Antibacterial Activity of the Lactoperoxidase System: a Review

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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 68583-0919

(Received for publication April 7, 1993) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/10/887/1664249/0362-028x-56_10_887.pdf by guest on 24 September 2021

ABSTRACT Lactoperoxidase is found in the mammary, salivary, and lachrymal glands of mammals and in their respective secretions, e.g., milk, The lactoperoxidase (LP) system is a naturally occurring system saliva, and tears. In milk and saliva, lactoperoxidase exists in a which was first discovered in raw milk. Different groups of bacteria soluble form, but within the cells of salivary and mammary glands, show a varying degree of resistance to the LP system. Gram-negative it is possible that the enzyme could be loosely bound to subcellular catalase-positive organisms, such as pseudomonads, coliforms, salmo- particles (18). This could influence its affinity for different sub nellae, and shigellae, are inhibited by the LP system. Depending on the strates and the relative rates of reactions it catalyses. Lactoperoxidase medium pH, temperature, incubation time, cell density, and the particu has a molecular weight of 77,500 (30) and is resistant in vitro to lar donor, these microorganisms may be killed. It has been shown that acidity as low as a pH of approximately 3 and to human gastric the LP system can increase storage times of raw milk by delaying juice (32). Lactoperoxidase is actually more active at acidic pH growth of psychrotrophs; perhaps this method could be used to extend values (37). the shelf life of other foods. Bovine milk lactoperoxidase is relatively heat resistant, with the enzyme being only partially inactivated by short-time pasteur The antibacterial activity/mechanism of the ization at 74°C, leaving sufficient activity to catalyze the reactions lactoperoxidase (LP) system is well-documented (30). It is between thiocyanate and hydrogen peroxide (32). Cow's milk the major intermediary product of the LP system reaction, contains from 1.2 to 19.4 units per ml and is about 20 times richer hypothiocyanite (OSCN), which oxidizes essential protein in peroxidase activity than human milk (16). Human milk sulfhydryls, resulting in altered cellular system functions and lactoperoxidase activity is low; values range from 0.06 to 0.97 units per ml (41). The highest content of lactoperoxidase (22 units per which causes inhibition of growth and/or death of the micro ml) has been reported for guinea pig milk (41). organism (30). The hypothiocyanite ion can be formed by In human saliva, lactoperoxidase has a role similar to that in mixing the components (lactoperoxidase, thiocyanate, and milk. As a component of the LP system, it is involved in the hydrogen peroxide) of the LP system together. inhibition of streptococci which promote dental carries (32). The The lactoperoxidase antimicrobial system is a naturally human infant already possesses salivary lactoperoxidase during the occurring system which has been proven to be both bacteriostatic first few days after birth (32). and bactericidal to a variety of gram-positive and gram-negative microorganisms (29). The LP system can alter many cellular Thiocyanate (SCN) systems, including the outer membrane, cell wall, cytoplasmic The thiocyanate anion is widely distributed in animal tissues membrane, transport systems, glycolytic enzymes, and nucleic and secretions. Thiocyanate is largely a constituent of the extracel acids. Nonpathogenic bacteria (5,8,9,23,26-28,36,39,40) as well lular fluid. It is, however, concentrated by certain cells of the body. as pathogenic bacteria (4,6,7,10,12-15,21,33,34,38) have been Whereas the blood serum concentration of thiocyanate is 0.1-0.3 mg%, the salivary concentration has been estimated at 1-27 mg% shown to be inhibited or killed by the LP system. (24). The level of thiocyanate is related to diet and habits such as This paper begins with a general discussion of the LP smoking. Thiocyanate is excreted mainly in the urine, and with system and then provides information about the antibacterial normal renal functions, the half-life of elimination is 2 to 5 d (32). activity of the LP system against specific nonpathogenic and The thiocyanate concentration of bovine milk, which varies with pathogenic bacteria. breed, species, and type of feed, has been estimated at 0.1-1.5 mg% (24). COMPONENTS OF THE LP SYSTEM There are two major dietary sources of thiocyanate, glucosinolates, and cyanogenic glucosides. Vegetables belonging to The LP system is made up of three components: the genus Brassica (family Cruciferae), such as cabbage, kale, lactoperoxidase, thiocyanate, and hydrogen peroxide. brussel sprouts, cauliflower, turnips and rutabaga, are particularly rich in glucosinolates, which upon hydrolysis yield thiocyanate in Lactoperoxidase (LP) addition to other reaction products (32). Peroxidases are defined as enzymes whose primary function is Cyanogenic glucosides are also found in cassava, potatoes, to oxidize molecules at the expense of hydrogen peroxide (32). maize, millet, sugar cane, peas, and beans. When hydrolyzed, glucosides release cyanide, which in a reaction with thiosulfate Published as Paper No. 10320, Journal Series, Nebraska Agricultural (metabolic product of sulfur-containing amino acids) is detoxified Research Division, Lincoln, NE 68583-0919. by conversion into thiocyanate (32). The latter reaction is catalyzed

JOURNAL OF FOOD PROTECTION, VOL. 56, OCTOBER 1993 WOLFSON AND SUMNER

+ by the enzyme rhodanase (52). Cyanide from tobacco smoke is Protein-S-SCN + H20 <- - - > Protein-S-OH + SCN' + H metabolized in the same way. Also, sulfenyl derivatives can undergo slow oxidation to sulfonic Hydrogen peroxide acids (35). The SCN moiety can be displaced from sulfenyl thio Hydrogen peroxide is the third component of the cyanate by reduction with a sulfhydryl compound, such as lactoperoxidase system. Hydrogen peroxide may be formed endo- dithiothreitol (35). When all the protein sulfhydryls are oxidized by genously. Many lactobacilli, lactococci, and streptococci produce (SCN)2 or SCN", tyrosine, tryptophan, and histidine residues are sufficient hydrogen peroxide under aerobic conditions to activate modified (35). Release of SCN" from sulfenyl thiocyanate is fa the LP system. Hydrogen peroxide may also be added or may be vored at low SCN" concentration (2). When SCN" is released, it can generated by the addition of one of a number of hydrogen peroxide- be reoxidized and participate in the oxidation of another sulfhydryl. generating systems. Among the latter are the oxidation of ascorbic As illustrated (Fig. 1) with OSCN", the oxidation of sulfhydr acid, the oxidation of glucose by glucose oxidase, the oxidation of yls to sulfenic acid does not consume SCN". Therefore, the amount hypoxanthine by xanthine oxidase, or the manganese-dependent of sulfhydryls oxidized does not depend on the amount of SCN" (2). aerobic oxidation of reduced pyridine nucleotides by peroxidase

(24). According to Klebanoff et al. (24), a hydrogen peroxide- H SCN, Protein-S-OH generating system is more effective than added hydrogen peroxide 2°2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/10/887/1664249/0362-028x-56_10_887.pdf by guest on 24 September 2021 as a component of the antimicrobial system. Lactoperoxidase Protein-S-SCN MODE OF ACTION

The lactoperoxidase catalyzed reaction yields short-lived inter t^O OSCN Protein-SH mediary oxidation products of SCN", which may be further oxi Figure 1. Oxidation of sulfhydryls. dized to end-products such as sulfate, C02, and ammonia or may be reduced back to SCN (32). Most researchers agree that the major THE HYPOTHIOCYANITE ION (OSCN) intermediary oxidation product is hypothiocyanite, OSCN" (2,19,20,24,30,32,35). It is proposed that peroxidase-catalyzed oxi As stated previously, the hypothiocyanite ion is believed to be dation of SCN" results in the accumulation of OSCN" (32). The the major intermediary oxidation product of the LP system. OSCN" hypothiocyanite ion can be produced by two different pathways. - can be considered the hypohalite of thiocyanogen (SCN)2, whose The oxidation of SCN may yield thiocyanogen (SCN)2, which chemical characteristics are similar to those of other hypohalites, hydrolyzes rapidly to yield hypothiocyanous acid (HOSCN), or including stability in ionic form but instability as the acid (20). OSCN. Many factors affect the stability of hypothiocyanite. The decomposition of OSCN" is strongly dependent on the pH of the Peroxidase solution; OSCN" is more stable at pH 7.5 than pH 5.0 (20). OSCN + 2SCN- + H202 + 2H >(SCN)2 + 2H20 solutions are sensitive to light, yet they are very heat stable (20). Stability of OSCN" solutions decrease on addition of metal ions (Fe, + (SCN)2 + H20 > HOSCN + SCN" + H Ni, Cu, Mn, etc.), glycerol, and ammonium sulfate, as well as the removal of lactoperoxidase (20). + HOSCN<- > H + OSCN Several analytical methods for the estimation of OSCN" have been used. These methods are based either on the reduction of Alternatively, SCN" may be oxidized directly to OSCN". 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 Peroxidase disulfide compound 5,5' dithiobis (2-nitrobenzoic acid) by OSCN" SCN + H202 > OSCN + H20 (20).

It is the intermediary oxidation product(s) that have antibacte GENERAL EFFECTS ON BACTERIA rial activities, such as inhibition of growth, oxygen uptake, and lactic acid production. In addition, it has been shown that bacterial The LP system can kill or inhibit the growth and metabolism enzymes, including hexokinase glyceraldehyde-3P-dehydrogenase, of many species of microorganisms. Many cellular systems (i.e., are inhibited (32). outer membrane, cell wall, cytoplasmic membrane, transport sys The oxidation of sulfhydryl (SH) groups of enzymes and other tems, glycolytic enzymes, and nucleic acids) can be altered by the proteins has been considered to be key to the antimicrobial action LP system. According to Pruitt and Reiter (29), for any particular of the LP system (32). Moreover, it appears that the bacterial microorganism, the antimicrobial effects depend upon the reaction cytoplasmic membrane is structurally damaged or changed because conditions. When adequate concentrations of lactoperoxidase are organisms exposed to the LP system immediately leak potassium, provided, bactericidal effects are greater at low temperatures (0- amino acids, and polypeptides into the medium (32). Subsequently, 5°C), at low pH (5 or less), and in the absence of reducing agents uptake of glucose, purines, pyrimidines, and amino acids as well as (29). synthesis of protein, DNA and RNA is also inhibited (32). The effectiveness of the LP system may vary with the medium Lactoperoxidase catalyzes the incorporation of SCN- into in which the test is conducted. For example, small molecular protein substrates. The reaction of (SCN)2 or OSCN' with proteins weight components present in brain heart infusion (BHI) broth oxidizes the protein sulfhydryls to sulfenyl thiocyanate derivatives interfere with the antimicrobial action of the LP system (20). A (35). given quantity of hydrogen peroxide appears to be more effective when it is supplied by the metabolism of the cells or by continuous + Protein-SH + (SCN)2 -> Protein-S-SCN + SCN' + H generation with glucose/glucose oxidase than when it is added Protein-SH + OSCN -> Protein-S-SCN + OH separately (32). When the components of the LP system are brought together in the presence of the target cells, killing or inhibition Sulfenyl thiocyanate derivatives can undergo further modifications, appears more effective than if the cells are added after the compo including reversible hydrolysis to yield sulfenic acids (35). nents are combined. Results obtained depend upon the state of the

JOURNAL OF FOOD PROTECTION, VOL. 56, OCCTOBER 1993 ANTIBACTERIAL ACTIVITY OF LACTOPEROXIDASE SYSTEM 889 cells (29). According to Pruitt and Reiter (29), resting cells or cells g of S. mutans. Inhibition was most effective at pH 5, whereas in the stationary phase of growth are more susceptible to killing or release of hydrogen peroxide and accumulation of the inhibitor inhibition than are metabolically active or growing cells, but other (OSCN) were highest at pH 8 (36). The results indicate that pH, studies show contrasting results (31,33). Bacteria grown anaerobi- amount of hydrogen peroxide, cell sulfydryl content, and carbohy cally appear to be more susceptible to the LP system than micro drate reserve determine susceptibility to inhibition (36). organisms grown aerobically (9). Carlsson et al. (9) grew strains of Streptococcus sanguis, It is possible that some microorganisms are capable, at least Streptococcus mitis, Streptococcus mutans, and Streptococcus partially, of "neutralizing" the oxidation product(s) of the LP salivarius under aerobic and anaerobic conditions to determine the system or of repairing the damage caused (32). According to a effects of the LP system on the rate of acid production and oxygen study by Aune and Thomas (7), removal of OSCN" by centrifuga- uptake by intact cells; the activity of glycolytic enzymes in cell-free tion permitted Escherichia coli to recover. After E. coli were extracts; and the levels of intracellular glycolytic intermediates. washed to remove OSCN", the sulfhydryl content of the cells Acid production, oxygen uptake, and, consequently, hydrogen per increased during incubation at 25°C. Also, the content of sulfenyl oxide excretion were inhibited in all the strains by the LP system. derivatives decreased. Therefore, recovery appeared to result from S. sanguis and S. mitis had a higher capacity to recover from the the reduction of sulfenyl derivatives back to sulfhydryls. Alterna inhibition than S. mutans and S. salivarius . According to Carlsson tively, the sulfenyl derivatives may have been degraded, and new et al. (9), higher activity in the former strains of an NADH-OSCN sulfhydryl components may have been synthesized. Under the same oxidoreductase, which converted OSCN" into thiocyanate, explained Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/10/887/1664249/0362-028x-56_10_887.pdf by guest on 24 September 2021 conditions, there was no increase in the sulfhydryl content of cells this difference. that had not been exposed to the LP system. Also, there was no increase in the number of viable cells. Therefore, the increase in THERMOPHILIC STARTER CULTURES, MILK, AND CHEESE sulfhydryl content was due to a repair process rather than to growth of cells (7). The effect of the LP system on the behavior of a thermophilic Various reports have been published concerning mechanisms starter culture commonly used in the dairy industry for cheesemaking of resistance to OSCN-. These include an enzyme that catalyzes the was investigated by DeValdez et al. (77). It was determined that the reduction of OSCN- by NADH (9) and an increase in cell sulfhy thermophilic starter culture was very sensitive to the LP system; the dryl groups that rapidly reduce OSCN" to SCN' (28). Other possible activity of the starter culture was strongly reduced. resistance mechanisms include novel OSCN" -resistant respiration Activation of the LP system in milk resulted in a substantial systems and a phosphoenolpyruvate-dependent phosphotransferase reduction of the bacterial flora and prevented the multiplication of system sugar transport mechanism which is resistant to OSCN" psychrotrophic bacteria for up to 5 d (5). Bjorck (5) concluded that (36). the treatment had no effect on the physiochemical properties of the milk and did not lead to the accumulation of resistant bacteria. EFFECTS ON SPECIFIC 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 un Streptococcus spp. changed for at least 104 h, whereas bacterial multiplication in the A chemically defined culture medium was used to study the controls started after 48 h. At 10°C, activation resulted in a lag effect of purified lactoperoxidase and thiocyanate on the growth of phase of at least 72 h, but at 17°C this was reduced to <24 h. several cultures of Streptococcus pyogenes and Streptococcus According to Zajac et al. (39), the results showed that by repeated agalactiae in a study by Mickelson (26). While not inhibited by activation of the LP system it is quite possible to store raw milk at either component alone, S. pyogenes was completely inhibited 4°C for extended periods of time. when both components were present in the medium. S. pyogenes The activated LP system in conjunction with pasteurization glyceraldehyde phosphate dehydrogenase was inhibited by extended the shelf life of milk held at 10°C by more than 20 d, lactoperoxidase when hydrogen peroxide was present. With S. compared to untreated milk (23). They found that observable agalactiae cultures, a delay in growth up to 6 h resulted instead of growth of surviving natural milk microflora started after 12 d in LP complete growth inhibition. The extent of growth inhibition was system-treated milk, compared to 4 d in untreated and H202-treated greatest in those strains which were unable to adapt to an oxidative milk. After 22 d, viable counts in untreated and H202-treated milk pathway for their energy supply (26). In another study by Mickelson reached 106-107 cells per ml compared to about 10' cells per ml in (27), transport of 2-deoxyglucose or glucose in S. agalactiae was LP system-treated milk (23). strongly inhibited if the cells were first exposed to a combination Zall et al. (40) made acceptable varieties of soft and hard of lactoperoxidase-thiocyanate-hydrogen peroxide. Interference of cheeses from chemically treated milk. Through the use of the LP the entry of glucose into cells of S. agalactiae by the LP system system, which avoids heating altogether, it was possible to compare could well account for its growth inhibitory properties with this cheese yields with products made from unheated and heated 8-d-old organism (27). The inhibition of glucose transport by the LP system milk. Chemically treated milk increased yields 2% as compared to and its reversibility with dithiothreitol suggest the modification of control cheese (40). functional sulfhydryl groups in the cell membrane as a cause of transport inhibition (27). Escherichia coli and Salmonella Results from a study by Oram and Reiter (28) showed that the In a study by Bjorck and Claesson (6), lactoperoxidase- growth of the LP-sensitive Streptococcus cremoris 972 (now catalyzed oxidation of thiocyanate ion resulted in a bactericidal Lactococcuss lactis var. cremoris) in a synthetic medium was effect against E. coli in a semisynthetic medium. According to inhibited by LP and SCN. The glycolysis and oxygen uptake of Bjorck and Claesson (6), the reaction produced an antibacterial suspensions of S. cremoris 972 in glucose or lactose was also agent that caused reversible inhibition of many gram-positive inhibited. It appears that the LP system completely inhibited the bacteria and an irreversible inhibition of gram-negative bacte hexokinases of nonmetabolizing suspensions of both strains (28). ria. The inhibition was reversible. Hexokinase and glycolytic activities Two strains of E. coli and one strain of Salmonella typhi- of S. cremoris 972 were restored by washing the inhibitor from murium were killed by the bactericidal activity of the LP system in cells (28). milk and in a synthetic medium (33). Hydrogen peroxide was Thomas et al. (36) studied the inhibition of bacterial metabo supplied exogenously by glucose oxidase, and glucose was added lism by the LP system with representatives of serotypes a through at a level which was itself noninhibitory. The bactericidal activity

JOURNAL OF FOOD PROTECTION, VOL. 56, OCTOBER 1993 890 WOLFSON AND SUMNER was greatest at pH 5 and below and depended on the thiocyanate dependent on the temperature of incubation, lasting 6 h at 30°C and concentration and the number of organisms. more than 20 and 100 h at 20 and 8°C, respectively. No difference According to Earnshaw et al. (13), a glucose/glucose oxidase was observed between the two organisms in their response to the activated LP system delayed the onset of exponential growth of S. LP system at 30°C. typhimurium and E. coli in infant formula milk. Addition of urea Effects of the LP system on L. monocytogenes (strains V7, peroxide with the LP system reduced the initial population size and Scott A, and California) were determined by El-Shenawy et al. (14) prevented growth of S. typhimurium and extended the lag period using a semisynthetic medium, raw milk and a buffer solution. before the onset of exponential growth of E. coli. Each medium was inoculated to contain three levels of the patho Results from a study by Purdy et al. (31) showed that the LP gen (low, 30 to 50 CFU/ml; medium, 104 CFU/ml; and high, 107 system was found to have both bacteriostatic and bactericidal CFU/ml) and incubated at 4 or 35°C. Low numbers of the bacteria activities against strains of S. typhimurium. Purdy et al. (31) were completely inactivated within 2 to 4 h (35°C) or 12 to 24 h believed that the bactericidal activity was clearly dependent on the (4°C); when substrates contained medium or high populations, a permeability of the bacterial cell envelope. Bacteria in log phase of limited bactericidal effect occurred with decreases in population of growth were more sensitive to the bactericidal effects than were one-half (4°C) or one (35°C) order of magnitude. The LP system those in stationary phase; growth phase had little influence on the failed to cause permanent injury of L. monocytogenes at 4 or 35°C. bacteriostatic effect (31). El-Shenawy et al. (14) believed that the efficacy of the LP system The LP system was investigated for its activity against salmo- as an antimicrobial agent was related to strain and number ofDownloaded from http://meridian.allenpress.com/jfp/article-pdf/56/10/887/1664249/0362-028x-56_10_887.pdf by guest on 24 September 2021 nellae in vivo and in vitro by Wray and McLaren (38). In acidified pathogen, suspending medium, and incubation temperature. raw milk, in which the LP system was supplemented with an The activity of a raw milk LP system or four L. monocyto exogenous supply of hydrogen peroxide, the number of salmonellae genes strains at refrigeration temperatures was studied by Gaya et decreased rapidly. According to Wray and McLaren (38), different al. (15). The LP system exhibited a bactericidal activity against L. salmonellae serotypes were affected to the same extent; rough monocytogenes at 4 and 8°C; the activity was dependent on strains, however, were more susceptible than smooth strains. Calves temperature, length of incubation, and strain of L. monocytogenes were fed fresh milk containing the LP system and challenged with tested (15). S. typhimurium; the clinical findings were similar to those of control calves fed on heated milk (38). Further field studies would Campylobacter be necessary to evaluate the LP system as a possible nonantibiotic Borch et al. (7) tested the antibacterial effect of the LP system system to control salmonellosis in calves. against strains of Campylobacter jejuni and Campylobacter coli isolated from poultry. The effect was studied at different pH values Listeria and Staphylococcus (5 to 7) and temperatures. The LP system had a strong bactericidal Earnshaw and Banks (12) found that a LP system activated by effect against C. jejuni and C. coli. Borch et al. (7) found that the glucose oxidase was bacteriostatic to Listeria monocytogenes in bactericidal effect was more rapid at 37°C compared to 20°C, and oculated into ultra-high temperature (UHT) milk supplemented that the effect of the LP system decreased with decreasing pH with glucose. The reasons for the nonlethal effect of the LP system values. The fastest reduction in viable numbers was obtained at pH were unclear. 6.6 and 37°C (7). Previous investigators (33) have demonstrated an The antibacterial activity of the LP system on the growth and enhanced effect of lactoperoxidase at lower pH values. The differ survival of L. monocytogenes in Trypticase soy broth, UHT milk, ent results with Campylobacter may be attributed to its and French soft cheese was determined by Denis and Ramet (10). microaerophilic nature. In Trypticase soy broth and UHT milk, presence of the LP system either inhibited growth or completely inactivated inoculated cells. MILK PRESERVATION According to Denis and Ramet (10), complete inactivation occurred at different times depending on initial inoculum concentration, In Western Countries milk is cooled and stored for increas culture medium, and storage temperature. The addition of the LP ingly long periods because of distribution circumstances. After 2 d, system to the surface of soft cheese led to elimination of viable such milk can deteriorate through the multiplication of psychrotrophic Listeria cells from cheeses previously inoculated to contain 10' to organisms (mainly pseudomonads), which produce extremely heat- 106 CFU/g. resistant lipases and proteases which survive pasteurization (25). Siragusa and Johnson (34) showed that the LP system inhib These enzymes can make the milk unpalatable or spoil dairy ited the growth of L. monocytogenes Scott A in a bacteriostatic but products such as butter and cheese. The enzyme lactoperoxidase not a bactericidal manner. Evidence of bacteriostasis was the remains active after some thermal treatments, such as normal extension of the lag period beyond that of the control flasks (H202 pasteurization (25). This suggests that subsequent reactivation of treatment or broth control). the LP system during storage of raw or pasteurized milk is possible. Kamau et al. (21) determined that the LP system, using the Gupta et al. (17) studied the effects of the LP system on the inherent milk lactoperoxidase, effectively inhibited the growth of L. preservation of milk. The LP system can be used to preserve infant monocytogenes and S. aureus at 35 and 37°C, respectively. In formula (3). It might also be possible to utilize the LP system to another study by Kamau et al. (22), the LP system was found to increase storage times of raw milk at ambient temperatures. The LP enhance thermal destruction of L. monocytogenes and S. aureus. system could be considered an alternative approach for preservation The most rapid killing of L. monocytogenes occurred when samples of raw milk in developing countries. were heated soon after activation of the LP system. According to Kamau et al. (22), activation of the LP system followed by heating SUMMARY can increase the margin of safety with respect to milkborne patho gens. According to Reiter and Harnulv (32), the strongest Survival and growth of L. monocytogenes and Listeria in- arguments against any undesirable side effects resulting from nocua in the presence of a reactivated LP system was investigated the activation of the LP system are based on: (i) the wide 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 distribution of all the components of the system in humans essentially bacteriostatic effect on both organisms. According to and animals; (ii) evidence of the in vivo activity of the LP Bibi and Bachmann (4), duration of the bacteriostatic effect was system in calves; (iii) the detection of one of the major

JOURNAL OF FOOD PROTECTION, VOL. 56, OCCTOBER 1993 ANTIBACTERIAL ACTIVITY OF LACTOPEROXIDASE SYSTEM 891

oxidation products, OSCN, in human saliva; and (iv) selec Prot. 53:170-172. tive damage to the bacterial cytoplasmic membrane but not to 14. El-Shenawy, M. A., H. S. Garcia, and E H. Marth. 1990. Inhibition and inactivation of Listeria monocytogenes by the lactoperoxidase mammalian cell membranes. The innocuous nature of the LP system in raw milk, buffer or a semi-synthetic medium. system is further supported by results from in vitro experi Milchwissenschaft. 45:638-641. ments with animal cell cultures showing no toxic effects of 15. Gaya, P., M. Medina, and M. Nunez. 1991. Effect of the the system (32). The LP system can, therefore, be regarded as lactoperoxidase system on Listeria monocytogenes behavior in raw milk at refrigeration temperatures. Appl. Environ. Microbiol. 57:3355- a naturally occurring antibacterial system that has evolved 3360. throughout the evolution of mammals to be a part of the 16. Gothefors, L., and S. Marklund. 1975. Lactoperoxidase activity in defense system against bacterial infections on mucosal sur human milk and in saliva of newborn infants. Infect. Immun. 11:1210- faces in, for example, the gastrointestinal tract (32). 1215. 17. Gupta, V. K., R. S. Patel, G. R. Patil, S. Singh, and B. N. Mathur. Indirect evidence of the importance of the LP system 1986. Preservation of milk with hydrogen peroxide and may be provided by the fact that nature has provided both lactoperoxidase/thiocyanate/hydrogen peroxide systems. Indian J. Dairy newborn calves and humans with this system (16). In the Sci. 39:269-276. former species it is provided by colostrum and to a much 18. Hogg, D. M., and G. R. Jago. 1970. The antibacterial action of lactoperoxidase; the nature of the bacterial inhibitor. Biochem. J. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/10/887/1664249/0362-028x-56_10_887.pdf by guest on 24 September 2021 smaller extent by saliva, and in the latter the quantities are 117:779-790. reversed (16). Furthermore, lactoperoxidase in milk is not 19. Hogg, D. M., and G. R. Jago. 1970. The oxidation of reduced inactivated by gastric juice. nicotinomide nucleotides by hydrogen peroxide in the presence of In addition to the antimicrobial properties, the LP system lactoperoxidase and thiocyanate, iodide or bromide. Biochem. J. 117:791-797. may also exert other biological functions in vivo. Among 20. Hoogendoorn, H., J. P. Pressens, W. Scholtes, and L. A. Stoddard. 1977. these are degradation of various carcinogens, which have Hypothiocyanite ion; the inhibitor formed by the system lactoperoxidase- been reported to occur in human saliva and protection of thiocyanate-hydrogen peroxide. Caries Res. 11:77-84. human cells from the toxicity of hydrogen peroxide (16). It 21. Kamau, D. N., S. Doores, and K. M. Pruitt. 1990. Antibacterial activity of the lactoperoxidase system against Listeria monocytogenes has been shown that the LP system can increase storage times and Staphylococcus aureus in milk. J. Food Prot. 53:1010-1014. of milk, particularly by delaying the growth of psychrotrophs 22. Kamau, D. N., S. Doores, and K. M. Pruitt. 1990. Enhanced thermal (25). Perhaps this method could be used to extend the shelf destruction of Listeria monocytogenes and Staphylococcus aureus by life of other foods. the lactoperoxidase system. Appl. Environ. Microbiol. 56:2711-2716. 23. Kamau, D. N., S. Doores, and K. M. Pruitt. 1991. Activation of the lactoperoxidase system prior to pasteurization for shelf-life extension REFERENCES of milk. Milchwissenschaft. 46:213-214. 1. Aune, T. M., and E. L. Thomas. 1977. Accumulation of 24. Klebanoff, S. J., W. H. Clem, and R. G. Luebke. 1966. 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Derr, com. from p. 886 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/10/887/1664249/0362-028x-56_10_887.pdf by guest on 24 September 2021 TX. Technology, Ames, IA. 31. Wierbicki, E. et al. 1986. Ionizing energy in food processing and 32. World Health Organization (WHO). 1992. Provisional edition report. pest control: I. Wholesomeness of food treated with ionizing energy. Review of the safety and nutritional adequacy of irradiated food. Report No. 109. pp. 1-50. Council for Agricultural Science and Geneva.

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