the minimal inhibitory concentration of 50%. BLIS was able to inhibit 60 and 100% of L. seeligeri and L. innocua, respectively, as well as, diluted 1x (v/v) in water was able to inhibit 100% growth of both Listeria. BLIS 17 showed also good results as food preservative when applied in ready-to-eat pork ham artificially contaminated with L. seeligeri in vacuum-package at 4oC during shelf life of 10 days. BLIS was able to maintain low Listeria multiplication, lower samples weight loss, low lipid peroxidation and good color parameters during samples storage. Results demonstrated the importance of optimizing the culture medium to increase microbial mass, to produce and to improve the activity of this antimicrobial molecule. Moreover, results also suggest the possible application of BLIS as a natural food preservative. / Bacteriocinas são peptídeos produzidos por várias espécies de bactérias, especialmente bactérias ácido-láticas (BALs) e apresentam um amplo espectro de ação contra bactérias deteriorantes e patógenos de origem alimentar. Entretanto, quando estas bacteriocinas não foram completamente caracterizadas quanto a sequência de seus nucleotídeos e do seu gene correspondente, é recomendada a denominação de substância semelhante a bacteriocina (BLIS). Para aumentar a atividade antimicrobiana de bacteriocinas, a habilidade de BALs probióticas, como Pediococcus pentosaceus, em fermentar diferentes fontes de carbono e nitrogênio tem sido estudado. Para o desenvolvimento de um meio de cultura melhorado, fontes de carbono e nitrogênio devem ser consideradas como nutrientes responsáveis pelo crescimento celular e pela produção de bacteriocina. A melhor condição, após 48 h de cultivo, para o crescimento (3,420 g/L) e para a produção de BLIS por P. pentosaceus ATCC 43200 foi em meio de cultivo Man, Rogosa e Sharp (MRS) suplementado com 1,5% de peptona, pH inicial 6,0 e sob as seguintes condições de cultivo: anaerobiose, 30oC e agitação de 200 rpm. Comparado ao controle (MRS sem suplementação), o crescimento de Pediococcus foi significativamente menor (1,995 g/L) assim, como também, reduziu significativamente o tempo de geração de 2,05 h (controle) para 1,28 h (MRS suplementado), uma redução de aproximadamente 62,5%. Além disso, a adição de peptona ao meio MRS promoveu redução de 4 h para o início da fase exponencial de Pediococcus. Quanto a atividade antimicrobiana de BLIS, a adição de fonte de nitrogênio ao meio MRS também foi bastante significativa. Através do método ágar difusão, BLIS apresentou halos de inibição entre 12,50 a 19,50 mm contra cepas de BALs (Lactobacillus sakei ATCC 15521, Lactobacillus plantarum CECT 221 e Carnobacterium piscicola CECT 4020). Contra cepas de Listeria (Listeria innocua NCTC 11288 e Listeria seeligeri NCTC 11289), a sua atividade inibitória foi melhor detectada em meio líquido, através da determinação da concentração mínima inibitória de 50%. BLIS sem diluição foi capaz de inibir 60 e 100% de L. seeligeri e L. innocua, 15 respectivamente, assim como, diluído 1x (v/v) em água foi capaz de inibir 100% o crescimento de ambas Listeria. BLIS também apresentou bons resultados como conservante de alimento quando aplicado em presunto contaminado artificialmente com L. seeligeri e armazenado a 4oC a vácuo por 10 dias. BLIS foi capaz de manter baixa a multiplicação de Listeria, menor perda de peso das amostras, baixa peroxidação lipídica e bons parâmetros de cor durante o armazenamento das amostras. Os resultados demonstraram a importância de se otimizar meio de cultivo tanto para o aumento da massa microbiana como para a produção e melhoramento da atividade desta molécula antimicrobiana. Além disso, os resultados também sugerem a possível aplicação de BLIS como conservante natural de alimentos.