887 Journal of Food Protection, Vol. 56, No. 10, Pages 887-892 (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 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'