Inf. Da& Journul5 (1995) 797-8 14 0 1995 Elsevier Science Limited Printed in Ireland. All rights reserved 0958-6946/95/$9.

50 + .OO 0958-6946(95)00034-S

Models and Mechanisms for Bacteriocin Action and Application

T. J. Montville,*

K. Winkowski

& R.D. Ludescher

Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers-The State University of New Jersey, New Brunswick, NJ 08903-0231, U.S.A.

ABSTRACT There is considerable research on bacteriocin genetics, purtfication, and properties. Less is known about the mechanism(s) by which bacteriocins kill pathogens, the physical chemistry of the bacteriocinlpathogen interaction, and of the variables which influence bacteriocins efficacy in foods. Such knowledge is prerequisite to the wider applications of bacteriocins and to increasing their efficacy by genetic engineering. Mechanistic studies using spores as bacteriocin targets are relatively_few. Empirical challenge studies in a variety of foods have had mixed results. Working with well defined model foods, we have determined that increasing protein or phospholipid concentrations decrease nisins effectiveness against Clostridium botulinurn growth jrom spore inocula. Nisin is also less effective at abuse compared to refrigerated temperatures. This may be a general characteristic of bacteriocins since increasing temperature decreases many bacteriocins inhibition of Listeria monocytogenes in ,foods. L. monocytogenes vegetative cells provide a better target for bacteriocin action than do C. botulinurn spores. Bacteriocins dissipate proton motive force (PMF) in L. monocytogenes, C. sporogenes and vegetative cells of other sensitive species. The cytoplasmic membrane is generally considered to be the site at which bacteriocins act. We have adopted fluorescence spectroscopy to characterize the interaction of bacteriocins with liposomes comprised of lipids extracted from L. monocytogenes membranes. The regulatory status of bacteriocins, various models ,for bacteriocin action, and,jicture prospects for their application are also discussed.

INTRODUCTION Interest in using bacteriocins from lactic acid bacteria to assure the safety and extend the shelf-life of refrigerated foods has increased dramatically over the last *Author to whom all correspondence should be addressed.

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T. J. Montville et al.

decade. Bacteriocin-producing lactic acid bacteria (LAB) are easily isolated from dairy products, meat, fish, and vegetables (Ahn et al., 1990; Flemning et al., 1975; Jimenez-Diaz et al., 1993; Lewus et al., 1991; Schillinger et al., 1991; Sobrino et al., 1991; Stoffels et al., 1992a, b; Uhlman et al., 1992), giving rise to hundreds of papers in this burgeoning field. Since authoritative comprehensive reviews on nisin and other bacteriocins have appeared in the 1960s (Reeves, 1965), 70s (Lipinska, 1977; Mayer-Harting et al., 1972; Tagg et al., 1976) 80s (Hurst, 1981; Klaenhammer, 1988), and 90s (Daeschel, 1990; Daeschel, 1993; DelvesBroughton, 1990; Hoover & Steenson, 1993; Styles & Hastings, 1991; Kim, 1993; Klaenhammer,l993; Nettles & Barefoot, 1993), this paper will make no attempt to be all inclusive. Narrow in scope, it will focus on applications of bacteriocins in foods, present mechanistic models of how bacteriocins work and emphasize research from our own laboratory. By examining some empirical studies on the application of bacteriocins in specific foods and comparing these with our empirical model of factors affecting the efficacy of nisin against C. botulinurn spores (Rogers & Montville, 1994), we hope to identify characteristics common to successful applications. Although it is generally accepted that bacteriocins act at the membrane (Chikindas et al., 1993; Klaenhammer 1993; Montville & Bruno, 1994), exactly how they work is unclear. Certainly, the depletion of proton motive force is a common mechanism of action (Bruno & Montville, 1994), but is this a primary or secondary event? Are bacteriocins a specific class of cytolytic pore forming proteins (Ojcius & Young, 1991)? Do they work as barrel stave poration complexes or do they destabilize the membrane in what might be viewed as a transient pore formation or even detergent-like action? There are many similarities between bacteriocins and other antimicrobial proteins such as thionins, magainins, yeast killer toxins and defensins (Montville & Kaiser 1993; Montville & Bruno, 1994). Current mechanistic models for bacteriocins draw heavily on the knowledge gained in these other systems. Ultimately, the empirical and mechanistic models should converge, leading to the rational design of genetically engineered bacteriocins and the rational application of bacteriocins in specific foods. Some preliminary remarks on the nature of bacteriocins are in order. In rapidly evolving fields, it can be difficult to determine which properties of a compound belong to the class of compounds of which it is a member, and which are unique to the specific compound under study. Thus, although the seven criteria suggested by Tagg et al. (1976) were initially widely used to characterize LAB antimicrobial proteins as bacteriocins, it is now clear that these criteria are not universal. Most proteins which inhibit the growth of related bacteria without affecting the producing strain are now considered bacteriocins (Montville & Kaiser, 1993) despite a proposal to restrict the use of bacteriocin to colicin-like proteins and categorize proteins meeting less restrictive criteria as Bacteriocin-Like Inhibitory Substances (BLIS proteins, Tagg, 1991). Proteins which act exclusively via enzymatic activity such as lysozyme are not classified as bacteriocins. Bacteriocins are classified in four major groups (Klaenhammer, 1993). Group I bacteriocins, lantibiotics like nisin, are small proteins containing lanthione (single sulfhydral) rings which are produced post-translationally. Group II bacteriocins, the small heat-stable proteins with a consensus leader sequence important for bacteriocin export, are subdivided into those having a -Tyr-Gly-Asn-Gly-ValXaa-Cys amino terminal consensus sequence, those requiring two different

Bacteriocin mechanisms and applications

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peptides for activity, and those requiring reduced cysteines for activity. Larger (> 30 kDa) heat labile proteins are classiIied as Group III bacteriocins. Bacteriocins having lipid or carbohydrate moieties are found in group IV. Bacteriocins from each of these classes deplete proton motive force in vegetative cells (Bruno & Montville, 1993), suggesting mechanism(s) that are at least similar.

ACTION Mechanistic studies

AGAINST

SPORES

There is less mechanistic information about bacteriocin action against spores than there is for vegetative cells. Most of these studies are older and deal primarily with nisin, which is sporostatic rather than sporocidal (Lipinska, 1977). Nisin is used in many countries as an adjunct to thermal processing to prevent spoilage by thermophilic spore formers (Delves-Broughton, 1990). It inhibits the step between botulinal spore germination (loss of heat resistance and refractility under phase microscopy) and pre-emergent swelling (Atwell et al., 1972). Bacillus species whose spore coats open by mechanical pressure are much more nisin sensitive than those whose spore coats are opened by lysis (Lipinska, 1977). Nisin modifies the sulfhydryl groups in the envelopes of germinated spores (Morris et al., 1984), presumably because the nisins dehydro residues act as electron acceptors (Gross & Morell, 1967; Gross & Morell, 1971). C. botulinurn is relatively nisin insensitive (Hurst, 1972), but nisin sensitivity among different strains varies considerably (Montville et al., 1992). For example, as little as 10 I.U. mL_ nisin is sporostatic to strain 169 spores, but 2500 I.U. spores mL - is required to inhibit strain 56A for 30 days at 35C. Heat-damaged are sensitized to nisin (Campbell et al., 1959; Denny et al., 1961; Heinemann et al., 1965). In basal medium, 10 I.U. mL_ nisin inhibits Bacillus licheniformis spores, but 100 I.U. mL_ is needed in the presence of 1% salt (Bell & De Lacy, 1985). This may be caused by salt interference with nisin absorption to the spore. The bacteriocins produced by Pediococcus pentosaceus (strains 43200 and 43201) and Lactobacillus plantarum (strains Lb75, Lb592, and BN) are also active against botulinal spores (Okereke & Montville, 1991a, b), but nothing about their mechanism(s) is known. Empirical studies in media and foods Although some studies have been done using Bacillus spores (c.f. Bell & De Lacy 1985; Jarvis & Farr, 1971; Delves-Broughton et al., 1992) most published work on nisin targets C. botulinum spores. Nisin increases the shelf life and delays toxin production by type E botulinal strains in fresh fish packaged in a carbon dioxide atmosphere. However, toxin can sometimes be detected before samples are obviously spoiled (Taylor et al., 1990). Studies using meat or cheese products are more common. Studies by Taylor at the University of Wisconsin were used to support the affirmation of nisin in pasteurized process cheese as Generally Recognized as Safe in the United State (Federal Register, 1988). Between 500 and 2000 I.U. mL_ of nisin inhibits by 50% botulinal spore outgrowth in tryptose peptone yeast extract

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glucose (TPYG) broth, but levels of up to 10,000 I.U. mL_ are ineffective in cooked meat medium (Scott & Taylor, 1981a, b). Nisin is more efficacious in TPYG at pH 5.3 than at pH 6.0. Temperature, pH and spore inoculum level all influence nisins efficacy (Scott & Taylor, 1991). Somers & Taylor (1987) examined nisins antibotulinal effectiveness in pasteurized process cheese spreads having moisture contents of 52 to 57%, NaCl at 0 to 2%, disodium phosphate at 1.4 to 2.5%. Nisin at 100 to 250 ppm allows salt reduction or increased moisture levels without elevating the risk of botulism. In these studies, nisin clearly serves as one part of a multiple barrier inhibition system. Some countries prohibit nisin addition to cheese, but allow nisin-producing starter cultures for cheese making. Pasteurized process cheese spread made from cheddar cheese manufactured with a nisin-producing culture has a shelf life of 87 days at 22C compared to 14 days for a conventional cheese spread (Roberts & Zottola, 1993). Once again, moisture content, pH, and spore load are important variables. The use of bacteriocins in meat as nitrite substitutes and to inhibit botulinal growth in sous vide type products has been only modestly successful. A nisinproducing L. Iactis, but not exogenously-added nisin, inhibits C. botulinum growth in chicken-a-la king (Salegh & Ordel, 1955). Nisin at 75 pg g- is more inhibitory to C. sporogenes PA 3679 spores than is nitrite at 150 ppm, but the inhibition decreases with increasing pH and spore load. Up to 550 ppm of nisin combined with 60 ppm nitrite fails to prevent outgrowth of C. botulinal spores in pork slurries at pH 5.8, although lowering the pH enhances activity (Rayman et al., 1983). Pediococcus acidilactici inhibits botulinal toxigenesis in temperatureabused chicken salad through acid production (Hutton et al., 1991). The contribution of bacteriocin to the inhibition was not considered in this study. During inoculated pack studies at 4 and 10C terminal pH values are often as low as 4.8 in sous vide beef that tests positive for botulinal toxin (Crandall et al., 1994). In model gravy systems containing 0.5% glucose, toxin production at 37C is not prevented by P. pentosaceus 43200 or L. plantarum strains Lb75 and Lb592. even though they reduce pH to <5.0 within 12 h (Crandall & Montville, 1993). Even the nisin producer L. lactis 11454, which acidifies to pH <4.7 within 6 h. cannot prevent toxigenesis in this system. An empirical model We have modeled nisins antibotulinal activity and quantified the influences of food ingredients on its efficacy (Rogers, 1991; Montville & Rogers, 1994). The model food was a medium of yeast extract, proteose peptone, and glucose supplemented with protein (0.075,0.75,7.5% w/v), phospholipid (0.075,0.75,7.5% w/v), or soluble starch (5, 17.5, 30% w/v); and was adjusted to pH 5.5,6.0, or 6.5 prior to inoculation with lo4 mL_ spores of C. botulinum 56A and incubation at 15,25, or 35C. The contribution of nisin to the delay in growth (AN, days) was calculated by subtracting the days required for visible growth in the absence of nisin from the days required for growth in the presence of nisin. Statistical analysis modeled the response (AN) against all combinations of experimental variables and identified temperature as the most significant variable. Nisin loses effectiveness with increasing temperature. Nisin and phospholipid concentrations have significant positive and negative linear effects, respectively.

Bacteriocin mechanisms and applications

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Some of the response surfaces developed from our model are presented in Fig. 1. Temperature and nisin concentration are the x and y axis in every plot. The measured response, or z axis, is AN (delay in growth, in days). Figure 1 compares the influence of pH 6.5 (top panels), pH 6.0 (middle panels) and pH 5.5 (bottom panels) on AN when all food components are at minimum (left panels) or maximum (right panels) concentrations. The influence of pH is more pronounced when the food components are at their highest level compared to their lowest level. Similarly the response surfaces are less nisin concentration-

Fig. 1. Response

surfaces illustrating the on the delay of botulinal growth (AN) (bottom), and when protein, starch, and lowest values (left) or

influence of temperature and nisin concentration at pH 6.5 (top), pH 6.0 (middle) and pH 5.5 phospholipid concentrations were fixed at their at their highest values (right).

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dependent when the levels of other food ingredients are low. In experiments with high concentrations of protein, carbohydrate, and lipid, there is a marked effect of nisin concentration. At pH 6.5 (top response surfaces), the organism grows faster in the control cultures, thereby increasing the difference between the (with and without nisin) endpoints. Clearly, at pH 6.5 and high food component concentrations, nisins ability to delay botulinal growth is highly dependent on nisin concentration and temperature. In contrast, at low ingredient concentrations, even low nisin concentrations delay growth. Although some suggest that bacteriocin-based preservation systems ultimately fail due to the growth of resistant organisms (Motlaagh et al., 1992, Foegeding et al., 1992), we have never isolated resistant organisms at the end of an inoculated pack study. We have, however, found low residual nisin levels and believe that the loss of nisin activity below a threshold inhibitory level is ultimately responsible for botulinal growth in systems where nisin is only sporostatic. To study this further, a model food system similar to that of Rogers & Montville (1994) was prepared with high levels of starch, albumen, lecithin, or carrageenan (Rogers, 1991). The loss of nisin activity over time was related to the onset of botulinal growth. When all ingredients in the model were at their highest level, the nisin threshold concentration (below which C. botulinum grew) decreased with decreasing temperature. Growth occurred when residual nisin concentrations fell below 154 I.U. mL_ at 35C 32 I.U. mL_ at 25C and 12 I.U. mL_ at 15C. In contrast, when the equations were solved using low concentrations of all food components, the nisin concentration intercepted the x axis near zero. This suggests the absence of a nisin threshold (i.e. that any nisin added to this system would inhibit C. botulinum).

ACTION Empirical studies in foods

AGAINST

VEGETATIVE

CELLS

Successful applications of bacteriocin action against L. monocytogenes are more common than antibotulinal ones. Lb. bavaricus MN (which produces bavaricin MN) co-inoculated with L. monocytogenes Scott A into a sterile model gravy, and incubated at 10C inhibits listerial growth. It is effective at temperatures as low as 4C when inoculated at lo3 cfu mL_ (IO-fold higher than the listerial inoculum) in the absence of a fermentable carbohydrate (Winkowski & Montville, 1992). Greater inhibition occurs when a fermentable carbohydrate is present. The antilisterial activity of Lb. bavaricus MN was studied further in formulations of sous vide beef cubes (Winkowski et al., 1993). L. monocytogenes (at lo3 cfu mL_) is inhibited at 4 and 10C in plain beef cubes, beef cubes in gravy and beef cubes in gravy having a fermentable carbohydrate when Lb. bavaricus is added at 10 or lo3 the presence of fermentable carbohydrates, and cfu mL_. Colder temperatures, the higher inoculum levels produce the greatest inhibition. In the best case scenario, L. monocytogenes viability declines lo-fold over six weeks at 4C when co-inoculated with Lb. bavaricus. In the absence of the lactobacilli, the listeria increase to > 10 cfu mL_ over the same period. Even in the worst case scenario (10C low lactobacillic inoculum, beef cubes without gravy), the bavaricin inhibits listeric growth for one week while they increase lOO-fold in the control beef.

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803

Carnobacterium piscicola LKS is more effective against L. monocytogenes in a variety of foods at 5C than at 19C (Buchanan & Klawitter, 1992a, b). Although it is ineffective against strains V7 and Murray B, C. piscicola LK5 can inhibit Scott A even when the listeria were present at lOO-fold higher concentrations. This is attributed to increased sensitivity of the listeria to the bacteriocin at low temperatures. L. monocytogenes cells are also more sensitive to lysozyme at refrigerated temperatures than they are under ambient conditions (Smith et al., 1991). Bacteriocinogenic pediococci appear especially effective. In situ pediocin production by P. acidilactici PAC 1.0 during the manufacture of fermented dry sausage reduces L. monocytogenes viability > lo-fold relative to the acid-induced decrease caused by nonbacteriocinigenic control (Foegeding et al., 1992). Both Pediocin AcH and the pediocin-producer, P. acidilactici, cause similar rates of L. monocytogenes death in wiener (hot dog) exudate at 4C. In untreated controls, the listeria population increases over lOOO-fold (Yousef et al., 1991). Pediocin AcH also inhibits Listeria spp. in sterile sausage, ground beef, cottage cheese and ice cream (Motlagh et al., 1992). The maximum bactericidal effect occurs within 1 h and is Listeria spp. -dependent; 1350 A.U. mL_ reduces viability from one to seven log cycles. The use of Pediocin PA-l (which is identical to Pediocin AcH) to kill L. monocytogenes inoculated at lo3 cfu mL_ in cottage cheese and cheese sauce is covered by an European patent (Vandenbergh et al., 1989). Mechanistic studies

Membrane permeability and depletion of PMF The cytoplasmic membrane of sensitive cells is the biological target of bacteriocins. The addition of these membrane-active peptides to vegetative cells causes a rapid and non-specific efflux of small molecular weight compounds. Exposure of sensitive cells to nisin (10 pg mL-i) causes efflux of pre-accumulated cations (Rb+) and amino acids from the cytoplasm of various Gram-positive bacteria (Ruhr & Sahl, 1985). This efflux is not specific for charge or size since different compounds have similar efflux velocities. Nisin (1.5 pg mL_) also causes rapid leakage of intracellular ATP in L. monocytogenes cells (Winkowski et al., 1994). The efflux of small molecular weight compounds is caused by other bacteriocins such as Las 5 (Zajdel et al., 1985) lactococcin A (Van Belkum et al., 1991) lactococcin B (Venema et al., 1993) lactacin F (Abee et al., 1994) pediocin PA-l (Chikindas et al., 1993) and mesentericin Y105 (Maftah et al., 1993). The changes in membrane permeability induced by bacteriocins decrease or deplete the proton motive force (PMF) of sensitive cells. PMF is an electrochemical gradient composed of a membrane potential (A$) and a pH gradient (ApH). These gradients serve as the driving force for many vital energy-dependent cellular processes. Bacteriocins from four LAB genera act similarly. Nisin (2.5 pg mL-), pediocin PA-l (20 pg mL_), leuconocin S (29.1 pg mL_) and lactacin F (13.5 ,ug mL_) dissipate the PMF in sensitive cells (Bruno et al., 1992; Bruno & Montville, 1993; Okereke & Montville, 1992). The ApH component of the PMF is depleted first by low concentrations of each bacteriocin tested, while higher concentrations are required to decrease the AP component. The PMF in sensitive cells is also depleted by lactococcin A (Van Belkum et al., 1991), lactococcin B (Venema et al., 1993) mesentericin Y105 (Maftah et al., 1993) and

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pediocin JD (Christensen & Hutkins, 1992) suggesting that the dissipation of the PMF is a common step by which different bacteriocins exert their antimicrobial action. However, some bacteriocins may require special conditions to dissipate PMF. While energized membranes enhance or are required by some bacteriocins to dissipate the PMF (as is the case of nisin and other related lantibiotics, Bruno & Montville, 1993; Schuller et al., 1989; Sahl, 1985), other bacteriocins (pediocin PA-l, leuconocin S, lactacin F and lactococcin A) act on deenergized cell membranes (Bruno & Montville, 1993; Van Belkum et al., 1991). Several bacteriocins cause a decrease in intracellular ATP levels. Both efflux and intracellular ATP hydrolysis contribute to the loss of intracellular ATP (Winkowski et al., 1994; Zajdel et al., 1985; Abee et al., 1994a, b). ATP hydrolysis may result from a shift in ATP equilibrium due to inorganic phosphate efflux or as a futile attempt of the cell to regenerate PMF (Abee et al., 1994 a, b; Winkowski et al., 1994). In toto, it is clear from the literature that treatment of cells with bacteriocins leads to the efflux of many intracellular compounds and to PMF dissipation. The latter event in turn, may render the cell unable to protect its cytoplasm from the surrounding medium, arrest all energy-dependent cellular processes and lead to growth inhibition, if not death. Models for pore formation versus membrane destabilization LAB bacteriocins are a heterogeneous group of peptides with different spectra of antimicrobial activity, different genetic determinants and distinct biochemical characteristics (Nettles & Barefoot, 1993; Klaenhammer, 1993). Since they share common mechanisms of action, it is important to identify common traits among these peptides that may explain their action. In this regard, most bacteriocins are amphiphilic and cationic. A common structural motif may underlay their antimicrobial activity as suggested for other antimicrobial peptides occurring in nature (Ojcius & Young, 1991). Based on bacteriocins amphiphilic characteristics, there are at least two different mechanisms which may explain their membrane-permeabilization action. Bacteriocins may act by a poration complex in which bacteriocin monomers bind, insert and oligomerize in the cytoplasmic membrane to form a pore with the hydrophilic residues facing inward and the hydrophobic ones facing the hydrophobic regions of phospholipid molecules in the interior of the membrane. Alternatively, bacteriocins may destabilize the integrity of the cytoplasmic membrane in a detergent-like fashion (Fig. 2). It is appropriate to say a few words about the binding step. Given the wide spectra of activity displayed by LAB bacteriocins, and that some strains are sensitive to some bacteriocins while insensitive to others, the question of a receptor arises. Some bacteriocins (e.g. nisin) are active both on cells and on lipid bilayers. Others, including lactococcin A, lactacin F and pediocin PA-1 are only active on whole cells or membrane vesicles, suggesting the need of a receptor to exert their antimicrobial action. To date, no bacteriocin receptor has been identified, nor has any active domain of the bacteriocin molecule been identified as the receptor binding site. While there is no direct physical proof in vegetative cells that bacteriocins form pores, results from many studies are clearly more consistent with pore formation than detergent-like action. We have observed that nisin (1.5 pg mL-) induces ATP efflux and PMF dissipation within 2 min. Furthermore, nisin action is

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805

::i

Fig. 2. Interaction

of bacteriocin monomers (ovals) with the cytoplasmic membrane according to the poration complex model (A) and the detergent disruption model (B).

concentration dependent and demonstrates saturation kinetics (Winkowski et al., 1994). The action of many other bacteriocins including PAl, lactococcin A, lactococcin B and lactacin F is concentration dependent and saturable favoring the idea that bacteriocins act by a poration complex (Chikindas et al., 1993; Van Belkum et al., 1991; Venema et al., Abee et al., 1994). A generalized solubilization mechanism would result in an all-or-none lysis of the cells, with a sudden collapse of the bioenergetic parameters and no saturation kinetics. Further evidence supporting pore formation as the mechanism comes from in vitro studies. Studies using black lipid membranes suggest that nisin, pep 5 and subtilin form transient multi-state pores with a predicted diameter of 0.2 to 2 nm and lifetimes of milliseconds to seconds (Kordel et al., 1988; Sahl et al., 1985; Schuller et al., 1989). To characterize the interaction of bacteriocins with lipid bilayers, we are currently using fluorescence spectroscopy. Liposomes made from

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lipids extracted from L. monocytogenes cells are loaded with the fluorophore carboxyfluorescein so that nisin-induced leakage of this small molecular weight compound (376 Da.) can be used to characterize the membranes permeabilization. Carboxyfluorescein efflux is dependent on the amount of bacteriocin molecules present as well as with the amount of lipid present. Increasing nisin concentrations or decreasing lipid concentration results in enhanced leakage rate, suggesting that the amount of peptide bound per lipid molecule determines the leakage rate. The critical number of bacteriocins required to induce efflux is also dependent on the pH, where fewer molecules are required at neutral pH compared to acidic pHs (unpublished results). Using a similar model system, Garcia-Garcera et al. (1993) found that nisin action causes a general disruption of liposomes. When liposomes loaded with high-molecular weight dextran molecules (MW of 4.4 or 9.4 kDa) are treated with nisin, no efflux occurs, suggesting that nisin forms putative pores with a defined size. The addition of pediocin PA-l at high concentrations, however, causes release of the dextrans, suggesting that the concentration of pediocin PA-l determines the size exclusion limit of the pores (Chikindas et al., 1993).

UNANSWERED Resistance

QUESTIONS

AND

CONCLUSIONS

Bacterial resistance to antibiotics is now a major societal problem. It is accelerating and increases morbidity, mortality and health-care costs (Cohen, 1992). More prudent use of antibiotics would reduce the selective pressure which favors the development of resistance (Levy, 1992). If indiscriminate antibiotic use continues, we may enter a post-antibiotic era marked by uncontrollable pneumonia, meningitis, typhoid fever, tuberculosis and rheumatic fever (Cohen, 1992). Food scientists should exercise care in bacteriocin applications and build resistance management into their plans just as entomologists have made resistance management an important component when using Bacillus thurengiengensis toxin as an agricultural biocide. The widespread emergence of antibiotic resistant pathogens was not inevitable (Levy, 1992). It could have been prevented and might still be reversed. An early understanding of how organisms become bacteriocin resistant will provide a rational basis for their application under conditions which do not promote selection of pathogens with high intrinsic resistance. Such research is currently under way in our laboratory. Bacteria can resist antibiotics by changing the antibiotic or changing themselves. The four most important resistance mechanisms are enzymatic degradation of the antimicrobial, covalent modification of it, alteration of target organisms antibiotic receptors, and changes in the organisms membrane permeability to antibiotics (Dever & Dermody, 1991). These are all applicable to bacteriocin resistance (Table 1). Nisin resistance (Nis) is usually plasmid-mediated and linked to another transmissible trait (Klaenhammer, 1993). Nisin dehydroreductase, which inactivates one of nisins dehydro residues (Jarvis & Farr, 1971), results in the Nis phenotype. The bacr phenotype similarly results from enzymes that modify or act on specific bacteriocins. Proteases might function in this fashion. While little is

Bacteriocin mechanisms and applications

TABLE 1 and Bacteriocin

807

Examples

of Antibiotic

Resistance

Mechanisms

Mechanism
Export

Action
specific

Antibiotics
tetracycline

Bacteriocins
not applicable, bacteriocins not in cytoplasm proteases, specific bacteriocinase dehydroreductase

Destruction

specific or general specific

j?-lactamases methylation of aminoglycosides penicillin binding proteins

Modification

Altered

receptors

specific

probable, but not reported demonstrated resistance

to date

Membrane composition

general

altered membranes E. coli and bacilli

in resistant

for nisin

know about the proteolytic systems of L. monocytogenes, their relatedness to the LAB which have elaborate proteolytic and peptidolytic systems (Thomas & Pritchard, 1987; Tan et al., 1993) suggests that proteases might play an important role in bacteriocin resistance. Spontaneous nisin-resistant mutants, designated Nis, are generated when nisin sensitive cells are exposed to high nisin concentrations (Klaenhammer, 1993). The Nis phenotype may involve some intrinsic cellular component involved with bacteriocin adsorption (i.e. the putative receptor, about which little is know) or membrane insertion. Genetically stable Nis L. monocytogenes occur at a frequency of lop6 (Harris et al., 1991; Davies & Adams, 1994). Nisin resistant isolates are generated from vegetative cells of Staphylococcus aureus, Bacillus licheniformis, B. subtilis, B. cereus (Ming & Daeschel, 1993) and C. botulinum (Crandall & Montville, unpublished data) at similar frequencies. The L. monocytogenes nisin-resistant isolates do not produce nisin-inactivating enzymes, but may have altered membrane compositions and appear to have a Nis phenotype (Ming & Daeschel, 1993). Membrane fluidity plays an important role in listeric resistance to other antimicrobials (Juneja & Davidson, 1993). L. monocytogenes cells grown in the presence of C14:O or C18:O fatty acids have higher Tc and increased resistance to four common antimicrobials relative to cells grown in the presence of C18:l which have lower Tc and greater antimicrobial sensitivity. Additional research in the area of bacteriocin resistance is required. Regulatory considerations The uncertain regulatory status of bacteriocins and bacteriocinogenic LAB casts a cloud over the whole field. Ingredients are regulated as food additives in the U.S. unless they are Generally Recognized as Safe (GRAS) (McNamara, 1986). The time and money required to obtain FDA approval of a new food additive is prohibitive and can only be justified for very novel and unique food additives. Scientists generally regard LAB which produce bacteriocins as save; however,

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their legal status as GRAS is unclear. GRAS status is predicated on safety and appropriateness for a specific use and can be based on documented use prior to 1958 or a consensus of scientific opinion. Although LAB are recognized as GRAS for producing fermented foods, they are not automatically GRAS as preservatives in, for example, (nonfermented) minimally processed refrigerated foods. Our demonstration that, in many cases, LAB are not efficacious antibotulinal agents argues against blanket affirmation of all bacteriocinogenic LAB for all applications. Providing efficacy data would not be a burden to those seeking GRAS affirmation. However, requiring extensive toxicological and safety data would increase the cost of approval to an unacceptably high level. Harlander (1993) provides an excellent and thorough discussion of these issues. She states, Bacteriocins are particularly attractive preservatives as they are naturally produced by many strains of lactic acid bacteria used for the production of fermented foods, and thus, have been consumed safely by humans for hundreds of years. In the U.S. one can self-affirm GRAS status, but most scientists in large food companies believe it prudent to seek FDA affirmation of GRAS status. Aplin and Barrett has done this for the use of nisin to increase the shelf-life of pasteurized processed eggs. In this case, the 1988 affirmation of nisin as GRAS for use in pasteurized processed cheese (which was supported by toxicological data) provides the foundation for subsequent expansions of GRAS applications. Under the U.S. National Labeling Education Act (NLEA), an ingredient may be added to a modified standard of identity food to restore other characteristics to levels found in the original product. Thus, the FDA accepted for tiling a request for GRAS affirmation of nisin to restore the shelf-life of reduced-fat pasteurized egg. Once this was accepted, an affirmation for use of nisin to extend the shelf-life of regular processed eggs was filed. Both of these applications limit use levels to the 10,000 I.U. g- for which toxicological and safety data were provided in the 1988 affirmation. Another case, which we believe would be acceptable under the NLEA, is one where a botulinal hazard is created (OMahony et al., 1990) when a maker of a hazelnut yoghurt changes from a sugar syrup (which because of low water activity inhibits botulinal toxigenesis) to a light, artificially-sweetened, syrup. The addition of nisin to this product (to restore the antibotulinal barrier) should be permitted. There is precedence for the use of cultured milk products as GRAS preservatives. Microgard TM is a cultured milk product added to much of the cottage cheese in the U.S. and is approved by the FDA as a GRAS food preservative (Daeschel, 1989). The fermentation of milk by Propionibacterium shermanii results in the production of acetate, propionic acid, and low molecular weight proteins; the propionic acid certainly plays a major role in its activity (Al-Zoreky et al., 1991). Since many foods are undoubtedly fermented by bacteria that happen to produce bacteriocins, and these fermented foods may be consumed directly or used as ingredients in other foods, it is difficult to see how their use as preservatives could be prohibited. When the unusual lanthionine containing bacteriocins were discovered early in the antibiotic era (Mattick & Hirsch, 1944), they were considered to be antibiotics (hence lantibiotics). The use of antibiotics as food preservatives is strictly prohibited. Thus, people concerned about the spread of antibiotic resistance (Neu, 1992) may campaign against the use of bacteriocins. We have discussed

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elsewhere (Montville & Kaiser, 1993) why bacteriocins are not antibiotics strict0 sensu. The prohibition against antibiotics in foods should not extend to bacteriocins because they have no clinical efficacy which might be threatened by development of resistance and are widely found in foods anyway (Garver & Muriana, 1993).

CONCLUSIONS There is voluminous literature that testifies to the efficacy of bacteriocins in multiple barrier preservations systems where pH, temperature, salt concentration and/or other preservatives can be tailored to provide synergism. There is equal evidence that bacteriocins do not work when used as stand-alone preservatives in refrigerated foods that contain no other barriers to microbial growth. Thus, we expect that applications of bacteriocin to food safety problems will unfold slowly and in a logical progression. Bacteriocin-producing LAB are already being used as starter cultures, whether intentionally or unintentionally. It seems logical that if LAB are already being used in a fermentation, the use of naturally-isolated bacteriocin-producers would add an extra degree of protection without posing any special problems. The use of cultures (the organisms, their substrates, and their products) as preservatives has precedence in MicrogardTM and NisaplinTM. GRAS affirmation of the generic use of LAB cultures as preservatives would accelerate the pace of research in this area. Regulatory approval for the use of purified bacteriocins is a larger issue. The approval of genetically-engineered bacteriocins is doubtful if they are considered as new proteins. We have not discussed the use of bacteriocins as selectable markers in genetic engineering nor their use to facilitate the dominance of starter cultures over indigenous microbiota in nonpasteurized fermented foods. Both of these applications are promising and should be investigated more thoroughly.

ACKNOWLEDGEMENTS This is manuscript D-10580-1-95 of the New Jersey Agricultural Experiment Station. Research in the authors laboratory and preparation of this manuscript was supported by state appropriations, U.S. Hatch Act Funds, the U.S. Israel Bilateral Research and Development Fund (BARD, Project No. US-21 13-92) and a grant from the United States Department of Agriculture CSRS NRI Food Safety Program (no. 91-37201-6796). This manuscript is an expansion of a paper presented at the International Dairy Lactic Acid Bacteria Conference, held in Palmerston North, New Zealand, February 19-23, 1995.

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