The most widely studied effects of the lactoperoxidase system have been carried out in bovine milk, and in various dairy products. The lactoperoxidase system has been demonstrated to work efficiently to preserve raw milk without refrigeration. The strategy, involving addition of low levels of H2O2, was advanced as a method for temporary preservation of raw milk in developing countries (Bjorck et al., 1979). Later, co-addition of equimolar concentrations of 0.25 mM SCN~ and H2O2 to milk, was found to maximise the activation of the lactoperoxidase system in preservation of unrefrigerated fresh milk (Wang et al., 1987). Other studies have indicated that use of 0.4 mM H2O2 and extra lactoperoxidase or addition of both lactoperoxidase (370 Enzyme Units/L), KSCN (0.3 mM), and H2O2 (0.3 mM) are required for optimal mobilisation of the lactoperoxidase system in milk (Siragusa and Johnson, 1989). However, in the latter case, the co-addition only delayed, but did not prevent the growth of, L. monocytogenes in milk stored at 20oC (Siragusa and Johnson, 1989). The keeping quality of milk is known to be better for milk pasteurised at 720C for 15 seconds than at 80°C for 15 seconds, which has been attributed to the heat shocking of spores at the higher temperature. However, since pasteurisation of milk at 80°C (15 seconds) completely inactivates bovine lactoperoxidase, while the residual lactoperoxidase is ^70% in low-pasteurised milk (720C, 15 seconds), it has been proposed that the lactoperoxidase system has a role in the shelf-life quality of pasteurised milk, and therefore that pasteurisation at a temperature of 720C may be a critical factor determining this effect (Barrett et al., 1999).
Addition of glucose with glucose oxidase to produce H2O2 (see enzymatic reaction in section 4.4) was used to boost the lactoperoxidase system to delay the onset of growth of Salmonella typhimurium in infant formula milk (Earnshaw et al., 1990). Similarly, in cottage cheese inoculated with Pseudomonas fragi, P. fluorescens, E. coli, and S. typhimurium, the addition of glucose and glucose oxidase activated the lactoperoxidase system to kill these organisms that were not detected in the cottage cheese during a 21-day storage period (Earnshaw et al., 1989). Equimolar addition of 25 mM KSCN and H2O2 as substrates for lactoperoxidase in pasteurised ewe's milk resulted in total inhibition of Aeromonas hydrophila, inoculated at 102cfu/mL, and reduced the level of psychrotrophs by more than 6 log cfu/g during 48 hours of refrigerated storage of fresh Spanish Villalon cheese (Santos et al., 1995). Other studies have shown that the lactoperoxidase system is not suitable to safeguard the preservation of cheese as activation of the naturally occurring lactoperoxidase in the cheese milk can result in decreased acidity production and prolonged coagulation time of the cheese as a result of inhibition of the lactic acid starter bacteria (Valdez et al., 1988).
An exogenous lactoperoxidase system treatment, comprising bovine lactoperoxidase (1 ^g/mL), KSCN (5.9 mM), H2O2 (2.5 mM) exerted antibacterial activity on S. typhimurium and psychrotrophic bacteria on Salmonella inoculated chicken legs in response to the time and temperature extension of the treatment (Wolfson et al., 1994). Maximum growth reduction achieved was a five-fold reduction - but not complete inhibition - of S. typhimurium cfu/g; this was achieved with 15 min. immersion of chicken legs into a 60°C water bath containing the lactoperoxidase system, which is thermostable at this temperature (Wolfson et al., 1994). Compared to lysozyme, the antibacterial spectrum is thus much wider for the lactoperoxidase system because of the lower specificity of the antibacterial mechanism. However, the antibacterial activity of the lactoperoxidase system, especially against Gram-positive organisms, appears strongly dependent on the relative amounts of available substrate, the pH, the medium, and also on the growth phase of the target organisms (Fuglsang et al., 1995). Streptococci appear to be relatively resistant to the antibacterial activity of the lactoperoxidase system compared to other bacteria, and it has been suggested that Streptococcus spp. may be able to reduce the oxidation products or repair the damage (Reiter and Harnulv, 1984). As will be discussed in section 4.5 the bacteriocidal activity of the lactoperoxidase system can be enhanced by combinatory strategies. In analogy to the comments given for the effects of lysozyme in food products (section 4.2.2) some more general, quantitative approaches to assess and model the antibacterial efficacy of lactoperoxidase are scarce. At present, it is therefore difficult to predict firmly how efficient the lactoperoxidase system is on specific types of bacteria in dairy foods.
Glucose oxidase (EC 18.104.22.168) catalyses the oxidation of ^-D-glucose by O2 producing 6-D-gluconolactone and H2O2. Since 6-D-gluconolactone hydrolyses spontaneously to D-gluconic acid (or rather, D-gluconate + H+), the net reaction catalysed becomes:
Glucose oxidase is produced by fungi of the genera Pénicillium and Aspergillus. Aspergillus niger strain NRRL 3 (ATCC 9029) is traditionally the most common source for the commercial production of glucose oxidase as the enzyme is a side-product of gluconic acid production by A. niger (Crueger and Crueger, 1990). Glucose oxidase has been employed industrially, notably in the US, since the early 1950s for removal of glucose in eggs prior to spray drying to prevent Maillard browning reactions (Szalkucki, 1993). Furthermore, in conjunction with catalase, glucose oxidase has been used as a flavour-protecting measure in citrus juices via removal of oxygen, but not as a bacteriocidal agent on a commercial scale (Szalkucki, 1993). The potential antibacterial effect of glucose oxidase action has nevertheless received steady research attention. The observed antibacterial action has been widely suggested to be primarily based on the production of H2O2 (Fuglsang et al., 1995). However, since catalase catalyses the disproportionation of H2O2 to water and oxygen, the antibacterial activity of glucose oxidase preparations should hence be strongly dependent on the (non)presence of catalase as side activity. Catalase is usually present as an impurity in commercial glucose oxidase preparations, as the coupled removal of H2O2 is often a prerequisite in non-antibacterial food processing applications of glucose oxidase (Szalkucki, 1993).
Since catalase is one of the fastest enzymes known with a hydrogen peroxide rate constant close to 107 seconds-1 mole-1, even low levels of catalase can retard the generation of H2O2 by glucose oxidase preparations. Likewise, in case H2O2 was the main antibacteriocidal effect, the susceptibility of various microorganisms to inhibition by glucose oxidase should depend on their ability to produce catalase (Fuglsang et al., 1995). However, an evaluation of the data obtained in various systems with commercial glucose oxidase preparations does not confirm these hypotheses. Rather, the data suggest, that especially the decrease in pH caused by the gluconate generation, perhaps coupled with the micro-anaerobic environment generated by the catalysed oxygen removal that may retard obligate aerobes, are more important to the antibacterial activity of glucose oxidase than H2O2 production (Dobbenie et al., 1995; Dondero et al., 1993). Again, some more thorough quantitative evaluations of the enzyme kinetics versus the antibacterial effects of glucose oxidase with or without catalase would improve our understanding of the mechanisms involved, and presumably allow a more focused approach to employing glucose oxidase as an antibacterial enzyme system.
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