Biopreservation and Protective Cultures

A growing demand for minimally preserved or preservative-free fresh produce has led to a search for alternatives to more traditionally used food antimicrobial compounds. Biopreservation, the use of antagonist or protective cultures, has shown potential for extension of produce storage life; natural microflora such as lactic acid bacteria (LAB) or bacterial metabolic byproducts such as organic acids or bacteriocins can serve as natural inhibitors of spoilage organisms. Application of antagonist organisms along with use of MAP technology and additional microbial control strategies can be synergistic in effect. For example, a prepackaging application to sweet cherry of the antagonist yeast Cryptococcus infirmo-miniatus (CIM) Pfaff and Fell followed by modified atmosphere storage at 2.8°C for 20 days or —0.5°C for 42 days resulted in significant reduction of the causal agent of brown rot, Monilinia fructicola G. Wint., an effect that was enhanced when a preharvest application of propiconazole was incorporated [28]. Prepackage application of organic acids to vegetables or fruits such as melon, papaya, or avocado, which are typically low-acid, results in a pH decline that is inhibitory to groups of spoilage organisms that grow best under neutral or near neutral pH environments.

Protective cultures such as LAB can be found naturally on produce, and thus may not significantly alter the typical or expected product taste or cause significant spoilage if applied directly. The most studied LAB bacteriocinogenic strains include Lactococcus lactis, Pediococcus acidilactici, and Lactobacillus sakei, which produce the antimicrobials nisin, pediocin, and sakacin, respectively [29]. Bacteriocins produced by LAB are typically antimicrobial towards Gram-positive spoilage organisms and pathogens, including L. monocytogenes and C. botulinum. Bacteriocins do not affect Gram-negative bacteria, which are a major spoilage group of concern; however LAB and associated bacteriocins may be used to target specific Gram-positive pathogens of concern or be used in combination with other technologies that reduce Gram-negative bacterial growth for a broader overall range of microbial control.

Some LAB strains may not grow well enough on produce at refrigeration temperatures in order to produce levels of bacteriocin necessary for antimicrobial activity; additionally, bacteriocins can be inactivated by bacterial proteolytic enzymes or by binding to food components, and target bacteria may become resistant. Bennik and others [30] isolated bacteriocinogenic strains of Pediococcus parvulus and Enterococcus mundtii from MAP endive and evaluated the ability of these strains to produce bacteriocin on mung bean sprouts at refrigeration temperatures of 4 to 8°C. E. mundtii was able to produce the bacteriocin mundticin on inoculated mung beans stored under MAP (1.5% O2, 20% CO2, balance N2) at 8°C, while P. parvulus did not survive under these conditions. When mundticin was extracted and used as a dip (200BUml_1) or incorporated into an alginate film (200BUml_1) on mung bean, the bacteriocin exhibited antimicrobial activity under refrigerated MAP storage [30]. Cai and others [31] isolated a strain of Lactococcus lactis subsp. lactis from mung bean sprouts that contained a gene for nisin-Z, an antilisterial compound. This isolate could survive on fresh-cut RTE Caesar salad at levels of 8 log10 CFU/g at 3 to 4.5°C for up to 20 days and could grow at 4°C and produce nisin-Z at 5°C. When co-incubated with 2 log10 CFU/g cells of Listeria monocytogenes on salad, L. monocytogenes populations were reduced by 1 to 1.4 log10CFU/g after 10 days' storage at 7 and 10°C. Thus bacteriocinogenic strains should be assessed for their ability to grow and produce bacteriocin on a target commodity under specific MAP conditions and storage temperatures. Additionally, bacteriocins directly applied should be assessed for their persistence and activity on a specific commodity during MAP shelf life.

Other microorganisms have been found to exhibit antimicrobial activity due to competition for nutrients, rapid growth rates, or production of inhibitory metabolites. Enterobacteriacea have been found to limit growth of L. monocytogenes on endive, most likely due to competition for nutrients. Mixed populations of nonbacteriocinogenic strains of Lactobacillus brevis and Leuconostoc citrium have been shown to inhibit competitively the growth of

L. monocytogenes on MAP MP vegetables [32]; Enterobacter cloacae and E. agglomerans were also found to be competitively inhibitive. This research group also found in challenge studies with MAP lettuce that increasing CO2 atmospheres decreased this inhibitory effect; when CO2 levels increased from 5 to 10 to 20%, a delayed inhibitory effect was increasingly observed [33]. The use of nonpathogenic strains naturally found on produce that competitively inhibit spoilage organisms and/or pathogens on produce under MAP storage conditions warrants further study as a promising biopreservative hurdle strategy.

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