Antibacterial Activity of the Lactoperoxidase System: A Review

Transcription

1 887 Journal of Food Protection, Vol. 56, No. 10, Pages (October 1993) Copyright, International Association of Milk, Food and Environmental Sanitarians Antibacterial Activity of the Lactoperoxidase System: A Review LISA M. WOLFSON and SUSAN S. SUMNER* Department of Food Science and Technology, University of Nebraska, Lincoln, Nebraska (Received for publication April 7, 1993) ABSTRACT The lactoperoxidase (LP) system is a naturally occurring system which was first discovered in raw milk. Different groups of bacteria show a varying degree of resistance to the LP system. Gram-negative catalase-positive organisms, such as pseudomonads, coliforms, salmonellae, and shigellae, are inhibited by the LP system. Depending on the medium ph, temperature, incubation time, cell density, and the particular donor, these microorganisms may be killed. It has been shown that the LP system can increase storage times of raw milk by delaying growth of psychrotrophs; perhaps this method could be used to extend the shelf life of other foods. The antibacterial activity/mechanism of the lactoperoxidase (LP) system is well-documented (30). It is the major intermediary product of the LP system reaction, hypothiocyanite (OSCN), which oxidizes essential protein sulfhydryls, resulting in altered cellular system functions and which causes inhibition of growth and/or death of the microorganism (30). The hypothiocyanite ion can be formed by mixing the components (lactoperoxidase, thiocyanate, and hydrogen peroxide) of the LP system together. The lactoperoxidase antimicrobial system is a naturally occurring system which has been proven to be both bacteriostatic and bactericidal to a variety of gram-positive and gram-negative microorganisms (29). The LP system can alter many cellular systems, including the outer membrane, cell wall, cytoplasmic membrane, transport systems, glycolytic enzymes, and nucleic acids. Nonpathogenic bacteria (5,8,9,23,26-28,36,39,40) as well as pathogenic bacteria (4,6,7,10,12-15,21,33,34,38) have been shown to be inhibited or killed by the LP system. This paper begins with a general discussion of the LP system and then provides information about the antibacterial activity of the LP system against specific nonpathogenic and pathogenic bacteria. COMPONENTS OF THE LP SYSTEM The LP system is made up of three components: lactoperoxidase, thiocyanate, and hydrogen peroxide. Lactoperoxidase (LP) Peroxidases are defined as enzymes whose primary function is to oxidize molecules at the expense of hydrogen peroxide (32). Published as Paper No , Journal Series, Nebraska Agricultural Research Division, Lincoln, NE Lactoperoxidase is found in the mammary, salivary, and lachrymal glands of mammals and in their respective secretions, e.g., milk, saliva, and tears. In milk and saliva, lactoperoxidase exists in a soluble form, but within the cells of salivary and mammary glands, it is possible that the enzyme could be loosely bound to subcellular particles (18). This could influence its affinity for different substrates and the relative rates of reactions it catalyses. Lactoperoxidase has a molecular weight of 77,500 (30) and is resistant in vitro to acidity as low as a ph of approximately 3 and to human gastric juice (32). Lactoperoxidase is actually more active at acidic ph values (37). Bovine milk lactoperoxidase is relatively heat resistant, with the enzyme being only partially inactivated by short-time pasteurization at 74 C, leaving sufficient activity to catalyze the reactions between thiocyanate and hydrogen peroxide (32). Cow's milk contains from 1.2 to 19.4 units per ml and is about 20 times richer in peroxidase activity than human milk (16). Human milk lactoperoxidase activity is low; values range from 0.06 to 0.97 units per ml (41). The highest content of lactoperoxidase (22 units per ml) has been reported for guinea pig milk (41). In human saliva, lactoperoxidase has a role similar to that in milk. As a component of the LP system, it is involved in the inhibition of streptococci which promote dental carries (32). The human infant already possesses salivary lactoperoxidase during the first few days after birth (32). Thiocyanate (SCN) The thiocyanate anion is widely distributed in animal tissues and secretions. Thiocyanate is largely a constituent of the extracellular fluid. It is, however, concentrated by certain cells of the body. Whereas the blood serum concentration of thiocyanate is mg%, the salivary concentration has been estimated at 1-27 mg% (24). The level of thiocyanate is related to diet and habits such as smoking. Thiocyanate is excreted mainly in the urine, and with normal renal functions, the half-life of elimination is 2 to 5 d (32). The thiocyanate concentration of bovine milk, which varies with breed, species, and type of feed, has been estimated at mg% (24). There are two major dietary sources of thiocyanate, glucosinolates, and cyanogenic glucosides. Vegetables belonging to the genus Brassica (family Cruciferae), such as cabbage, kale, brussel sprouts, cauliflower, turnips and rutabaga, are particularly rich in glucosinolates, which upon hydrolysis yield thiocyanate in addition to other reaction products (32). Cyanogenic glucosides are also found in cassava, potatoes, maize, millet, sugar cane, peas, and beans. When hydrolyzed, glucosides release cyanide, which in a reaction with thiosulfate (metabolic product of sulfur-containing amino acids) is detoxified by conversion into thiocyanate (32). The latter reaction is catalyzed JOURNAL OF FOOD PROTECTION, VOL. 56, OCTOBER 1993

2 WOLFSON AND SUMNER by the enzyme rhodanase (52). Cyanide from tobacco smoke is metabolized in the same way. Hydrogen peroxide Hydrogen peroxide is the third component of the lactoperoxidase system. Hydrogen peroxide may be formed endogenously. Many lactobacilli, lactococci, and streptococci produce sufficient hydrogen peroxide under aerobic conditions to activate the LP system. Hydrogen peroxide may also be added or may be generated by the addition of one of a number of hydrogen peroxidegenerating systems. Among the latter are the oxidation of ascorbic acid, the oxidation of glucose by glucose oxidase, the oxidation of hypoxanthine by xanthine oxidase, or the manganese-dependent aerobic oxidation of reduced pyridine nucleotides by peroxidase (24). According to Klebanoff et al. (24), a hydrogen peroxidegenerating system is more effective than added hydrogen peroxide as a component of the antimicrobial system. MODE OF ACTION The lactoperoxidase catalyzed reaction yields short-lived intermediary oxidation products of SCN", which may be further oxidized to end-products such as sulfate, C0 2, and ammonia or may be reduced back to SCN (32). Most researchers agree that the major intermediary oxidation product is hypothiocyanite, OSCN" (2,19,20,24,30,32,35). It is proposed that peroxidase-catalyzed oxidation of SCN" results in the accumulation of OSCN" (32). The hypothiocyanite ion can be produced by two different pathways. The oxidation of SCN - may yield thiocyanogen (SCN) 2, which hydrolyzes rapidly to yield hypothiocyanous acid (HOSCN), or OSCN. Peroxidase 2SCN- + H H + >(SCN) 2 + 2H 2 0 (SCN) 2 + H 2 0 > HOSCN + SCN" + H + HOSCN<- > H + + OSCN Alternatively, SCN" may be oxidized directly to OSCN". Peroxidase SCN + H > OSCN + H 2 0 It is the intermediary oxidation product(s) that have antibacterial activities, such as inhibition of growth, oxygen uptake, and lactic acid production. In addition, it has been shown that bacterial enzymes, including hexokinase glyceraldehyde-3p-dehydrogenase, are inhibited (32). The oxidation of sulfhydryl (SH) groups of enzymes and other proteins has been considered to be key to the antimicrobial action of the LP system (32). Moreover, it appears that the bacterial cytoplasmic membrane is structurally damaged or changed because organisms exposed to the LP system immediately leak potassium, amino acids, and polypeptides into the medium (32). Subsequently, uptake of glucose, purines, pyrimidines, and amino acids as well as synthesis of protein, DNA and RNA is also inhibited (32). Lactoperoxidase catalyzes the incorporation of SCN- into protein substrates. The reaction of (SCN) 2 or OSCN' with proteins oxidizes the protein sulfhydryls to sulfenyl thiocyanate derivatives (35). Protein-SH + (SCN) 2 Protein-SH + OSCN -> Protein-S-SCN + SCN' + H + -> Protein-S-SCN + OH Sulfenyl thiocyanate derivatives can undergo further modifications, including reversible hydrolysis to yield sulfenic acids (35). Protein-S-SCN + H 2 0 <- - - > Protein-S-OH + SCN' + H + Also, sulfenyl derivatives can undergo slow oxidation to sulfonic acids (35). The SCN moiety can be displaced from sulfenyl thiocyanate by reduction with a sulfhydryl compound, such as dithiothreitol (35). When all the protein sulfhydryls are oxidized by (SCN) 2 or SCN", tyrosine, tryptophan, and histidine residues are modified (35). Release of SCN" from sulfenyl thiocyanate is favored at low SCN" concentration (2). When SCN" is released, it can be reoxidized and participate in the oxidation of another sulfhydryl. As illustrated (Fig. 1) with OSCN", the oxidation of sulfhydryls to sulfenic acid does not consume SCN". Therefore, the amount of sulfhydryls oxidized does not depend on the amount of SCN" (2). H 2 2 t^o Lactoperoxidase SCN, OSCN Figure 1. Oxidation of sulfhydryls. Protein-S-SCN THE HYPOTHIOCYANITE ION (OSCN) Protein-S-OH Protein-SH As stated previously, the hypothiocyanite ion is believed to be the major intermediary oxidation product of the LP system. OSCN" can be considered the hypohalite of thiocyanogen (SCN) 2, whose chemical characteristics are similar to those of other hypohalites, including stability in ionic form but instability as the acid (20). Many factors affect the stability of hypothiocyanite. The decomposition of OSCN" is strongly dependent on the ph of the solution; OSCN" is more stable at ph 7.5 than ph 5.0 (20). OSCN solutions are sensitive to light, yet they are very heat stable (20). Stability of OSCN" solutions decrease on addition of metal ions (Fe, Ni, Cu, Mn, etc.), glycerol, and ammonium sulfate, as well as the removal of lactoperoxidase (20). Several analytical methods for the estimation of OSCN" have been used. These methods are based either on the reduction of OSCN" to SCN", which can be assayed by the iron-complex method, or on the oxidation of 5-thio-2-nitrobenzoic acid to the colorless disulfide compound 5,5' dithiobis (2-nitrobenzoic acid) by OSCN" (20). GENERAL EFFECTS ON BACTERIA The LP system can kill or inhibit the growth and metabolism of many species of microorganisms. Many cellular systems (i.e., outer membrane, cell wall, cytoplasmic membrane, transport systems, glycolytic enzymes, and nucleic acids) can be altered by the LP system. According to Pruitt and Reiter (29), for any particular microorganism, the antimicrobial effects depend upon the reaction conditions. When adequate concentrations of lactoperoxidase are provided, bactericidal effects are greater at low temperatures (0-5 C), at low ph (5 or less), and in the absence of reducing agents (29). The effectiveness of the LP system may vary with the medium in which the test is conducted. For example, small molecular weight components present in brain heart infusion (BHI) broth interfere with the antimicrobial action of the LP system (20). A given quantity of hydrogen peroxide appears to be more effective when it is supplied by the metabolism of the cells or by continuous generation with glucose/glucose oxidase than when it is added separately (32). When the components of the LP system are brought together in the presence of the target cells, killing or inhibition appears more effective than if the cells are added after the components are combined. Results obtained depend upon the state of the JOURNAL OF FOOD PROTECTION, VOL. 56, OCCTOBER 1993

3 ANTIBACTERIAL ACTIVITY OF LACTOPEROXIDASE SYSTEM 889 cells (29). According to Pruitt and Reiter (29), resting cells or cells in the stationary phase of growth are more susceptible to killing or inhibition than are metabolically active or growing cells, but other studies show contrasting results (31,33). Bacteria grown anaerobically appear to be more susceptible to the LP system than microorganisms grown aerobically (9). It is possible that some microorganisms are capable, at least partially, of "neutralizing" the oxidation product(s) of the LP system or of repairing the damage caused (32). According to a study by Aune and Thomas (7), removal of OSCN" by centrifugation permitted Escherichia coli to recover. After E. coli were washed to remove OSCN", the sulfhydryl content of the cells increased during incubation at 25 C. Also, the content of sulfenyl derivatives decreased. Therefore, recovery appeared to result from the reduction of sulfenyl derivatives back to sulfhydryls. Alternatively, the sulfenyl derivatives may have been degraded, and new sulfhydryl components may have been synthesized. Under the same conditions, there was no increase in the sulfhydryl content of cells that had not been exposed to the LP system. Also, there was no increase in the number of viable cells. Therefore, the increase in sulfhydryl content was due to a repair process rather than to growth of cells (7). Various reports have been published concerning mechanisms of resistance to OSCN -. These include an enzyme that catalyzes the reduction of OSCN - by NADH (9) and an increase in cell sulfhydryl groups that rapidly reduce OSCN" to SCN' (28). Other possible resistance mechanisms include novel OSCN" -resistant respiration systems and a phosphoenolpyruvate-dependent phosphotransferase system sugar transport mechanism which is resistant to OSCN" (36). EFFECTS ON SPECIFIC BACTERIA Streptococcus spp. A chemically defined culture medium was used to study the effect of purified lactoperoxidase and thiocyanate on the growth of several cultures of Streptococcus pyogenes and Streptococcus agalactiae in a study by Mickelson (26). While not inhibited by either component alone, S. pyogenes was completely inhibited when both components were present in the medium. S. pyogenes glyceraldehyde phosphate dehydrogenase was inhibited by lactoperoxidase when hydrogen peroxide was present. With S. agalactiae cultures, a delay in growth up to 6 h resulted instead of complete growth inhibition. The extent of growth inhibition was greatest in those strains which were unable to adapt to an oxidative pathway for their energy supply (26). In another study by Mickelson (27), transport of 2-deoxyglucose or glucose in S. agalactiae was strongly inhibited if the cells were first exposed to a combination of lactoperoxidase-thiocyanate-hydrogen peroxide. Interference of the entry of glucose into cells of S. agalactiae by the LP system could well account for its growth inhibitory properties with this organism (27). The inhibition of glucose transport by the LP system and its reversibility with dithiothreitol suggest the modification of functional sulfhydryl groups in the cell membrane as a cause of transport inhibition (27). Results from a study by Oram and Reiter (28) showed that the growth of the LP-sensitive Streptococcus cremoris 972 (now Lactococcuss lactis var. cremoris) in a synthetic medium was inhibited by LP and SCN. The glycolysis and oxygen uptake of suspensions of S. cremoris 972 in glucose or lactose was also inhibited. It appears that the LP system completely inhibited the hexokinases of nonmetabolizing suspensions of both strains (28). The inhibition was reversible. Hexokinase and glycolytic activities of S. cremoris 972 were restored by washing the inhibitor from cells (28). Thomas et al. (36) studied the inhibition of bacterial metabolism by the LP system with representatives of serotypes a through g of S. mutans. Inhibition was most effective at ph 5, whereas release of hydrogen peroxide and accumulation of the inhibitor (OSCN) were highest at ph 8 (36). The results indicate that ph, amount of hydrogen peroxide, cell sulfydryl content, and carbohydrate reserve determine susceptibility to inhibition (36). Carlsson et al. (9) grew strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Streptococcus salivarius under aerobic and anaerobic conditions to determine the effects of the LP system on the rate of acid production and oxygen uptake by intact cells; the activity of glycolytic enzymes in cell-free extracts; and the levels of intracellular glycolytic intermediates. Acid production, oxygen uptake, and, consequently, hydrogen peroxide excretion were inhibited in all the strains by the LP system. S. sanguis and S. mitis had a higher capacity to recover from the inhibition than S. mutans and S. salivarius. According to Carlsson et al. (9), higher activity in the former strains of an NADH-OSCN oxidoreductase, which converted OSCN" into thiocyanate, explained this difference. THERMOPHILIC STARTER CULTURES, MILK, AND CHEESE The effect of the LP system on the behavior of a thermophilic starter culture commonly used in the dairy industry for cheesemaking was investigated by DeValdez et al. (77). It was determined that the thermophilic starter culture was very sensitive to the LP system; the activity of the starter culture was strongly reduced. Activation of the LP system in milk resulted in a substantial reduction of the bacterial flora and prevented the multiplication of psychrotrophic bacteria for up to 5 d (5). Bjorck (5) concluded that the treatment had no effect on the physiochemical properties of the milk and did not lead to the accumulation of resistant bacteria. In a study by Zajac et al. (39), results showed that at 4 C the standard plate count in LP-activated milk remained basically unchanged for at least 104 h, whereas bacterial multiplication in the controls started after 48 h. At 10 C, activation resulted in a lag phase of at least 72 h, but at 17 C this was reduced to <24 h. According to Zajac et al. (39), the results showed that by repeated activation of the LP system it is quite possible to store raw milk at 4 C for extended periods of time. The activated LP system in conjunction with pasteurization extended the shelf life of milk held at 10 C by more than 20 d, compared to untreated milk (23). They found that observable growth of surviving natural milk microflora started after 12 d in LP system-treated milk, compared to 4 d in untreated and H treated milk. After 22 d, viable counts in untreated and H treated milk reached cells per ml compared to about 10' cells per ml in LP system-treated milk (23). Zall et al. (40) made acceptable varieties of soft and hard cheeses from chemically treated milk. Through the use of the LP system, which avoids heating altogether, it was possible to compare cheese yields with products made from unheated and heated 8-d-old milk. Chemically treated milk increased yields 2% as compared to control cheese (40). Escherichia coli and Salmonella In a study by Bjorck and Claesson (6), lactoperoxidasecatalyzed oxidation of thiocyanate ion resulted in a bactericidal effect against E. coli in a semisynthetic medium. According to Bjorck and Claesson (6), the reaction produced an antibacterial agent that caused reversible inhibition of many gram-positive bacteria and an irreversible inhibition of gram-negative bacteria. Two strains of E. coli and one strain of Salmonella typhimurium were killed by the bactericidal activity of the LP system in milk and in a synthetic medium (33). Hydrogen peroxide was supplied exogenously by glucose oxidase, and glucose was added at a level which was itself noninhibitory. The bactericidal activity JOURNAL OF FOOD PROTECTION, VOL. 56, OCTOBER 1993

4 890 WOLFSON AND SUMNER was greatest at ph 5 and below and depended on the thiocyanate concentration and the number of organisms. According to Earnshaw et al. (13), a glucose/glucose oxidase activated LP system delayed the onset of exponential growth of S. typhimurium and E. coli in infant formula milk. Addition of urea peroxide with the LP system reduced the initial population size and prevented growth of S. typhimurium and extended the lag period before the onset of exponential growth of E. coli. Results from a study by Purdy et al. (31) showed that the LP system was found to have both bacteriostatic and bactericidal activities against strains of S. typhimurium. Purdy et al. (31) believed that the bactericidal activity was clearly dependent on the permeability of the bacterial cell envelope. Bacteria in log phase of growth were more sensitive to the bactericidal effects than were those in stationary phase; growth phase had little influence on the bacteriostatic effect (31). The LP system was investigated for its activity against salmonellae in vivo and in vitro by Wray and McLaren (38). In acidified raw milk, in which the LP system was supplemented with an exogenous supply of hydrogen peroxide, the number of salmonellae decreased rapidly. According to Wray and McLaren (38), different salmonellae serotypes were affected to the same extent; rough strains, however, were more susceptible than smooth strains. Calves were fed fresh milk containing the LP system and challenged with S. typhimurium; the clinical findings were similar to those of control calves fed on heated milk (38). Further field studies would be necessary to evaluate the LP system as a possible nonantibiotic system to control salmonellosis in calves. Listeria and Staphylococcus Earnshaw and Banks (12) found that a LP system activated by glucose oxidase was bacteriostatic to Listeria monocytogenes inoculated into ultra-high temperature (UHT) milk supplemented with glucose. The reasons for the nonlethal effect of the LP system were unclear. The antibacterial activity of the LP system on the growth and survival of L. monocytogenes in Trypticase soy broth, UHT milk, and French soft cheese was determined by Denis and Ramet (10). In Trypticase soy broth and UHT milk, presence of the LP system either inhibited growth or completely inactivated inoculated cells. According to Denis and Ramet (10), complete inactivation occurred at different times depending on initial inoculum concentration, culture medium, and storage temperature. The addition of the LP system to the surface of soft cheese led to elimination of viable Listeria cells from cheeses previously inoculated to contain 10' to 10 6 CFU/g. Siragusa and Johnson (34) showed that the LP system inhibited the growth of L. monocytogenes Scott A in a bacteriostatic but not a bactericidal manner. Evidence of bacteriostasis was the extension of the lag period beyond that of the control flasks (H treatment or broth control). Kamau et al. (21) determined that the LP system, using the inherent milk lactoperoxidase, effectively inhibited the growth of L. monocytogenes and S. aureus at 35 and 37 C, respectively. In another study by Kamau et al. (22), the LP system was found to enhance thermal destruction of L. monocytogenes and S. aureus. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LP system. According to Kamau et al. (22), activation of the LP system followed by heating can increase the margin of safety with respect to milkborne pathogens. Survival and growth of L. monocytogenes and Listeria innocua in the presence of a reactivated LP system was investigated by Bibi and Bachmann (4) in skim milk at 30 C, and for L. innocua additionally at 20 and 8 C. The LP system was found to have an essentially bacteriostatic effect on both organisms. According to Bibi and Bachmann (4), duration of the bacteriostatic effect was dependent on the temperature of incubation, lasting 6 h at 30 C and more than 20 and 100 h at 20 and 8 C, respectively. No difference was observed between the two organisms in their response to the LP system at 30 C. Effects of the LP system on L. monocytogenes (strains V7, Scott A, and California) were determined by El-Shenawy et al. (14) using a semisynthetic medium, raw milk and a buffer solution. Each medium was inoculated to contain three levels of the pathogen (low, 30 to 50 CFU/ml; medium, 10 4 CFU/ml; and high, 10 7 CFU/ml) and incubated at 4 or 35 C. Low numbers of the bacteria were completely inactivated within 2 to 4 h (35 C) or 12 to 24 h (4 C); when substrates contained medium or high populations, a limited bactericidal effect occurred with decreases in population of one-half (4 C) or one (35 C) order of magnitude. The LP system failed to cause permanent injury of L. monocytogenes at 4 or 35 C. El-Shenawy et al. (14) believed that the efficacy of the LP system as an antimicrobial agent was related to strain and number of pathogen, suspending medium, and incubation temperature. The activity of a raw milk LP system or four L. monocytogenes strains at refrigeration temperatures was studied by Gaya et al. (15). The LP system exhibited a bactericidal activity against L. monocytogenes at 4 and 8 C; the activity was dependent on temperature, length of incubation, and strain of L. monocytogenes tested (15). Campylobacter Borch et al. (7) tested the antibacterial effect of the LP system against strains of Campylobacter jejuni and Campylobacter coli isolated from poultry. The effect was studied at different ph values (5 to 7) and temperatures. The LP system had a strong bactericidal effect against C. jejuni and C. coli. Borch et al. (7) found that the bactericidal effect was more rapid at 37 C compared to 20 C, and that the effect of the LP system decreased with decreasing ph values. The fastest reduction in viable numbers was obtained at ph 6.6 and 37 C (7). Previous investigators (33) have demonstrated an enhanced effect of lactoperoxidase at lower ph values. The different results with Campylobacter may be attributed to its microaerophilic nature. MILK PRESERVATION In Western Countries milk is cooled and stored for increasingly long periods because of distribution circumstances. After 2 d, such milk can deteriorate through the multiplication of psychrotrophic organisms (mainly pseudomonads), which produce extremely heatresistant lipases and proteases which survive pasteurization (25). These enzymes can make the milk unpalatable or spoil dairy products such as butter and cheese. The enzyme lactoperoxidase remains active after some thermal treatments, such as normal pasteurization (25). This suggests that subsequent reactivation of the LP system during storage of raw or pasteurized milk is possible. Gupta et al. (17) studied the effects of the LP system on the preservation of milk. The LP system can be used to preserve infant formula (3). It might also be possible to utilize the LP system to increase storage times of raw milk at ambient temperatures. The LP system could be considered an alternative approach for preservation of raw milk in developing countries. SUMMARY According to Reiter and Harnulv (32), the strongest arguments against any undesirable side effects resulting from the activation of the LP system are based on: (i) the wide distribution of all the components of the system in humans and animals; (ii) evidence of the in vivo activity of the LP system in calves; (iii) the detection of one of the major JOURNAL OF FOOD PROTECTION, VOL. 56, OCCTOBER 1993

5 ANTIBACTERIAL ACTIVITY OF LACTOPEROXIDASE SYSTEM 891 oxidation products, OSCN, in human saliva; and (iv) selective damage to the bacterial cytoplasmic membrane but not to mammalian cell membranes. The innocuous nature of the LP system is further supported by results from in vitro experiments with animal cell cultures showing no toxic effects of the system (32). The LP system can, therefore, be regarded as a naturally occurring antibacterial system that has evolved throughout the evolution of mammals to be a part of the defense system against bacterial infections on mucosal surfaces in, for example, the gastrointestinal tract (32). Indirect evidence of the importance of the LP system may be provided by the fact that nature has provided both newborn calves and humans with this system (16). In the former species it is provided by colostrum and to a much smaller extent by saliva, and in the latter the quantities are reversed (16). Furthermore, lactoperoxidase in milk is not inactivated by gastric juice. In addition to the antimicrobial properties, the LP system may also exert other biological functions in vivo. Among these are degradation of various carcinogens, which have been reported to occur in human saliva and protection of human cells from the toxicity of hydrogen peroxide (16). It has been shown that the LP system can increase storage times of milk, particularly by delaying the growth of psychrotrophs (25). Perhaps this method could be used to extend the shelf life of other foods. REFERENCES 1. Aune, T. M., and E. L. Thomas Accumulation of hypothiocyanite ion during peroxidase-catalyzed oxidation of thiocyanate ion. Eur. J. Biochem. 80: Aune, T. M., and E. L. Thomas Oxidation of protein sulfhydryls by products of peroxidase-catalyzed oxidation of thiocyanate ion. Biochemistry. 17: Banks, J. G., and R. G. Board Preservation by the lactoperoxidase system (LP-S) of a contaminated infant milk formula. Lett. 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