Bacteriocins: Biological tools for bio-preservation and shelf-life extension

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International Dairy Journal 16 (2006) 1058–1071 www.elsevier.com/locate/idairyj

Review

Bacteriocins: Biological tools for bio-preservation and shelf-life extension Lucy H. Deegana, Paul D. Cottera, Colin Hilla,c,, Paul Rossb,c a

Microbiology Department, University College Cork, Cork, Ireland b Moorepark Biotechnology Centre, Teagasc, Ireland c Alimentary Pharmabiotic Centre, Cork, Ireland Received 28 April 2005; accepted 19 October 2005

Abstract The lactococcal bacteriocin named nisin (or group N inhibitory substance) was first marketed in England in 1953 and since then has been approved for use in over 48 countries. The successful development of nisin from an initial biological observation through regulatory approval to commercial application is a model that has stimulated significant resurgence in bacteriocin research in recent years, but similar success is yet to be repeated on the same scale. In spite of this sobering fact, we remain convinced that bacteriocins can be exploited in foods in a variety of imaginative and commercially significant applications in bio-preservation and shelf-life extension. However, in order to fully realise this potential, it is necessary to understand the biology of bacteriocins; in particular, to elucidate structure–function relationships, production, immunity, regulation and mode of action. In this paper, we will discuss some of the advances, made mainly with other lactococcal bacteriocins, in improving food safety, food quality and preventing food spoilage. r 2006 Elsevier Ltd. All rights reserved. Keywords: Spoilage; Pathogen; Inhibitor; Food grade

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of lactic acid bacteria bacteriocins . . . . 2.1. Class I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Class II . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Bacteriocin production and export . . . . . . . . . 2.4. Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Mode of action . . . . . . . . . . . . . . . . . . . . . . 3. Using bacteriocins to improve food safety . . . . . . . . 3.1. Bacteriocin preparations as ingredients. . . . . . 3.2. Bacteriocin-producing cultures. . . . . . . . . . . . 4. Bacteriocins in combination with additional hurdles . 5. Bacteriocins for improving food quality and flavour . 6. Conclusion/future prospects . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author. Microbiology Department, University College Cork, Cork, Ireland. Tel.: +353 21 4901373; fax: +353 21 4903101.

E-mail address: [email protected] (C. Hill). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.10.026

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1. Introduction Bacterial fermentation of perishable raw materials has been used for centuries to preserve the nutritive value of food and beverages over an extended shelf-life. In a number of food fermentations, the key event is the conversion of sugars to lactic acid by lactic acid bacteria (LAB, which include the genera Lactococcus, Streptococcus, Lactobacillus and Pediococcus, among others). Lactic acid and other end products of LAB metabolism, including hydrogen peroxide, diacetyl, acetoin and other organic acids, act as bio-preservatives by altering the intrinsic properties of the food to such an extent as to actually inhibit spoilage microorganisms (Fig. 1). While the role of these metabolic end products has long been appreciated, the contribution of LAB-derived bacteriocins may frequently have been overlooked. The widespread ability of LAB to produce bacterocins implies an important biological role maintained over many generations and the precise nature of this role has been the subject of intensive research in recent times. Bacteriocin production could be considered as advantageous to the producer as, in sufficient amounts, these peptides can kill or inhibit bacteria competing for the same ecological niche or the same nutrient pool (Fig. 1). This role is supported by the fact that many bacteriocins have a narrow host range, and are likely to be most effective against related bacteria competing for the same scarce resources. Although bacteriocins Nutrient

Acid

Producer bacterium

H2O2, Diacetyl, other metabolites Bacteriocin

Target bacterium

Fig. 1. Lactic acid bacteria can produce a number of antimicrobial compounds, but bacteriocins are often the most potent inhibitors of related bacteria. A bacteriocin producer (top) can be identified by the zones of inhibition produced in a lawn of sensitive indicator cells.

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are produced by many Gram-positive and Gram-negative species, those produced by the LAB are of particular interest to the food industry (Nettles & Barefoot, 1993), since these bacteria have generally been regarded as safe (GRAS status). Furthermore, as the majority of bacteriocin-producing LAB are natural food isolates, they are ideally suited to food applications. Although various methods other than bacteriocin production are employed for the preservation of food/beverages, an increasingly, health conscious public may seek to avoid foods that have undergone extensive processing or which contain chemical preservatives. Therefore, the production of bacteriocins by LAB is not only advantageous to the bacteria themselves but could also be exploited by the food industry as a tool to control undesirable bacteria in a food-grade and natural manner, which is likely to be more acceptable to consumers. To date, the only commercially produced bacteriocins are nisin (or group N inhibitory substance), produced by Lactoccocus lactis, and pediocin PA-1, produced by Pediococcus acidilactici, marketed as NisaplinTM (product description-PD45003-7EN; Danisco, Copenhagen, Denmark) and ALTATM 2431 (Kerry Bioscience, Carrigaline, Co. Cork, Ireland), respectively. Nisin is used in over 48 countries, has Food and Drug Administration approval and NisaplinTM is sold as a natural food protectant. Nisin has been shown to be effective in a number of food systems, inhibiting the growth of a wide range of Grampositive bacteria, including many important foodborne pathogens such as Listeria monocytogenes (Tagg, Dajani, & Wannamaker, 1976). It is used predominantly in canned foods and dairy products and is especially effective when utilised in the production of processed cheese and cheese spreads where it protects against heat-resistant sporeforming organisms such as those belonging to the genera Bacillus and Clostridium. This has particular significance in the case of preventing contamination with Clostridium botulinum as there can be serious repercussions resulting from toxin formation by this species. There are also a number of yet-to-be commercialised bacteriocins reported in the scientific literature, such as lacticin 3147 and lacticin 481, which have shown the potential for exploitation as natural food bio-preservatives and flavour enhancers. Intensive studies to elucidate the fundamental structural and functional properties of bacteriocins have been valuable. However, applied research carried out with a view to determining the impact of food components and processing methods on the structure, solubility and activity of bacteriocins is of extreme importance when considering potential food applications. In addition to the ongoing study of existing bacteriocins, the discovery of new bacteriocins, combined with imaginative developments regarding their application, can only be beneficial and will increase the likelihood that the use of these peptides can be optimised to fulfil their potential in food applications. This review will focus mainly on evaluating both non-commercialised LAB bacteriocins and those that are

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already used within the food sector to improve food safety and quality. 2. Classification of lactic acid bacteria bacteriocins Bacteriocins can be distinguished from peptide antibiotics in that they are ribosomally synthesised rather than secondary metabolites (Hurst, 1981) and the genes responsible for production and immunity are generally found clustered in operons (Jack, Bierbaum, Hiedrich, & Sahl, 1995). While as many as five classes of bacteriocins have been proposed (Cotter, Hill, & Ross, 2005a; Klaenhammer, 1993; Nes et al., 1996), the majority fall into Classes I and II. As these bacteriocins feature predominantly in this review, the following section will describe their biosynthesis, export, immunity and mode of action. 2.1. Class I Class I LAB bacteriocins (reviewed by Chatterjee, Paul, Xie, & van der Donk, 2005) are small ðo5 kDaÞ, heatstable peptides that are extensively modified after translation, resulting in the formation of characteristic thioether amino acids lanthionine (Lan) and methyllanthionine (MeLan) (Jack & Sahl, 1995). These arise via a two-step process: firstly, gene-encoded serine and threonine can be subject to enzymatic dehydration to give rise to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively. Subsequently, thiol groups from neighbouring cysteines attack the double bond of Dha or Dhb yielding either Lan or MeLan, respectively. This condensation between two neighbouring residues results in the formation of covalently closed rings within the formerly linear peptide, conferring both structure and functionality (Ingram, 1969). The LAB lantibiotics were initially divided into two subclasses based on structural similarities. Subclass Ia included relatively elongated, flexible and positively charged peptides and they generally act by forming pores in the cytoplasmic membranes of sensitive target species. The prototypic lantibiotic nisin is a member of this group. Subclass Ib peptides are characteristically globular, more rigid in structure and are either negatively charged or have no net charge (Klaenhammer, 1993). They exert their action by interfering with essential enzymatic reactions of sensitive bacteria. For example, the non-LAB IIb-type lantibiotic mersacidin interferes with cell wall biosynthesis by binding to the peptidoglycan precursor lipid II (Brotz, Bierbaum, Leopold, Reynolds, & Sahl, 1998). More recently, the subclassification of lantibiotics on the basis of mode of action has been somewhat confused by the discovery that nisin is active through dual mechanisms. In addition to pore formation, it can also inhibit cell wall biosynthesis through the binding of lipid II—a step which in fact probably precedes pore formation (Breukink et al., 1999; Weidemann et al., 2001). Furthermore, the existence of a number of two-component lantibiotics that function through the combined activity of two peptides has further

confused the classification of these peptides. To reflect this considerable diversity within the group, the lantibiotics have been subclassified into 11 subgroups based on the alignment of the unmodified structural peptides (Cotter, Hill, & Ross, 2005b). These are named to reflect the prototypic lantibiotic in each case, i.e. nisin, epidermin, streptin, pep5, lacticin 481, mersacidin, LtnA2, cytolysin, lactocin S, cinnamycin and sublancin. 2.2. Class II Class II bacteriocins are also small ðo10 kDaÞ and heat stable but do not contain Lan residues. A number of different subclasses of Class II bacteriocins have been suggested (Diep & Nes, 2002; Nes et al., 1996; van Belkum & Stiles, 2000), but their heterogeneous nature makes subclassification difficult. Two subclasses are common to all classification systems: the subclass IIa pediocin-like (or Listeria-active) and the subclass IIb two-component bacteriocins. Among the former group, pediocin AcH/ PA-1 was the first to be extensively studied. Members of the pediocin-like peptides show a high degree of homology (40–60%) when the corresponding amino acid sequences are aligned. In particular, the cationic N-terminal domain contains the homologous region YGNGVXCXXXXCXV or ‘‘pediocin box’’ with the two cysteine residues forming a disulphide bridge (Eijsink, Skeie, Middelhoven, Brurberg, & Nes, 1998). Class IIb refers to two-component bacteriocins that require two peptides to work synergistically (Nissen-Meyer, Holo, Harvastein, Sletten, & Nes, 1992). Singly these peptides have little or no activity and there appears to be no sequence similarities between complementary peptides. Both lactacin F and lactococcin G are members of this subgroup. 2.3. Bacteriocin production and export The genes responsible for bacteriocin production are frequently associated with mobilisable elements, e.g. on the chromosome in association with transposons or on plasmids. As is the case for most bacteriocins, the lantibiotics are initially synthesised with an N-terminal leader peptide. In general, the pre-peptide (generically designated LanA) is modified by the action of other proteins encoded by the bacteriocin gene cluster before export. The formation of Lan bridges is also enzyme mediated. In some cases (e.g. nisin), a LanB protein performs the initial dehydrations of serines and threonines, while LanC is required for Lan formation (reviewed by McAuliffe, Ross, & Hill, 2001). Alternatively, some lantibiotics (e.g lacticin 481) have a single enzyme, LanM, which performs both of these tasks. In the special case of two-component lantibiotics (e.g. lacticin 3147), two LanM enzymes may be required, one for each peptide (McAuliffe, Hill, & Ross, 2000, McAuliffe et al., 2001). Export of lantibiotics is through a dedicated ATP-binding cassette (ABC) transporter, LanT, consisting of two domains, one

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which acts to recognise the pre-peptide and one that hydrolyses ATP to fuel export (Higgins, 1992; Walker, Saraaste, Runswick, & Gay, 1982). For subclass Ia lantibiotics, a serine protease (LanP) removes the leader peptide before or after export from the cell (GeiXler, Gotz, & Kupke, 1996; Meyer et al., 1995), whereas the leaders of subclass Ib lantibiotics, which contain a double glycine cleavage site, are cleaved concomitantly with export by LanT. The genes required for the processing and export of Class II bacteriocins differ somewhat from those for lantibiotics due to the absence of post-translational modifications. For the purpose of this review, the genes associated with pediocin PA-1/AcH production, i.e. pedA, B, C and D, are used as an example. Like the majority of Class II bacteriocins, pediocin PA-1/AcH is synthesised as a pre-peptide, with a leader peptide containing a conserved double glycine cleavage site, encoded by pedA. PedB is responsible for producer self-protection and its role is discussed further below. The ABC transporter (encoded by pedD) along with an essential accessory protein (PedC) are involved in cleavage of the leader in combination with export of the bacteriocin (Havarstein, Diep, & Nes, 1995). 2.4. Immunity Self-evidently, bacteriocin producers must protect themselves from the action of their bacteriocins. This is accomplished by the production of dedicated immunity proteins. Two types of immunity have been described for lantibiotics, one reliant on a specific immunity protein, LanI, while the other depends on a separate dedicated multi-component ABC transporter (LanEFG). Some lantibiotic clusters have only the single immunity protein, as is the case for Pep5 (Reis & Sahl, 1991), whereas others, such as nisin, possess both mechanisms (Kuipers, Beerthuyzen, Siezen, & de Vos, 1993; Seigers & Entian, 1995). It is thought that the LanI protein antagonises the action of lantibiotics, somehow preventing the insertion of the lantibiotic in the cell membrane, while it has been suggested that the ABC transporter works by expelling the antimicrobial peptide from the cell or the membrane (Peschel & Gotz, 1996; Stein, Heinzmann, Dusterhaus, Borchert, & Entian, 2005). Class II bacteriocins generally have a single-cell membrane-associated immunity protein that provides complete immunity (Quadri et al., 1995). Based on the study of carnobacteriocin B2 (Class IIa), it appears that the majority of the immunity protein is located inside the cytoplasm and that it may interact with another protein, perhaps a receptor, at the cytoplasmic side of the cell membrane (Quadri et al., 1995; Sprules, Kawulka, & Vederas, 2004). 2.5. Mode of action While LAB bacteriocins can work via different mechanisms to exert an antimicrobial effect, the cell envelope is generally the target. Nisin has a broad spectrum of activity

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against most Gram positives and also inhibits the outgrowth of Bacillus spp. and Clostridium spp. spores (Gross & Morell, 1971), while in contrast lactococcin A is narrow spectrum, being active only against other lactococcal strains (Stoddard, Petzel, van Belkum, Kok, & McKay, 1992). The initial electrostatic attraction between the target cell membrane and the bacteriocin peptide is thought to be the driving force for subsequent events. While many bacteriocins have been shown to induce pore formation in sensitive microorganisms, the mechanism of action of nisin has been studied in greatest detail. Nisin forms pores that disrupt the proton motive force and the pH equilibrium causing leakage of ions and hydrolysis of ATP resulting in cell death (Benz, Jung, & Sahl, 1991; Sahl, Kordel, & Benz, 1987). Other lantibiotics that also form pores include lacticin 3147, Pep5, subtilin and epidermin (Brotz et al., 1998, Kordel, Benz, & Sahl, 1988, McAuliffe et al., 1998; Schuller, Benz, & Sahl, 1989). However, it has long been recognised that nisin also interferes with cell wall biosynthesis (Linnett & Strominger, 1973; Reisinger, Seidel, Tschesche, & Hammes, 1980). It has now been established that this phenomenon is mediated by the ability of nisin to bind lipid II, a peptidoglycan precursor, thus inhibiting cell wall biosynthesis. Such binding is also intrinsic to the ability of nisin to form pores. The possession of dual mechanisms of action renders nisin active at nM concentrations (Breukink et al., 1999). As pointed out previously, mersacidin also binds to lipid II, although at a different position to that of nisin, and does not exhibit pore-forming activity (Brotz et al., 1998). Class II bacteriocins predominantly act by forming pores, causing dissipation of the cell membrane, depletion of intracellular ATP and leakage of amino acids and ions. The mannose permease EIIman of the phosphotransferase t system, a sugar uptake/phosphorylation system, has been suggested as a putative receptor for Class IIa bacteriocins based on a number of lines of evidence. This theory was initially fuelled by the existence of spontaneous bacteriocin-resistant mutants displaying reduced expression of certain subunits, which constitute EIIman , i.e. subunit IIAB t was found not to be produced in a leucocin A-resistant L. monocytogenes (Ramnath, Beukes, Tamura, & Hastings, 2000), while mutation of mptD, encoding another membrane subunit of EIIman , in a strain of L. monocytogenes t also led to resistance to mesentericin Y105 (Dalet, Cenatiempo, Cossart, Hechard, & the European Listeria Genome Consortium , 2001). However, the role of EIIman t as a target for Class IIa bacteriocins has not been fully elucidated. 3. Using bacteriocins to improve food safety Bacteriocins have often been mooted as potentially valuable biological tools to improve the food safety and reduce the prevalence of foodborne illnesses. It is usually suggested that bacteriocins should not be used as the primary processing step or barrier to prevent the growth or

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survival of pathogens, but rather that they could provide an additional hurdle to reduce the likelihood of foodborne disease. There already exist many control measures within the food industry to prevent or minimise bacterial contamination, including good manufacturing practices, effective sanitation and hygiene measures with respect to raw materials, the food plant, the food products, the food processing personnel (Moberg, 1989) and other basic fundamentals of an effective Hazard Analysis Critical Control Point (HACCP) programme. These measures facilitate the identification, evaluation and control of food safety hazards (National Advisory Committee on Microbiological Criteria for Foods (NACMCF), 1998). However, despite these precautions, foodborne outbreaks do occur alarmingly frequently. L. monocytogenes is of particular concern to the food industry and susceptible consumers that include pregnant women, infants, immunocompromised individuals and the elderly. L. monocytogenes is ubiquitous in the environment and is extremely resilient, surviving refrigeration temperatures and high salt concentrations. Statistics for Wales and England show 194 hospital cases of listeriosis in 2000, of which 68 resulted in death (Adak, Long, & O’Brian, 2002). Within the United States, the microorganism is estimated to be responsible for 2500 cases, resulting in 500 deaths annually (www.cdc.gov/ ncidod/dbmd/diseaseinfo/listeriosis_t.htm). A policy of zero tolerance in ready to eat foods has been implemented in the United States for L. monocytogenes, but outbreaks continue to occur. A recent outbreak associated with contamination of hot dogs resulted in 101 cases and 21 deaths (www.cdc.gov/ncidod/disease/foodborn/lister.htm) and the recall of 500; 000 lbs of contaminated hot dogs and meats (Donnelly, 2001). This highlights the considerable burden this food pathogen can place on consumers and the food industry. It would seem particularly important to provide an additional hurdle in food to prevent such outbreaks, and bacteriocins would be an economically feasible option. L. monocytogenes is not the only concern, there exists a substantial list of food pathogens that result in foodborne illness every year, including many Gram-negative pathogens such as Escherichia coli VTEC 0157, Campylobacter and Salmonella among others (Adak et al., 2002). Although the nature of the Gram-negative cell wall restricts the activity of LAB bacteriocins, bacteriocins may be used in combination with other treatments, such as high hydrostatic pressure (HHP), to increase their effectiveness. Thus, bacteriocins may be best applied when providing an extra obstacle to prevent the growth of pathogenic and spoilage bacteria, especially in situations where contaminationcould occur post-production. There are at least three ways in which bacteriocins can be incorporated into a food to improve its safety (Fig. 2), i.e., using a purified/semi-purified bacteriocin preparation as an ingredient in food, by incorporating an ingredient previously fermented with a bacteriocin-producing strain, or by using a bacteriocin-producing culture to replace all or

Live culture

Fermented food

Ingredient

Non-fermented food

Fig. 2. Bacteriocins can be incorporated directly into fermented foods by using a bacteriocin producer as a starter or adjunct culture. Alternatively, the producer can be used to make a food-grade fermentate, which can be dried to make a powdered ingredient. This powder can be then incorporated into either fermented or non-fermented foods.

part of a starter culture in fermented foods to produce the bacteriocin in situ. The use of purified bacteriocins is not always attractive to the food industry, as in this form they may have to be labelled as additives and require regulatory approval. Nisin is utilised as an additive and was assigned the number E234 (EEC, 1983 EEC commission directive 83/463/EEC). The two other alternatives (fermented ingredient/starter culture) do not require regulatory approval or preservative label declarations. These options are frequently regarded as more attractive routes through which bacteriocins can be incorporated into a food. These two options are described in greater detail below and a number of representative examples, primarily relating to the use of nisin, pediocin PA-1/AcH and lacticin 3147, are described. 3.1. Bacteriocin preparations as ingredients Bacteriocins can be incorporated into foods as a concentrated, though not purified, preparation made with food-grade techniques. For example, NisaplinTM is prepared by initially fermenting non-fat milk with a nisinproducing Lactococcus lactis. The resulting fermentate is subsequently concentrated and separated, spray dried and milled to yield small particles. The end product (consisting of nisin ð1
106 IU g1 Þ, 74.4% NaCl, 23.8% denatured milk solids and 1.7% moisture) has found many applications in food processing and a large body of data on the use of nisin in food systems has been generated over the past 50 years. While nisin has been found to be extremely effective as an additive to prevent spoilage and increase shelf-life in a number of foods (Chen & Hoover, 2003; Choi & Park, 2000; Cutter & Siragusa, 1995; Davies et al., 1999) its effectiveness has been more variable with respect to other applications (Bell & De Lacy, 1985; Jones, 1974; Jung, Bodyfelt, & Daeschel, 1992; Scott & Taylor, 1981a,b). The fact that situations have been described where nisin is

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ineffective highlights why it would be attractive to explore the use of other bacteriocins. When investigating novel candidates, there are many considerations that will determine their usefulness in food systems. One of the most significant criteria is an ability to withstand thermal processing. Thermal processing is used extensively within the food manufacturing process and can have adverse effects on the bio-active capability of a bacteriocin, potentially rendering it less effective. The chemical and physical properties of a food, e.g. pH, and fat content, can also have a significant role in the suitability of a particular bacteriocin. This is exemplified by the fact that nisin is very active at acid pH, but loses activity above pH 7 (DelvesBroughton, Blackburn, Evans, & Hugenholtz, 1996), whereas another lactococcal lantibiotic, lacticin 3147, retains activity at neutral pH and significantly is particularly heat stable at low pH (McAuliffe et al., 1998). It should also be noted that nisin is generally not as effective in the preservation of meat as it is in dairy products. This is thought to be due to interference by meat components such as phospholipids that limit its activity, especially where there may be a high-fat content. The corollary is also true

Infant formula

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in that nisin has been shown to be effective in the preservation of Bologna-type sausages with a lower fat content (Davies et al., 1999). The Class IIa bacteriocin pediocin PA-1/AcH has received attention due to its ability to protect fresh and fermented meat from contamination by L. monocytogenes. Its use seems especially appropriate when combined with modified atmosphere packaging (MAP). In fact, the use of MAP to package meat under higher levels of carbon dioxide to reduce the incidence of Gram-negative spoilage bacteria can be especially conducive towards bacteriocin production as it favours the proliferation of non-spoilage LAB that can contribute to increasing the shelf-life of refrigerated meats (Hitchener, Egan, & Rogers, 1982; Shaw & Harding, 1984; Stiles & Hasting, 1991). The LAB microflora found in chilled meat predominantly consists of lactobacilli and pedicocci, and significantly many pediocin PA-1/AcH-producing strains have been isolated from meat (Bhunia, Johnson, & Ray, 1988; Gonzalez & Kunka, 1987; Rodriguez et al., 1997). Pediocin PA-1/AcH has also been tested as a bio-active ingredient in various dairy systems, thus demonstrating its effectiveness over a range of

Cottage cheese

Yoghurt

14 12

Survival (%)

10 8 6 4 2 0

100

200

0

100

200

0

75

150

Time (min)

(a) Cottage cheese

Surface of mould cheese

Log cfu.mL-1

8 7 6 5 4 3 0

(b)

2

4

6 0 Time (days)

1

2

3

Fig. 3. Control of Listeria monocytogenes using (a) lacticin 3147 powder as an ingredient at 0 (black), 5 (grey) and 10% (white), and (b) a lacticin 3147producing culture (black) and non-producing culture (white).

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different pH and in varying levels of fat (for a review see Rodriguez, Martinez, & Kok, 2002). Lacticin 3147 has also been produced and tested as a bio-preservative. A producing strain, Lc. lactis DPC3147, was used to ferment reconstituted demineralised whey (10% solids), which was pasteurised, concentrated and spray dried to produce a bioactive lacticin 3147 powder (Morgan, Galvin, Kelly, Ross, & Hill, 1999). This powder was subsequently tested to investigate its effectiveness against L. monocytogenes Scott A and Bacillus cereus in natural yoghurt, cottage cheese and soup showing the potential of lacticin 3147 as an aid to eliminate pathogenic organisms (Fig. 3). Furthermore, lacticin-containing powder has also been tested in infant milk formula, which is of particular significance as infants are more vulnerable to the effects of listeriosis. Ominously, counts of L. monocytogenes increased 700% in control samples over a 3 h period, whereas the addition of either 15 or 10% lacticin powder resulted in 99% kill after the same period (Fig. 3). Although there is no doubt that lacticin 3147 powder has the ability to kill pathogenic organisms in the foods tested, the optimisation of lacticin 3147 powder to increase specific activity is needed, as it may not be feasible to replace 10% of the product with lacticin powder (Morgan et al., 1999, Morgan, Galvin, Ross, & Hill, 2001). Large-scale production of this lantibiotic could be possible through immobilisation of DPC3147 to calcium alginate beads to improve the stability and productivity of bacteriocin over conventional cell-free fermentations in a lab setting. Some initial investigations in this regard have yielded promising results (Scannell et al., 2000a,b). Variacin, a bacteriocin produced by Kocuria varians, has also been used to produce a milk-based fermented ingredient and was used to evaluate its effectiveness against B. cereus in chilled dairy products, vanilla and chocolate desserts. The fermented ingredient was tested using either 1, 3 or 10% and over a range of abusive temperatures 8, 12 and 30  C. A correlation between temperature abuse and the level of bio-active ingredient required to be effective was very evident. Although the use of the fermentate at lower percentages showed potential at lower temperatures, the use of 10% fermentate was more effective overall (O’Mahony, Rekhif, Cavadini, & Fitzgerald, 2001). Further, showing the much needed investment into optimising the production of these and other bio-active fermentates. 3.2. Bacteriocin-producing cultures The possibility of including live bacteriocin-producing LAB in foods has been touched on previously in this review. This system of incorporating a bacteriocin-producing culture into a food gives it its own built in biopreservation, thereby returning to a more natural method of shelf-life extension and improving the safety of food (for a review see O’Sullivan, Ross, & Hill, 2002). There can, however, also be problems associated with the utilisation of bacteriocin-producing cultures in fermen-

ted foods, the most obvious being a lack of compatibility between the bacteriocin-producing strain and other cultures required in the fermentation. This was found to be the case for nisin-producing cultures, which inhibit the starter cultures required for cheese making. In an attempt to overcome this problem, the viability of using nisinproducing starters was assessed, but unfortunately these starters suffered from poor acid development, possessed inadequate proteolytic activity and often displayed an enhanced susceptibility to bacteriophage attack. As a consequence, without intervention or troubleshooting, nisin-producing cultures appear to be unsuitable as a biopreservative in cheese production. However, the manipulation of multi-strain starters can be adopted by using bacteriocin-producing strains in concert with other cultures possessing desirable traits such as efficient acid production (Lipinska, 1973). The use of cultures to produce bacteriocins in situ as a means of bio-preservation has received a great deal of interest in recent times. The inhibition of L. monocytogenes in dry-fermented sausage using lyophilised bacteriocin-producing cultures as a protective adjunct has been investigated. To reflect realistic pathogen contamination levels, sausage meat was artificially spiked with a four strain cocktail at 102 –103 cfu g1 . Lb. curvatus was more successful than Lc. lactis, as following 4 h fermentation, L. monoctogenes was reduced to o10 cfu g1 . Also recovery proceeding enrichment was not possible after day 8 of drying. Lc. lactis also decreased listeria to below the detectable limit after day 15 of drying. Furthermore, pH was not regarded as a factor in elimination of L. monocytogenes in this study as there was no significant difference between samples containing bacteriocin-producing cultures and those that did not contain these cultures (Benkerroum et al., 2005). The utilisation of Carnobacterium strains to combat listerial contamination in cold-smoked salmon (CSS) has been studied and has shown potential, but due to the emergence of resistance to Class II bacteriocins there has been an increased interest in the use of bacteriocin-negative Carnobacterium strains as protective cultures (Nilsson et al., 2004). However, a recent study highlights the potential of C. divergens V41 for effective bacteriocin production in situ for bio-preservation of CSS. C. divergens when compared with two other bacteriocin-producing Carnobacterium strains was more efficient as during a 4-week period of vacuum storage levels of L. monocytogenes were substantially reduced to o50 cfu g1 regardless of the pathogens sensitivity. In addition, this strain colonised the CSS to a higher level than the other strains, which could be a contributing factor to its effectiveness against all L. monocytogenes strains tested. This study also shows the different sensitivities to bacteriocins that can be encountered with pathogens (Brillet, Pilet, Prevost, Bouttefroy, & Leroi, 2004). The strains used in this study were isolated directly from salmon in the environment and salmon products, which is undoubtedly an important aspect when considering the use of a bacteriocin to be applied to a certain food system.

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Lacticin 3147-producing strains have also been added to foods. As the genetic determinants for lacticin 3147 are encoded on a large conjugative plasmid pMRC01, they can also be transferred in a food-grade manner to many commercially useful starter cultures (Coakley, Fitzgerald, & Ross, 1997). pMRC01 also contains genes that encode resistance to bacteriophage attack, an industrially important trait in the manufacture of cheese. A lacticin 3147 transconjugant was used successfully in a study where its effectiveness against L. monocytogenes Scott A in cottage cheese was investigated (McAuliffe, Hill, & Ross, 1999). The ability of the producing strain to reduce numbers of L. monocytogenes ð104 cfu mL1 Þ artificially introduced into cottage cheese (pH 5.2) at 4, 18 and 30  C was tested over a duration of 1 week. Reductions of 99.9% were seen in cottage cheese held at 4  C after only 5 days (Fig. 3), whereas at the elevated temperatures the reductions occurred more promptly. Mould-ripened cheese (pH 6.5–8.0) is a favourable setting to support the growth of L. monocytogenes. A lacticin 3147 transconjugant showed a positive protective effect, reducing L. monocytogenes 1000fold when applied to the surface of the cheese (Ross, Stanton, Hill, Fitzgerald, & Coffey, 2000). In contrast, this protective effect was not apparent when a nisin-producing culture was used, most probably due to pH instability (Ross et al., 1999). These studies are representative of similar observations associated with a variety of different bacteriocin producers. Recently, a food-grade strain has been developed to produce both lacticin 3147 and lacticin 481. This strain addresses both the food safety and food improvement aspects of bacteriocin production. Significantly, the killing effect of this double producer was more pronounced, when tested against Lb. fermentum and L. monocytogenes, than either bacteriocin producer alone (O’Sullivan, Ross, & Hill, 2003). The use of strains that produce multiple bacteriocins could be advantageous to limit the potential emergence of bacteriocin-resistant populations. Resistance can occur naturally and it has been reported especially with regards to Class IIa bacteriocins such as pediocin PA-1 and mesentericin Y105 among others (Ennahar, Sashihara, Sonomoto, & Ishizaki, 2000). Pediocin-acquired resistance has been reported at levels of 104 –106 (Dykes & Hastings, 1998), while nisin-resistant mutants occur at a frequency of 102 –108 (Gravesen, Jydegaard Axelson, Mendes de Silva, Hansen, & Knochel, 2002; Mazzotta, Modi, & Montville, 2000, Ming & Daeschel, 1993). In contrast, lacticin 3147-resistant strains have not been found in the presence of high levels of the bacteriocin (Coakley et al., 1997; Dodd, 1996; Klijn, 2001; Ryan, Meaney, Ross, & Hill, 1998). Given the manner in which bacteriocins are likely to be used in food, i.e. as one of a number hurdles, it is unlikely that the food industry will see the emergence of bacteriocin-resistant bacteria in the extent to which antibiotic-resistant pathogens have emerged in hospitals. However, the utilisation of double bacteriocinproducing strains would provide an additional barrier to

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ensure that the emergence of resistant populations is even less likely. As stated previously, food producers and consumers may frequently serendipitously benefit from the presence of bacteriocin-producing LAB in foods. One particularly interesting study has demonstrated that of 48 different vacuum-packed meat products, bacteriocin-producing colonies were isolated from 46% of the packages examined (Budde, Hornbaek, Jacobsen, Barkholt, & Koch, 2003). This study confirms that bacteriocin-producing cultures are frequently found in some commercially available foods. In summary, the use of bacteriocin-producing cultures in foods can be very beneficial. While the choice of bacteriocin-producing culture may have to vary to meet with the particular microenvironment associated with certain foods, the net result of this technology can be the production of foods that have a built-in preservation system. 4. Bacteriocins in combination with additional hurdles Hurdle technology refers to the manipulation of multiple factors (intrinsic and extrinsic) designed to prevent bacterial contamination or control growth and survival in food. A combination of preservation methods may work synergistically or at least provide greater protection than a single method alone, thus improving the safety and quality of a food. While in certain foods intrinsic properties such as high salt (see a review by Leistner, 2000) may provide adequate protection, the conscious addition of an extra hurdle(s) can ensure safety. The role of bacteriocins as hurdles in food systems has been documented in this, and other, reviews. However, it is also apparent that a bacteriocin alone in a food is not likely to ensure comprehensive safety. This is of particular significance with regards to Gram-negative pathogenic bacteria that are protected by the presence of an outer membrane. When the outer membrane is impaired by agents such as the foodgrade chelating agent ethylene diamine tetraacetate (EDTA), which acts by binding to Mg2þ ions in lipopolysaccharide, the outer membrane is disrupted rendering Gram-negatives sensitive to bacteriocins (Stevens, Sheldon, Klapes, & Klaenhammer, 1991). Non-thermal treatments such as HHP and pulsed electric field (PEF) represent other methods of food preservation. As these technologies are non-thermal, they are advantageous as they have little or no effect on food functionality and nutritional qualities compared to thermal methods which affect vitamin content. As a consequence of using these non-thermal methods, the bacterial cell membrane becomes destabilised, thus interfering with essential cell functions (Jeyamkondan, Jayas, & Holley, 1999). While the utilisation of HHP or PEF alone is not always economically viable, lower levels could be used if combined with other treatments such as bacteriocins. The benefits of combining nisin with HHP (Capellas, Mor-Mur, Gervilla, Yuste, & Guamis, 2000; Lopez-Pedemonte, Roig-Sagues,

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Trujillo, Capellas, & Guamis, 2003; Stewart, Dunne, Sikes, & Hoover, 2000) and to a lesser extent PEF (Pol, Mastwijk, Bartels, & Smid, 2000; Terebiznik, Jagus, Cerrutti, de Huergo, & Pilosof, 2000) has been demonstrated extensively. Lacticin 3147 was also used in concert with HHP at pressures of 150–275 MPa to investigate the effects on Staphylococcus aureus and L. innocua. Using 10; 000 AU mL1 and the lower pressure of 150 MPa resulted in a 2:1 log kill of S. aureus relative to a control lacking lacticin where a o0:5 log kill was observed. A more pronounced 46 log kill was observed when lacticin 3147 was combined with a higher pressure of 275 MPa. Similar, although less marked, trends were seen for L. innocua (Morgan, Ross, Beresford, & Hill, 2000). More recently, the ability of seven bacteriocin-producing LAB have been investigated in combination with HHP at 300 and 500 MPa in raw cheese that had been contaminated with E. coli 0157:H7. In all cases, HHP- and bacteriocinproducing adjuncts where synergistic resulting in the increased inactivation of E. coli 0157:H7 compared to each hurdle individually. In particular, HHP at 300 MPa on day 50 (of 60-day-old cheeses) in combination with adjuncts producing nisin A, bacteriocin TAB 57 and enterocin I or AS-48 resulted in the complete elimination of E. coli 0157:H7 (Rodriguez, Arques, Nunez, Gaya, & Medina, 2005). Similarly, S. aureus was inactivated using bacteriocin-producing cheese adjuncts with HHP, which was also synergistic (Arques et al., 2005). Bacteriocins used in combination with organic acids or other antimicrobials can also result in enhanced inactivation of bacteria. This phenomenon has been demonstrated for pediocin following artificial contamination of beef franks with 107 cfu g1 L. monocytogenes; pediocin in the form of ALTA 2341 was used in combination with sodium diacetate (SD) and sodium lactate (SL) as dipping solutions. It was found to be very effective in controlling L. monocytogenes in vacuum packed beef franks stored at 4  C. Pediocin ð6000 AUÞ reduced levels of L. monocytogenes by o1 log cfu g1 unit, following storage for 14 and 21 days. This activity was enhanced by the addition of antimicrobials SD and SL, after the same period of storage L. monocytogenes was reduced by 1.5 to 2:5 log cfu g1 , respectively (Uhart, Ravishankar, & Maks, 2004). In a more recent comprehensive study, nisin and pediocin are used in combination with a number of antimicrobials including citric acid and potassium sorbate to combat L. monocytogenes on fresh-cut produce. The synergistic nature of hurdle technology was evident from this study as in nearly all cases combinations were better than nisin and pediocin alone. Nisin with phytic acid was most effective overall at reducing L. monocytogenes (Bari et al., 2005). In a study by Scannell et al. it was shown that lacticin 3147 activity was improved in fresh pork sausage with either SL or sodium citrate (SC). In particular Clostridium perfringens was reduced by 3:1 log with only lacticin 3147 but was not recovered for up to 4 days with the addition of either SL or SC. The use of lacticin 3147 or nisin with SC reduced

Salmonella enterica var. Kentucky by 1 and 1:6 log, respectively, again highlighting that the use of combinations of preservation techniques can limit the risk posed by Gram-negative pathogens (Scannell, Ross, Hill, & Arendt, 2000). Bio-active packaging is a further potential application in which bacteriocins can be incorporated into packaging destined to be in contact with food. This system combines the preservation function of bacteriocins with conventional packaging materials, which protects the food from external contaminants. Spoilage of refrigerated foods usually begins with microbial growth on the surface, which reinforces the attractive use of bacteriocins being used in conjunction with packaging to improve food safety and improve shelf-life (Collins-Thompson & Hwang, 2000). Bio-active packaging can be prepared by directly immobilising bacteriocin to the food packaging, or by addition of a sachet containing the bacteriocin into the packaged food, which will be released during storage of the food product. Studies investigating the effectiveness of bio-active cellulose-based packaging inserts and a vacuum packaging pouch made with polyethylene/polyamide to improve shelflife and safety aspects have proved promising. When considering bio-active packaging, the stability and the ability to retain activity while immobilised to the packaging film is of vital importance. Studies with lacticin 3147 and nisin have shown that lacticin 3147 did not absorb effectively to plastic, whereas nisin in the form of NisaplinTM absorbed to the surface tightly, resulting in effective bio-active films that reduced levels of non-starter LAB (NSLAB), L. innocua and S. aureus in vacuum and MAP packaged cheddar cheese, and also showed protection towards ham that had been packaged under MAP. During this study, it was also demonstrated that the inserts were stable at ambient and refrigeration temperatures and that the bio-active packaging for optimal protection must be in direct contact with the food. (Scannell et al., 2000b). While bacteriocins may also be incorporated into an edible film or coating that can be applied to the food either by dipping or spraying, this approach has legislative implications as it cannot be described as active packaging only. In a study by Coma et al. whereby nisin was added to a filmforming solution, although, L. innocua and S. aureus were inhibited, it was apparent that other constituents, such as stearic acid added to the film to improve water vapour barriers, can have adverse effects on the activity of incorporated bacteriocins (Coma, Sebti, Pardon, Deschamps, & Pichavant, 2001). Methods involving the incorporation of chelating agents such as EDTA (already described in this review) could also be an option for active packaging designed to provide protection against Gramnegative spoilage/pathogenic contamination. This has been investigated by Siragusa, Cutter, and Willett (1999) and was effective, when used with nisin, in inhibiting E. coli in beef carcasses. It would seem apparent that this might be an effective way of delivering bacteriocins to the surface of food to improve preservation and shelf-life. Further, the continued research in this area taking into consideration

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both the positive and negative aspects with new developments in polymeric packaging could lead to an advanced system of bio-active packaging. Thus, while many LAB bacteriocins possess significant antimicrobial qualities that could greatly enhance the safety of a food, it may yet emerge that industrially they will be most frequently applied as a ‘final-hurdle’ in a food system where another hurdle(s) already exists to eliminate pathogens and spoilers that survive only in adventitious circumstances. 5. Bacteriocins for improving food quality and flavour Bacteriocins can also be used to improve the quality and sensory attributes of certain foods (Fig. 2). During the manufacturing of cheese, bacteriocin-producing LAB can be utilised to control adventitious flora such as NSLAB and also in the induction of cell lysis, thereby increasing the rate of proteolysis in cheese (see a review by Guinane, Cotter, Hill, & Ross, 2005) The role of NSLAB in cheese ripening has not been entirely elucidated but it is apparent that they are responsible for defects such as the formation of calcium lactate crystals (due to an ability to racemise Llactate to D-lactate), slit formation and off-flavour development, but they are likely to also have a positive effect on flavour. Using bacteriocins as tools to influence the development of NSLAB in cheese could prove to be very advantageous by giving cheese manufacturers more control over this normally uncontrolled flora. A lacticin 3147 transconjugant, used as the starter culture in the production of low-fat cheddar cheese, proved to be efficient in controlling NSLAB during ripening (Fenelon et al., 1999). The control of NSLAB in cheese at an elevated ripening temperature could prove to be very economically attractive to the cheese industry. Lactococci-producing lactococcin ABM (a combination of three Class IIa plasmid-encoded bacteriocins) have a narrow spectrum with bacteriolytic and bacteriostatic effects on sensitive lactococci (Morgan, Ross, & Hill, 1995). Although these bacteriocins have a narrow spectrum and do not exhibit activity against pathogens, ABM producers do possess substantial potential with respect to improving cheese quality and flavour as the presence of lactococcin ABM accelerates the rate-limiting step of releasing intracellular proteinases and peptidases from the starter culture, contributing to the flavour. A shortcoming associated with the use of bacteriocins for lysing starter culture cells is reduced acidification, especially in cases where a bacteriocin- sensitive starter is employed. It can also be extremely difficult to get the correct balance between lysis and appropriate levels of acidification. As a result, a three starter system was developed using Lc. lactis HP, Lc. lactis DPC3286 and a Streptococcus thermophilus DPC1842 strain that is not susceptible to lactococcin ABM and which contributes to acid production (Morgan, O’Sullivan, Ross, & Hill, 2002). Although there are commercial enzymes available to carry out the procedure

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of starter cell lysis, they can be costly and inefficient, whereas bacteriocin-producing cultures can perform this process without adverse cost implications and with an improvement in the overall quality and flavour of the cheese. Danisco have formulated a freeze-dried culture of Pediococcus acidilactici marketed as CHOOZITTM Lyo. Flav 43 (product description—PD203800-1EN). CHOOZITTM which can be used for ‘‘cheddar cheese and semi-hard cheese’’ as an ‘‘adjunct, which accelerates and enhances strong and sweet flavour compounds, due to the production of bacteriocins.’’ The ability to induce the release of intracellular enzymes has also been associated with the lacticin 3147 producing strain Lc. lactis IFPL359 resulting in the transamination of branched chain amino acids into the volatile compound 2methylbutanal in cheese. The formation of this compound is desirable in cheese production as it is responsible for flavour enhancement (Fernandez de Palencia et al., 2004). Similarly, a lacticin 481 (a medium spectrum lantibiotic with activity against other LAB and Cl. tyrobutryricum) producing adjunct has also shown great potential with respect to the control of NSLAB and the acceleration of starter cell lysis without acid production by the starter being compromised (O’Sullivan, Ryan, Ross, & Hill, 2003). Avila et al. have also shown the potential of using lacticin 481- or a plantaracin-producing adjunct with the highly peptidolytic Lb. helveticus for the manufacture of cheese due to the improvement of cell-free aminopeptidases (Avila, Garde, Medina, & Nunez, 2005). 6. Conclusion/future prospects Civilisation has reaped the benefits of bacteriocins unknowingly for 1000s of years, yet nisin is the only bacteriocin bio-preservative that has received acceptance in countries worldwide. Knowing that other bacteriocins exist and can work at least as effectively as nisin (if not more so) with respect to particular foods/target bacteria, the question is often posed why more have not been exploited to the same extent. In addition to the difficulties that can arise with respect to legislation, there may be a reluctance to commit financially to the development/formulation of commercial products akin to NisaplinTM . Despite this, it should be apparent that a wide array of means exist through which a bacteriocin can be incorporated into a food, all of which are at the disposal of the food sector. It would be naive to believe that bacteriocins represent the ultimate solution to food safety problems. However, given the effectiveness of bacteriocins, the existence of economically viable means through which they can be incorporated and a consumer desire for minimally processed food, they may represent an excellent alternative for use in combination with other natural preservatives. There is a need to attract consumer attention to the existence of natural substances that can protect against food-related illness. The acceptance of probiotics by the consumer was aided greatly when such bacteria were marketed as natural

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cultures that aid in digestion/health. In the same way, bacteriocins and bacteriocin-producing cultures should be attractive, especially as a consequence of consumer distrust of chemical preservatives. While the application of bioengineered/modified bacteriocins may be counterproductive with respect to the marketing of bacteriocins as natural products, the creation of such derivatives has been beneficial with respect to our understanding of these peptides. Such techniques could be utilised to improve the stability and production of bacteriocins so they may be more applicable in food products. There have already been examples of such successes, for example, a single amino acid change in nisin Z, whereby a methionine at position 21 was changed to a lysine, resulted in a 5-fold increase in the solubility of the peptide at pH 8 compared to wild-type nisin Z plus an extension of its antimicrobial spectrum to encompass Gram-negative bacteria (Yuan, Zhang, Chen, Yang, & Huan, 2004). As some of the food matrices and processes employed by the food industry can affect the activity/ stability of bacteriocins, the generation of derivatives with enhanced features could be very rewarding from a food safety and economic perspective, but only if any associated consumer concerns could be addressed. Continued research on bacteriocins will undoubtedly lead to our increased understanding, and with the emergence of new bacteriocins, new potential bio-preservatives. However, this potential will only be fulfilled if bacteriocins are appropriately used and marketed successfully.

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