Interactions between bacteriocin-producing lactic acid bacteria and the autochthonous microbiota from charqui

O charque é um produto cárneo tipicamente brasileiro, salgado e seco ao sol, ainda produzido de maneira artesanal. Durante sua produção há uma etapa de fermentação, realizada pela microbiota naturalmente presente na matéria-prima, o que dificulta a padronização do produto, e pode influenciar negativamente em suas características sensoriais e qualidade microbiológica. O controle da etapa de fermentação do charque seria uma alternativa para minimizar este problema e, neste contexto, as bactérias láticas produtoras de bacteriocinas se enquadram de forma interessante. A microbiota autóctone de charque inclui principalmente bactérias láticas e micro-organismos halofílicos e halotolerantes, sendo assim, este produto apresenta potencial como fonte para o isolamento de novas bactérias láticas produtoras de bacteriocinas. Assim, este trabalho teve por objetivo isolar e identificar culturas de bactérias láticas produtoras de bacteriocinas naturalmente presentes no charque, caracterizar parcialmente as bacteriocinas produzidas por essas culturas, avaliar seu potencial de aplicação neste produto para a melhoria de sua qualidade microbiológica e avaliar seu efeito na ecologia microbiana do charque, nas diferentes etapas de sua fabricação. Através da técnica de tripla camada em ágar foi isolada uma cepa de Lactococcus lactis subsp. lactis apresentando o gene codificador para nisina Z e com capacidade de inibir, in vitro, micro-organismos medianamente e altamente halotolerantes isolados de charque, além de outros micro-organismos deteriorantes e patogênicos importantes em alimentos, como Lactobacillus spp., Listeria monocytogenes e Staphylococcus aureus. A bacteriocina produzida pela cepa isolada neste estudo também possui características interessantes para sua aplicação na bioconservação de alimentos, como resistencia ao calor, presença de agente químicos e altos teores de NaCl, além de não ser afetada pelo pH. A aplicação dessa cepa em charque modelo resultou na redução de até 2 ciclos log na população de micro-organismos halotolerantes, indicando apresentar um potencial de aplicação como agente de bioconservação do produto. Os ensaios de avaliação da ecologia microbiana, empregando DGGE, indicaram que a fermentação natural do charque ocorreu com a participação de bactérias láticas dos gêneros Lactobacillus, Streptococcus, Lactococcus e de micro-organismos halotolerantes do gênero Staphylococcus. Além disso, os estudos referentes à dinâmica populacional demonstraram que a adição da cepa bacteriocinogênica ao charque não influenciou, de forma qualitativa, as populações presentes no produto.Charqui is a Brazilian traditional meat product, salted and sun-dried, still manufactured without control of the fermentation step, which is performed by the indigenous microbiota. This fact interferes on the standardization of the product and can negatively affect the sensorial properties and microbiological quality. The application of a known microbiota would be an alternative to minimize this problem and the bacteriocin-producing lactic acid bacteria can can fit in this purpose. The charqui indigenous microbiota mainly includes lactic acid bactéria and halophilic and halotolerant microorganisms, therefore, this product presents a potencial as a source for the isolation of new bacteriocin-producing lactic acid bacteria. The aim of the present work was to isolate and identify bacteriocin-producing lactic acid bacteria from charqui, characterize the bacteriocins produced by the isolated culture, evaluate its potential as biopreservative in charqui and its influence on the microbial populations during the manufacture of the product. A bacteriocinogenic Lactococcus lactis subsp. lactis strain was isolated from charqui through the triple-layer agar technique. This strain produces a nisin-like bacteriocin capable to inhibit in vitro medium and highly halotolerant bacteria isolated from charqui and other food-borne pathogenic and spoilage microorganisms. The application of this strain for charqui manufacturing caused a reduction of up to 2 log in the halotolerant bacteria population, evidencing its potential application for charqui biopreservation. Studies in the populational dynamics using DGGE indicated that the presence of the bacteriocinogenic strain did not affect the microbial populations in the product

Influence of the food matrix on the antimicrobial effect of oregano essential oil and nisin against Listeria monocytogenes: evaluation in meat models

Listeria monocytogenes é um microrganismo patogênico psicrotrófico e consequentemente de grande importância para alimentos conservados em refrigeração. Vários estudos têm demonstrado que é possível utilizar antimicrobianos naturais, como bacteriocinas e compostos fenólicos, para retardar a multiplicação desse patógeno em produtos cárneos refrigerados. Entretanto, sabe-se que a atividade antimicrobiana desses compostos pode ser influenciada pelos componentes da matriz alimentar. Esse trabalho de mestrado pretendeu avaliar a influência dos ingredientes de lingüiça frescal de suíno na ação inibitória de óleo essencial de orégano (OEO) e nisina sobre Listeria monocytogenes. Foram realizados testes com diferentes modelos alimentares preparados com carne suína moída adicionada de cada um dos ingredientes utilizados na fabricação de lingüiça frescal (sais de cura, açúcar, antioxidante, emulsificante, condimentos e NaCl) separadamente, comparando-se os resultados com os obtidos no modelo contendo todos os ingredientes simultaneamente. Pretendeu-se também avaliar a interferência do tempo de contato da nisina e óleo essencial de orégano com os diferentes modelos alimentares na ação antimicrobiana sobre L. monocytogenes. Os resultados indicaram que os antimicrobianos foram capazes de inibir a multiplicação de L. monocytogenes em todos os modelos analisados armazenados à 5ºC por até 72h. Comparando-se todos os modelos compostos por carne adicionada de apenas um dos ingredientes, a maior atividade antilisterial foi observada no modelo composto de carne com antioxidante. Entretanto, essa atividade inibitória foi menor do que a obtida no modelo contendo todos os ingredientes em conjunto. Quanto à influência do tempo prévio de contato entre os antimicrobianos e os modelos estudados na ação antilisterial, observou-se que na formulação com todos os ingredientes a ação foi mais intensa quando o contato foi de 2h. Entretanto, neste modelo o efeito mais duradouro foi obtido com 12h de contato prévio. Nos demais modelos, o maior efeito foi obtido no tempo zero. Os resultados permitem concluir que cada um dos ingredientes da formulação da lingüiça contribui com uma parcela na ação antilisterial do OEO e nisina nesse produto, e que essa ação é influenciada pelo tempo de contato prévio entre os modelos e os agentes antimicrobianos.Listeria monocytogenes is psychrotrophic pathogen and, consequently, very important for cold-preserved foods. Many studies have demonstrated that natural antimicrobials such as bacteriocins and phenolic compounds can be used to inhibit the growth of this pathogen in refrigerated meat products. However, it is known that the antimicrobial activity of these antimicrobials may be influenced by components of the food matrix. The aim of the present study was to evaluate the influence of the ingredients of \"lingüiça\" in the inhibitory activity of O.E.O. and nisin against L. monocytogenes. The study was carried out with different food models prepared with minced raw pork meat added separately of each ingredient used for manufacturing \"lingüiça\" (curing salts, sugar, antioxidants, emulsifiers, condiments and NaCl), and results were compared to those obtained in a model containing all ingredients simultaneously. The study also aimed to evaluate the interference of the contact time between the food models and the antimicrobials on the effect against L. monocytogenes. Results indicated that the antimicrobials added to the models were able to inhibit the growth of the pathogen at 5ºC up to 72h. Comparing the models, the highest antilisterial activity was observed in the model containing pork meat and antioxidants. However, this inhibitory activity was lower than that observed in the model with all ingredients. What concerns the influence of contact time in antilisterial activity, we observed that in the model containing all ingredients the antilisterial activity was more intense after 2h of contact, but the more lasting effect was observed after 12h of contact. In the other models, the most intense effect was noted in time zero. The conclusions of the study are that each ingredient used in manufacture of \"lingüiça\" has its share in the antilisterial activity of nisin and OEO in this product and that this activity is influenced by the contact time between the food matrix and the antimicrobials

Interactions between bacteriocin-producing lactic acid bacteria and the autochthonous microbiota from charqui

O charque é um produto cárneo tipicamente brasileiro, salgado e seco ao sol, ainda produzido de maneira artesanal. Durante sua produção há uma etapa de fermentação, realizada pela microbiota naturalmente presente na matéria-prima, o que dificulta a padronização do produto, e pode influenciar negativamente em suas características sensoriais e qualidade microbiológica. O controle da etapa de fermentação do charque seria uma alternativa para minimizar este problema e, neste contexto, as bactérias láticas produtoras de bacteriocinas se enquadram de forma interessante. A microbiota autóctone de charque inclui principalmente bactérias láticas e micro-organismos halofílicos e halotolerantes, sendo assim, este produto apresenta potencial como fonte para o isolamento de novas bactérias láticas produtoras de bacteriocinas. Assim, este trabalho teve por objetivo isolar e identificar culturas de bactérias láticas produtoras de bacteriocinas naturalmente presentes no charque, caracterizar parcialmente as bacteriocinas produzidas por essas culturas, avaliar seu potencial de aplicação neste produto para a melhoria de sua qualidade microbiológica e avaliar seu efeito na ecologia microbiana do charque, nas diferentes etapas de sua fabricação. Através da técnica de tripla camada em ágar foi isolada uma cepa de Lactococcus lactis subsp. lactis apresentando o gene codificador para nisina Z e com capacidade de inibir, in vitro, micro-organismos medianamente e altamente halotolerantes isolados de charque, além de outros micro-organismos deteriorantes e patogênicos importantes em alimentos, como Lactobacillus spp., Listeria monocytogenes e Staphylococcus aureus. A bacteriocina produzida pela cepa isolada neste estudo também possui características interessantes para sua aplicação na bioconservação de alimentos, como resistencia ao calor, presença de agente químicos e altos teores de NaCl, além de não ser afetada pelo pH. A aplicação dessa cepa em charque modelo resultou na redução de até 2 ciclos log na população de micro-organismos halotolerantes, indicando apresentar um potencial de aplicação como agente de bioconservação do produto. Os ensaios de avaliação da ecologia microbiana, empregando DGGE, indicaram que a fermentação natural do charque ocorreu com a participação de bactérias láticas dos gêneros Lactobacillus, Streptococcus, Lactococcus e de micro-organismos halotolerantes do gênero Staphylococcus. Além disso, os estudos referentes à dinâmica populacional demonstraram que a adição da cepa bacteriocinogênica ao charque não influenciou, de forma qualitativa, as populações presentes no produto.Charqui is a Brazilian traditional meat product, salted and sun-dried, still manufactured without control of the fermentation step, which is performed by the indigenous microbiota. This fact interferes on the standardization of the product and can negatively affect the sensorial properties and microbiological quality. The application of a known microbiota would be an alternative to minimize this problem and the bacteriocin-producing lactic acid bacteria can can fit in this purpose. The charqui indigenous microbiota mainly includes lactic acid bactéria and halophilic and halotolerant microorganisms, therefore, this product presents a potencial as a source for the isolation of new bacteriocin-producing lactic acid bacteria. The aim of the present work was to isolate and identify bacteriocin-producing lactic acid bacteria from charqui, characterize the bacteriocins produced by the isolated culture, evaluate its potential as biopreservative in charqui and its influence on the microbial populations during the manufacture of the product. A bacteriocinogenic Lactococcus lactis subsp. lactis strain was isolated from charqui through the triple-layer agar technique. This strain produces a nisin-like bacteriocin capable to inhibit in vitro medium and highly halotolerant bacteria isolated from charqui and other food-borne pathogenic and spoilage microorganisms. The application of this strain for charqui manufacturing caused a reduction of up to 2 log in the halotolerant bacteria population, evidencing its potential application for charqui biopreservation. Studies in the populational dynamics using DGGE indicated that the presence of the bacteriocinogenic strain did not affect the microbial populations in the product

Application of Lactic Acid Bacteria in Time-Temperature Integrators

International audienceBiological Time-Temperature Integrators (TTI) offer a novel approach for improving food safety and preventing spoilage. These smart tools relay, by an irreversible color shift, the cumulative effect of time and temperature on the microbial quality of food they are attached to. Among various types of TTI developed to date, biological TTI have the advantage of reproducing microbiological spoilage reactions that take place in food. They are based on the pH decline of a medium contained within the label, resulting from lactic acid bacteria (LAB) growth and acidification. When developing LAB-based TTI, careful LAB strain selection, research, and development efforts on TTI production are necessary to closely match the behavior of both spoilage and pathogenic microorganisms that grow during the storage of perishable foods. Covering a wide range of time-temperature profiles is a challenging goal involving research in different domains (microbiology, food science, modelling, etc.). This chapter describes the design and working principle of LAB-based TTI, how they are parametrized to be able to track a wide range shelf-lives and how their performance is evaluated. Current applications and future prospects of this innovative way of using lactic acid bacteria are also discussed

Bacteriocin producing Enterococcus faecalis isolated from chicken gastrointestinal tract originating from Phitsanulok, Thailand: Isolation, screening, safety evaluation and probiotic properties

A total of 230 isolates of lactic acid bacteria were isolated from gastrointestinal tracts (GIT) of indigenous chickens purchased from local market in Bang Rakam district, Phitsanulok, Thailand. Among 230 isolates, only 7 isolates, named CM6CR07, CM6CR11, CF1GI15, CF1GI14, CF1GI17-1, CF1GI17-2 and CF1GI 19 showed an inhibition zone against indicator strains. Bacteriocin activity was completely inactivated when Neutralized Cell Free Supernatants (NCFS) of the isolates were treated with proteases, confirming the proteinaceous nature of the bacteriocin component. NCFS of all selected bacteriocin producing lactic acid bacteria (SBP-LAB) retained their bacteriocin activity after heating at 100 C-omicron for 30 min. All of SBP-Lwere identified as Enterococcus faecalis. These Ent. faecalis isolates harbored the same virulence genes. All strains were positive for gelE (gelatinase), efaA(fs) (cell wall adhesins), hyl (hyaluronidase), ace (adhesin of collagen protein), cylL(L) (cytolysin structural subunit) and cylL(s) (cytolysin structural subunit) and were negative for asal (aggregation substance), agg (aggregation protein involved in adherence to eukaryotic cells), and esp (enterococcal surface protein). These Ent. faecalis showed a wide range of inhibitory spectrum against food-spoilage and food-borne pathogens when studied by agar spot test. The CFS and NCFS also inhibited some of food-spoilage and food-borne pathogenic indicator strains when checked by agar well diffusion assay. All of these Ent. faecalis survived simulated stomach conditions, whereas all of them could survive in conditions of simulated intestinal juice

Effect of indigenous lactic acid bacteria isolated from goat milk and cheeses on folate and riboflavin content of fermented goat milk

The aim of the present study was to isolate riboflavin- and folate-producing lactic acid bacteria (LAB) from raw goat milk and cheeses, identify them and evaluate their capability to increase the content of these vitamins in fermented goat milk, envisaging potential application for development of novel bioenriched goat milk products. From 179 LAB isolates obtained, 151 (84%) were capable to produce at least one of these vitamins. The average production of total folate and riboflavin in vitamin-free media was 138 ng/ml and 364 ng/ml, respectively. Based on RAPD-PCR and 16S rDNA sequencing, 19 different genetic profiles were obtained and 7 species were identified, with predominance of Streptococcus thermophilus (7), Weissella paramensenteroides (6), and Lactococcus lactis (4). Seven isolates that produced folate and riboflavin above the average were tested for vitamins production in UHT goat milk. Five isolates were capable to increase four to six fold the original amount of folate in the milk in 24 h. Folate content in milk fermented with Lc. lactis FP368 for 24 h was 313 ng/ml that could provide 19% of the recommended daily intake of this vitamin. In addition, St. thermophilus FP268 increased the folate concentration in the milk almost four fold in only 6 h.Fil: Pacheco da Silva, Fabiana Fernanda. Universidade de Sao Paulo; BrasilFil: Biscola, Vanessa. Universidade de Sao Paulo; BrasilFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Gombossy de Melo Franco, Bernadette Dora. Universidade de Sao Paulo; Brasi

Bacteriocin-producing <em>Enterococcus faecalis</em> KT2W2G isolated from mangrove forests in southern Thailand: Purification, characterization and safety evaluation

International audienceThe aim of this work was to purify and characterize the bacteriocin produced by Enterococcus faecalis KT2W2G isolated from the mangrove forest in southern Thailand, in order to evaluate its potential as a new food protective agent. The active peptide from the cell-free supernatant of Ent. faecalis KT2W2G was purified in 4 steps: (i) precipitation with 70% saturated ammonium sulfate, (ii) elution on a reversed phase cartridge (Sep-Pak C8) using different concentrations of acetonitrile, (iii) cation-exchange chromatography and (iv) final purification by reversed phase-HPLC on a C8 column. Each purification step increased the specific activity and reduced the amount of contaminating non-bacteriocin proteins. The specific activity of purified bacteriocin was 13,470.53 AU/mg of protein, which corresponded to a 48.10- fold increase. TricineeSDS-PAGE of the purified bacteriocin gave molecular weight ranging between 3.5 and 6.5 kDa. The activity of the partially purified bacteriocin was unaffected by pH (2.0e12.0) and thermostable, but was sensitive to proteolytic enzymes. This bacteriocin maintained full stability after storage at 20, 4 and 37 C for 2 months. It was stable when incubated for 1 month at 4 C in 0e30% NaCl. Inhibitory spectrum of this bacteriocin showed a wide range of activities against other LAB, foodspoilage and food-borne pathogens. Ent. faecalis KT2W2G was sensitive to kanamycin, tetracycline and vancomycin but resistant to ampicillin, gentamicin and penicillin. PCR amplification demonstrated that Ent. faecalis KT2W2G does not harbor virulence genes cylA, cylB and esp but has virulence genes ace, asa1and efaAfs. The bacteriocin and its producing strain may find application as bio-preservatives for reduction of food-spoilage and food-borne pathogens in food products

Evaluation of the proteolytic activity of <em>Enterococcus faecalis</em> FT132 and <em>Lactobacillus paracasei</em> FT700, isolated from dairy products in Brazil, using milk proteins as substrates

International audienceLactic acid bacteria (LAB) have been used by mankind from immemorial times due to their technological properties and ability to improve sensorial properties of foods. Some Lstrains are also able to hydrolyze milk proteins, which increase their digestibility and contribute to the production of desirable flavors. Moreover, proteolytic activity of Lon milk proteins may release bioactive peptides with different activities such as antihypertensive, antioxidant, antimicrobial, immunomodulatory and mineral-binding activities. This study aimed to evaluate the proteolytic activity of Enterococcus faecalis FT132 and Lactobacillus paracasei FT700, isolated from Brazilian dairy products. The proteolytic activity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography using milk proteins as substrates in different conditions. Both E. faecalis FT132 and L. paracasei FT700 were proteolytic at pH 6.5 in the range of 37-42 A degrees C, and these activities were due to metalloproteases. L. paracasei FT700 could then be used as adjunct culture in fermented dairy products in order to increase their digestibility. On the contrary, E. faecalis FT132, which harbors three virulence genes asa1, ace and gelE and which is resistant to erythromycin and tetracycline, cannot be added to food products. However, the peptides produced after hydrolytic activity on milk proteins by both strains might be used as ingredients in purified formulas