Addition of nisin together with lactoperoxidase and glucose/glucose-oxidase to pre-sterilised milk inoculated with L. monocytogenes was demonstrated to exert a pronounced synergistic and lasting bacteriocidal effect on L. monocytogenes as no cells were detected in the inoculated milk during 15 days of incubation of the milk at 250C (Boussel et al., 2000). Likewise, designed additions of nisin and bacteriocin-producing lactic acid bacteria in conjunction with activation of the lactoperoxidase system gave a more pronounced decrease of L. monocytogenes counts in skim milk and raw milk than those observed for the activated lactoperoxidase system alone (Zapico et al., 1998; Rodriguez et al., 1997). Addition of monolaurin and concomitant activation of the lactoperoxidase system resulted in improved growth inhibition of foodborne pathogens including Escherichia coli 0157:H7 and Staphylococcus aureus by lactoperoxidase in milk (McLay et al., 1998).
As already highlighted above, pasteurisation of milk at 720C for 15 seconds versus at 80°C for 15 seconds gave more residual lactoperoxidase activity in milk, suggesting that the lactoperoxidase system may be active in ensuring a better keeping quality of low-pasteurised milk as compared to milk pasteurised at higher temperatures (Barrett et al., 1999). In contrast, a high hydrostatic pressure treatment of milk combined with lactoperoxidase addition to milk was shown to result in an improved bacteriocidal effect on Escherichia coli strains and Listeria innocua in milk (Garcia-Graells et al., 2000).
As with other food-processing aids and additives, the application of enzymes in food manufacture is obviously limited by safety and toxicology requirements and therefore strongly regulated by legislation. It is beyond the scope of this chapter to discuss the general legal aspects of the use of enzymes in production of foods and beverages but a few key points deserve mention: In the United States, several enzyme preparations have GRAS status, where GRAS designates Generally Recognized As Safe. The GRAS enzyme preparations may be used as catalysts in several different food and beverage processes. European food legislation on enzyme applications, however, differentiates between the use of enzymes as processing aids and as food additives, respectively (AMFEP 2001; Directive 95/2/EC). When enzymes are employed as processing aids it implies that the added enzymes do not exert any activity nor any technological function in the final product. Each approval concerns the use of a certain type of enzyme preparation for one (or more) specific type of application (AMFEP, 2001).
Recently, the European Commission proposed to clarify this legislation by laying down specific provisions with respect to the use of enzymes as food additives and processing aids (Commission of the European Communities, 2000 and 2002). Despite this proposal and other recent harmonisation efforts at the EU level, there is at present no harmonised EU legislation on the use of enzymes as processing aids in food and beverage manufacture. Notable differences in the legislation on application of enzymes thus exist at the National levels in Europe and between the US and Europe. However, even though some differences in enzyme approvals remain at the national levels in Europe, a large number of enzyme preparations have obtained general approval for use as food processing aids with each enzyme preparation being allowed for use in specific types of processes (AMFEP, 2001).
In the EU, lysozyme from hen egg white is the only enzyme that has status as a food additive and hence has an E-number, E 1105 (Directive 95/2/EC). As a food additive, lysozyme has been permitted for use since 1995 as a preservative agent in ripened cheeses to prevent 'gas blowing' from growth of Clostridium tyrobutyricum (Directive 95/2/EC). Recently, the Commission proposed also that the addition of lysozyme to wine to prevent growth of lactic acid bacteria should be included as an authorised use of lysozyme (E 1150) (Commission of the European Communities, 2002).
Food quality and safety are an increasingly important concern. In particular, the apparent rise in cases of foodborne illness and the scale of outbreaks of foodborne illness underline the need to ensure that the available food preservation principles and antibacterial safeguards are employed optimally in all steps of food manufacture. The trend in consumption and availability of more industrially processed food, including meals manufactured on a larger scale in catering businesses and restaurants, only strengthens the need for careful selection and rational development of efficient food preservation methods. Paradoxically, consumers demand more 'natural' and 'minimally processed' foods, that are able to remain 'fresh' for extended periods of time! As a result there is great interest in naturally produced antimicrobial agents in both industry and academia.
Enzymes represent one type of natural food preservative agent. If employed as antibacterial agents, enzymes may most likely be used as extra safeguards in hurdle approaches, to preserve foods effectively. These applications will involve selected, optimised combinations of the enzyme and additional additives, e.g., bacteriocins, and specific treatments and combinations of physicochemical parameters that impart the highest antibacterial potency of the combination. The future challenges will involve continued research on the efficacy of rational combinations of new food preservative agents against spoilage and pathogenic bacteria in foods. Notably, a more quantitative approach, rather than the present empirical avenue, is warranted in the future investigations of enzyme-based food-preservation principles.
Many new results are currently achieved in research laboratories all over the world. Although it is advisable to keep an eye on new patents in the field, monitoring of the research literature is especially recommended. As is obvious from the reference list to this chapter, data on the efficacy of novel food preservatives are published in many different scientific journals, and it is recommended to attempt to evaluate all the available information critically and carefully. Additional information on the antibacterial mechanism of lysozyme and lactoperoxidase can be found in:
Natural Food Antimicrobial Systems (Naidu, N, ed) Woodhead Publishers, 2000
Natural Antimicrobial Systems and Food Preservation (V.M. Dillon, R.G Board, eds) CAB Intl. 1994
While a broader view on minimal processing of foods can be found in:
Minimal Processing Technologies in the Food Industry (Ohlsson T and Bengtsson N, eds) Woodhead Publishing Ltd (2002).
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