A BBREVIATIONS
C. Class III
1.13. Methods to detect bacteriocin activity
Isolation and screening of bacteriocins producing lactic acid bacteria essentially depends on the various culture-dependent methods. A variety of techniques have been used to determine the potency of bacteriocin preparations. The methods like pour plating assay, agar well diffusion assay, spot on lawn assay has been developed to determine the inhibitory effect of bacteriocin producing LAB or its culture filtrate (Tagg and McGiven, 1971; Kekessy and Piguet, 1970). These have been based either upon broth dilution methods or the more frequently used plate assays. In the latter, the bacteriocin diffuses radially through an agar layer from circular cups cut into the gel or from containers or filter-paper discs placed on the surface of the medium seeded with the sensitive indicator strain. To compare quantitative production of nisin by natural isolates of Lactococcus lactis subsp. lactis and thus compare the variability in the production of nisin agar plate diffusion assay was applied (De Vuyst, 1994). Comparative analysis of bacteriocins assays was performed to evaluate the applicability of particular assay method (Parente et al., 1995). Further, such assays were adopted and applied on routine basis to quantitate and compare the amount of bacteriocin production by natural antagonistic isolates of LAB.
Traditionally used plating-based microbiological assays on selective medium require long incubation time and provide limited information (Pucci et al., 1988). With the increasing knowledge of chemistry of fluorophore, various fluorescence probe has been developed, which can rapidly assess the cell physiology and could easily differentiate between live and dead bacterial cells (Berney et al., 2007; Bunthof et al., 2001).
Antimicrobial effect of pediocin on carboxyfluorescein diacetate (cFDA) labelled Lactobacillus casei NCFB 2714 has been studied by spectrofluorometer measurement of
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effluxed carboxyfluorescein dye (Budde et al., 2001). The method was found to be rapid and sensitive to quantitate the microbicidal activity of bacteriocin.
1.14. Food applications of bacteriocin producing LAB/bacteriocin molecule
Preservation of foods by fermentation is a well practiced technology since ancient times.
Fermentation ensures not only increased shelf life and microbiological safety of a food but may also make some foods more digestible and reduces their toxicity to the consumer (Caplice et al., 1999; Fleet et al., 1999; Holzapfel et al., 1995; Ross et al., 2002). The antimicrobial activity of bacteriocinogenic LAB is essential in their applications in the food fermentation industry. Bacteriocin producing LAB strains have been widely reported as an inherent LAB population in milk, vegetable and meat products (Castellano et al., 2008;
Galvez et al., 2007; Jones et al., 2008).
In the past 20 years, numerous studies have focused on bacteriocins produced by lactic acid bacteria. This interest was inspired by the possibility of using bacteriocins as food preservatives. The review of Galvez et al. (2007) has discussed in detail the application of bacteriocins in food preservation. Bacteriocin preparation can be added to food as a concentrate additive or ingredient to extend the shelf-life of fermented food products or they can be produced in situ by bacteriocinogenic starters, adjunct or protective LAB cultures.
The autochthonous or allochthonous bacteriocinogenic LAB or their secreted bacteriocins in fermented food samples are primarily involved as biopreservative to increase the shelf-life of cereal, vegetable, dairy and meat fermented or non-fermented products. The application of bacteriocins provides an alternative to satisfy the increasing consumer’s demands for safe, fresh-tasting, ready-to-eat and minimally processed food.
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Cereal grains constitute a major source of dietary nutrition with increased nutritional, sensorial and functional qualities after fermentation. In cereal based fermentations, like formation of sourdough bread, the autochthonous bacteriocinogenic LAB population plays crucial role in the increased shelf-life (Caplice et al., 1999; Holzapfel et al., 1995). The most important group of bacterial strains involved in sourdough fermentation includes strains from Lactobacillus genus. Bacteriocin produced by the starter culture as well was the adjuvant cultures have also played substantial role as a bio-preservative in vegetable fermentation (Settani and Corsetti, 2008). Bacteriocin-producing LAB have been also applied as starter culture or additives for the improvement and safety of dairy food products (O’Sullivan et al., 2002; Jagannath et al., 2001). Bacteriocins have been also applied as bio- protective agents in combination with hurdle technology to increase the shelf-life and quality of fermented sausages (Jones et al., 2008; Tyopponen et al., 2003). In addition to bacteriostatic or bactericidal effect conferred by bacteriocins produced by LAB, other metabolic products of LAB have been also studied for their antagonistic activity against various foodborne pathogens.
Although many fermentations are traditionally dependent on inoculation from a previous batch starter cultures known as backslopping. But, for large scale commercial application of the starter culture, strain identification and other technological and functional parameters of the starter strains need to be defined to ensure the increased shelf-life and organoleptic property of the fermented product (Coolbear et al., 2008). Further, application of antagonistic LAB strains as a starter culture demands critical strain identification with molecular tools. The next section of this literature review highlights the approach and discusses the use of molecular tools for the identification of LAB strains.
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LAB drives food fermentation process and thus necessitates a routine basis of monitoring of fate of LAB strain during the course of fermentation along with other technological parameter of safety and quality control of fermented food products (Saarela et al., 2000). In this context, reliable monitoring and identification of LAB remains a point of crucial concern. The 16S rRNA and 23S rRNA gene as well as an intergenic spacer (IGS) region gene have been the target of choice for molecular identification and genetic characterization of LAB strains (Acquilanti et al., 2007; Chen et al., 2008; Dolci et al., 2008). The reasons justified for the selection of these targets in the molecular identification and taxonomic studies include universal abundance, evolutionary and phylogenetic properties, high discriminatory potential, multiple-copy nature and extensive availability of sequences in public databases (Juste et al., 2008).
Over the past decade, the scientific community has paid special attention in the molecular identification of bacteria applied in health benefit and food industry. Molecular studies on LAB diversity and community analysis in various fermented product with culture- dependent and culture-independent molecular tools has been thoroughly studied (Delbes et al., 2007; Miambi et al., 2003; Temmerman et al., 2004).
The demand of rapid identification technique has been contented with rapid, robust and culture-independent method without compromising their detection limits. Some of the molecular techniques used for LAB detection are discussed under the following sections:
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