ANTAGONISM ACTIVITY OF LACTIC ACID BACTERIA ISOLATED FROM TRADITIONAL FERMENTED MEAT PETIS

The research of antagonism activity of lactic acid bacteria to pathogen bacteria, spoilage bacteria and histamin producer bacteria was done. The goal of the research was to know the influence of lactic acid bacteria on antagonistic activity to pathogen bacteria, spoilage bacteria, and histamin-producer bacteria. The research used exploration experimental method with 4 replications. This research used lactic acid bacteria which was isolated from traditional fermented meat petis The code of lactic acid bacteria were Pediococcus YDA1 sp., Pediococcus YDA2 sp., Pediococcus YDA3 sp., Pediococcus YDA4 sp., Pediococcus YDA5 sp., Pediococcus YDA6 sp., Pediococcus YDA7 sp., Pediococcus YDA8 sp., Pediococcus YDA9 sp., Pediococcus YDA10 sp., and Pediococcus YDA11 sp. The spoilage bacteria was Bacillus substilis FNCC 0061. The pathogen bacteria were Salmonella typimurium FNCC 0134, Escherchia coli FNCC 0047, Staphylococcus aureus FNCC 0097, and Vibrio parahaemolyticus FNCC 0166. While the codes of the histamin-producer bacteria were P1, P2, and P3. The measurement of antagonistic activity of lactic acid bacteria used well diffusion modification method. This was represent by clear zone diametres as capability of inhibitory. The result of research was that 5 lactic acid bacteria had antagonism activity to pathogen bacteria, spoilage bacteria and histamin-producer bacteria. They were Pediococcus YDA1 sp., Pediococcus YDA3 sp., Pediococcus YDA4 sp., Pediococcus YDA10 sp., and Pediococcus YDA11 sp. Keywords : Pediococcus, Fermented Meat, Antagonism Activit

Identification and biotechnological characterization of lactic acid bacteria isolated from chickpea sourdough in northwestern Argentina

Chickpea, a relevant legume worldwide, can be nutritional and functionally improved by fermentation with lactic acid bacteria (LAB). In order to select suitable autochthonous starter cultures, we isolated and identified LAB from kabuli chickpeas cultivated and consumed in northwestern Argentina, and screened their relevant techno-functional properties. Chickpeas were milled and spontaneously fermented with daily back-slopping at 37 °C for 6 days and evolution of microbial populations were followed by plate counting. Phenotypic and genotypic methods including (GTG)5-based PCR fingerprinting and 16S rDNA sequencing were used to differentiate and identify the isolates to species level. A marked increase of LAB counts was observed throughout fermentation raising from 0.88 ± 0.35 log CFU/g of unfermented flours to 9.61 ± 0.21 log CFU/g after 5 backslopping steps with a concomitant pH decline from 6.09 ± 0.05 to 4.40 ± 0.03. Eighteen strains belonging to four LAB genera and six species: Enterococcus durans, E. mundtii, Lactococcus garvieae, Pediococcus pentosaceus, Weissella cibaria and W. paramesenteroides were identified in chickpea sourdoughs. Based on their abilities, Weissella cibaria CRL 2205 (acidification capacity), W. paramesenteroides CRL 2191 (proteolytic activity), Pediococcus pentosaceus CRL 2145 (gallate decarboxylase and peptidase activities), Lactococcus garviae CRL 2199 (α-galactosidase activity) and E. durans CRL 2193 (antimicrobial activity), were selected to design novel fermented chickpea products.Fil: Saez, Gabriel Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad San Pablo Tucumán; ArgentinaFil: Saavedra, Maria Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Zarate, Gabriela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad San Pablo Tucumán; Argentin

Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli

peer-reviewedThe bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli TunerTM (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinations of these genes, revealed that the structural and the transporter genes alone are sufficient to permit heterologous production and secretion in this host. Even though the accessory protein, usually associated with optimal disulfide bond formation, was not required for bacteriocin synthesis, its presence did result in greater pediocin PA-1 production. The simplicity of the system and the fact that the associated bacteriocins could be recovered from the extracellular medium provides an opportunity to facilitate protein engineering and the overproduction of biologically-active bacteriocins at industrial scale. Additionally, this system could enable the characterization of new bacteriocin operons where genetic tools are not available for the native producers

Incorporating a mucosal environment in a dynamic gut model results in a more representative colonization by lactobacilli

To avoid detrimental interactions with intestinal microbes, the human epithelium is covered with a protective mucus layer that traps host defence molecules. Microbial properties such as adhesion to mucus further result in a unique mucosal microbiota with a great potential to interact with the host. As mucosal microbes are difficult to study in vivo, we incorporated mucin-covered microcosms in a dynamic in vitro gut model, the simulator of the human intestinal microbial ecosystem (SHIME). We assessed the importance of the mucosal environment in this M-SHIME (mucosal-SHIME) for the colonization of lactobacilli, a group for which the mucus binding domain was recently discovered. Whereas the two dominant resident Lactobacilli, Lactobacillus mucosae and Pediococcus acidilactici, were both present in the lumen, L. mucosae was strongly enriched in mucus. As a possible explanation, the gene encoding a mucus binding (mub) protein was detected by PCR in L. mucosae. Also the strongly adherent Lactobacillus rhamnosus GG (LGG) specifically colonized mucus upon inoculation. Short-term assays confirmed the strong mucin-binding of both L. mucosae and LGG compared with P. acidilactici. The mucosal environment also increased long-term colonization of L. mucosae and enhanced its stability upon antibiotic treatment (tetracycline, amoxicillin and ciprofloxacin). Incorporating a mucosal environment thus allowed colonization of specific microbes such as L. mucosae and LGG, in correspondence with the in vivo situation. This may lead to more in vivo-like microbial communities in such dynamic, long-term in vitro simulations and allow the study of the unique mucosal microbiota in health and disease

Contribution of natural milk culture to microbiota, safety and hygiene of raw milk cheese produced in alpine malga

Processing of alpine milk in malga farms is carried out under conditions that can favor contamination by coliforms, coagulase-positive staphylococci, or pathogens such as Listeria monocytogenes. With the aim to improve the hygienic characteristics and safety of cheese produced in four malga farms the use of lyophilized Natural Milk Culture prepared with selected strains was tested.. Two cheesemaking tests were carried out in the same day always starting from the same milk: in the first case following the malga recipe that uses either Natural Whey Culture or without the addition of a starter, in the second one using a Natural Milk Culture. Cheesemaking were carried out in four malga farms located in the west area of Trentino region within the same week. For hygienic and safety evaluation, aerobic colony count, coagulase-positive staphylococci, Escherichia coli, staphylococcal toxins, Listeria monocytogenes, and Salmonella spp, pH and aw were determined in raw milk from evening and morning milking, curd in vat, curd after extraction and two months-ripened cheese. Pathogens or toxins, high values of coagulase- positive staphylococci and E. coli were not found in cheese samples. However, in the curd coagulase-positive staphylococci reached values almost of 5 Log CFU/g in the two malga without starter cultures. The use of Natural Milk Culture reduced E. coli counts. In addition, DNA was extracted from cheese samples and from Natural Milk Culture and the composition of the microbial community determined by Next Generation Sequencing method. The determination of cheese microbial communities demonstrated that the use of Natural Milk Culture exerted different effects in the different malga, in any case preserving bacterial biodiversity

Occurrence of Pectinatus and Megasphaera in the major UK breweries

The occurrence of beer spoilage bacteria belonging to the genera Pectinatus and Megasphaera in ten major UK breweries was investigated. The sampling points were selected from fermentation areas, beer conditioning areas and beer bottling and canning sites. Multiplex PCR methodology was used for detection of three Pectinatus and three Megasphaera species using species specific primers. The presence of six Lactobacillus species was also examined. Overall, 117 samples were analysed from ten breweries; six samples were positive for the presence of Pectinatus species and three samples were positive for the presence of Megasphaera species, while 34 samples were positive for the presence of Lactobacillus species. Lactobacillus species appeared to be the major potential spoilage microorganisms. Although none of the actual beer samples were found to be positive for Pectinatus and Megasphaera species, their occurrence in aerobic brewery environments indicates sanitation problems and revealed the presence of highly established biofilms in some breweries

Use of Carnobacterium piscicola to limit the growth of Listeria monocytogenes in mussel products : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Bacteria were screened in order to find an organism antagonistic to Listeria monocytogenes which could be applied to mussel products and enhance their safety, especially when temperature-abused. A Listeria monocytogenes isolate from the seafood industry was selected as the target organism. Strains of Lactobacillus reuteri and Enterococcus fecium were screened on plates incubated at 35°C and 10°C for anti-listerial compounds, but none were found. A non-bacteriocinogenic strain of Carnobacterium piscicola, A9b- was selected as the antagonist for detailed examination of growth in broth, agar and mussel systems at 10°C. This temperature was chosen to represent temperature abuse of refrigerated products. To distinguish between the growth of the Carnobacterium piscicola strain and wild-type Listeria monocytogenes a "semi-selective" agar was developed using phenol-red indicator, and mannitol as the sole carbohydrate source. Growth rates of Carnobacterium piscicola and Listeria monocytogenes were compared when grown alone and as a co-culture in agar and broth. Growth rates of Listeria monocytogenes when grown alone, and in the presence of Carnobacterium piscicola, were determined on mussels. Regression analyses were done for the inhibition of Listeria monocytogenes by Carnobacterium piscicola. In all cases Carnobacterium piscicola significantly inhibited the growth of Listeria monocytogenes (P broth = 0.018, P agar <0.001, P mussels < 0.001). Growth of both organisms was faster in broth, than on mussels or agar. The greatest inhibition of Listeria monocytogenes was observed in broth reaching log₁₀4.8 at 41 hours of incubation, prior to decreasing after this time. In agar and mussels the inhibition lasted longer and had not decreased at the end of the trial. The log₁₀ reduction in growth of Listeria monocytogenes in agar was measured at 3.4 and in mussels measured at 1.6. These results were statistically significant (P<0.001 for all). Inhibition of wild type Listeria monocytogenes was also shown in broth when a much lower concentration of Carnobacterium piscicola was used. These results should be considered as preliminary and further confirmatory work should be done. However, Carnobacterium piscicola A9b- shows promise as an antagonistic organism to assist in the control of Listeria monocytogenes in mussel products along with industry-accepted good hygienic practices

Fluorescence in situ hybridisation detection of Lactobacillus plantarum group on olives to be used in natural fermentations

At present there are very few studies on the bacterial diversity of olives and on the importance of the microbial species for the fermentation of olives aimed to table olives production. Most of the authors report on the occurrence of Lactobacillus plantarum as principal member of these communities or at least as the species responsible for the fermentation. In this study, fluorescence in situ hybridisation (FISH) with 16S rRNA probes was used to evaluate the occurrence of L. plantarum in olives. A 18-bp oligonucleotide probe was used in FISH experiments to evaluate the specificity of detection among Lactobacillus species. The probe was tested against 30 Lactobacillus species and appeared to be specific for L. plantarum, L. paraplantarum and L. pentosus. The probe was then used to investigate the occurrence of these species in 25 samples of olives (cultivar “Leccino”) collected in Campania region (Southern Italy). The olives were washed in a saline solution and the suspensions were then analysed by FISH and observed by fluorescence microscopy. No hybridisation signal was detected in at least 30 fields of observation when the L. plantarum-specific probe was used, probably due to the low sensitivity of the FISH method. Olive samples were plated on Rogosa agar and about 40% of the samples did not give growth after 5 days. When colony growth was observed, bulk cells from Rogosa agar plates were collected and analysed by DNA extraction followed by 16S rDNA Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE). The different microbial species were identified by direct sequencing of DGGE bands. Leuconostoc pseudomesenteroides was the most frequently found species, occurring in more than 50% of the samples that had shown growth on Rogosa agar. The closest relatives of the species of the genera: Leuconostoc, Pediococcus, Pseudomonas and Raoultella were also identified suggesting that guided fermentation by using selected LAB starters is advisable for a safe and desired table olives production

Bacteriocins: Novel Solutions to Age Old Spore-Related Problems?

peer-reviewedBacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, which have the ability to kill or inhibit other bacteria. Many bacteriocins are produced by food grade lactic acid bacteria (LAB). Indeed, the prototypic bacteriocin, nisin, is produced by Lactococcus lactis, and is licensed in over 50 countries. With consumers becoming more concerned about the levels of chemical preservatives present in food, bacteriocins offer an alternative, more natural approach, while ensuring both food safety and product shelf life. Bacteriocins also show additive/synergistic effects when used in combination with other treatments, such as heating, high pressure, organic compounds, and as part of food packaging. These features are particularly attractive from the perspective of controlling sporeforming bacteria. Bacterial spores are common contaminants of food products, and their outgrowth may cause food spoilage or food-borne illness. They are of particular concern to the food industry due to their thermal and chemical resistance in their dormant state. However, when spores germinate they lose the majority of their resistance traits, making them susceptible to a variety of food processing treatments. Bacteriocins represent one potential treatment as they may inhibit spores in the post-germination/outgrowth phase of the spore cycle. Spore eradication and control in food is critical, as they are able to spoil and in certain cases compromise the safety of food by producing dangerous toxins. Thus, understanding the mechanisms by which bacteriocins exert their sporostatic/sporicidal activity against bacterial spores will ultimately facilitate their optimal use in food. This review will focus on the use of bacteriocins alone, or in combination with other innovative processing methods to control spores in food, the current knowledge and gaps therein with regard to bacteriocin-spore interactions and discuss future research approaches to enable spores to be more effectively targeted by bacteriocins in food settings.KE, DF, CH, PC, MR, RR are supported by the Irish Government under the National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Fisheries and Food, Ireland (DAFM 13/F/462) to PC and MR, a Science Foundation Ireland (SFI) Technology and Innovation Development Award (TIDA 14/TIDA/2286) to DF, SFI-PI funding (11/PI/1137) to PDC and the APC Microbiome Insitute under Grant Number SFI/12/RC/2273

Real-time detection of riboflavin production by Lactobacillus plantarum strains and tracking of their gastrointestinal survival and functionality in vitro and in vivo using mCherry labeling

Some strains of lactic acid bacteria (LAB) produce riboflavin, a water-soluble vitamin of the B complex, essential for human beings. Here, we have evaluated riboflavin (B2 vitamin) production by five Lactobacillus plantarum strains isolated from chicha, a traditional maize-based fermented alcoholic beverage from north-western Argentina and their isogenic riboflavin-overproducing derivatives previously selected using roseoflavin. A direct fluorescence spectroscopic detection method to quantify riboflavin production in bacterial culture supernatants has been tested. Comparison of the efficiency for riboflavin fluorescence quantification with and without prior HPLC fractionation showed that the developed method is a rapid and easy test for selection of B2 vitamin-producing strains. In addition, it can be used for quantitative detection of the vitamin production in real time during bacterial growth. On the basis of this and previous analyses, the L. plantarum M5MA1-B2 riboflavin overproducer was selected for in vitro and in vivo studies after being fluorescently labeled by transfer of the pRCR12 plasmid, which encodes the mCherry protein. The labeling did not affect negatively the growth, the riboflavin production nor the adhesion of the strain to Caco-2 cells. Thus, L. plantarum M5MA1-B2[pRCR12] was evaluated for its survival under digestive tract stresses in the presence of microbiota in the dynamic multistage BFBL gut model and in a murine model. After exposure to both models, M5MA1-B2[pRCR12] could be recovered and detected by the pink color of the colonies. The results indicated a satisfactory resistance of the strain to gastric and intestinal stress conditions but a low colonization capability observed both in vitro and in vivo. Overall, L. plantarum M5MA1-B2 could be proposed as a probiotic strain for the development of functional foods.Fil: Mohedano, Mari Luz. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Hernández Recio, Sara. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Yépez, Alba. Universidad de Valencia; EspañaFil: Requena, Teresa. Consejo Superior de Investigaciones Científicas. Instituto de Investigación en Ciencias de la Alimentación; EspañaFil: Martínez Cuesta, M. Carmen. Consejo Superior de Investigaciones Científicas. Instituto de Investigación en Ciencias de la Alimentación; EspañaFil: Peláez, Carmen. Consejo Superior de Investigaciones Científicas. Instituto de Investigación en Ciencias de la Alimentación; EspañaFil: Russo, Pasquale. Università di Foggia; ItaliaFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Spano, Giuseppe. Università di Foggia; ItaliaFil: Aznar, Rosa. Universidad de Valencia; España. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; EspañaFil: López, Paloma. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; Españ