<p>Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of <i>Salmonella enterica</i> serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic <i>Escherichia coli</i>.</p><p>The immune response elicited using secreted proteins from <i>S. enterica</i> serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant <i>Salmonella</i> atypical fimbriae (SafB/D) complexes, a conserved structure within <i>Salmonella</i> species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. </p><p>Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. </p><p>Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.</p>

Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of Salmonella enterica serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic Escherichia coli. The immune response elicited using secreted proteins from S. enterica serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant Salmonella atypical fimbriae (SafB/D) complexes, a conserved structure within Salmonella species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.

Orientador: Isabel Cristina Boleli / Banca: Silvana Martinez Baraldi Artoni / Banca: Nilce Maria Soares Queiroz Gama / Resumo: O experimento foi realizado com o objetivo de avaliar os pesos corporal (PC), do fígado (PF) e da bursa de Fabrício (PB), o perfil hematológico (eritrograma, leucograma e glicemia), a integridade e o desenvolvimento da mucosa intestinal de pintos de corte de submetidos ao tratamento por exclusão competitiva (EC) antes e após infecção experimental por Salmonella Enteritidis (SE). Foram utilizados 128 pintos, distribuídos num delineamento ao acaso com esquema fatorial 2 x 4 [2 sexos e 4 tratamentos (NI: não infectados com SE; I: infectados com SE; IEC: infectados com SE e tratados com EC 24 h após infecção; ECI: tratados com EC e infectados com SE 24 h após tratamento)]. Os animais foram sacrificados com 1, 3, 5 e 7 dias pós-infecção (dpi) para obtenção do conteúdo cecal (microbiológico), fígado, bursa de Fabrício e fragmentos do intestino delgado. Os dados demonstram que o uso de EC após infecção não evita a colonização cecal por SE, mas que a mesma é evitada quando a EC é realizada antes da infecção. A partir do 7° dpi as aves infectadas apresentaram os menores valores de PC e PB, e não se evidenciou diferenças na glicemia e no PF. As alterações do eritrograma ficaram restritas aos três primeiros dpi e os resultados do leucograma mostraram uma resposta sexo-específica frente à infecção por SE. Em relação à integridade, o presente trabalho mostrou que os vilos do intestino delgado dos pintos de corte apresentam um grande processo de renovação celular ao final da primeira semana de vida e que EC acelera este processo. O desenvolvimento intestinal variou de acordo com o segmento, o sexo, o tratamento e a idade analisada. / Abstract: The present experiment was carried out with the objective to evaluate the weights corporal 0NC), of the liver 0NF) and bursa of Fabrício (WB), the hematology profile (erytrogram, leucogram and glycemia), the integrity and the development of the intestinal mucosa by young chickens submitted to competitive exclusion treatment (CE) before and after experimental infection with Salmonella Enteritidis (SE). 128 young chickens, distributed in completely randomized design in 4 x 2 factorial arrangement [had been used 2 sex and 4 treatments (NI: not infection with SE - Control; I: infection with SE; ICE: infection with SE and treated with CE 24 h after infection; CEI: treated with CE and infection with SE 24 h after treatment)]. The animais had been sacrificed with 1, 3, 5 and 7 days post infection (dpi) for attainment of the cecal content (microbiological), liver, bursa of Fabrício and fragments of the thin intestine. The data demonstrate that the CE use after infection does not prevent the cecal settling for SE, but that the same one is prevented when the CE is carried through before the infection. From 7° dpi the infections birds had presented the lesser values of WC and WB, and it did not prove differences in the glycemia and the WF. The alterations of the erytrogram had been restricted to three first dpi, and the results of the leucogram had shown to a sex-specific reply front to the infection for SE. In relation to the integrity, the present work showed that the vilos of the thin intestine of young chickens present a great process of cellular renewal to the end of the first week of life and that CE speeds up this processo The intestinal development in accordance with varied the segment, the sex, the treatment and the analyzed age. / Mestre

Salmonella é uma das principais causas de Doenças Transmitidas por Alimentos em todo o mundo, sendo que no Estado do Rio Grande do Sul (RS) esse microrganismo tem sido apontado como um dos principais agentes de toxinfecções alimentares nos últimos anos. Neste trabalho foram caracterizados isolados de Salmonella envolvidas em salmoneloses ocorridas no RS, no período de 2007 a 2013. Entre os 163 isolados investigados, 138 (84,7%) foram sorotipificados com S. Enteritidis, enquanto os outros isolados foram S. Schwarzengrund (n = 9 – 5,5 %), S. Typhimurium (n = 6 – 3,7%), S. Infantis (n = 1 – 0,6 %), S. Agona (n = 1 – 0,6 %), S. Derby (n = 1 – 0,6 %), S. London (n = 1 – 0,6 %), S. Give (n = 1 – 0,6 %), S. Panama (n = 1 – 0,6 %) e S. enterica (n = 4 – 2,5 %). Os principais alimentos envolvidos nos surtos foram maionese caseira (17,39%), seguido dos produtos de confeitaria (15,94 %) e carnes (12,32 %). A resistência da S. Enteritidis a 12 agentes antimicrobianos também foi investigada. As maiores porcentagens de resistência foram encontradas em relação à nitrofurantoína (94,2 %) e ao ácido nalidíxico (89,1 %). A resistência para duas drogas foi verificada em 80,43 % dos isolados. Sendo que a multirresistência para três ou cinco antimicrobianos foi verificada em quatro e dois isolados, respectivamente. Quando os isolados foram submetidos à PCR-Ribotipificação, apenas um perfil de bandas foi identificado. Os resultados de PCR-Ribotipificação sugerem que uma mesma cepa de S. Enteritidis foi isolada a partir de alimentos envolvidos em salmoneloses ocorridas em diferentes municípios do Estado do RS no período de 2007 a 2013. Uma vez que o mesmo perfil de bandas foi identificado em S. Enteritidis causadoras de salmoneloses, durante 1999 a 2006, os resultados indicam que a mesma cepa de S. Enteritidis tem causado surtos alimentares no RS, durante o período de 1999 a 2013. / Salmonella is a major cause of Foodborne Diseases worldwide, and in the State of Rio Grande do Sul (RS) this microorganism has been identified as the main agent of foodborne diseases in last years. In this work, Salmonella isolates responsible for salmonellosis occurred in the State of RS, in the period 2007 to 2013 were characterized. Among the 163 isolates investigated, 138 (84.7 %) were serotyped as S. Enteritidis, whereas the other isolates were S. Schwarzengrund (n = 9 – 5.5 %), S. Typhimurium (n = 6 – 3.7 %), S. Infantis (n = 1 – 0.6 %), S. Agona (n = 1 – 0.6 %), S. Derby (n = 1 – 0.6%), S. London (n = 1 – 0.6 %), S. Give (n = 1 – 0.6 %), S. Panama (n = 1 – 0.6 %) and S. enterica (n = 4 – 2.5 %). The main food vehicles identified were homemade mayonnaise (17.39 %), followed by pastry products (15.94 %) and beef (12.32 %). The S. Enteritidis resistance to 12 antimicrobial agents was investigated. The highest percentages of resistance were found to nitrofurantoin (94.2 %) and nalidixic acid (89.1 %). The resistance to two different drugs was observed in 80.43 % of the isolates. Multidrug-resistance for three to five antimicrobials was observed in four and two isolates, respectively. When the isolates were analysed by PCR-Ribotyping, only one banding profile was identified. The results of PCR-Ribotyping suggest that the same strain of S. Enteritidis was isolated from foods involved in salmonelloses occurred in different municipalities of the State of RS in the period 2007-2013. Since the same banding pattern was found in strains involved in salmonellosis outbreaks of 1999 to 2006, results indicated that the same strain of S. Enteritidis has caused salmonellosis outbreaks in RS, during the period of 1999 to 2013.

Salmonella é uma das principais causas de Doenças Transmitidas por Alimentos em todo o mundo, sendo que no Estado do Rio Grande do Sul (RS) esse microrganismo tem sido apontado como um dos principais agentes de toxinfecções alimentares nos últimos anos. Neste trabalho foram caracterizados isolados de Salmonella envolvidas em salmoneloses ocorridas no RS, no período de 2007 a 2013. Entre os 163 isolados investigados, 138 (84,7%) foram sorotipificados com S. Enteritidis, enquanto os outros isolados foram S. Schwarzengrund (n = 9 – 5,5 %), S. Typhimurium (n = 6 – 3,7%), S. Infantis (n = 1 – 0,6 %), S. Agona (n = 1 – 0,6 %), S. Derby (n = 1 – 0,6 %), S. London (n = 1 – 0,6 %), S. Give (n = 1 – 0,6 %), S. Panama (n = 1 – 0,6 %) e S. enterica (n = 4 – 2,5 %). Os principais alimentos envolvidos nos surtos foram maionese caseira (17,39%), seguido dos produtos de confeitaria (15,94 %) e carnes (12,32 %). A resistência da S. Enteritidis a 12 agentes antimicrobianos também foi investigada. As maiores porcentagens de resistência foram encontradas em relação à nitrofurantoína (94,2 %) e ao ácido nalidíxico (89,1 %). A resistência para duas drogas foi verificada em 80,43 % dos isolados. Sendo que a multirresistência para três ou cinco antimicrobianos foi verificada em quatro e dois isolados, respectivamente. Quando os isolados foram submetidos à PCR-Ribotipificação, apenas um perfil de bandas foi identificado. Os resultados de PCR-Ribotipificação sugerem que uma mesma cepa de S. Enteritidis foi isolada a partir de alimentos envolvidos em salmoneloses ocorridas em diferentes municípios do Estado do RS no período de 2007 a 2013. Uma vez que o mesmo perfil de bandas foi identificado em S. Enteritidis causadoras de salmoneloses, durante 1999 a 2006, os resultados indicam que a mesma cepa de S. Enteritidis tem causado surtos alimentares no RS, durante o período de 1999 a 2013. / Salmonella is a major cause of Foodborne Diseases worldwide, and in the State of Rio Grande do Sul (RS) this microorganism has been identified as the main agent of foodborne diseases in last years. In this work, Salmonella isolates responsible for salmonellosis occurred in the State of RS, in the period 2007 to 2013 were characterized. Among the 163 isolates investigated, 138 (84.7 %) were serotyped as S. Enteritidis, whereas the other isolates were S. Schwarzengrund (n = 9 – 5.5 %), S. Typhimurium (n = 6 – 3.7 %), S. Infantis (n = 1 – 0.6 %), S. Agona (n = 1 – 0.6 %), S. Derby (n = 1 – 0.6%), S. London (n = 1 – 0.6 %), S. Give (n = 1 – 0.6 %), S. Panama (n = 1 – 0.6 %) and S. enterica (n = 4 – 2.5 %). The main food vehicles identified were homemade mayonnaise (17.39 %), followed by pastry products (15.94 %) and beef (12.32 %). The S. Enteritidis resistance to 12 antimicrobial agents was investigated. The highest percentages of resistance were found to nitrofurantoin (94.2 %) and nalidixic acid (89.1 %). The resistance to two different drugs was observed in 80.43 % of the isolates. Multidrug-resistance for three to five antimicrobials was observed in four and two isolates, respectively. When the isolates were analysed by PCR-Ribotyping, only one banding profile was identified. The results of PCR-Ribotyping suggest that the same strain of S. Enteritidis was isolated from foods involved in salmonelloses occurred in different municipalities of the State of RS in the period 2007-2013. Since the same banding pattern was found in strains involved in salmonellosis outbreaks of 1999 to 2006, results indicated that the same strain of S. Enteritidis has caused salmonellosis outbreaks in RS, during the period of 1999 to 2013.

Salmonella é uma das principais causas de Doenças Transmitidas por Alimentos em todo o mundo, sendo que no Estado do Rio Grande do Sul (RS) esse microrganismo tem sido apontado como um dos principais agentes de toxinfecções alimentares nos últimos anos. Neste trabalho foram caracterizados isolados de Salmonella envolvidas em salmoneloses ocorridas no RS, no período de 2007 a 2013. Entre os 163 isolados investigados, 138 (84,7%) foram sorotipificados com S. Enteritidis, enquanto os outros isolados foram S. Schwarzengrund (n = 9 – 5,5 %), S. Typhimurium (n = 6 – 3,7%), S. Infantis (n = 1 – 0,6 %), S. Agona (n = 1 – 0,6 %), S. Derby (n = 1 – 0,6 %), S. London (n = 1 – 0,6 %), S. Give (n = 1 – 0,6 %), S. Panama (n = 1 – 0,6 %) e S. enterica (n = 4 – 2,5 %). Os principais alimentos envolvidos nos surtos foram maionese caseira (17,39%), seguido dos produtos de confeitaria (15,94 %) e carnes (12,32 %). A resistência da S. Enteritidis a 12 agentes antimicrobianos também foi investigada. As maiores porcentagens de resistência foram encontradas em relação à nitrofurantoína (94,2 %) e ao ácido nalidíxico (89,1 %). A resistência para duas drogas foi verificada em 80,43 % dos isolados. Sendo que a multirresistência para três ou cinco antimicrobianos foi verificada em quatro e dois isolados, respectivamente. Quando os isolados foram submetidos à PCR-Ribotipificação, apenas um perfil de bandas foi identificado. Os resultados de PCR-Ribotipificação sugerem que uma mesma cepa de S. Enteritidis foi isolada a partir de alimentos envolvidos em salmoneloses ocorridas em diferentes municípios do Estado do RS no período de 2007 a 2013. Uma vez que o mesmo perfil de bandas foi identificado em S. Enteritidis causadoras de salmoneloses, durante 1999 a 2006, os resultados indicam que a mesma cepa de S. Enteritidis tem causado surtos alimentares no RS, durante o período de 1999 a 2013. / Salmonella is a major cause of Foodborne Diseases worldwide, and in the State of Rio Grande do Sul (RS) this microorganism has been identified as the main agent of foodborne diseases in last years. In this work, Salmonella isolates responsible for salmonellosis occurred in the State of RS, in the period 2007 to 2013 were characterized. Among the 163 isolates investigated, 138 (84.7 %) were serotyped as S. Enteritidis, whereas the other isolates were S. Schwarzengrund (n = 9 – 5.5 %), S. Typhimurium (n = 6 – 3.7 %), S. Infantis (n = 1 – 0.6 %), S. Agona (n = 1 – 0.6 %), S. Derby (n = 1 – 0.6%), S. London (n = 1 – 0.6 %), S. Give (n = 1 – 0.6 %), S. Panama (n = 1 – 0.6 %) and S. enterica (n = 4 – 2.5 %). The main food vehicles identified were homemade mayonnaise (17.39 %), followed by pastry products (15.94 %) and beef (12.32 %). The S. Enteritidis resistance to 12 antimicrobial agents was investigated. The highest percentages of resistance were found to nitrofurantoin (94.2 %) and nalidixic acid (89.1 %). The resistance to two different drugs was observed in 80.43 % of the isolates. Multidrug-resistance for three to five antimicrobials was observed in four and two isolates, respectively. When the isolates were analysed by PCR-Ribotyping, only one banding profile was identified. The results of PCR-Ribotyping suggest that the same strain of S. Enteritidis was isolated from foods involved in salmonelloses occurred in different municipalities of the State of RS in the period 2007-2013. Since the same banding pattern was found in strains involved in salmonellosis outbreaks of 1999 to 2006, results indicated that the same strain of S. Enteritidis has caused salmonellosis outbreaks in RS, during the period of 1999 to 2013.

<p>An introduction to bacterial polysaccharides and the methods for structural determination are described in the first two parts of the thesis.</p><p>In a structural elucidation of bacterial polysaccharides NMR experiments are important as is component analysis. A short description of immunochemical methods such as enzyme immunoassays is included. Two NMR techniques used for interaction studies, trNOE and STD NMR, are also discussed. </p><p>The third part of the thesis discusses and summarizes the results from the included papers. The structures of the exopolysaccharides produced by two lactic acid bacteria are determined by one- and two dimensional NMR experiments. One is a heteropolysaccharide produced by <i>Streptococcus thermophilus</i> and the other a homopolysaccharide produced by <i>Propionibacterium freudenreichii</i>. The structure of an acidic polysaccharide from a marine bacterium with two serine residues in the repeating unit is also investigated. The structural and immunological relationship between two O-antigenic polysaccharides from <i>Escherichia coli</i> strain 180/C3 and O5 is discussed and investigated. Finally, interaction studies of an octasaccharide derived from the <i>Salmonella enteritidis</i> O-antigen and a bacteriophage are described which were performed with NMR experiments.</p>

An introduction to bacterial polysaccharides and the methods for structural determination are described in the first two parts of the thesis. In a structural elucidation of bacterial polysaccharides NMR experiments are important as is component analysis. A short description of immunochemical methods such as enzyme immunoassays is included. Two NMR techniques used for interaction studies, trNOE and STD NMR, are also discussed. The third part of the thesis discusses and summarizes the results from the included papers. The structures of the exopolysaccharides produced by two lactic acid bacteria are determined by one- and two dimensional NMR experiments. One is a heteropolysaccharide produced by Streptococcus thermophilus and the other a homopolysaccharide produced by Propionibacterium freudenreichii. The structure of an acidic polysaccharide from a marine bacterium with two serine residues in the repeating unit is also investigated. The structural and immunological relationship between two O-antigenic polysaccharides from Escherichia coli strain 180/C3 and O5 is discussed and investigated. Finally, interaction studies of an octasaccharide derived from the Salmonella enteritidis O-antigen and a bacteriophage are described which were performed with NMR experiments.

El consum de fruites fresques tallades i sucs de fruites no pasteuritzats s'haincrementat significativament en els darrers anys degut a la creixent demanda deproductes sans de baix contingut calòric i característiques similars a les del productefresc. Tot i això, aquests productes poden contaminar-se amb microorganismespatògens o deterioratius per una inadequada manipulació o emmagatzematge, donantlloc a riscs de malalties microbianes i deteriorament del producte. De fet, el nombrede brots i casos de malalties causades pel consum d'aquests productes ha augmentatnotablement, motiu pel qual l'ús de composts naturals que garanteixin la seguretat iqualitat de les fruites fresques tallades i sucs de fruites no pasteuritzats haaugmentat. L'objectiu principal d'aquesta recerca ha estat avaluar l'efectivitat dediferents antimicrobians per a controlar microorganismes patògens tals com Listeriamonocytogenes, Salmonella Enteritidis i Escherichia coli O157:H7 i allargar la vidaútil de fruites fresques tallades. Inicialment les concentracions mínimes inhibitòries(CMI) i bactericides (CMB) d'àcid màlic i diferents olis essencials (EOs) i els seuscompostos actius van ser determinats en sucs de poma, pera i meló. Desprésaquestes substàncies han estat aplicades en combinació amb compostsestabilitzadors de la qualitat directament per immersió o indirectament a través derecobriments comestibles a base d'alginat a pomes, peres i melons frescs tallats per agarantir la seva seguretat i qualitat. Els sucs de poma, pera i meló van ser inhibitorisper a L. monocytogenes, S. Enteritidis i E. coli O157:H7 a 5ºC, no obstant a 20 i35ºC concentracions mínimes d'àcid màlic de 0,2% per a L. monocytogenes i S.Enteritidis i 0,4% per a E. coli O157:H7 van ser requerides per a inhibir els seuscreixements en suc de meló. En general, concentracions d'àcid màlic de 2% en sucsde poma i pera i de 2,5% en suc de meló van ser requerits per a reduir les poblacionsde L. monocytogenes, S. Enteritidis i E. coli O157:H7 en més de 5 cicles log a 5ºC,mentre a 20 i 35ºC concentracions més baixes van ser suficients per a aconseguiraquest efecte. D'altra banda, els olis essencials de canyella, clan, herba de llimona ipalmarosa així com també els seus compostos actius van mostrar activitatantimicrobiana sobre els microorganismes patogènics però en diferents graus. Aixíl'efectivitat d'aquestes substàncies va dependre del tipus de microorganismes, tipusde suc i concentració de la substància. Una combinació d'àcid màlic al 2,5%, Nacetil-L-cisteina a l'1% glutatione a l'1% i lactat de calci a l'1% aplicada perimmersió va reduir les poblacions de L. monocytogenes i S. Enteritidis inoculades enpomes i peres fresques tallades en més de 5 cicles log el mateix dia de la preparacióde les mostres (t = 0 dies), mentre que la població de E. coli O157:H7 va mostraruna major àcid-resistència, aconseguint aquesta reducció a partir dels 3 i 14 dies enaquestes fruites respectivament. A més a més, aquesta combinació de substàncies vaoriginar una disminució en la tasa de creixement i una prolongació de la fase lag deles poblacions d'aerobis mesòfils, psicròfils i floridures i llevadures, allargant lavida útil des d'un punt de vista microbiològic fins a 23 dies en peces de poma i permés de 30 dies en peces de pera. Així mateix, un menor consum d'O2 i una menorproducció de CO2, etilé i etanol, com també un millor manteniment de la textura i elcolor foren aconseguits amb l'ús d'aquesta combinació de substàncies, aconseguintaixí un allargament de la vida útil fisicoquímica. D'altra banda, la incorporaciód'àcid màlic junt amb composts estabilitzadors de la qualitat en recobrimentcomestibles a base d'alginat va ser efectiva per a reduir les poblacions d'E. coliO157:H7 i S. Enteritidis inoculades en peces de poma i meló així com també per ainhibir el creixement de la flora nativa, tot i això un major efecte sobre aquellsmicroorganismes es va aconseguir quan ambdós àcids màlic i EOs o els seusprincipals composts actius van ser incorporats en els recobriments, mostrant ser l'oliessencial d'herba de llimona la substància antimicrobiana més efectiva. Aquestefecte antimicrobià fou a més a més potenciat amb l'augment de les concentracionsd'EOs. Una extensió de la vida útil microbiològica de peces de poma i meló foutambé aconseguida amb la incorporació d'aquestes substàncies, no obstant algunescaracterístiques fisicoquímiques i sensorials de les fruites com textura, color, olor igust van ser afectades. Els resultats demostraven que l'ús d'antimicrobians naturalsper immersió o a través de la seva incorporació en recobriments comestibles a based'alginat és una bona alternativa per a garantir la seguretat i qualitat de fruitesfresques tallades. / El consumo de frutas frescas cortadas y zumos de frutas no pasteurizadosha incrementado significativamente en los últimos años debido a la crecientedemanda de productos sanos con bajo contenido calórico y características similaresa las del producto fresco. No obstante, estos productos pueden contaminarse conmicroorganismos patógenos o deteriorativos por una inapropiada manipulación oalmacenamiento, dando lugar a riesgos de enfermedades microbianas y deterioro delproducto. De hecho, el número de brotes y casos de enfermedades causadas porconsumo de esos productos ha aumentado notablemente, razón por la cual el uso decompuestos naturales que garanticen la inocuidad y calidad de las frutas frescascortadas y zumos de frutas no pasteurizados ha aumentado. El objetivo principal deesta investigación fue evaluar la efectividad de diferentes antimicrobianos paracontrolar microorganismos patógenos tales como Listeria monocytogenes,Salmonella Enteritidis y Escherichia coli O157:H7 y extender la vida útil de frutasfrescas cortadas. Primeramente se determinaron las concentraciones mínimasinhibitorias (CMI) y bactericidas (CMB) de ácido málico y diferentes aceitesesenciales (EOs) y sus compuestos activos en zumos de manzana, pera y melón.Después, esas sustancias fueron aplicadas en combinación con compuestosestabilizadores de la calidad directamente por inmersión o indirectamente a través derecubrimientos comestibles a base de alginato a manzanas, peras y melones frescoscortados para garantizar su inocuidad y calidad. Los zumos de manzana, pera ymelón fueron inhibitorios para L. monocytogenes, S. Enteritidis y E. coli O157:H7 a5ºC, sin embargo a 20 y 35ºC, se necesitaron concentraciones mínimas de ácidomálico de 0,2% para L. monocytogenes y S. Enteritidis y 0,4% para E. coli O157:H7para inhibir su crecimiento en zumo de melón. En general, concentraciones de ácidomálico de 2% en zumos de manzana y pera y 2,5% en zumo de melón fueronrequeridas para reducir las poblaciones de L. monocytogenes, S. Enteritidis y E. coliO157:H7 en más de 5 ciclos log a 5ºC, mientras a 20 y 35ºC concentraciones másbajas fueron suficientes para alcanzar ese efecto. Por otra parte, los aceitesesenciales de canela, clavo, hierba de limón y palmarosa así como también suscompuestos activos mostraron actividad antimicrobiana sobre los microorganismospatogénicos pero en diferentes grados. Así la efectividad de esas sustancias dependiódel tipo de microorganismo, tipo de zumo y concentración de la sustancia. Unacombinación de ácido málico al 2,5%, N-acetíl-L-cisteína al 1% glutatione al 1% ylactato de calcio al 1% aplicada por inmersión redujo las poblaciones de L.monocytogenes y S. Enteritidis inoculadas en manzanas y peras frescas cortadas enmás de 5 ciclos log el mismo día de la preparación de las muestras (t = 0 días),mientras que la población de E. coli O157:H7 mostró una mayor ácido-resistencia,alcanzando esa reducción a partir de los 3 y 14 días en esas frutas respectivamente.Además esa combinación de sustancias causó una disminución en la tasa decrecimiento y una prolongación de la fase lag de las poblaciones de aerobiosmesófilos, psicrófilos y mohos y levaduras, extendiendo la vida útil desde un puntode vista microbiológico hasta 23 días en piezas de manzanas y por más de 30 días enpiezas de peras. Así mismo, un menor consumo de O2 y una menor producción deCO2, etileno y etanol, así como también un mejor mantenimiento de la textura y elcolor fueron logradas con el uso de esa combinación de sustancias, alcanzándose asíuna extensión de la vida útil fisicoquímica. Por otra parte la incorporación de ácidomálico junto con compuestos estabilizadores de la calidad en recubrimientoscomestibles a base de alginato fue efectiva para reducir las poblaciones de E. coliO157:H7 y S. Enteritidis inoculadas en piezas de manzana y melón así comotambién para inhibir el crecimiento de la flora nativa, sin embargo un mayor efectosobre esos microorganismos fue logrado cuando ambos ácido málico y EOs o susprincipales compuestos activos fueron incorporados en los recubrimientos,mostrando ser el aceite esencial de hierba de limón la sustancia antimicrobiana másefectiva. Este efecto antimicrobiano fue además potenciado con el aumento de lasconcentraciones de EOs. Una extensión de la vida útil microbiológica de piezas demanzana y melón también fue alcanzada con la incorporación de estas sustancias,sin embargo algunas características fisicoquímicas y sensoriales de las frutas comotextura, color, olor y sabor fueron afectadas. Los resultados demostraron que el usode antimicrobianos naturales por inmersión o a través de su incorporación enrecubrimientos comestibles a base de alginato es una buena alternativa paragarantizar la inocuidad y calidad de frutas frescas cortadas. / The consumption of fresh-cut fruits and unpasteurized juices hassubstantially risen over the last years mostly due to the increasing demand of healthyfood with low caloric contents and fresh-like characteristics. Nonetheless, thoseproducts can contaminate with pathogenic and deteriorative microorganisms as aconsequence of inappropriate manipulation and storage conditions, resulting in risksof microbial diseases and spoilage of the product. In fact, the number of outbreaksand cases of illness caused by consumption of fresh-cut fruits and unpasteurizedjuices has notably increased reason by which, the use of natural compounds thatassure the safety and quality of fresh-cut fruits and unpasteurized fruit juices hasincreased. To evaluate the effectiveness of different antimicrobials to controlpathogenic microorganisms such as Listeria monocytogenes, Salmonella Enteritidisand Escherichia coli O157:H7 and extend the shelf-life of fresh-cut fruits was themain objective of this research. Minimal inhibitory (MIC) and bactericidalconcentrations (MBC) of malic acid and essential oils (EOs) and their activecompounds against those pathogens were firstly determined in apple, pear andmelon juices. Then, those substances were applied in combination with qualitystabilizing compounds directly by dipping treatments or indirectly through thealginate-based edible coatings to fresh-cut apple, pear and melon to assure theirsafety and quality. Apple, pear and melon juices were inhibitory for L.monocytogenes, S. Enteritidis and E. coli O157:H7 at 5ºC, however at 20 and 35ºCminimal concentrations of 0.2% of malic acid for L. monocytogenes and S.Enteritidis and 0.4% for E. coli O157:H7 were required to inhibit their growth inmelon juice. In general, concentrations of malic acid of 2% in apple and pear juicesand 2.5% in melon juice were necessary to reduce more than 5 log cycles of L.monocytogenes, S. Enteritidis and E. coli O157:H7 at 5ºC, whereas at 20 and 35ºClower concentrations were enough to reach that effect. On the other hand, EOs ofcinnamon, clove, lemongrass and palmarosa as well as their active compoundsshowed antimicrobial activity over pathogenic microorganisms but in differentgrade. Thus, the effectiveness of those substances depended on the microorganismtype, kind of juice and concentration of the substance, thus resulting, the essential oilof lemongrass the substance more effective to inactivate those microorganisms. Acombination of 2.5% D-L malic acid, 1% N-acetyl-L-cysteine, 1% glutathione and1% calcium lactate applied through dipping treatment reduced the populations of L.monocytogenes and S. Enteritidis inoculated in fresh-cut apples and pears in morethan 5 log cycles the same day of sample preparation (t = 0 days), whereas thepopulation of E. coli O157:H7 showed a higher acid-resistance, reaching thatreduction from 3 and 14 days in those fruits respectively. In addition, thatcombination provoked a decrease of the growth rate and prolonged the lag phase ofmesophilic aerobic, psychrophilic and yeast and molds populations, extending theshelf-life from a point of view microbiological until 23 days in apple pieces and bymore than 30 days in fresh-cut pears. Likewise, a lower consumption of O2 and alower production of CO2, ethylene and ethanol, as well as a better maintenance ofthe firmness and color were achieved with the use of that substances combination,thus reaching a physicochemical shelf-life extension. On the other hand, theincorporation of malic acid with quality stabilizing compounds into alginate-basededible coatings was effective to reduce the populations of E.coli O157:H7 and S.Enteritidis inoculated in apple and melon pieces as well as to inhibit the native floragrowth. However, a higher effect over those microorganisms was achieved whenboth malic acid and EOs or their main active compounds were added into coatings,showing the essential oil of lemongrass to be the most effective antimicrobialsubstance. That antimicrobial effect was also intensified with the increase of theEOs concentrations. An extension of the microbiological shelf-life of apple andmelon pieces was also reached with the incorporation of those substances.Nonetheless, some physicochemical and sensory characteristics of the fruits such asfirmness, color, flavour and taste were affected. The results demonstrated that theuse of natural antimicrobials by immersion or through their incorporation intoalginate-based edible coatings is a good alternative to assure the safety and qualityof the fresh-cut fruits.

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