Isolation, Identification and Mode of action of Partially Purified Bacteriocins from Lactic Acid Bacteria in Fermented Cassava Grits

The study on the isolation, identification and mode of action of partially purified bacteriocin from lactic acid bacteria found in fermented cassava grits was carried out. Fermented cassava grits were collected from different garri processing plants and transported with cold box to the laboratory for analysis. The viable microbial count after the partially purified bacteriocin from the various lactic acid bacteria isolates were grown against the food borne bacteria (Staphylococcus aureus and Bacillus subtilis) ranged from 0.98 x 103 CFU/ml for partially purified bacteriocin from isolate 6 at 8 hrs to 9.2 x103 CFU/ml for isolate 3 at 24 hrs. Similar results were obtained against Bacillus subtilis with microbial counts that ranged from 1.02 x102 CFU/ml for isolate 3 at 8 hrs to 9.2 x 102 CFU/ml at 24 hrs. Isolates 6, 7, 10 and 11 were bactericidal to both Staphylococcus aureus and Bacillus subtilis while isolate 3 was bacteriostatic. The viable microbial count after the partially purified bacteriocin was grown against the foodborne bacteria (Escherichia coli and Salmonella typhi) ranged from 1.0 x102 CFU/ml for partially purified bacteriocin from isolate 3 at 8 hrs to 7.1 x 102 CFU/ml for partially purified bacteriocin from isolate 6 at 24 hours. The microbial count against Salmonella typhi ranged from 6.50 x 102 CFU/ml for isolate 6 at 8 hrs to 8.5 x 102 CFU/ml for isolate 7 at 24 hrs. Partially purified bacteriocins from isolates 3 and 7 were bacteriostatic while isolates 6, 10 and 11 were bactericidal to Escherichia coli and Salmonella typhi. This result showed that the partially purified bacteriocins were very efficacious in killing or inhibiting the growth of some foodborne pathogens which can be applied in biopreservation.

Control del deterioro microbiológico de filetes de tilapia mediante la aplicación de bacterias lácticas / Control Microbiological Deterioration of Tilapia Fillets by the Application of Lactic Acid Bacteria / Controle de Deterioração Microbiológica da Tilápia Arquiva Através da Aplicação de Bactérias Láticas

Resumen En los últimos años se ha reconocido el potencial de las bacterias ácido lácticas (BAL) como agentes bioconservantes de productos cárnicos, incluido el pescado, al reducir el deterioro microbiológico y químico. El objetivo de este trabajo fue determinar el efecto bioconservante de dos cepas de BAL y el tiempo de impregnación en la calidad microbiológica y química de filetes de tilapia. Se evaluaron dos tiempos de impregnación (1h y 2h) y las BAL Lactobacillus plantarum JCM 1149 y Lactobacillus acidophilus ATCC4356. Los filetes bioconservados (FB) se almacenaron a 5°C y se evaluaron los cambios en el recuento de BAL, mesófilos, psicrófilos y coliformes totales durante 30 días. Los recuentos en los FB fueron de 5.94 de Log UFC/g. de BAL, y <2.7 log UFC/g de coliformes y psicrófilos. Los filetes control presentaron recuentos de 1.2 log UFC/g de BAL mientras los coliformes totales y psicrófilos superaron el límite permitido para consumo humano luego de 10 días. Luego de 10 días. El tratamiento que mantuvo la calidad microbiológica y química de los FB por más tiempo fue L. plantarum con inmersión de una hora. Las BAL estudiadas mostraron un efecto inhibitorio de la microflora deteriorante del pescado y una reducción en la formación de nitrógeno volátil, siendo un método viable para la conservación del pescado.

Abstract In recent years, it has recognized the potential of lactic acid bacteria (LAB) as biopreservatives agents for meat products, including fish, by reducing the microbiological and chemical spoilage. The aim of this study was to determine the effect of two strains biopreservative LAB and impregnation time on the microbiological and chemical quality of tilapia fillets. Two impregnation time (1h and 2h) and two LAB (Lactobacillus plantarum JCM 1149 and Lactobacillus acidophilus ATCC4356 were evaluated. The biopreserved fillets (BF) were stored at 5 ° C and the changes of the LAB count, mesophilic, psychrophilic and total coliforms., were evaluated for 30 days. The microbiological analysis show for the BF 5.94 log CFU / g. of LAB and <2.7 log CFU / g of coliform and psychrophilic bacteria. The control Fillets showed 1.2 log CFU / g of LAB while the psychrophile and total coliforms exceeded the allowed limit for human consumption after 10 days. The treatment kept the microbiological and chemical quality of the BF longer was L. plantarum with one hour of impregnation. The LAB studied showed an inhibitory effect of the spoilage microflora of fish and a reduction in the formation of volatile nitrogen compounds, being a viable fish preservation method.

Resumo Nos últimos anos tem se reconhecido o potencial das bactérias ácido lácticas (BAL) como agentes bioconservantes de pro dutos carnicos, incluído o peixe ao diminuir o deterioro microbiológico e químico. O propósito deste estudo foi determinar o efeito bioconservante de duas cepas de BAL e a avaliação do tempo de impregnação requerido para obter uma boa qua lidade microbiológica e química de filetes de tilapia. Foram avaliados dois tempos de impregnação (1h e 2h) com as BAL Lactobacillus plantarum JMC 1149 e Lactobacillus acidophilus ATCC4356. Os filetes biopreservados (FB) foram estocados a 5oC e valoradas as modificações na contagem de microorganismos BAL, mesófilos, psicrófilos, coliformes totais durante 30 dias. As contagens microbianas nos FB foram de 5.94 de log UFC/g e <2.7 log UFC/g de coliformes e psicrofilos. Os filetes controle apresentaram valores de 1.2 log UFC/g de BAL, não entanto os coliformes totais e psicrófilos superaram o limite permitido para consumo humano após 10 dias de estocagem. O tratamento que manteve a qualidade microbiológica e química dos FB por maior tempo foi de imersão com Lactobacillus plantarum por uma hora. As BAL estudadas amostraram um efeito inibitório da microflora deteriorante dos peixes e uma redução na formação de nitrogênio volátil se apresentan do como um método viável para a preservação de filete de peixe

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain (Lc. lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.

Bacteriocinogenic Lactococcus lactis subsp: lactis DF04Mi isolated from goat milk: Evaluation of the probiotic potential

Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its probiotic potential. Lc. lactis DF4Mi was resistant to acidic pH and oxbile, presented co-aggregation with Listeria monocytogenes, and was not affected by several drugs from different generic groups, being sensitive to most tested antibiotics. These properties indicate that this Lc. lactis strain can be used for enhancement of dairy foods safety and quality, in combination with potential probiotic properties.

Technological Properties of Lactococcus lactis subsp. lactis I23 Isolated from Nigerian Beef and Characterization of Bacteriocin Produced by It.

Aim: The present study reports the phenotypic and molecular characterization of Lactococcus strains isolated from Nigerian beef. The technological properties of selected strains were evaluated in order to test their suitability as candidates of starter cultures for food preservation. Study Design: Experimental microbiological testing. Place and Duration of Study: Microbiology Department, University of Ibadan, Nigeria and Division of Food Sciences, The University of Nottingham, UK; between January 2005 and February 2009. Methodology: Phenotypic method was adopted in the initial characterization of the presumptive Lactococcus strains which were later characterized using pulsed field gel electrophoresis. The use of 16S rDNA sequencing was adopted in the full identification of selected strains. High performance liquid chromatography and Gas chromatography were employed in determining production of organic acids and diacetyl respectively by the strains of Lactococcus. Testing and identification of bacteriocin production by the strains were observed using in vitro and polymerase chain reaction methods. The antimicrobial activities of the strains were evaluated against known spoilage and pathogenic organisms of food origin. Results: Three strains, Lc. lactis L11, Lc. lactis subsp. lactis I23 and Lc. lactis subsp. hordinae E91 showed better production of diacetyl and lactic acid than others; concentrations in excess of 31.15 μg/10-7cfu and 17.56 g/107cfu at 18 and 24 h of incubation were recorded for the respective compounds. Result of their antimicrobial activities indicates that varying levels of inhibition were recorded against the spoilage/pathogenic organisms; however no inhibition was recorded against Brochothrix thermosphacta except by Lc. lactis subsp lactis I23 which was shown to encode gene for production of bacteriocin, nisin. The nucleotide sequence of the bacteriocin was deposited at the genBank under accession number EU667387. Conclusion: The ability of the three Lactococcus strains to combat spoilage and pathogenic organisms could help promote safety in food processing and preservation in Nigeria; moreover, production of bacteriocin by Lc. lactis subsp. lactis I23 may confer added advantage during choice of candidacy for starter cultures in food biopreservation.

Antibacterial activity of bacteriocin-like substance P34 on Listeria monocytogenes in chicken sausage

The antimicrobial activity of the bacteriocin-like substance (BLS) P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g-1) previously inoculated with a suspension of 10² cfu g-1 of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe) against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products.

Bio-preservation of ground beef meat by Enterococcus faecalis CECT7121

Meat and particularly ground beef is frequently associated with Food Poisoning episodes and breeches in Food Safety. The main goal of this research was to evaluate the bactericide effect of the probiotic Enterococcus faecalis CECT7121, against different pathogens as: Escherichia coli O157:H7, Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes, inoculated in ground beef meat. Three studies were performed to evaluate the inhibition of E. faecalis CECT7121 on ground beef meat samples inoculated with pathogens: Study I: Samples (100 g meat) were inoculated with pathogens (10³ CFU/g)) and E. faecalis CECT7121 (10(4) CFU/g) simultaneously. Study II: Samples were inoculated with E. faecalis CECT7121 24 h before the pathogens. Study III: E. faecalis CECT7121were inoculated 24 h after pathogens. The viable counts were performed at 0, 24, 48 and 72 h post-inoculation. The simultaneous inoculation of E. faecalis CECT7121 with E. coli O157:H7 strains resulted in the absence of viable counts of bacteria at 72 h post-treatment. However, when the probiotic was added 24 h before and 24 h after the pathogen E. coli O157:H7, viable cells were not detected at 24 h and 48 h post-treatment, respectively. Consistently, neither S. aureus nor Cl. perfringens viable bacteria were detected at 48 h in whole assays when inoculated with E. faecalis CECT7121. The same trend than described before was obtained after applying the 3 models assayed for L. monocytogenes. The current assays demonstrated the bactericide activity of E. faecalis CECT7121 strain on bacterial pathogens in ground beef meat.

Interações entre bactérias láticas produtoras de bacteriocinas e a microbiota autóctone de charque / 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

Antimicrobial activity of Enterococcus faecium FAIR-E 198 against gram-positive pathogens

This study investigated the antimicrobial activity of Enterococcus faecium FAIR-E 198 against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. Using the critical-dilution method, the bacteriocin produced by E. faecium FAIR-E 198 inhibited all L. monocytogenes strains evaluated (1,600 to 19,200 AU mL-1). However, none of the B. cereus and S. aureus strains investigated were inhibited. The maximum activity of this bacteriocin (800 AU mL-1) was observed in MRS broth, while the activity in milk was 100 AU mL-1. In the co-cultivation test in milk, B. cereus K1-B041 was reduced to below the detection limit (1.00 log CFU mL-1) after 48 h. E. faecium reduced the initial L. monocytogenes Scott A population by 1 log CFU mL-1 after 3 h at 35ºC. However, the pathogen regained growth, reaching 3.68 log CFU mL-1 after 48 h. E. faecium did not influence the growth of S. aureus ATCC 27154 during the 48 h of co-cultivation. Therefore, it can be concluded that the effectiveness of the antimicrobial activity of E. faecium FAIR-E 198 is strictly related to the species and strain of the target microorganism and to the culture medium.

Evaluación de bacteriocinas como medio protector para la biopreservación de la carne bajo refrigeración / Evaluation of bacteriocines as protective means for the biopreservation of refrigerated meat

The objective was to evaluate shelf-life extension of fresh beef meat applying crude extract of bacteriocines produced by a native strain of Lactobacillus plantarum LPBM10. Fillets of rib (longissimus dorsi) were stocked during 12 days at 3° C and analyzed by means of microbiology analysis, pH, texture, weight loss and sensorial. Significant differences were found among treatments for psychrotrophics and fecal coliforms, being better the treatment with crude bacteriocin extract that had germicide effect. Fecal coliforms were inhibited by the crude bacteriocin extract during most of the period. The mesophilic count didn’t present difference among treatments. The pH changes and the weight loss present statistical variations among treatments and between days. Cutting force diminished during storage time in all treatments improving meat tenderness without presenting significant difference. The appearance, color and aroma diminished the acceptance value as the storage time advanced.

El objetivo del presente trabajo fue evaluar la extensión de la vida útil de carne refrigerada mediante la aplicación de extracto crudo de bacteriocinas producido por Lactobacillus plantarum LPBM10. Se utilizaron filetes de solomo redondo (longissimus dorsi) almacenados durante 12 días a 3º C y analizados por medio de análisis microbiológicos, pH, textura, pérdidas de peso y sensoriales. Se encontraron diferencias significativas entre los tratamientos para psicrotrófilos y coliformes totales, resultando mejor el tratamiento con extracto crudo de bacteriocinas que tuvo efecto bactericida. Los coliformes fecales también fueron inhibidos por el extracto crudo de bacteriocinas. Los cambios de pH y pérdida de peso presentan variaciones estadísticas entre tratamientos y entre días. La fuerza de corte disminuyó durante el tiempo de almacenamiento en todos los tratamientos mejorando la terneza de la carne sin presentar diferencia significativa. La apariencia, color y aroma disminuyeron el valor de aceptación a medida que avanzo el tiempo.

Utilización de sustancias antimicrobianas producidas por bacterias acido lácticas en la conservación de la carne / Use of antimicrobian substances produced by acid lactic bacterias on meat conservation

The use of lactic acid bacteria (LAB) for food bio-preservation has taken great importance nowadays due to its capacity for controlling pathogenic and spoilage microorganisms. Application of bi-o preservative strains as well as extracts and metabolites produced by them have demonstrated to control diverse undesirable microorganisms improving the enlargement of foods shelf-life and safety against bacteria that can affect the consumerís health. This review involves aspects of food bio-preservation and specifically of meat and meat products susceptible of alterations and attacks of diverse microorganisms. Bio-preservation methodologies of common use in foods are detailed as well as more outstanding aspects of metabolites produced by LAB, making special emphasis on bacteriocins, antimicrobial substances that have demonstrated its effectiveness to control diverse microorganisms and have had successful application on foods. The use of bio-preservation is revised, considered as a technology barrier that combined with other conservation methods like refrigeration and joint to good manufacturing practices can be an interesting option to diminish the addition of chemical preservatives, providing safe foods naturally preserved.

El uso de bacterias ácido lácticas (BAL) en la biopreservación de alimentos ha tomado gran importancia en los últimos años debido a la capacidad para controlar microorganismos patógenos y alterantes. La aplicación de cepas biopreservantes así como de los extractos y metabolitos producidos por ellas, han demostrado tener control sobre diversos microorganismos no deseados consiguiendo alargar la vida útil de los alimentos y dar seguridad contra bacterias que puedan afectar la salud del consumidor. En esta revisión se abarca aspectos de la biopreservación en alimentos y específicamente en carne y productos cárnicos, susceptibles de alteración y ataques de diversos microorganismos. Se detallan las metodologías de biopreservación más comunes utilizadas en alimentos, así como aspectos mas relevantes de los metabolitos producidos por las bacterias ácido lácticas, haciendo especial énfasis en las bacteriocinas que son sustancias antimicrobianas que han demostrado ser eficaces en el control de diversos microorganismos y han tenido exitosa aplicación a los alimentos. Se revisa el uso de la biopreservación considerado como una tecnología de barrera que combinado con otros métodos de conservación como la refrigeración y ligado a Buenas Prácticas de Manufactura pueden ser una opción interesante para disminuir la adición de preservantes químicos, proporcionando alimentos seguros preservados naturalmente.