Discovery of novel biopreservation agents with inhibitory effects on growth of food-borne pathogens and their application to seafood products

Institut National des Sciences et Technologies de la Mer (INSTM), Rue 2 Mars 1934, 2025 Salammbô, Tunis, Tunisia.
Research in Microbiology (Impact Factor: 2.71). 01/2012; 163(1):44-54. DOI: 10.1016/j.resmic.2011.08.005
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ABSTRACT Selection of protective cultures is relevant in order to biopreserve and improve the functional safety of food products, mainly through inhibition of spoilage and/or pathogenic bacteria. Accordingly, the present study investigated potential applications of lactic acid bacteria (LAB) in the biopreservation of fish and shellfish products. For this purpose, a collection of 84 LAB strains isolated from sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) was identified and characterized for their inhibitory activities against the most relevant seafood-spoilage and pathogenic bacteria potentially present in commercial products. The bioactive strains belonged to the genus Enterococcus and exhibited inhibition against Carnobacterium sp, Bacillus sp, Listeria monocytogenes, Aeromonas salmonicida, Aeromonas hydrophila and Vibrio anguillarum. Treatment of cell-free extracts of the LAB strains with proteases revealed the proteinaceous nature of the inhibition. Interestingly, the cell-free extracts containing bacteriocins remained 100% active after treatment up to 100 °C for 30 min or 121 °C for 15 min. Molecular analysis led to identification of the bacteriocins investigated, including enterocins A, B, L50 and P. All of these proteins demonstrated remarkable anti-Listeria activity and were found to be heat-resistant small class IIa bacteriocins. The results presented in this work open the way for potential applications of these LAB strains to the biopreservation of minimally-processed seafood products.

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Available from: Monia El Bour, Feb 23, 2014
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    • "This is why many industrials are moving toward the use of protective microbial cultures, mainly LAB, able to produce antagonistic metabolites such as bacteriocins, peptides and/or low-weight non-proteinaceous compounds (organic acids, fatty acids, H 2 O 2 , etc.). Many scientific evidences or proof-of-concept in literature underline the great potential of such an approach to combat pathogenic or spoilage microorganisms in various food products such as meat (Budde et al., 2003; Vermeiren et al., 2004; Castellano et al., 2008), fish (Brillet et al., 2005; Tomé et al., 2008; Chahad et al., 2012), bakery products (Dal Bello et al., 2007; Gerez et al., 2009; Ryan et al., 2011) and vegetables (Trias et al., 2008; Randazzo et al., 2009). However, in contrast to probiotics (Gregoret et al., 2013), only a limited number of commercial protective cultures are marketed today, and this statement is especially true for antifungal bioprotective cultures in dairy products. "
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    ABSTRACT: Few antifungal protective cultures adapted to fermented dairy products are commercially available because of the numerous constraints linked to their market implementation. Consumer's demand for naturally preserved food products is growing and the utilization of lactic acid bacteria is a promising way to achieve this goal. In this study, using a 2(5-1) factorial fractional design, we first evaluated the effects of fermentation time, of initial sucrose concentration and of the initial contamination amount of a spoilage yeast, on antifungal activities of single and mixed cultures of Lactobacillus rhamnosus K.C8.3.1I and Lactobacillus harbinensis K.V9.3.1Np in yogurt. L. harbinensis K.V9.3.1Np, the most relevant strain with regard to antifungal activity was then studied to determine its minimal inhibitory inoculation rate, its antifungal stability during storage and its impact on yogurt organoleptic properties. We showed that L. harbinensis K.V9.3.1Np maintained a stable antifungal activity over time, which was not affected by initial sucrose, nor by a reduction of the fermentation time. This inhibitory activity was an all-or-nothing phenomenon. Once L. harbinensis K.V9.3.1Np reached a population of ∼2.5 × 10(6) cfu/g of yogurt at the time of contamination, total inhibition of the yeast was achieved. We also showed that an inoculation rate of 5 × 10(6) cfu/ml in milk had no detrimental effect on yogurt organoleptic properties. In conclusion, L. harbinensis K.V9.3.1Np is a promising antifungal bioprotective strain for yogurt preservation. Copyright © 2014 Elsevier Ltd. All rights reserved.
    Food Microbiology 02/2015; 45 Pt A(Pt A):10-7. DOI:10.1016/ · 3.33 Impact Factor
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    • "Bacteriocin-producing enterococci are widespread in nature. They have been isolated from numerous sources, such as dairy products (Foulqui e Moreno et al. 2006), fermented sausages (Cocolin et al. 2007), fish (Bourouni Chahad et al. 2012), vegetables (Bennik et al. 1998; Zendo et al. 2005) and mammalian gastrointestinal tract (Carina Audisio et al. 2000; Brand~ ao et al. 2010). "
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    ABSTRACT: Unlabelled: A total of 300 isolates of Enterococcus, from different sources including faeces of poultry, cow and sheep, raw milk, ricotta cheese and water, in Tunisia, were screened for their antibacterial activity. Amongst them, 59 bacteriocin-producing strains were detected and identified by molecular methods. Genes encoding for entA, entP, entB, entL50A/B, AS-48 and bac31 bacteriocins were targeted by PCR. The bacteriocin-producing strains were assigned to the species Enterococcus faecium, Enterococcus faecalis, Enterococcus hirae, Enterococcus mundtii and Enterococcus durans, respectively, 34, 19, 3, 2 and 1 isolates. Antimicrobial activity was specifically observed against different spoilage and pathogenic micro-organisms, such as Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Escherichia coli, Ent. faecalis, Staphylococcus aureus, Salmonella enterica serovar Enteritidis and Paenibacillus larvae. The inhibitory activity was totally lost after proteinase K treatment, thereby revealing the proteinaceous nature of the antimicrobial compound. Only three bacteriocin genes, namely entP, entA and entL50A/B were detected in the isolates included in this study. Enterocins A and P were the most frequent genes and they were found in 55 (93.2%) and 39 isolates (66.1%), respectively, followed by enterocin L50A/B present in 27 isolates (45.7%). These newly identified bacteriocin-producing enterococci have the potential to be used in bio-preservation of food as well as biological control of foulbrood disease. Significance and impact of the study: Enterococci possess interesting properties not only for the food industry, but also for animal and human health. The antimicrobial potential of these bacteria includes principally bacteriocin-like molecules. With the aim of identifying bacteriocinogenic strains, a collection of 300 enterococci isolated from different origins were screened and their spectrum of action, as well as the gene encoding the bacteriocin, was determined. Fifty-nine bacteriocin-producing Enterococcus showed high activity against Listeria monocytogenes and Paenibacillus larvae, the causative agent of American foulbrood. Enterocins A, P and L50A/B were found in various combinations. The most important finding of this study is the growth inhibition of P. larvae due to bacteriocin-producing Enterococcus, which opens up the possibility to use these strains to control the disease in honeybees.
    Letters in Applied Microbiology 02/2014; 59(1). DOI:10.1111/lam.12239 · 1.66 Impact Factor
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    • "In a recent study, Chahad, El Bour, Calo-Mata, Boudabous, and Barros-Velàzquez (2012) showed the presence of high bacteriocin-producing enterococci in farmed marine fish. The acquisition of these strains, already acclimated to seafood habitats, could be advantageous for biopreservation in aquatic foodstuffs (Chahad et al., 2012). Similarly, due to their implication in cheese ripening and aroma development, dairy enterococcal strains with bacteriocinogenic activity and a proved harmless nature could be included in starter cultures for many cheeses (Ogier & Serror, 2008). "
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    ABSTRACT: Enterococcus faecalis is frequently associated with raw milk cheeses of Mediterranean area. The genetic diversity of 38 E. faecalis obtained from raw milk products in Italy was assessed through Randomly Amplified Polymorphic DNA PCR (RAPD-PCR) and repetitive extragenic palindromic PCR (rep-PCR). The strains were screened for their antimicrobial activity against 5 food-borne spoilage and pathogenic bacteria and 13 lactic acid bacteria (LAB), commonly used as starter cultures. Investigation was made to identify the bacteriocinogenic potential by searching for bacteriocin-encoding genes. Inhibitory effects against undesirable bacteria, including Bacillus cereus (44.7% of strains), Escherichia coli (18.4%), Listeria monocytogenes (15.8%), Staphylococcus aureus (2.6%), and Clostridium sporogenes (21.1%), were detected. Moderate antagonism towards LAB was found. One strain producing enterocin AS-48 was identified, suggesting that the antimicrobial activity of the phenotypically positive isolates should be necessarily due to another non-enterocin factor. A deeper insight intobiopreservation potential of dairy E. faecalis was provided, highlighting the influence of this species on cheese microbial community.
    CyTA - Journal of Food 11/2013; 12(3). DOI:10.1080/19476337.2013.825327 · 0.82 Impact Factor
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