Food applications of bacteriocins
Biopreservation refers to the use of antagonistic microorganisms or their metabolic products to inhibit or destroy undesired microorganisms in foods to enhance food safety and extend shelf life.
Three approaches are commonly used in the application of bacteriocins for biopreservation of foods:
- Inoculation of food with lactic acid bacteria that produce bacteriocin in the products. The ability of the lactic acid bacteria to grow and produce bacteriocins in the products is crucial for its successful use.
- Addition f purified or semi-purified bacteriocins as food preservatives
- Use of a product previously fermented with a bacteriocin-producing strain as an ingredient in food processing.
Biopreservation of Meat Products
Listeria monocytogenes is a Gram-positive, non-spore forming, facultative anaerobic rod widely distributed in the natural environment. It can grow over a pH range of 4.1 to 9.6 and a temperature range of 0 to 45 0C. Moreover, it is relatively resistant to desiccation and can grow at water activity values as low as 0.90 (Chen and Hoover, 2003).
The ubiquitous nature of Listeria monocytogenes, its hardiness and ability to grow at refrigeration temperatures and anaerobic conditions make it a threat to safety of foods. It is regarded as a major food safety problem because it can cause serious illnesses and death. It has been detected in a variety of foods and implicated in several food-borne outbreaks, such as turkey franks. Many studies have been carried out to control L. monocytogenes in meat products since it is common within slaughterhouse and meat-packing environments and has been isolated from raw meat, cooked and ready-to-eat meat products.
The use of bacteriocinogenic protective cultures to control Listeria monocytogenes in meat products is shown in Table 1.
Table 1- Use of bacteriocinogenic protective cultures to control Listeria monocytogenes in meat products (Chen and Hoover, 2003)
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Meat products Protective culture
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Meats
minced meat and comminuted cured raw Lactobacillus sake Lb 706
pork filled into casings
Vacuum packaged
minimally heat-treated beef cubes Lactobacillus bavaricus MN
wieners Pediococcus acidilactici JBL 1095
frankfurters Pediococcus acidilactici JD1-23
Fermented
dry fermented sausage Staphylococcus xylosus DD-34
Pediococcus acidilactici PA-2, and
Lactobacillus bavaricus MI-401
dry fermented sausages Lactobacillus sake CTC494
chicken summer sausages Pediococcus acidilactici
salami Lactobacillus plantarum MCS
turkey summer sausage Pediococcus acidilactici JBL 1095
Modified atmosphere packaged
Brazilian sausage Lactobacillus sake 2a
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Biopreservation of Dairy Products
Listeria monocytogenes has been the documented cause of a number of outbreaks associated with dairy products, such as pasteurized milk and cheese and nisin has been shown to be effective against Listeria monocytogenes in dairy products. The shelf life of the nisin-containing pasteurized process cheese (301 and 387 IU nisin/g) was significantly greater than that of the control cheese spreads. In cold pack cheese spreads, nisin (100 and 300 IU/g) significantly reduced the numbers of Listeria monocytogenes, Staphylococcus aureus and heat-shocked spores of Clostridium sporogenes.
Another problem in cheese production is the Clostridium- associated butyric acid fermentation. Nisin is commonly added to pasteurized processed cheese spreads to prevent the outgrowth of clostridial spores, such as Clostridium tyrobutyricum (Chen and Hoover, 2003).
Application of lacticin 3147 to cheddar cheese and nisin to ricotta-type cheese has also been seen to prolong the shelf life of the dairy products (Chen and Hoover, 2003).
Biopreservation of Seafood Products
The effectiveness of bacteriocins and protective cultures to control growth of Listeria monocytogenes in vacuum-packed, cold-smoked salmon has been demonstrated by several researchers. Katla et al., (2001) examined the inhibitory effect of sakacin P and/or Lactobacillus sake cultures (sakacin P producer) against Listeria monocytogenes in cold-smoked salmon.
In a study using vacuum-packed cold-smoked rainbow trout, Nykanen et al., (2000) examined the inhibition of Listeria monocytogenes and mesophilic anaerobic bacteria by nisin, sodium lactate, or their combination. Trout samples were stored at 80C for 17 days and at 30C for 29 days. Both nisin and lactate inhibited the growth of Listeria monocytogenes from 3.3 to 1.8 log10 CFU/g over 16 days of storage at 80C. The level of Listeria monocytogenes remained almost constant (4.7 to 4.9 log10 CFU/g) for 29 days at 30C in the samples injected before smoking and which contained both nisin and sodium lactate.
The following applications have been seen to prolong the shelf life of the sea food products: Sakacin P to vacuum-packed salmon; nisin to CO2-packed, cold-smoked salmon; nisin Z to brined shrimp and nisin and lactate to vacuum-packed, cold-smoked rainbow trout (Chen and Hoover, 2003)
Hurdle technology to enhance food safety
The major functional limitations for the application of bacteriocins in foods are relatively narrow activity spectra and moderate antibacterial effects. Moreover, they are generally nit active against gram-negative bacteria. To overcome these limitations, more and more researchers use the concept of hurdle technology to improve shelf life and enhance food safety (Tables 2-3). It is well documented that gram-negative bacteria become sensitive to bacteriocins if the permeability barrier properties of their outer membrane are impaired. For example, chelating agents, such as EDTA, can bind magnesium ions from the lipopolysaccharide layer and disrupt the outer membrane of gram negative bacteria, thus allowing nisin to gain access to the cytoplasmic membrane.
It is well documented that nisin enhances thermal inactivation bacteria, thus reducing the treatment time and resulting in better food qualities (Table 2).
The synergistic effect between bacteriocins and other processing technologies on the inactivation of microorganisms has also been frequently reported in the literature (Table 2).
There has been continued interest in the food industry in using non-thermal processing technologies, such as high hydrostatic pressure (HP) and pulsed electric field (PEF) in food preservation. It is frequently observed that bacteriocins, in combination with these processing techniques, enhance bacterial inactivation (Table 3). In addition, Gram-negative bacteria that are usually insensitive to lactic acid bacteria bacteriocins, such as Escherichia coli O157:H7 and Salmonella typhimurium, become sensitive following HP/PEF treatments that induce sublethal injury to bacterial cells. Studies in our laboratory also demonstrate that nisin enhances the pressure inactivation of spores of Bacillus caogulans, Bacillus subtilis and Clostridium sporogenes (Stewart et al., 2000)
Table 2- Hurdle technology to enhance food safety (Chen and Hoover, 2003)
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Bacteriocins Inactivation effects
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In combination with heat
nisin Nisin (1000 IU/g) enhances inactivation of Listeria monocytogenes in lobster by mild heat (60 or 65 oC).
nisin, pediocin AcH Both bacteriocins reduced the viability of gram-negative and
gram-positive bacterial cells surviving sublethal stresses.
In combination with chelating agents
nisin When used with EDTA, citrate, or lactate, nisin (2000 IU/ml) is effective against gram-negative bacteria (Salmonella typhimurium and Escherichia coli O157:H7), in combination with modified atmosphere packaging (MAP)
In combination with antimicrobials
nisin The combined use of potassium sorbate (0.3%) and nisin (400 UI/ml) inhibited the growth of L. monocytogenes.
pediocin AcH Synergistic effects between sodium diacetate (0.3 and 0.5%) and pediocin (5000 AU/ml) against Listeria monocytogenes.
In combination with lactoperoxidase system
nisin A synergistic and lasting bactericidal effect on L. monocytogenes between nisin (100 or 200 IU/ml) and lactoperoxidase system.
In combination with other bacteriocins
pediocin AcH When used with nisin, lacticin 481, or lactacin F, pediocin AcH produced synergistic effects.
leucocin F10 In combination with nisin, leucocin F10 provides greater activity against L. monocytogenes.
curvaticin Simultaneous or sequential additions of nisin (50 IU/ml) and curvaticin 13 (160 AU/ml) induces a greater inhibitory effect against L. monocytogenes than the use of a single bacteriocin.
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Table 3- Simultaneous application of bacteriocins and high hydrostatic pressure or pulsed electric field (PEF) to enhance food safety (Chen and Hoover, 2003).
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Bacteriocins Inactivation effects
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In combination with high hydrostatic pressure
nisin Nisin (1000 IU/g) increases pressure (155 to 400 MPa) inactivation of Escherichia coli, Salmonella enteritidis, Salmonella typhimurium, Shigella sonnei, Psseudomonas fluorescens, and Staphylococcus aureus.
pediocin AcH Pediocin AcH (3000 AU/ml) enhances pressure (345 MPa) inactivation of Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Escherichia coli O157:H7, Lactobacillus sake, Leuconostoc mesenteroides, Serratia liquefaciens, and P. fluorescens.
In combination with PEF
nisin Nisin (2.4 IU/ml) enhances inactivation of vegetative cells of Bacillus cereus by PEF treatment (16.7 kV/cm, 50 pulses each of 0.0002-ms duration).
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Bacteriocins in packaging film
Incorporation of bacteriocins into packaging films to control spoilage and pathogenic organisms has been an area of active research for the last decade. Antimicrobial packaging film prevents microbial growth on food surface by direct contact of the package with the surface of foods, such as meats and cheese. For this reason, for it to work, the antimicrobial packaging film must contact the surface of the food so that bacteriocins can diffuse to the surface. The gradual release of bacteriocins from a packaging film to the food surface may have an advantage over dipping and spraying foods with bacteriocins. In the later processes, antimicrobial activity may be lost or reduced due to inactivation of the bacteriocins by food components or dilution below active concentration due to migration into foods (Appendini and Hotchkiss, 2002).
Two methods have been commonly used to prepare packaging films with bacteriocins (Appendini and Hotchkiss, 2002). One is to incorporate bacteriocins directly into polymers. Examples include incorporation of nisin into biodegradable protein films. Secondly, packaging film-forming methods, heat-press and casting, were used to incorporate nisin into films made from soy protein and corn zein in this study. Both cast and heat-press films formed excellent films and inhibited the growth of L. plantarum. Compared to the heat-press films, the cast films exhibited larger inhibitory zones when the same levels of nisin were incorporated. Incorporation of EDTA into the films increased the inhibitory effect of nisin against Escherichia coli. Nisin was incorporated into a polyethylene-based plastic film that was used to vacuum-pack beef carcasses. Nisin retained activity against Lactobacillus helveticus and B. thermosphacta inoculated in carcass surface tissue sections. An initial reduction of 2-log10 cycles of B. thermosphacta was observed with nisin-impregnated packed beef within the first two days of storage at 40C. After 20 days of refrigerated storage at 4 to 120C (to simulate temperature abuse), B. thermosphacta populations from nisin-impregnated plastic-wrapped samples were significantly less than control (without nisin). Coma et al., (2001) incorporated nisin into edible cellulose films made with hydroxypropylmethylcellulose by adding nisin to the film-forming solution. Inhibitory effect could be demonstrated against Listeria innocua and Staphylococcus aureus, but film additives such as stearic acid, used to improve the water vapor barrier properties of the film, significantly reduced inhibitory activity. It was noted that desorption from the film and diffusion into the food required further optimization for nisin to function more effectively as a preservative agent in the packaged food.
Another method to incorporate bacteriocins into packaging films is to coat or adsorb bacteriocins to polymer surfaces. Examples include nisin/methylcellulose coatings for polyethylene films and nisin coatings for poultry, adsorption of nisin on polyethylene, ethylene vinyl acetate, polypropylene, polyamide, polyester, acrylics, and polyvinyl chloride (Appendini and Hotchkiss, 2002). Nisin adsorbed onto silanized silica surfaces inhibited the growth of Listeria monocytogenes and the contacting surfaces were evaluated at 4-hours interval for 12 hours. Cells on surfaces that had been in contact with a high concentration of nisin (4000 IU/ml) had a smaller degree of inhibition. In contrast, surfaces contacted with films of heat-inactivated nisin allowed Listeria monocytogenes to grow. Listeria innocua and Staphylococcus aureus (along with Lactococcus lactis subsp. lactis) were also used in a study by Scannell et al., (2000) of cellulose-based bioactive inserts and antimicrobial polyethylene/polyamide pouches. Lacticin 3147 and nisin were the tested bacteriocins. Although lacticin 3147 adhered poorly to plastic film, nisin bound well and the bioactive film made with nisin was stable for 3 months with or without refrigeration. Bacterial reductions of up to 2log10 CFU/g cycles in vacuum-packed cheese were seen in combination with modified atmosphere packaging (MAP) with storage at refrigeration temperatures. Cellulose-based bioactive inserts were placed between sliced products of cheese and ham under MAP. Inserts with immobilized nisin reduced Listeria innocua (starting inocula of 2.0 to 4.0x105 CFU/g) by > 3log10 CFU/g in cheese after 5 days at 40C, and by approximately 1.5 log10 CFU/g in sliced ham after 12 days, while S. aureus (starting inocula of 2.0 to 4.0x105 CFU/g) was reduced by1.5 and 2.8 log10 CFU/g in cheese and ham, respectively. The efficacy of bacteriocins coatings on the inhibition of pathogens has also been demonstrated in other studies. For example, coating of pediocin onto cellulose casings and plastic bags has been found to completely inhibit growth of inoculated Listeria monocytogenes in meats and poultry through 12-week storage at 40C. Coating of solutions containing nisin, citric acid, EDTA, and Tween 80 onto polyvinyl chloride, linear low-density polyethylene, and nylon films reduced the counts of Salmonella typhimurium in fresh broiler drumstick skin by 0.4- to 2.1-log10 cycles after incubation at 40C for 24 hours (Natrajan and Sheldon, 2000).
Conclusion and Recommendations
Although intensive studies over the last decade have greatly advanced our knowledge base about bacteriocins, further work is needed before we are able to fully understand the molecular mechanisms, structure-function relationships, and mechanisms of action of bacteriocins. In the biosynthesis of lantibiotics, the function of the enzymes responsible for modification reactions is still not clearly understood. The mechanism of producer immunity remains to be answered. Research in these areas is critical for the effective applications of bacteriocins and would help develop methods to genetically engineer bacteriocins with better activity, solubility, and stability.
Genetic engineering or chemical modifications of bacteriocins to improve their activity and properties can be expected to persist and possibly thrive. For example, the solubility and stability of nisin Z was improved by replacing Asn-27 or His-31 with lysine; the stability of pediocin PA-1 at 40C and room temperature was improved by replacing Met-31 with alanine, isoleucine or leucine; a pediocin PA-1 chimeric protein mutant displayed approximately 2.8-fold-higher activity against an indictor strain, L. plantarum (Johnsen et al., 2000). Examples such as these indicate solutions to some of the problems related to bacteriocin application; however, getting the regulatory approval for the use of engineered bacteriocins would be very difficult if they are considered as new proteins.
Although many bacteriocins have been isolated and characterized, only a few have demonstrated commercial potential in food application. At the time of this writing, nisin is the only purified bacteriocin approved for food use in the U.S. It has been used as a food preservative in more than 50 countries, mainly in cheese, canned vegetables, various pasteurized dairy, liquid egg products, and salad dressings (Guder et al., 2000). The applications of other bacteriocins in food preservation have been studied intensively. The use of pediocin PA-1 for food Biopreservation has been commercially exploited and is covered by several U.S. and European patents (Ennahar et al., 2000; Rodriguez et al., 2002). Fermentate containing pediocin PA-1, AltaTM, is commercially available and used as a food preservative to increase shelf life and inhibit the growth of bacteria, especially Listeria monocytogenes in ready-to-eat meats (Rodriguez et al., 2002). Lacticin 3147, which is active over a wider pH range than nisin, is expected to find application in non-acid foods.
Since bacteriocins for use as food preservatives have relatively narrow activity spectra and are generally not active against Gram-negative bacteria, it can be expected that nisin and other bacteriocins will continue to be incorporated and developed into hurdle concept technologies for food preservatives. The simultaneous application of bacteriocins and non-thermal processing technologies usually improve shelf life of foods is attractive since foods produced using these non-thermal technologies usually have better sensory and nutritional qualities compared with products using conventional thermal processing methods.
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