Biopreservation The Food Preservation Biology Essay

Biopreservation entails the preservation of food and food products utilising biological substances (Caplice and Fitzgerald, 1999). It refers to the extension of the shelf-life and the improvement of the safety of foods using microorganisms and/or their metabolites or fermentation products to control spoilage and pathogenic bacteria. It is perceived to be a sustainable and more natural way of food preservation as compared to the use of chemical food preservatives (Caplice and Fitzgerald, 1999; Cleveland et al., 2002; Ross et al., 2002).

Food preservation is paramount to food safety and storage and has an impact on food security (Caplice and Fitzgerald, 1999). Food-borne pathogenic and spoilage micro-organisms such as Bacillus spp, Clostridium spp, Staphylococcus aureus, Escherichia coli and Salmonella spp can be controlled and minimised to acceptable and non-infective doses through food preservation (Cleveland et al., 2001; Dalton, 2002; Mufandaedza et al., 2006). Synthetic chemical substances have been widely applied in the food industry, particularly in food preservation. Such preservatives may act as antimicrobial preservatives, which inhibit/arrest the growth of bacteria and/or fungi or as antioxidants such as oxygen absorbers, which inhibit the oxidation of food constituents thereby enabling the food to last longer. Common antimicrobial preservatives include sorbic acid and its salts, benzoic acid and its salts, calcium propionate, sodium nitrite (and sodium nitrate which converts to sodium nitrite "in situ"), sulphites (sulphur dioxide, sodium bisulphite, potassium hydrogen sulphite, etc.) and disodium EDTA (Dalton, 2002; McCann et al, 2007; FDA, 2009).

1.1.2 Problem with chemical food preservatives

The benefits and safety of many artificial food additives (including preservatives) is the subject of debate amongst academics and regulators specializing in food science, toxicology, and biology (Rodford, 1997; Ross et al., 2002). Chemical food preservatives such as the sulphites which are used in the preservation of dried fruits and vegetables have been implicated to be allergenic and intolerant to some consumers (Parke and Lewis, 1992). Sodium nitrite is a preservative commonly used in maintaining the stability of cured fish and meat (Dalton, 2002). The University of Minnesota Extension found that while sodium nitrite is useful in preserving meats, under certain circumstances, such as cooking meat at a high temperature, sodium nitrite may create cancer-causing substance called nitrosamine which is both a mutagen and carcinogen. A number of food product recalls have occurred due to the presence of nitrosamine in the food products (Scotter and Castle, 2004; McCann et al, 2007). According to Centre for Science in the Public Interest (CSPI), some food preservatives may encourage the growth of tumours. An example is Butylated hydroxyanisole (BHA), a food preservative commonly added to cereals and potato chips; it has been shown to cause tumours in rats, rabbits and hamsters (Chung, 1999; Dalton, 2002; Daniells, 2006; McCann et al, 2007). While it has not been scientifically proven to cause tumours in humans, there is a strong correlation to the effects in humans when a chemical causes problems in three different species (Dalton, 2002).

Despite the facts on their potential harm, chemical food preservatives are still in use and it is from such a background that they have been met with some consumer fears. This has prompted research in alternative and safer methods of food preservation of which biopreservation has been perceived as a potential substitute (Caplice and Fitzgerald, 1999; Ross et al., 2002). A number of microorganisms and other biological agents have been envisaged to be crucial in biopreservation of foods. Lactic acid bacteria have been shown to elicit antimicrobial activities and are perceived to be potentially applicable as food biopreservatives (Gibson and Fuller, 2000; Cleveland et al., 2001; Holzapfel et al., 2001). The lactic acid bacteria have the potential to inhibit growth of several food-borne pathogens and spoilage microorganisms, thereby improving the hygienic quality and shelf life of various food products (Mufandaedza et al., 2006).

1.1.3 Potential use of lactic acid bacteria in biopreservation

The lactic acid bacteria (LAB) comprise a large group of physiologically related Gram-positive non-sporulating cocci, coccobacilli or rods (Mutukumira et al., 2008). These include many species in the genera Lactobacillus, Leuconostoc, Pediococcus, Lactococcus and Streptococcus which grow in complex media where fermentable carbohydrates and higher alcohols are used as an energy source primarily to produce lactic acid only or to produce lactic acid, CO2 and ethanol in equimolar amounts (Carr et al., 2002; Mathara et al., 2004; Mutukumira et al., 2008).

Lactic acid bacteria are amongst the most important groups of microorganisms used in food fermentations and are responsible for many spontaneous food fermentation processes. They contribute to the taste and texture of fermented products and inhibit food spoilage bacteria by producing growth-inhibiting substances and large amounts of lactic acid (Chelule et al., 2010; Endo et al, 2011). As agents of fermentation, LAB are involved in making yoghurt, cheese, cultured butter, sour cream, sausage, cucumber pickles, olives and sauerkraut, but some species may spoil beer, wine and processed meats. Lactic acid bacteria are also found on non-fermented foods such as dairy products and meat products (De Vuyst and Deegest, 1999; Chelule et al., 2010).

Spontaneous fermentation processes take place in a mixed colony of microorganisms such as moulds, bacteria and yeasts. Lactic acid bacteria have been found to be the dominant microorganisms and therefore, lactic acid fermentation is considered to be the major contributor to the beneficial characteristics observed in most fermented foods (Caplice and Fitzgerald, 1999; Mufandaedza et al., 2006). This makes such foods to be potential sources for LAB isolates with crucial preservative properties (Rodriguez et al., 2003). At domestic conditions, dairy products have been prepared from cow milk, but also from sheep’s, goat’s and buffalo-cow’s milk. These include fermented milk, cheese, curds etc. From these products the species composition of lactic acid bacteria is more various and inconstant when compared with those of the trade/ commercial products (Stiles and Holzapfel, 1997; Todorov et al., 2007; Todorov, 2008). Lactic acid bacteria (LAB) are known to produce antimicrobial substances such as bacteriocins, which have great potential for use as natural preservatives (Stiles and Holzapfel, 1997). Bacteriocins are of interest to the food industry and their potential application in food biopreservation has prompted the need to isolate and identify best performing LAB species in terms of bacteriocin production and for their subsequent degree of antagonism to pathogenic and food spoilage micro-organisms (Silva et al., 2002).

Isolation and screening of microorganisms from naturally occurring processes has always been the most powerful means for obtaining useful cultures for scientific and commercial purposes (Togo et al., 2002). This certainly holds true for lactic acid bacteria (LAB), which are used throughout the world for manufacture of a wide variety of traditional fermented foods. Since they are involved in numerous food fermentations known to man for a millennia, it is assumed that most representatives of this group do not pose any health risks to man and are designated as GRAS (Generally Recognized As Safe) organisms (Carr et al., 2002; Chelule et al., 2010). In this regard, bacteriocins of LAB are also considered to be safe since they can also be degraded by proteases in the gastrointestinal tract (Cleveland et al., 2001).

Bacteriocins are extracellularly released peptides or protein molecules, with a bactericidal or bacteriostatic mode of action against closely related species mostly the Gram-positive bacterium (Jack et al., 1995; Nes et al., 1996; Cleveland et al., 2001). The inhibitory spectrum of some bacteriocins includes food spoilage and/or food-borne pathogenic microorganisms. The discovery of nisin produced by Lactococcus lactis, the first bacteriocin to be used on a commercial scale as a food preservative dates back to the first half of last century but research on bacteriocins of LAB has expanded in the last two decades, searching for novel bacteriocin producing strains from dairy, meat and plant products, as well as traditional fermented products (Delves-Broughton et al., 1996; Ennahar et al., 2000; Jack and Jung, 2000; De Vuyst et al., 2002).

Many bacteriocins have been isolated and characterised, but most lack a broad antimicrobial activity spectrum and a few are effective against Gram-negative pathogens hence the need to bioprospect for LAB with broad spectrum antimicrobial activity for use in biopreservation (Nettles and Barefoot, 1993; Settanni and Corsetti, 2007). The core of this study focuses on the isolation of LAB from spontaneously fermented milk (Amasi) and characterising them for bacteriocin production, subsequent antimicrobial activity and for biopreservative potential by investigating suitability in food systems. These LAB "wild" strains, in biotechnological aspect are perspective as antimicrobials and bacteriocin producers.

1.2 Project rationale

There are growing global safety and health concerns over the use of synthetic chemical and artificial food preservatives and additives such as nitrites and sulphites in foods since they have been found to be mutagenic and to trigger allergies and intolerances respectively. This has called for the need for more natural and safer approaches to food preservation and a lot of effort has been put towards moving away from the use of chemical food preservatives. Lactic acid bacteria and their metabolites are envisaged to be potential alternatives since they exhibit antimicrobial effects against spoilage and pathogenic bacteria. In addition, lactic acid bacteria are safer to consumers since they have a ‘generally recognized as safe’ (GRAS) status. Bacteriocins of LAB have a narrow antimicrobial activity spectrum and are particularly active against microbes closely related to the species producing them. In some cases, bacteriocins with a broad spectrum of activity have been discovered hence the need to bioprospect for LAB spp with a broad spectrum of antimicrobial activity. Furthermore, the indigenous genetic diversity of these microbes still remains largely unexploited hence the need for use molecular techniques in the characterisation of these LAB spp.

1.3 Objectives

1.3.1 Broad objective of the study

To determine the biopreservative potential of bacteriocins produced by lactic acid bacteria isolates in spontaneously fermented milk (Amasi).

1.3.2 Specific objectives

To isolate and identify lactic acid bacteria species in spontaneously fermented milk (Amasi).

To determine the antimicrobial activity of the lactic acid bacteria isolates cell free supernatants against pathogenic and food spoilage microbes.

To determine the biopreservative activity of the partially purified bacteriocins in a food system.

CHAPTER TWO: LITERATURE REVIEW

This study sought to isolate bacteriocin producing lactic acid bacteria from spontaneously fermented milk, to identify the bacteriocins with effective antimicrobial activity and to determine their suitability in selected food systems.

2.1 Fermented milk (Amasi)

LAB were first isolated from milk (Metchnikoff, 1908; Sandine et al., 1972; Carr et al., 2002) and have since been found in other foods and fermented products such as meat, milk products, vegetables, beverages and bakery products. Fermented milk (Amasi) is a product produced from unpasteurised bovine (cow’s) milk which is allowed to ferment spontaneously in an earthenware (clay) pot or gourd ("calabash") for two to three days at ambient temperature. The microbial flora responsible for the fermentation is derived from the air, raw milk and walls of the containers. After coagulation, the whey is drained through a plugged hole at the bottom of the container to leave behind the coagulated milk curd which is the fermented milk (Amasi) (Caplice and Fitzgerald, 1999; Beukes et al., 2001; Mutukumira et al., 2008).

Fermentation of milk is dominated by lactic acid bacteria, typically in the order of 108 CFU/ml (Beukes, et al., 2001; Mutukumira et al., 2008). Strains isolated from Amasi produced in South Africa were identified as members of Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Leuconostoc lactis, Leuconostoc citreum, Leuconostoc mesenteroides subsp. dextranicum and Lactobacillus delbrueckii subsp. lactis (Beukes, et al., 2001). Amasi produced with milk from Northern Zimbabwe contained strains of Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus casei subsp. casei and Lactobacillus casei subsp. pseudoplantarum (Feresu & Muzondo, 1990; Todorov et al., 2007; Mutukumira et al., 2008). In a similar product (kule naoto), produced by the Maasai tribe in Kenya, Lactobacillus plantarum was reported as the dominant species, among Lactobacillus fermentum, Lactococcus lactis subsp. lactis, Lactobacillus casei subsp. casei, Lactobacillus acidophilus and Leuconostoc mesenteroides subsp. mesenteroides (Mathara et al., 2004). It is from such a background that fermented milk is considered rich in diverse natural lactic acid bacteria which can be isolated for investigation as biopreservatives.

2.2 Lactic acid bacteria (LAB)

2.2.1 Common features of lactic acid bacteria

The lactic acid bacteria (LAB) comprise a large group of physiologically related Gram-positive, catalase negative and oxidase negative non-sporulating cocci, coccobacilli or rods. These include many species in the genera Lactobacillus, Leuconostoc, Pediococcus, Aerococcus, Carnobacterium, Enterococcus, Lactococcus and Streptococcus which grow in complex media where fermentable carbohydrates and higher alcohols are used as an energy sources primarily to produce lactic acid or to produce equimolar proportions of lactic acid, CO2 and ethanol (Fooks et al., 1999; Jay, 2000). Homofermentative LAB such as the genera Lactococcus and Streptococcus degrade hexoses mainly to lactate, whereas heterofermentative LAB such as genera Leuconostoc and Weissella degrade hexoses to lactate and other additional products such as acetate, ethanol, CO2, formate, or succinate (Stiles and Holzapfel, 1997; Gibson and Fuller, 2000; Holzapfel et al., 2001). All LAB grow anaerobically, but unlike most anaerobes, they can also grow in the presence of O2 as "aerotolerant anaerobes".

2.2.2 Identification of lactic acid bacteria

Lactic acid bacteria are nutritionally fastidious, requiring carbohydrates, amino acids, peptides, nucleic acid derivatives and vitamins for growth. Different species of lactic acid bacteria have adapted to grow under widely different environmental conditions and are widespread in nature i.e. in the soil, water, plants and animals (De Vuyst and Deegest, 1999). Lactic acid bacteria (LAB) can be identified through phenotypic techniques. Phenotypic methods alone are not always reliable and have a low taxonomic resolution for the identification of lactic acid bacteria species (Stiles and Holzapfel, 1997; Temmerman et al., 2004). Identification through morphological and biochemical characteristics is considered to be having a relatively poor reproducibility and a low taxonomic resolution. This implies that more accurate identification should combine conventional identification methods based on microbiological and biochemical features, along with genotypic methods (Berthier and Ehrlich, 1998; Togo et al., 2002; Temmerman et al., 2004; Ehrmann and Vogel, 2005).

Several molecular methods exhibiting various levels of discriminatory power have been developed for LAB and have been recently applied for more reliable identification (Torriani et al., 2001). These methods are 16S rRNA gene sequencing (De Vuyst et al., 2002; Catzeddu et al., 2006), randomly amplified (RAPD-PCR) analysis (De Vuyst et al., 2002; Catzeddu et al., 2006), PCR followed by denaturing gradient gel electrophoresis (DGGE) (Meroth et al., 2003) and by temperature gradient gel electrophoresis (TGGE) (Ferchichi et al., 2007).

Furthermore, molecular analysis has allowed for the identification of new species such as Lactococcus rossiae (Corsetti et al., 2005), Lactobacillus acidifarinae, Lactobacillus zymae (Vancanneyt et al., 2005), Lactobacillus hammesii (Valcheva et al., 2005), Lactobacillus nantensis (Valcheva et al., 2006) and Lactobacillus sigilinis (Aslam et al., 2006).

Direct identification based on PCR amplification of targeted genes, due to rapid and easy performance is a very useful method for identifying species of LAB. Several LAB species identification methodologies that use primers targeting different sequences have been reported in the literature such as the 16S rRNA- or 23S encoding genes (Temmerman et al., 2004), the more variable 16S-23S rRNA intergenic spacer region (ISR) (Berthier and Ehrlich, 1998), as well as the recA and (lactate dehydrogenase D) ldh D genes (Torriani et al., 2001). In addition to highly conserved primer binding sites, 16S rRNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid, accurate alternative to phenotypic methods of bacterial identification. Although it was originally used to identify bacteria, 16S sequencing was subsequently found to be capable of reclassifying bacteria into completely new species, or even genera. It has also been used to describe new species that have never been successfully cultured (Berthier and Ehrlich, 1998; Zoetendal et al., 1998).

The taxonomy of LAB based on comparative 16S ribosomal RNA (rRNA) sequencing analysis has revealed that some taxa generated on the basis of phenotypic features do not correspond with the phylogenetic relations. Molecular techniques, especially polymerase chain reaction (PCR) based methods, such as rep-PCR fingerprinting and restriction fragment length polymorphism (RFLP) as well as pulse-field gel electrophoresis (PFGE), are regarded important for specific characterization and detection of LAB strains (Gevers et al., 2001; Holzapfel et al., 2001). Recently, culture-independent approaches have been applied for the detection of intestinal microbiota (Zoetendal et al., 2002). Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) analysis of faecal 16S rDNA gene and its rRNA amplicons have shown to be powerful approaches in determining and monitoring the bacterial community (Zoetendal et al., 1998; Zoetendal et al., 2002).

Monoplex PCR strategies are suitable when some information is available on bacterial genes in order to choose the appropriate genus or species-specific primers. Multiplex PCR using simultaneous amplification of more than one locus have the advantage to detect several bacteria in a single reaction and was developed to offer more convenient method for the study of complex microbial community (Settanni and Corsetti, 2007).

2.2.3 Uses of lactic acid bacteria in foods

It is important to be noted that LAB are responsible for many spontaneous food fermentation processes and are also commonly found on non-fermented foods such as dairy products, meat products, crops and in humans and animals. They have been used as protective cultures in the food industry for the production of fermented foods, including dairy (yogurt and cheese), meat (sausages), fish, cereals and bread, beverages such as beer, fruit (malolactic fermentation processes in wine production) and vegetables (sauerkraut and silage) (Stiles and Holzapfel, 1997; Ross et al., 2002; Togo et al., 2002). Most LAB are considered as ‘generally recognised as safe’, GRAS and can be utilised to ensure safety, preserve food quality, develop characteristic new flavours and improve the nutritional qualities of food (Cleveland et al., 2001; Silva et al. 2002, Rodriguez et al. 2003).

Lactic acid bacteria exert antagonistic activity against many food contaminating microorganisms such as Listeria, Clostridium, Staphylococcus and Bacillus species as a result of the production of organic acids, hydrogen peroxide, diacetyl, inhibitory enzymes and bacteriocins (Ross et al., 2002; Gadaga et al., 2007; Settanni and Corsetti, 2007). Amongst these inhibitory substances, bacteriocins have received considerable attention during recent years for their possible use as biopreservatives in foods in place of chemical food preservatives. The application of bacteriocinogenic LAB with antimicrobial activity against spoilage and pathogenic microorganisms can thus be used to ensure the microbial safety of food products. Currently, many studies are being completed on the production and use of these bacteriocins as natural food biopreservatives to control spoilage and pathogenic bacteria. However, before a bacteriocin is considered for application in food industry, information on its antimicrobial spectrum, biochemical and genetic characteristics, effectiveness in food systems and regulatory implications should be known (Nettles and Barefoot, 1993; McAuliffe et al., 2001; Mufandaedza et al., 2006; Settanni and Corsetti, 2007).

Spoilage and pathogenic bacteria are responsible for the short shelf-life of food products and for causing food-borne illnesses such as bacterial food-poisoning and gastroenteritis. The bacterial pathogens that account for many of these cases include Salmonella, Campylobacter jejuni, Escherichia coli 0157:H7, Listeria monocytogenes, Staphylococcus aureus and Clostridium botulinum (Jay, 2000; Mufandaedza et al., 2006). Until recently, approaches to seek improved food safety have relied on the search for more efficient chemical food preservatives or on the application of more drastic physical treatments (e.g. high temperatures) (Dalton, 2002). Nevertheless, these types of solutions have many drawbacks: the proven toxicity of many of the commonest chemical food preservatives (e.g. nitrites), the alteration of the organoleptic and nutritional properties of foods and especially recent consumer trends in purchasing and consumption which demands for safe but minimally processed products without additives (Dalton, 2002; McCann et al., 2007). It is from such background that biopreservation has received much attention.

2.3 Application of lactic acid bacteria in biopreservation

2.3.1 Use of lactic acid bacteria in biopreservation

Biopreservation, which is defined as the extension of shelf life and enhanced safety of foods by the use of natural or controlled microbiota and/or antimicrobial compounds, is an innocuous and ecological approach to the problem of food preservation and has gained increasing attention in recent years (Caplice and Fitzgerald, 1999; Fooks et al., 1999; Cleveland et al., 2001). Consequently, certain lactic acid bacteria (LAB), with demonstrated antimicrobial properties commonly associated with foods, are being assayed to increase the safety and/or prolong the shelf life of foods. The antagonistic properties of LAB derive from competition for nutrients and the production of one or more antimicrobial active metabolites such as organic acids (lactic and acetic), hydrogen peroxide, and antimicrobial peptides (bacteriocins). The use of LAB bacteriocins is considered an integral part of hurdle technology. Their combined use allows most pathogenic and spoilage bacteria to be controlled and to extend inhibitory activity spectrum to such intrinsically resistant organisms such as the Gram-negative bacteria (Nettles and Barefoot, 1993; Delves-Broughton et al., 1996; Cleveland et al., 2001).

A bacterium that is a suitable candidate for use as a biopreservative does not necessarily have to ferment the food, but if conditions are suitable for microbial growth, then a biopreservative bacterium will compete well for nutrients with the spoilage and pathogenic bacteria in the food (Delves-Broughton et al., 1996). As products of its metabolism, it should also produce acids and other antimicrobial agents, particularly bacteriocins. In addition, biopreservative bacteria, such as lactic acid bacteria, must be harmless to humans (Ennahar et al., 2000; Kuipers et al., 2000; Jack et al., 1995).

To harmonize consumer demands with the necessary safety standards, traditional means of controlling microbial spoilage and safety hazards in foods are being replaced by combinations of innovative technologies that include biological antimicrobial systems such as lactic acid bacteria (LAB) and/or their bacteriocins (Nettles and Barefoot, 1993). The use of LAB and/or their bacteriocins, either alone or in combination with mild physicochemical treatments and low concentrations of traditional and natural chemical food preservatives, may be an efficient way of extending shelf life and food safety through the inhibition of spoilage and pathogenic bacteria without altering the nutritional quality of raw materials and food products. This has seen intensive investigation on LAB and their antimicrobial products to discover new bacteriocinogenic LAB strains that can be used in food preservation (Ross et al., 2002; Settanni and Corsetti, 2007).

2.3.2 Potential application of lactic acid bacteria bacteriocins

Bacteriocins are ribosomally-synthesized peptides with antimicrobial activity (Guder et al., 2000). They are produced by many different bacteria, yet bacteriocins produced by LAB have received particular attention due to their potential application in the food industry as natural preservatives (Rodriguez et al., 2003). A large number of bacteriocins have been isolated and characterized from lactic acid bacteria and have been considered to be potential food preservatives (Ennahar et al., 2000).

More than 300 different bacteriocins have been described for the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Enterococcus (Kuipers et al. 2000). The important ones are nisin, diplococcin, acidophilin, bulgarican, helveticins, lactacins and plantaricins (Nettles and Barefoot, 1993; Jack and Jung, 2000). The lantibiotic nisin which is produced by different GRAS Lactococcus lactis spp is the only bacteriocin that is applied as a food additive in at least 48 countries, particularly in processed cheese, dairy products and canned foods (Delves-Broughton et al., 1996; Ennahar et al., 2000). The FDA-approved bacteriocin, nisin is undoubtedly, the most extensively studied bacteriocin which has gained widespread applications in the food industry. Nisin is effective against food-borne pathogens such as L. monocytogenes and many other Gram-positive spoilage microorganisms. It is listed in Spain as E-234 and may also be cited as nisin preservative or natural preservative (Jack et al., 2000; Jack and Jung, 2000).

Strain AMA-K, isolated from naturally fermented milk produced in Gwanda, Kafusi area, Zimbabwe, was identified as Lactobacillus plantarum based on sugar fermentation reactions (API 50 CHL) and PCR with species-specific primers. The cell-free supernatant containing bacteriocin AMA-K inhibited the growth of Listeria innocua, Enterococcus faecalis, Escherichia coli and Klebsiella pneumoniae (Todorov et al., 2007; Todorov, 2008). Antimicrobial properties of lactic acid bacteria isolated from spontaneously fermented milk have also been shown against Escherichia coli and Salmonella enteritidis strains (Mufandaedza et al., 2006).

The bacteriocins of lactic acid bacteria have many attractive characteristics that make them suitable candidates for use as food preservatives and these include; the fact that the bacteriocins are protein in nature and can be inactivated by proteolytic enzymes of the gastrointestinal tract, they are non-toxic to laboratory animals tested and are generally non-immunogenic. In addition, the bacteriocins are inactive against eukaryotic cells (Cleveland et al., 2001; McAuliffe, 2001). The bacteriocins are generally thermo-resistant such that they can maintain antimicrobial activity after pasteurization and sterilization. Some bacteriocins have a broad bactericidal activity affecting most of the Gram-positive bacteria and some Gram-negative bacteria including various pathogens such as L. monocytogenes, Bacillus cereus, S. aureus and Salmonella (Guder, 2000; Mufandaedza et al., 2006). The genetic determinants for the bacteriocins are generally located in the plasmid; this facilitates genetic manipulation to increase the variety of natural peptide analogues with desirable characteristics. For these reasons, the use of bacteriocins has attracted considerable interest for use as biopreservatives in foods, which has led to the discovery of an ever-increasing potential of these peptides (Jack et al., 1995; Ross et al., 2002).

In general, the following features should be considered when selecting bacteriocin-producing strains for food applications; the producing strain should preferably have GRAS status, the bacteriocin should have a broad spectrum of inhibition that includes pathogens or else high specific activity, thermostability, beneficial effects and improved safety and no adverse effect on quality and flavour (Ross et al., 2002; Silva et al., 2002; Settanni and Corsetti, 2007).

The effectiveness and application of bacteriocins, particularly nisin, in food systems is still under review. It is now known that the activity of bacteriocins in foods can be influenced by many factors and these include physical conditions and chemical composition of food (e.g. pH, temperature, nutrients, etc). Nisin, for example, is 228 times more soluble at pH 2 than at pH 8 (Nettles and Barefoot, 1993; Delves and Broughton et al., 1996). The effectiveness of bacteriocin activity in food is negatively affected by the development of resistance to the bacteriocin by the pathogens and inadequate environmental conditions for the biological activity; higher retention of the bacteriocin molecules by food system components (e.g. fat); inactivation by other additives; slower diffusion and solubility and/or irregular distribution of bacteriocin molecules in the food matrix ((Nettles and Barefoot, 1993; Caplice and Fitzgerald, 1999; Ennahar et al., 2000; De Vuyst et al., 2002).

2.4 Types and classes of lactic acid bacteria bacteriocins

The bacteriocins produced by Gram-positive bacteria such as LAB are small peptides, 3-6 kDa up to 10 KDa in size (Nes et al., 1996). Depending on the producer organism and classification criteria, bacteriocins can be classified into several groups in which classes I and II are the most thoroughly studied (Ennahar et al., 2000; Jack and Jung, 2000; Cleveland et al., 2001; McAuliffe et al., 2001). Class I bacteriocins are termed lantibiotics, constitute a group of small peptides that are characterized by their content of several unusual amino acids (Guder et al., 2000). The class II bacteriocins are small, nonmodified, heat stable peptides and class III bacteriocins are large heat labile bacteriocins. A fourth class of bacteriocins is composed of an undefined mixture of proteins, lipids and carbohydrates. The existence of the fourth class was supported mainly by the observation that some bacteriocin activities obtained in cell free supernatant, exemplified by the activity of Lb. plantarum LPCO 10 were abolished not only by protease treatments, but also by glycolytic and lipolytic enzymes (Jimenez-Diaz et al., 1993; Ennahar et al., 2000; Guder et al., 2000; Jack and Jung, 2000).

Most bacteriocins of Gram positive bacteria are membrane active compounds that increase the permeability of the cytoplasmic membrane (Jack et al., 1995). They often show a much broader spectrum of bactericidal activity than the colicins (Gram negative bacteriocins which are produced by Escherichia coli). Bacteriocins fall within two broad classes; these are the lantibiotics (Jack et al., 1995) and the non lantibiotic bacteriocins (Nes et al., 1996). Nisin prevents clostridial spoilage of processed and natural cheeses, inhibits the growth of some psychrotropic bacteria in cottage cheese, extends the shelf life of milk in warm countries, prevents the growth of spoilage lactobacilli in beer and wine fermentations and provides additional protection against Bacillus and clostridial spores in canned foods (Delves and Broughton et al., 1996; Cleveland et al., 2001; Mufandaedza et al., 2006).

2.5 Applications of bacteriocin-producing lactic acid bacteria in different foods

2.5.1 Strategies of application of lactic acid bacteria in foods

The strategies for the application of LAB and/or their bacteriocins in foods are diverse and involve the following; inoculation of food with LAB (starter cultures or protective cultures) where bacteriocins are produced in situ, use of food previously fermented with the bacteriocin-producing strains as an ingredient in food processing (e.g. Nisaplin TM, Microgard TM, Alta TM 2341) or addition of purified or semi-purified bacteriocins in food. The purified bacteriocins are considered additives and always require express authorization for their use (Delves and Broughton et al., 1996; Caplice and Fitzgerald, 1999; Cleveland et al., 2001; Ross et al., 2002).

The potential of bacteriocin-producing LAB and their bacteriocins, especially Lactococci, Pediococci, Lactobacilli and Enterococci, to control undesirable microorganisms in food has been evaluated by a number of research groups (Jack et al., 1995; Kuipers et al., 2000). Although most bacteriocins have been isolated from food-associated LAB, they are not necessarily effective in all food systems. However, several bacteriocins certainly do have potential in food applications when used under the proper conditions (Ross et al., 2002; Settanni and Corsetti, 2007). The subsections 2.5.1.1, 2.5.1.2, 2.5.1.3, 2.5.1.4 below will cite examples where bacteriocin-producing cultures or their bacteriocins which show potential for future applications have been successfully employed to inhibit pathogenic microorganisms in a variety of food systems.

2.5.1.1 Applications in dairy products

Several researchers have demonstrated the effectiveness of nisin and/or nisin-producing strains against pathogenic bacteria such as Clostridium botulinum in cheese and against L. monocytogenes in cheeses such as Camembert, Ricotta and Manchego (Delves and Broughton et al., 1996). Other bacteriocins have been tested in milk and dairy products for example the bacteriocin pediocin AcH in milk, Cheddar and Munster cheeses acts against L. monocytogenes, S. aureus and E. coli O157:H7, the bacteriocin lacticin 3147 acts against undesirable LAB, L. monocytogenes and B. cereus in Cheddar, Cottage cheeses and yoghurt, and the bacteriocin enterocin AS-48 acts against B. cereus, S. aureus and L. monocytogenes in milk and Manchego cheese (Caplice and Fitzgerald, 1999; Ross et al., 2002; Mufandaedza et al., 2006).

2.5.1.2 Applications in meat products

When evaluating a bacteriocin-producing culture for sausage fermentation and/or biopreservation, one must bear in mind that meat and meat products are complex systems with a number of factors influencing microbial growth and metabolite production. Therefore, the influence of formula and fermentation technology on the performance of bacteriocin-producing cultures needs to be assayed (Nettles and Barefoot, 1993; Ross et al., 2002).

The most-studied bacteriocins in meat and meat products include nisin, enterocin AS-48, enterocins A and B, sakacin, leucocin A and especially pediocin PA-l/AcH, alone or in combination with several physicochemical treatments, modified atmosphere packaging, high hydrostatic pressure (HHP), heat and chemical food preservatives, act as an additional hurdle to control the proliferation of L. monocytogenes and other pathogens (Delves and Broughton et al., 1996; Cleveland et al., 2001). Furthermore, several bacteriocinogenic LAB have been used as bioprotective cultures for food manufacturing processes in attempts to control these pathogens (Ross et al., 2002).

The data available on the use of nisin in cured and fermented meat are equivocal. Compared to dairy products, nisin use in meat products has not been very successful because of its low solubility, irregular distribution, and lack of stability. Pediocin PA-l/AcH is more suitable for use in meat and meat products than nisin (Delves and Broughton et al., 1996; Ross et al., 2002).

2.5.1.3 Applications in vegetable products

Tests of bacteriocins in vegetable products include nisin in tinned vegetables and fruit juices, pediocin PA-1/AcH in salad and fruit juice and enterocin AS-48 against B. cereus in rice and vegetables and in fruit juices against other pathogens such as E. coli O157:H7, S. aureus and the spoilage bacterium Alicyclobacillus acidoterrestris (Delves and Broughton et al., 1996; Settanni and Corsetti, 2007).

2.5.1.4 Applications in fish

The deterioration of fresh fish is generally caused by Gram-negative microorganisms; however, in vacuum-packed fresh fish and seafood, pathogenic organisms such as Clostridium botulinum and L. monocytogenes can also cause problems (Settanni and Corsetti, 2007). Little work has been focused on incorporating live bacteriocin-producing cultures into these products or on the addition of concentrated bacteriocin preparations. The combination of nisin and Microgard (a natural antimicrobial produced by fermenting selected food grade cultures on dairy and/or sugar based ingredients) reduces the total aerobic bacteria populations of fresh chilled salmon, increases its shelf-life and also reduces the growth of inoculated L. monocytogenes in frozen-thawed salmon. The inhibition of L. monocytogenes has also been confirmed with other bacteriocin-producer cultures such as Carnobacterium divergens. It has also been demonstrated that the synergistic effect of the combination of lactic acid, sodium chloride, and/or nisin in rainbow trout controls pathogenic microorganisms in fish (Delves and Broughton et al., 1996; Nes et al., 1996; Ross et al., 2002).

2.6 Hurdle technology for food preservation

2.6.1 Hurdle concept and technology

The hurdle concept was introduced by L. Leistner in 1978 and stated that the microbial safety, stability, sensorial and nutritional qualities of foods are based on the application of combined preservative factors (called hurdles) to which microorganisms present in the food are unable to overcome (Leistner, 1978; Jay, 2000). Hurdle technology thus refers to the combination of different preservation methods and processes to inhibit microbial growth. An intelligent application of this technology requires a better understanding of the occurrence and interaction of different hurdles in foods as well as the physiological responses of microorganisms during food preservation. Using an adequate mix of hurdles is not only economically attractive; it also serves to improve not only microbial stability and safety, but also the sensory and nutritional qualities of a food (Leistner, 1978; Leistner, 1994; Dalton, 2002; Jay, 2000; Ross et al., 2002).

Novel concept, multitarget food preservation has emerged in relation to hurdle technology based on the proven fact that, at times, different hurdles in food have not just an additive effect on microbial stability, but a synergistic one (Ross et al., 2002). This approach may afford a nonaggressive but more effective preservation of foods by the application of multiple soft treatments that disturb homeostasis and metabolic exhaustion and avoid stress reactions by bacteria. In practical terms, this means that it is more effective to employ different small-intensity preservation factors than one large-intensity preservation factor (Leistner, 1994; Jay, 2000; Kuipers et al., 2000).

2.6.2 Applications of bacteriocins in hurdle technology

It is recommended to use bacteriocins combined with other preservation methods to create a series of hurdles during the manufacturing process to reduce food spoilage caused by microorganisms (Delves and Broughton et al., 1996; Cleveland et al., 2001). In fact, it has been proven that the application of chemical food preservatives, physical treatments (heat) or new mild non-thermal physical methods (pulsed electric field, high hydrostatic pressure (HHP), vacuum or modified atmosphere packaging) which increase the permeability of cell membranes, positively affects the activity of many bacteriocins (Settanni and Corsetti, 2007). Combined treatments of bacteriocins with selected hurdles affecting outer-membrane (OM) permeability increase the effectiveness of some LAB bacteriocins against Gram-negative cells which are generally resistant. The growth of Gram-negative pathogens such as E. coli O157:H7 and Salmonella species can also be controlled when metal chelators such as EDTA, sodium tripolyphosphate (STPP) or physical methods such as heat and HHP are used in combination with bacteriocins (Delves and Broughton et al., 1996; Cleveland et al., 2001; Dalton, 2002).

2.7 Chemical food preservatives

2.7.1 Uses and limitations of chemical food preservatives

Foods in their natural, raw state perish quickly. Chemical food preservatives are added to foods to extend their shelf-life and maintain their appearance by retarding and preventing their deterioration, according to the U.S. Food and Drug Administration (FDA, 2002; IFIC and FDA, 2004). While the Centre for Science in the Public Interest concedes that many chemical food preservatives are safe for consumption, some have been shown to produce harmful effects on health. Even though the FDA regulates the use of preservatives, some chemicals have shown tumour growth in laboratory animals and breathing issues in humans. A high intake of certain additives has been associated with a heightened risk of heart disease, stroke, cancer and diabetes (Parke and Lewis, 1992; Rodford, 1997; Scotter and Castle, 2004).

Chemical food preservatives are used for different purposes in many foods. The U.S. Food and Drug Administration (FDA) requires that, when an approved chemical preservative is added to a food, the preservative's common name and function must be included in the ingredient list. Common functions for chemical food preservatives in food include colour retention, flavour protection, mould inhibition, spoilage retardation and general preservation (FDA, 2002; IFIC and FDA, 2004; Daniells, 2006; FDA, 2009). Some examples of chemical food preservatives are stated in the subsections 2.7.1.1, 2.7.1.2, 2.7.1.3, 2.7.1.4, 2.7.1.5 below together with their uses and health effects.

2.7.1.1 Sulphites

Sulphites are used in light-coloured foods such as golden raisins or mashed potato flakes to retain their light appearance. They are also used to inhibit bacterial growth in wine and on grapes. Dried and dehydrated foods often contain sulphites (Chemical and Engineering News, 2002; Larson-Duyff, 2002). Examples of sulphites are; sodium sulphite, sulphur dioxide, Sodium Metabisulphite (FDA, 2002; FDA, 2009).

The FDA estimated that, more than one million asthmatics are sensitive or allergic to sulphites. Symptoms related to sulphite consumption include trouble breathing, hives, stomach ache and anaphylactic shock. A 1986 FDA ruling mandated food packagers to list all chemical food preservatives, including sulphites, on product packaging. That same year the agency also banned the use of sulphites on salad bar foods in restaurants and fresh produce displays at grocery stores. The FDA cautions asthmatics to bring their inhalers if they go out to eat (FDA, 2002; IFIC and FDA, 2004; Daniells, 2006; FDA, 2009).

2.7.1.2 Butylated hydroxyanisole (BHA)

BHA is a food additive commonly found in foods such as cereals, gum, potato chips and vegetable oil. It is an antioxidant that curbs spoilage in foods high in fat (Wurtzen, 1990; Chung, 1999). When given in high dosages, BHA is carcinogenic and causes tumours in laboratory test rats, fish, hamsters and mice. BHA is "reasonably anticipated to be a human carcinogen." The FDA still, has approved the use of BHA as a food additive despite the research that proves it could be harmful (Chung, 1999; Kirlin et al., 1999).

2.7.1.3 Nitrates and Nitrites

Sodium nitrate and nitrite are food additives used to colour and preserve meats and to improve on their safety by reducing the growth of botulism-causing bacteria. Nitrates are harmless, but when they are converted into nitrites in the body they react with amino acids and can form nitrosamines, which are cancer-causing chemicals (Daniells, 2006). According to the Centre for Science in the Public Interest, some studies have shown that eating cured meats and nitrites in children, pregnant women and adults may cause these groups of people to become more susceptible to certain types of cancer (Scotter and Castle, 2004; Daniells, 2006).

Nitrites occurring in cured meats including sausages, bacon and hot dogs have been found to increase the risk of lung disease, according to scientists from Columbia University (Daniells, 2006; FDA, 2009). The study linked regular consumption of cured meats to a 71 percent higher risk of chronic obstructive pulmonary disease (COPD). The disease, which causes excessive mucus production in the lungs, has no cure and can cause death. The study found an increased risk of the disease among people who ate meat treated with nitrites 14 times each month. Doctors have traditionally linked COPD to smokers, but Columbia assistant professor R. Graham Barr stated that 10 percent of people who die from the disease have never smoked (Rodford, 1997; Daniells, 2006).

2.7.1.4 Sodium Benzoate

Sodium benzoate is commonly used in fruit juices, salad dressings, jams and pickles (FDA, 2002; FDA, 2009). According to the Centre for Science in the Public Interest, people can safely ingest the preservative sodium benzoate except when used in combination with ascorbic acid, also known as Vitamin C. In an acidic substance, the two chemicals can mix and form benzene, which the centre links to cancers such as leukaemia. The risk is small, but the centre requires people not be exposed to any risk (Parke and Lewis, 1992; Scotter and Castle, 2004).

The Food and Drug Administration has recommended that food and beverage manufacturers refrain from using sodium benzoate in conjunction with ascorbic acid, but it hasn't banned the combination (Rodford, 1997; Scotter and Castle, 2004).

2.7.1.2 Natamycin

Natamycin is a chemical additive added to meats and cheeses as an antimicrobial agent (FDA, 2002; FDA, 2009). It kills bacteria and prevents mould growth, while it causes dangerous effects of nausea, vomiting, diarrhoea and skin inflammation (Chemical and Engineering News, 2002; Larson-Duyff, 2002).

It is from such a background that some chemical food preservatives have been implicated in the following; circulatory health problems (strokes, heart attacks and heart diseases), cancer and diabetes. Based on this overview, there is a need to identify and characterise LAB and bacteriocins that can be suitably applied in the food industry to minimise the use chemical food preservatives, part of which is an investigation of this research.