Enterocins in food preservation

Abstract

The Enterococcus genus, a member of the Lactic Acid Bacteria (LAB) is found in various environments, but more particularly in the intestines of humans and other animals. Although sometimes associated with pathogenicity these bacteria have many benefits. They have been found in traditional artisanal fermented products, are used as probiotic cultures and nowadays extensively studied for the production of bacteriocins — the enterocins. Many of these enterocins have been found to be active against Listeria monocytogenes, and a few have also been reported to be active even against Gram negative bacteria, an unusual property for the bacteriocins produced by LAB. These properties have resulted in many studies describing the use of enterocins as preservatives in foods of animal and vegetable origin. This review covers the most recent information on the use of enterocins as food preservatives, either produced in-situ by the addition of enterocin producing strains or as external preservatives in the form of purified or semi-purified extracts, to prevent the growth of spoilage and pathogenic microorganisms.

Introduction

Lactic Acid Bacteria (LAB) is a diverse group of beneficial bacteria which have been inadvertently used by mankind for thousands of years. They have been used as starter cultures for preparation of an array of fermented dairy products which include yoghurt, cheese, buttermilk, kefir and many products indigenous to various regions of the world. The preparation of these products has been documented in archaic texts of various regions such as ancient Iraq (Stiles and Holzapfel, 1997). The starter cultures not only provide peculiar taste to fermented products, but also serve to extend the shelf-life of the product mainly by the conversion of lactose to lactic acid.

The LAB have been divided into many genera, and those important in food include Lactobacillus, Lactococcus, Enterococcus, Pediococcus, Leuconostoc and Streptococcus. Among them the genus Enterococcus is of particular interest. The bacteria belonging to this genus are found in the intestine of humans and other animals, where they live as commensals and also provide many beneficial effects.

The enterococci as a group were first described by (Thiercelin, 1899) who defined them as ‘enterocoque’ owing to their enteral habit. Later the genus Enterococcus was proposed by (Thiercelin and Jouhaud, 1903). However, when (Lancefield, 1933) developed a serological system for further classification of streptococci, the streptococci of intestinal origin (earlier classified in genus Enterococcus) were placed in group D as they possessed the group D antigen. Based on this typing system (Sherman, 1937) divided the streptococci into four groups viz viridans, pyogenes, lactic and enterococcus. Therefore, the enterococci formed a sub-division of the genus Streptococcus. In 1984, however, the genus Enterococcus was revived (Schleifer and Kilpper-Bälz, 1984), based on DNA:DNA and DNA:rRNA hybridization techniques which proved that these bacteria are distinct from other streptococci and four species of enteric origin were placed in this genus. To date more than 25 species belonging to the genus Enterococcus have been described but the most important ones are Enterococcus faecium and Enterococcus faecalis which are commonly found in the intestinal tract of animals and humans (Klein, 2003).

Although enterococci have a long history of use as artisanal cultures for preparation of various types of cheeses, they are sometimes associated with pathogenicity. They have been reported to produce endocarditis, bacteraemia and urinary tract infections (Franz et al., 1999, Kayser, 2003). However, when enterococcal isolates from various sources are tested for virulence factors, it has been found that incidence of virulence and pathogenicity is highest in clinical isolates, followed by animal and food isolates. When (Mannu et al., 2003) compared the occurrence of virulence factors and antibiotic resistance between E. faecium strains of dairy, animal and clinical origin they observed similar results. Therefore, it can be concluded that enterococcal strains lacking hemolytic activity and not carrying cytolysin nor vancomycin resistance genes may be regarded as safe and can be used as starter cultures, co-cultures, or probiotics. (De Vuyst et al., 2003).

The members of the genus Enterococcus are found in many food products. They are most frequently present in many traditional European cheeses prepared in the Mediterranean countries, mostly from raw ewes' or goats' milk, where they are thought to have initially made their way as contaminants from the faeces of animals, water or milking equipment and storage tanks, and thereafter became an important component of artisanal cultures (Foulquié Moreno et al., 2006). These bacteria now play a fundamental role in the ripening of these cheeses, probably by proteolysis, lipolysis and citrate breakdown, hence adding a unique taste and flavour to these products (Manolopoulou et al., 2003). In addition to providing the peculiar taste and flavour, the enterococci also act as protective agents against various pathogens, such as Listeria monocytogenes, a common pathogen found in meat and dairy products. This is due to their ability to produce antimicrobial peptides called bacteriocins (De Vuyst and Vandamme, 1994), which have been defined as ribosomally synthesized peptides and proteins which are antagonistic to other bacteria, but confer immunity to the host strain (Klaenhammer, 1993). These bacteriocins are produced by many species of LAB, and nowadays extensively studied for their food preservation properties in a variety of food products of animal and vegetable origin, against a range of foodborne pathogenic and spoilage bacteria, and these studies have been recently reviewed (Gálvez et al., 2008). They are considered most suitable alternatives to chemical preservatives because they are harmless to eukaryotic cells, are usually pH and heat tolerant and are readily digested by proteolytic enzymes in the stomach due to their proteinaceous nature (Gálvez et al., 2007).

The bacteriocin producing activity of enterococci (at that time classified as group D streptococci) was first noted by (Kjems, 1955) while studying bacteriophages. Since then a large number of bacteriocins produced by enterococci (enterocins) have been described and many have been fully characterized at the biochemical and genetic level, which have been grouped into a simplified classification scheme (Franz et al., 2007). According to this scheme the enterocins have been divided into four major classes: Class I consisting of post-translationally modified lantibiotic enterocins, Class II consisting of linear, unmodified peptides having molecular mass less than 10 kDa, Class III consisting of cyclic peptides and Class IV encompassing the large, heat labile proteins. Out of these four classes, the enterocins of Class II and III (particularly enterocin AS-48) have attracted considerable interest for their potential use as natural and non-toxic food preservatives as they inhibit not only closely related species but also Gram positive food-spoilage and foodborne pathogens (Cotter et al., 2005). In particular, enterocins with high anti-listerial activity are promising candidates as useful antimicrobial agents in food preservation. Many of these enterocins have been tested as additives in foods to act as preservatives against spoilage and pathogenic microorganisms. The following account briefly describes the use of some important enterocins investigated by various researchers as food preservatives.