Biopreservation

Meat Preserving And Curing Guide

A Cured Meat Guide for Everyone

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Bruna C. Gomes, Lizziane K. Winkelstroter, Fernanda B. dos Reis, and Elaine C. P. De Martinis

Introduction

In the last decades important changes have been observed in the food science area, with increasing consumers demand for ready-to-eat (RTE) and minimally processed foods, as a reflection of the increasing awareness of the risks derived not only from foodborne pathogens but also from artificial chemical preservatives used to control them (Castellano, Belfiore, Fadda, & Vignolo, 2008; Parada, Caron, Medeiros, & Soccol, 2007; Rodriguez, Martinez, Horn, & Dodd, 2003; Schuenzel & Harrison, 2002). This tendency allied to strict government requirements for food safety has faced food producers with conflicting challenges (Settanni & Corsetti, 2008). The preservation techniques used in early days relied, without any understanding of the microbiology, on the inactivation of undesirable microorganisms through drying, salting, heating, or fermentation. These methods are still used today, combining various hurdles to inhibit growth of microorganisms, but some of the classic preservation techniques are not suitable for fresh meats and RTE products (Gram et al., 2002; Quintavalla & Vicini, 2002; Rao, Chander, & Sharma, 2008).

Meat is a nutrient-rich matrix that provides a suitable environment for proliferation of many spoilage microorganisms and foodborne pathogens (Ananou, Garriga, et al., 2005; Aymerich, Picouet, & Monfort, 2008; Hugas, 1998). Microbial contamination of meats has been implicated with the most serious foodborne outbreaks and recalls from the food marketplace (Ananou, Garriga, et al., 2005; Sofos, 2008). Major causes of concern and product recalls associated with fresh meat products are Escherichia coli O157:H7 and related enteric pathogens such as Salmonella, while the Gram-positive Listeria mono-cytogenes is the pathogen of concern in RTE meat and poultry products. L. monocytogenes is a psychrotrophic bacterium that can grow during

Faculdade de Cieencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe; s/n 14040903, Ribeirao Preto, Brazil e-mail: [email protected]

F. Toldra (ed.), Safety of Meat and Processed Meat,

Food Microbiology and Food Safety, DOI 10.1007/978-0-387-89026-5_11,

© Springer Science+Business Media, LLC 2009

refrigerated storage of meats, even after a lethality treatment, if recontamination occurs during slicing, packaging, peeling, or handling (Sofos, 2008; Trivedi, Reynolds, & Chen, 2008).

To reduce the level of microbial contamination on raw meats and animal carcasses, processing facilities of all sizes in the United States are currently required to establish Sanitation Standard Operating Procedures (SSOP) as well as the Hazard Analysis and Critical Control Points (HACCP) program (Aymerich et al., 2008; Trivedi et al., 2008). Several carcass decontamination methods have been validated for use as critical control points to reduce bacterial populations on meat and poultry, including steam/hot water vacuuming, spray washing, and steam pasteurization (Trivedi et al., 2008).

The need for alternatives to extend the shelf life of foods without changing their sensory properties has launched research on biopreservation technologies, which are based on the use of non-pathogenic microorganisms and/or their metabolites to retard food spoilage and/or to improve food safety (De Martinis, Publio, Santarosa, & Freitas, 2001; Ross, Morgan, & Hill, 2002).

Methods of Biopreservation

Some pathogens present in foods may be inhibited or even eliminated by the action of competitors or antagonistic microbiota, improving the shelf life and safety of products without the need of using elevated levels of chemical additives (Schuenzel & Harrison, 2002). The presence of a competitive microbiota is a promising alternative also to prevent biofilm formation by some pathogens in food processing equipments (Jeong & Frank, 1994; Minei, Gomes, Ratti, D'Angelis, & De Martinis, 2008).

Lactic acid bacteria (LAB) have a major potential for use in biopreservation because they have a long history of safe consumption and they naturally dominate the microflora of many foods during storage. Moreover, in raw meats and fish that are chill stored under vacuum or under elevated CO2 concentration, LAB become the dominant population and preserve the meat with a "hidden" fermentation (Stiles, 1996). The same rationale applies to processed meats if LAB survive heat treatment or if they are reintroduced in the product after heat treatment (Stiles, 1996).

Biopreservation by Lactic Acid Bacteria

LAB constitute a group of Gram-positive, catalase-negative cocci or rods with similar characteristics and the ability to produce lactic acid as the main product of the fermentation of carbohydrates. Many of these microorganisms are considered "food grade" and may exert their antimicrobial properties against pathogens, spoilage bacteria, yeasts, and molds by different ways such as

(a) production of volatile acids, hydrogen peroxide (H2O2), carbon dioxide (CO2), diacetyl, acetaldehyde; (b) competitive exclusion; and (c) production of bacteriocins (Naidu, Bidlack, & Clemens, 1999; Settanni & Corsetti, 2008). Bacteriocins are antimicrobial peptides produced by numerous Gram-positive and Gram-negative organisms, but bacteriocins from LAB are of special interest in the food science area because LAB present a positive association with foods and have a long history of safe consumption, as part of the natural microbiota of meat, milk, vegetable and fish products (Rodríguez et al., 2003; Aymerich et al., 2008). Moreover, the use of LAB and/or their metabolites for food preservation is generally accepted by consumers as something "natural" and "health promoting" (Deegan, Cotter, Hill, & Ross, 2006; Rodriguez et al., 2003). Bacteriocins of LAB present potential applications in meats, as shown in Table 11.1.

Bacteriocins can be applied in meat systems by two basic methods: by adding crude, purified, or semi-purified bacteriocin preparations or by inoculation with pure cultures of the bacteriocinogenic strains. Both approaches offer advantages and disadvantages, and the choice of either one will depend on the bacteriocin, the producer strain, the food system, and the target microorganism (Ananou, Maqueda, Martínez-Bueno, Galvez, & Valdivia, 2005; Hugas, 1998). In meat environments, a higher concentration of bacteriocin-producing cells may be necessary to compensate adsorption of bacteriocin molecules to the meat matrix (Ananou, Garriga, et al., 2005).

Before using a given bacteriocin for biopreservation, it is necessary to study its efficacy for each particular food system, to determine the concentrations of bacteriocin required to achieve an efficient control of foodborne pathogens, or the capacity of bacteriocinogenic strains for growth and bacteriocin production in the food system (Ananou, Garriga, et al., 2005).

The naturally occurring LAB strains in meat and meat products include Carnobacterium piscícola and C. divergens; Lactobacillus sakei, Lb. curvatus, and Lb. plantarum; Leuconostoc mesenteroides subsp. mesenteroides, Lc. gelidum, and Lc. carnosum. LAB play an important role in fermented foods, causing flavor and texture changes together with a preservative effect resulting in products with increased shelf life (Hugas, 1998; Settanni & Corsetti, 2008).

LAB such as Carnobacterium spp., Lactobacillus spp., and Leuconostoc spp. are those related to meat spoilage, but a selective promotion of growth of LAB capitalizing on their ability to control meatborne pathogens with a preferential growth of benign strains would minimize detrimental effects (Settanni & Corsetti, 2008).

In fermented meat products, Enterococcus spp., especially E.faecium, represents one of the LAB species that can be found in relatively high numbers during fermentation and they may contribute to the flavor of products by their glyco-lytic, proteolytic, and lipolytic activities (Ananou, Garriga, et al., 2005). Bacteriocin-producing enterococci are widespread in nature and strains with strong anti-listerial activity have been isolated from numerous fermented meat products and have been well characterized (Belgacem, Ferchichi, Prévost, Dousset, & Manai, 2008).

Table 11.1 Some examples of microorganisms that may be controlled by biopreservation techniques

Target microorganism

Meat products

Biopreservative agent

References

Listeria monocytogenes

Package

Bacteriocin 32Y from

Ercolini et al.

ground

Lactobacillus curvatus

(2006); Mauriello

beef and

et al. (2004)

pork steak

Total aerobic bacteria

Frankfurters

Nisin

Guerra et al. (2005)

and fresh

veal meat

L. monocytogenes

Ready-to-eat

Nisin plus lysozyme

Mangalassary et al.

turkey

(2007)

bologna

Lb. sakei and Lb. curvatus

Bologna-

Nisin

Davieset al. (1999)

type

sausage

Clostridium perfringens,

Fresh pork

Lacticin 3147 from

Scannell et al.

Salmonella Kentucky,

sausage

Lactococcus lactis DPC

(2000)

and L. innocua

3147

L. monocytogenes

Brazilian

Lb. sakei 2a bacteriocin

De Martinis and

sausage

producer

Franco (1998)

L. monocytogenes

Fish peptone

Carnobacterium piscicola

Alves, De Martinis,

model

Destro,

system

Fonnesbech, and

Gram (2005)

L. monocytogenes

Cooked ham

Lb. sakei 1 bacteriocin

Alves, Martinez,

producer

Lavrador, and

De Martinis

(2006)

L. monocytogenes

Meat gravy

Lb. sakei 1 bacteriocin

Alves, Lavrador,

system

producer

and De Martinis

(2003)

L. monocytogenes

Cooked ham

Enterocins A and B, sakacin

Jofre et al. (2007)

K and nisin plus lactate

L. monocytogenes,

Vacuum-

Lb. sakei and Lact. lactis

Jones, Hussein,

Brochothrix thermosphacta,

packed

Zagorec,

Campylobacter jejuni and

chill-

Brightwell,

Cl. estertheticum

stored

and Tagg (2008)

meat

L. monocytogenes and

Dry sausage

Lactic acid bacteria

Tyopponen, Petaja,

Escherichia coli O157:H7

and Mattila-

Sandholm (2003)

L. monocytogenes

Meat

Lactic acid bacteria

De Martinis et al.

(2001)

Total plate count

Brined

Nisin Z, camocin UI49 from

Einarsson and

shrimp

Cb. piscicola and bavaracin

Lauzon (1995)

A from Lb. bavaricus MI

401

Bacteriocins of Lactic Acid Bacteria

Bacteriocins produced by LAB may be very attractive for biopreservation due to (i) production by strains generally recognized as safe, (ii) lack of action against eukaryotic cells, (iii) inactivation by digestive proteases, which preserve the gut microbiota, showing no cross-resistance with antibiotics, (iv) tolerance to pH and heat, (v) mostly bactericidal mode of action, and (vi) genetic determinants usually plasmid encoded, facilitating genetic manipulations (Galvez, Abriouel, Lopez, & Omar, 2007).

However, many bacteriocins have not been fully characterized and, consequently, cannot be extensively used in the food industry. To date, the only bacteriocin licensed as a food preservative is nisin (Cleveland, Montville, Nes, & Chikindas, 2001; Galvez et al., 2007; Sobrino-Lopez & Martin-Belloso, 2008). This antimicrobial peptide is produced by Lactococcus lactis subsp. lactis and marketed under the trade name Nisaplin, which contains ca. 2.5% of nisin (product description PD45003-7EN; Danisco, Copenhagen, Denmark). Alternatively, strains producing bacteriocins can be added to concentrates originating from a food-grade substrate (milk or whey), with commercial applications such as pediocin PA-1 produced by Pediococcus acidilactici commercialized as ALTAtm 2341 (Kerry Bioscience, Carrigaline, Co. Cork, Ireland) (Deegan et al., 2006; Galvez et al., 2007; Sobrino-Lopez & Martin-Belloso, 2008). Commercial bioprotective cultures (Chr. Hansen, Denmark) have been developed to reduce the incidence of spoilage microbiota and some pathogens during processing of meat products: SafePro® B-2 (containing non-bacteriocin-producing Lb. sakei BJ33) and SafePro® B-SF-43 (containing bacteriocin-producing Lc. carnosum 4010). Also, several patents have been deposited dealing with biopreservation (Cleveland et al., 2001).

Pediocin PA-1 is a plasmid-encoded class II bacteriocin with a broad inhibitory spectrum against E.faecalis, Staphylococcus aureus, Clostridium perfrin-gens and particularly effective against L. monocytogenes (Guerra, Bernardez, & Castro, 2007; Reviriego et al., 2005; Sobrino-Lopez & Martin-Belloso, 2008).

Although pediocin PA-1 is mainly used in vegetables and meat products, the extension of its application to dairy products is being evaluated due to its anti-listerial activity, stability in aqueous solutions, wide pH range for activity, and the fact that it is unaffected by heating or freezing (Sobrino-Lopez & MartinBelloso, 2008; Reviriego et al., 2005).

Nisin was discovered in 1928, after observations that certain lactococcal strains inhibited other LAB in dairy fermentations, and it is currently approved for use in over 50 countries (McAuliffe, Ross, & Hill, 2001; Ross et al., 2002; Delves-Broughton, 2005; Deegan et al., 2006). This bacteriocin is effective in a number of food systems, inhibiting the growth of a wide range of Gram-positive bacteria and their spores, but it does not inhibit the growth of yeasts and molds (Deegan et al., 2006).

Stevens, Sheldon, Klapes, and Klaenhammer (1991) hypothesized that the cell wall of Gram-negative bacteria, composed of lipopolysaccharides, acts as a permeability barrier, preventing nisin from reaching the target cytoplasmic membrane. Those authors affirmed that chelators, hydrostatic pressure, or cell injury may destroy the cell wall, rendering the Gram-negative bacteria sensitive to the bacteriocin. However, the application of nisin to meats may be limited due to its low solubility in meat pH, the inability of the producer organism to grow in meats, and to its inefficiency to inhibit all the spoilage and pathogenic microorganisms associated with meats (Stiles & Hastings, 1991).

Moreover, Rose, Palcic, Sporns, and Mc Mullen (2000) demonstrated that nisin may be inactivated by the enzyme glutathione S-transferase of raw beef, confirming that the use of this bacteriocin in raw meats may be limited. Nisin use for partial replacement of nitrite in cured meats has been investigated and only high and uneconomic levels of nisin may promote good control of C. botulinum (Delves-Broughton, 2005). However, better results have been achieved for the use of nisin to overcome post-processing contamination of meat products where LAB can cause spoilage (Aymerich et al., 2008; Delves-Broughton, 2005). The application of nisin in vacuum-packed cooked sausage has achieved regulatory approval in the United States (Delves-Broughton, 2005). In Brazil, nisin was approved for use in cheeses and also for spraying on the surface of frankfurters at the end of the thermal processing step. In fresh meat, nisin has also been tested as spray to sanitize the surface of red meat carcasses (Aymerich et al., 2008).

The use of nisin in meats is still controversial, although it has been reported to present better action in products with lower fat levels (Castro, 2002; Davies et al., 1999; Delves-Broughton, 2005; El-Katheib, Yousef, & Ockerman, 1993; Fang & Lin, 1994)

It has been postulated that bacteriocins and/or protective cultures may be more effective if used in the hurdle technology approach, in combination with other barriers such as modified atmosphere packaging, hydrostatic pressure, high temperature, chelating agents, antimicrobials, and lactoperoxidase system (Chen & Hoover, 2003; Cleveland et al., 2001).

According to Garcia, Martin, Sanz, and Hernandez (1995) and Mc Mullen and Stiles (1996) the most suitable strains to be used in biopreservation of a certain food product are likely those isolated from the same type of food where they are intended to be used. They attributed this probability to competitive advantage of the previously adapted strains. Based on this premise, several studies for the isolation of bacteriocinogenic LAB from meats have been conducted in several countries (De Martinis, Alves, & Franco, 2002).

Some bacteriocins presenting anti-listerial activity in meat homogenates have been applied experimentally as ingredients in several meat products, such as enterocin A, enterocin B, and sakacin K (Jofre, Garriga, & Aymerich, 2007).

Enterocin AS-48, produced by E. faecalis S-48, exhibits bactericidal activity against a wide variety of Gram-positive bacteria, including food spoilage and pathogenic bacteria such as Bacillus cereus, C. botulinum, C. difficile, C. perfringens, S. aureus, and L. monocytogenes. It also shows activity against some Gram-negative species (Abriouel, Valdivia, Martínez-Bueno, Maqueda, & Galvez, 2003; Ananou, Garriga, et al., 2005; Lucas et al., 2006). Some features of AS-48 such as (i) broad spectrum of antimicrobial activity, (ii) stability in a wide range of temperature and pH, and (iii) sensitivity to digestive proteases render this bacteriocin a promising alternative to chemical preservatives (Ananou, Garriga, et al., 2005; Ananou, Maqueda, et al., 2005, Lucas et al., 2006).

Recently, a database containing calculated or predicted physicochemical properties of diverse bacteriocins was created and named BACTIBASE (http://bactibase.pfba-lab.org), which can be an efficient tool to facilitate future food biopreservation studies (Hammami, Zouhir, Hamida, & Fliss, 2007). Besides, the elucidation of the mode of action of these antimicrobial peptides can help to optimize their food applications.

Mode of Action of Bacteriocins

The family of bacteriocins includes a diversity of proteins in terms of size, microbial targets, mode of action, and immunity mechanism (Riley & Wertz, 2002). Bacteriocins are proteins ribosomally synthesized and are often confused in literature with antibiotics (Cleveland et al., 2001). They differ from antibiotics because they have a relatively narrow killing spectrum and are only toxic to bacteria closely related to the producer strain (Riley & Wertz, 2002). Other differences are that antibiotics are generally considered secondary metabolites and are not ribosomally synthesized (Cleveland et al., 2001; Deegan et al., 2006).

As a group, bacteriocins act on target cells by various mechanisms: (i) permeabilization of the cytoplasmic membrane followed by leakage of low-molecular-weight cellular compounds and dissipation of the proton motive force (PMF); (ii) cell lysis; (iii) degradation of vital macromolecules such as DNA and RNA; and (iv) inhibition of biological processes such as synthesis of protein, DNA, RNA, and peptidoglycan (De Martinis et al., 2002; Motta, Flores, Souto, & Brandell, 2008).

Bacteriocins were first characterized in Gram-negative bacteria (Cleveland et al., 2001). Colicins of E. coli are well studied and can act as membrane-depolarizing agents, DNA or RNA endonucleases, translation blocker, or inhibition of murein synthesis (Cursino, Smarda, Chartone-Souza, & Nascimento, 2002; Kolade et al., 2002).

Bacteriocins may be classified into four classes based on their biochemical and genetic properties (Deegan et al., 2006; Drider, Fimland, Hechard, McMullen, & Prevost, 2006; Naghmouchi, Kheadr, Lacroix, & Fliss, 2007). Class I peptides are the lantibiotics, which are small and characterized by unusual amino acids, such as lanthionine, and nisin is included in this class. Class I is subdivided into class Ia and Ib. In general, class Ia bacteriocins consist of cationic and hydrophobic peptides and class Ib bacteriocins are globular peptides with no net charge or a net negative charge (Cleveland et al., 2001). Class II comprise small heat-stable, non-modified peptides and are subdivided into three subclasses, namely, class IIa (pediocin-like bacteriocins), IIb (two different peptides), and IIc (one-peptide bacteriocins). The class III peptides are large and thermosensitive (Cleveland et al., 2001; Drider et al., 2006; Oppegard, Rogne, Emanuelsen, Kristiansen, Fimland, & Nissen-Meyer, 2007). A fourth class contains complex bacteriocins that are composed of protein plus one or more chemical moieties (lipid, carbohydrate) required for activity: plantaricin S, leuconocin S, lactocin 27, pediocin SJ-1 (De Martinis et al., 2002). Class IV is currently the subject of discussion and not formally recognized since this class has not been studied sufficiently at the biochemical level. Studies have principally focused on members of class I and class II due to the abundance of these peptides and their potential for commercial applications (McAuliffe et al., 2001; Naghmouchi, Drider, & Fliss, 2007).

According to Parisien, Allain, Zhang, Mandeville, and Lan (2008) lantibio-tics inhibit target cells by forming pores in the membrane, depleting the transmembrane potential and/or the pH gradient, resulting in the leakage of cellular materials (Cleveland et al., 2001; Deegan et al., 2006; McAuliffe et al., 2001). The electrostatic interactions between the positive charge of bacteriocins and the negative charge of phosphate groups on target cell membranes are thought to contribute to the initial binding with the target membrane. It is likely that the hydrophobic portion inserts into the membrane, forming pores (Cleveland et al., 2001; Deegan et al., 2006). There are two models for pore forming, the barrel stave and the wedge. In barrel stave model, each nisin molecule orients itself perpendicular to the membrane forming an ion channel that spans the membrane (Fig. 11.1A). According to the wedge model, after a critical number of nisin molecules associate with membrane, they insert concurrently, forming a wedge (Fig. 11.1B, C) (Cleveland et al., 2001; McAuliffe et al., 2001; Bauer & Dicks, 2005).

Besides pore formation, it is believed that nisin also mediates inhibition of cell wall biosynthesis, by forming a complex with lipid II, the bactoprenol-bound peptidoglycan precursor (Deegan et al., 2006; Hechard & Sahl, 2002; McAuliffe et al., 2001). It suggests that nisin may use lipid II as a docking molecule for facilitating the interaction with the bacteriocin and specific membranes (Fig. 11.2) (Cleveland et al., 2001). The combination of two killing mechanisms, inhibition of the peptidoglycan synthesis and the pore formation, renders nisin active at nanomolar concentrations (Deegan et al., 2006; Hechard & Sahl, 2002).

Class II bacteriocins predominantly act by inducing permeabilization of the target cell membrane, probably by forming ion-selective pores which cause dissipation of the proton motive force, depletion of intracellular ATP, and leakage of amino acids and ions (Deegan et al., 2006; Drider et al., 2006).

Class IIa is the largest and most extensively studied subgroup of class II bacteriocins that are especially strong inhibitors of L. monocytogenes. Because of this anti-listerial effectiveness class IIa bacteriocins have significant potential as biopreservatives in a large number of foods (Ennahar, Sashihara, Sonomoto, & Ishizaki, 2000). Pediocin PA-I and other identical bacteriocins produced by P. acidilactici (pediocin AcH, pediocin SJ-I, pediocin JD) are the most extensively studied class II bacteriocins. This bacteriocin was found to induce the leakage of K + , amino acids, and other low-molecular-weight molecules, which lead to rapid depletion of intracellular bacterial ATP (Drider et al., 2006). Pediocin PA-I dissipates the membrane potential and causes release of amino acids accumulated

Bacteriocins Diagram

Fig. 11.1 Models of non-targeted pore formation by nisin. (A) Barrel stave pore. (B, C) General models for pore formation. Step 1: binding of nisin via its C-terminal. Step 2: insertion of nisin into the membrane. The depth of insertion depends on the percentage of anionic lipids and nisin concentration. Step 3: wedge/magainin-like pore. Diagrams B and C represent pore formation initiated by translocation of the C-terminus and N-terminus, respectively. Step 4: translocation of the peptide to the inside of the membrane (Bauer & Dicks, 2005)

Fig. 11.1 Models of non-targeted pore formation by nisin. (A) Barrel stave pore. (B, C) General models for pore formation. Step 1: binding of nisin via its C-terminal. Step 2: insertion of nisin into the membrane. The depth of insertion depends on the percentage of anionic lipids and nisin concentration. Step 3: wedge/magainin-like pore. Diagrams B and C represent pore formation initiated by translocation of the C-terminus and N-terminus, respectively. Step 4: translocation of the peptide to the inside of the membrane (Bauer & Dicks, 2005)

either in a proton motive force (PMF)-dependent or -independent manner (Héchard & Sahl, 2002).

There is less information about bacteriocin action mechanism against spores than there is for vegetative cells. Most of these studies deal with nisin, which is sporostatic rather than sporocidal. It was found that nisin modifies the sulfhy-dryl groups in the envelopes of germinative spores, acting as electron acceptors (Montville, Winkowski, & Ludescher, 1995).

Several factors influence the bacteriocin activity on the target bacterial cell (Hechard & Sahl, 2002; Motta et al., 2008). These include the structure and amount of the substance, the composition of the cytoplasmic membrane, the structure and the expression level of a protein with an immunity function, and the chemical composition of the environment (Ennahar et al., 2000; Hechard &

Environment Safety Drawing

Fig. 11.2 Model for lipid Il-mediated inhibition of peptidoglycan biosynthesis. Lantibiotics (marked by shading) such as nisin and the mersacidin subtype bind to lipid II, thereby blocking the polymerization of the peptidoglycan. The recognition site for nisin is MurNAc, whereas mersacidin interacts with GlcNAc. Interaction with the pyrophosphate (PP) moiety of lipid II may be involved in stabilizing the transmembrane orientation of the peptides (Bauer & Dicks, 2005)

Fig. 11.2 Model for lipid Il-mediated inhibition of peptidoglycan biosynthesis. Lantibiotics (marked by shading) such as nisin and the mersacidin subtype bind to lipid II, thereby blocking the polymerization of the peptidoglycan. The recognition site for nisin is MurNAc, whereas mersacidin interacts with GlcNAc. Interaction with the pyrophosphate (PP) moiety of lipid II may be involved in stabilizing the transmembrane orientation of the peptides (Bauer & Dicks, 2005)

Sahl, 2002). Thus, the effective use of bacteriocins in food preservation requires the understanding of their mode of action and inhibitory action under different biochemical conditions naturally occurring in foods (Motta et al., 2008).

There is concern on the development of resistance to bacteriocins and also that exposure to bacteriocins renders target microbial cells more resistant to antibiotics (Martinez, Obeso, Rodriguez, & Garcia, 2008). Genetically stable bacteriocin-resistant organisms have been generated with a frequency of 1 in 106 cells under optimal growth conditions (Harris, Fleming, & Klaenhammer, 1991; Ming & Daeschel, 1993). Nisin-resistant cells have already been observed for L. monocytogenes, S. aureus, C. botulinum, and B. cereus. However, no cross-resistance to antibiotics has been observed, likely due to different modes of action of bacteriocins and antibiotics (Cleveland et al., 2001). It has been shown that bacteriocin resistance results from physiological changes in target cell membrane or production of an enzyme that degrades bacteriocin, while antibiotic resistance is generally associated with genetic determinants. Cross-resistance between different class Ila bacteriocins has been reported and it seems to be related to changes in phosphotransferase systems (PTSs), responsible for the uptake and concomitant phosphorylation of a number of sugars in bacteria (Gravesen et al., 2002). Moreover, according to Gravesen et al. (2002) since food systems are inherently heterogeneous, many interacting factors will influence the development of bacteriocin resistance and need to be further investigated.

Future Perspectives

The application of bacteriocins as food additives demands an exhaustive evaluation. Before being legally accepted, their use and efficacy must be shown and they must be chemically identified and characterized. Moreover, manufacturing process and assays used for quantification and standardization of peptide must be described; in addition, toxicological data and fate of molecule after ingestion are also needed (Cleveland et al., 2001; Sobrino-Lopez & Martin-Belloso, 2008). The potential applications of bacteriocins from LAB in the food and health care sectors are evident. However, for effective commercial application and for production in large scale, both genetic and fermentative protocols need to be optimized (Guerra, Agrasar, Macias, Bernardez, & Castro, 2007; Kim & Mills, 2007).

Nowadays researchers are focusing on the application of bacteriocins in foods as part of packaging films, since microbial contamination of meat products occurs primarily at the surface, due to post-processing handling (Coma, 2008). One strategy for reducing contamination is to entrap the antimicrobials in an edible film matrix packaging, which allows a slow migration to the food surface and helps to maintain high concentrations of the biopreservative as needed (Cagri, Ustunol, & Ryser, 2004). Antimicrobial packaging films have been studied to deliver bacteriocins as an additional barrier to control microbial growth (Cagri et al., 2004; Cha & Chinnan, 2004; Ercolini, Storia, Villani, & Mauriello, 2006; Guerra, Macias, Agrasar, & Castro, 2005; Mauriello, Ercolini, La Storia, Casaburi, & Villani, 2004; Ming, Weber, Ayres, & Sandine, 1997; Quintavalla & Vicini, 2002; Siragura, Cutter, & Willet, 1999).

Recent trends in bacteriocin research also involve heterologous production of LAB bacteriocins to construct multi-bacteriocinogenic strains or to confer antimicrobial properties to strains of technological interest, such as those used as starter cultures (Rodriguez et al., 2003). Cloning and expression of bacter-iocin genes in new hosts have allowed to constitute production and even overexpression of bacteriocins, therefore overcoming bacteriocin regulation systems (Ennahar et al., 2000).

E. coli has long been considered the primary prokaryotic host for cloning and expressing heterologous genes due to its extensive genetic characterization (Billman-Jacobe, 1996). Consequently, this bacterium has invariably been selected as the first host for cloning a variety of genes involved in the biosynthesis of many LAB bacteriocins, but alternative food-grade organisms must be employed when production of recombinant proteins in industrial food products is desired. Many LAB species or strains are potentially useful for the hetero-logous production of commercially important proteins or peptides since they fulfill this requirement of food-grade organisms (Rodriguez et al., 2003).

As an example, pediocin PA-1-producing bacteria are pediococci, usually associated with vegetables and meat products but not suitable for production of dairy products. Since this species is unable to ferment lactose, it is metabolically and technologically unsuitable for dairy fermentation. Attempts have been made to achieve the heterologous expression of pediocin PA-1 in Lact. lactis or of acidocin A in Lb. casei for production of the bacteriocin during the lactic fermentation process (Reviriego, Fernandez, Kuipers, Kok, & Rodriguez, 2007; Reviriego, Fernandez, & Rodriguez, 2007). Heterologous expression creates interesting possibilities for further development and extension of bac-teriocin applications as preservatives in various food industries.

References

Abriouel, H., Valdivia, E., Martínez-Bueno, M., Maqueda, M., & Gálvez, A. (2003). A simple method for semi-preparative-scale production and recovery of enterocin AS-48 derived from Enterococcus faecalis subsp. liquefaciens A-48-32. Journal of Microbiological Methods, 55, 599-605.

Alves, V. F., De Martinis, E. C. P., Destro, M. T., Fonnesbech, B., & Gram, L. (2005). Antilisterial activity of a Carnobacterium piscicola isolated from Brazilian smoked fish (surubim [pseudoplatystoma sp.]) and its activity against a persistent strain of Listeria monocytogenes isolated from surubim. Journal of Food Protection, 68, 2068-2077.

Alves, V. F., Lavrador, M. A. S., & De Martinis, E. C. P. (2003). Bacteriocin exposure and food ingredients influence on growth and virulence of Listeria monocytogenes in a model meat gravy system. Journal of Food Safety, 23, 201-217.

Alves, V. F., Martinez, R. C. R., Lavrador, M. A. S., & De Martinis, E. C. P. (2006). Antilisterial activity of lactic acid bacteria inoculated on cooked ham. Meat Science, 74, 623-627.

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