Enterocin AS-48 Research Articles

The Effects of Cultivating Lactic Starter Cultures with Bacteriocin-Producing Lactic Acid Bacteria

The effects of bacteriocins produced by six strains of lactic acid bacteria on 9 mesophilic and 11 thermophilic commercial starter cultures were investigated in mixed cultures of commercial starters with bacteriocin-producing strains in milk. The bacteriocins produced by the test organisms were nisin A, nisin Z, lacticin 481, enterocin AS-48, a novel enterocin, and a novel plantaricin. Mesophilic commercial starters were in most cases tolerant of bacteriocins, with only two of the starters being partially inhibited, one by four and the other by two bacteriocins. The aminopeptidase activities of mesophilic starters were generally low, and only one of the combinations of mesophilic starter–bacteriocin producer gave double the aminopeptidase activity of the starter culture without the bacteriocin producer. Thermophilic commercial starters were more sensitive to bacteriocins than mesophilic starters, with six thermophilic starters being partially inhibited by at least one of the bacteriocins. Their aminopeptidase activities were generally higher than those of the mesophilic starters. The aminopeptidase activities of seven thermophilic starters were increased in the presence of bacteriocins, by factors of up to 9.0 as compared with the corresponding starter cultures alone. Bacteriocin-producing strains may be used as adjunct cultures to mesophilic starters for the inhibition of pathogens in soft and semihard cheeses, because mesophilic starters are rather tolerant of bacteriocins. Bacteriocin producers may also be used as adjunct cultures to thermophilic starters of high aminopeptidase activity, more sensitive to lysis by bacteriocins than mesophilic starters, for the acceleration of ripening in semihard and hard cheeses.

Diversity of bacteriocins produced by lactic acid bacteria isolated from raw milk

Bacteriocin-producing lactic acid bacteria were isolated from 298 samples of raw ewes', goats’ or cows’ milk. Eighty-two bacteriocin producers were phenotypically and genotypically identified as L. lactis subsp. lactis (59 isolates), L. lactis subsp. cremoris (2 isolates), L. lactis subsp. lactis biovar diacetylactis (6 isolates), E. faecalis (7 isolates), E. faecium (1 isolate), L. paracasei subsp. paracasei (4 isolates), L. plantarum (1 isolate) and Leuconostoc spp. (2 isolates). By means of PCR-techniques, nisin was characterized in 39 of the 67 bacteriocin-producing lactococci and lacticin 481 in 23 isolates, some of which presented antilisterial activity. Enterocin AS-48 was produced by four enterococcal isolates. Four non-identified bacteriocins produced by 16 isolates showed a broad inhibitory spectrum. Nisin-producing lactic acid bacteria were the most abundant, but lacticin 481-producing lactococci and AS-48-producing enterococci were found at relatively high rates.

Detection of specific bacteriocin-producing lactic acid bacteria by colony hybridization.

A colony hybridization method for detecting lactic acid bacteria encoding specific bacteriocins was developed. Specific PCR-generated probes were used to detect colonies of pediocin PA-1, lactococcin A, enterocin AS-48, nisin A and lacticin 481 producing strains. The probes were shown to be sensitive and specific for sequences belonging to the structural genes of the respective bacteriocins.

Detection of enterocin AS-48-producing dairy enterococci by dot-blot and colony hybridization.

The enterococci have traditionally been used as indicators of faecal contamination because they are common inhabitants of the human and animal intestinal tract. In addition, some strains are well documented as opportunistic pathogens, and have been implicated in endocarditis, infant diarrhoea and other conditions. However, other strains are widespread in foods, particularly in milk and dairy products, where they are considered desirable microflora. In fact, through their proteolytic and lipolytic abilities, they play an important role in cheese ripening, contributing to the development of the organoleptic properties characteristic of certain cheeses (Villani et al. 1993).Production of antimicrobial substances is one of the mechanisms by which microorganisms can exert a probiotic effect in a host. In this context, a significant number of bacteriocin-producing enterococci of dairy origin have been isolated in recent years (Giraffa, 1995). Production of these antimicrobial peptides or proteins is a common phenotype among lactic acid bacteria, and this is the application of enterococcal bacteriocins of special interest in dairy systems. Firstly, they show activity against a broad spectrum of spoilage and pathogenic organisms of concern in dairy industries, such as Listeria monocytogenes. Secondly, they are inactivated by human gastric enzymes but not by some enzymes that, like rennet, are frequently used in dairy plants. Finally, their marked heat stability enables them to be used in a wide variety of dairy products (Giraffa, 1995).The genes that encode the biosynthesis of some enterocins or enterococcins, such as enterocin AS-48 (Martínez-Bueno et al. 1994), have been sequenced, allowing their rapid detection by molecular biology techniques such as the polymerase chain reaction (PCR) (Joosten et al. 1997). Enterococcal bacteriocins that have been genetically characterized have been shown to be plasmid-encoded (Clewell, 1993).In this paper, we report a simple method for the isolation of plasmid DNA from dairy enterococci, using a combination of lysozyme and glass beads (Frère, 1994; Reinkemeier et al. 1996) to achieve cell lysis. Plasmid DNA was used in dot-blot and Southern hybridization analyses to identify enterocin AS-48-encoding dairy enterococci by using a specific PCR-generated probe. In addition, a more rapid detection method based on colony hybridization was also developed.