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Journal of Food Protection, Vol. 47. No. 9, Pages 724-732 (September 1984) Copyright®, International Association of Milk, Food, and Environmental Sanitarians

Lactoperoxidase Antibacterial System: Natural Occurrence, Biological Functions and Practical Applications

BRUNO REITER1 and GORAN HARNULV2* Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/9/724/1650811/0362-028x-47_9_724.pdf by guest on 29 September 2021 National Institute for Research in Dairying, Shinfield, Reading, RG2 9AT, England and Alfa-Laval Agri International AS, P.O. Box 39, S-I47 00 Tumba, Sweden

(Received for publication January 30, 1984)

ABSTRACT (37,80). The various biological functions of the LP sys­ tem, which have now been established, will be discussed In the present review dealing with the antibacterial lac­ below. toperoxidase (LP) system, it is shown that the two reactants In recent years, much work has been devoted to practi­ (SCN~) and (H202) as well as cal applications of the LP system. The effect against oral the catalytic lactoperoxidase (LP) are widely distributed streptococci (5,19,33,101,105,107) has led to the de­ in nature and that evidence for the activity of the LP system velopment and marketing of a toothpaste containing nec­ in animals, including man, is accumulating. The in vitro effects on bacterial and animal cells are discussed and the unique ac­ essary ingredients to activate the LP system. Promising tion of the LP system on the bacterial cytoplasmic membrane results have also been obtained when including activating is pointed out. Some practical applications are also presented, components in the feed to calves (81,83,86) with the aim with particular emphses on the possibility of utilizing the LP of potentiating the LP system in the intestinal tract. system to preserve the quality of raw, cooled as well as un- Partly because most of the early work on the LP sys­ cooled milk. It is concluded that the addition of minute quan­ tem was focused on bacteria associated with milk, the tities of SCN and H202 (ca. 12 and 8 ppm, respectively) to possible utilization of the LP system in milk quality pre­ secure an optimum activity of the LP system should be harm­ servation soon became obvious and is at present being less to the consumer of milk and milk products treated in this discussed within the International Dairy Federation (4). way. [Note that this method of milk quality preservation, based

on the addition of minute amounts of SCN~ and H202, should not be confused with the "traditional" method of As early as 1924, Hanssen (27) observed that freshly using H 0 in concentrations up to 800 ppm (46,102)]. drawn milk was bactericidal against certain types of bac­ 2 2 It was established that refrigerated storage of milk on the teria and ascribed this effect to the presence of oxidizing farm could be considerably extended by activating the LP . Several years later, it was found that lac­ system (14,79,83,85). A few years earlier, Reiter [cited toperoxidase (LP) was involved in the inhibition of lactic by Tentoni et al. (702)] had suggested the LP system as streptococci (cheese starters) (66,120) and subsequently a possible means of increasing the keeping quality of shown that hydrogen peroxide (H 0 ) was necessary for 2 2 milk at high ambient temperature on the farm and during the reaction (34). Soon afterwards, it was established subsequent collection. Field experiments in Kenya (75), (82,84) that a third factor (besides LP and H 0 ) was 2 2 Sri Lanka (30) and Pakistan (29) have since proven that involved. The compound was eventually isolated from treatment of uncooled milk with the LP system is feasible milk by ion-exchange chromatography (which made the under practical conditions. In spite of high ambient tem­ milk non-inhibitory) and identified by IR spectroscopy as perature and transportation over long distances, undue thiocyanate (SCN"). The two reactants (H 0 and SCN") 2 2 acid development and thus spoilage of the milk could be and catalyst (LP) of the LP system were thus identified. prevented. LP-treated milk passed also various other The wide distribution of enzymes and SCN keeping quality tests, whereas untreated milk samples in biological fluids other than milk early suggested a role failed under identical conditions. for the LP system in, for example, and tears Additives to milk are rightly strictly controlled and it is therefore necessary' to ascertain that such compounds are harmless. This annotation is based on the past re­ 'National Institute for Research in Dairying, (Retired). search on the LP system as well as on literature pertain­ 2Alfa-Laval Agri International AS. ing to it. It documents the wide distribution of all three

JOURNAL OF FOOD PROTECTION, VOL. 47, SEPTEMBER 1984 LACTOPEROXIDASE ANTIBACTERIAL SYSTEM 725 components of the LP system, its mode of action on bac­ Milk of all species tested so far shows peroxidative ac­ teria, including, in particular, its unique action on the tivity. Bovine milk is rich in LP (26,35,39,40,97,121) cytoplasmic membrane. Most importantly, it is shown and milk can be diluted up to 100 times without affecting that the complete LP system occurs naturally in man and the antibacterial activity of the LP system (76). It is diffi­ animals, where it functions as an indigenous antibacterial cult to compare the literature data on peroxidase activities system promoting a balanced flora in the oral cavity and because different methods of analysis have been used. the intestinal tract (38,40,69,74-76,78,80,101). Using a recently developed method based on the oxida­ tion of 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) (92), an average value of about 1.4 units/ LACTOPEROXIDASE ml has been reported for bovine milk (97) and 0.9 unit/ ml for buffalo's milk (30). In the following, values of metabolism leads to toxic end-products, such LP activities are given as ABTS units/ml. as free oxygen radicals and H 0 , in both procaryotic 2 2 In human milk, the peroxidase activity is much lower and eucaryotic cells unless they are protected by enzymes

than in bovine milk and generally decreases rapidly dur­ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/9/724/1650811/0362-028x-47_9_724.pdf by guest on 29 September 2021 such as superoxide dismutase, and . ing the first week after delivery (26,35,76,121). Contrary The first enzyme converts the superoxide into to this, an average value of 0.23 unit/ml was found in H 0 , the second reduces the H 0 to water and oxygen, 2 2 2 2 samples of milk from Gambian mothers, 1 to 9 months and the peroxidases catalyze the reduction of H 0 by 2 2 post-partum. Since 0.02 unit/ml was determined to be the a variety of electron donors (25). limit for bactericidal activity of the LP system, the great Peroxidases (EC 1.11.1.7.) are defined as enzymes majority of the samples (53 of 60) fell above this level whose primary function is to oxidize molecules at the ex­ (76). Recent research using exclusion chromatography pense of H 0 . Probably because of their wide distribu­ 2 2 and immunological cross-reaction has, however, indicated tion and their dramatic of the formation of col­ that the peroxidative activity of human milk is not due ored products, these enzymes have been among the most to LP but to derived from milk leuco­ extensively studied since the beginning of enzymology. cytes (58). In addition to SCN", and , The peroxidase in the mammary, salivary and lacrimal myeloperoxidase can also oxidize chloride in the presence glands have been found to be immunologically and chem­ of H 0 . It appears, therefore, that the reason for the ically similar (61,108). The peroxidase isolated from milk 2 2 persistence of peroxidase in the above Gambian milk was given the trivial name lactoperoxidase (LP) and be­ samples is probably because of infection of the mammary cause of the similarity mentioned above, the salivary glands causing infiltration of leucocytes. peroxidase is often also referred to as lactoperoxidase. Other mammalian peroxidases include neutrophil The highest concentration of LP so far detected in milk myeloperoxidase (88), (115), is 22 units/ml which was observed in guinea pigs (97). (3), intestine mucosa peroxidase (96) It is interesting to note that the saliva of guinea pigs con­ and uterine peroxidase (93). tains very little LP. Considering that bovine and guinea pig milk are rich in LP but the corresponding salivary It is now established that a peroxidase in the thyroid secretions low and the reverse is true with humans, it gland is involved in the iodination of thyroglobulin to indicates that either milk or saliva can be a source of thyroxine and that defects in the peroxidative activity are LP for the newborn. responsible for some of the goiter syndromes (62). In cer­ LP is resistant in vitro to acidity down to a pH of vical mucus, the peroxidase inhibits spermatozoal activity about 3 (720) and to human gastric juice (26). Moreover (77,95), but during ovulation the enzyme appears to be it was shown that in the abomasum of the calf, the LP absent (45,94). In saliva, the LP has a role similar to of the ingested milk can survive for several hours. Before that in milk. As a component of the LP system, it is feeding, only traces of LP could be detected (81). Bovine involved in the inhibition of lactic acid bacteria which milk LP is relatively heat resistant, with the enzyme promote dental caries. being only partially inactivated by short time pasteuriza­ There is great variation in the distribution of LP in the tion at 74°C (120,122), leaving sufficient activity to salivary glands of different animals (60,61,97,108,111). catalyze the reaction between SCN and H 0 . The human infant possesses salivary LP already during 2 2 the first few days after birth, but there is marked varia­ tion in consecutive specimens from the same individual HYDROGEN PEROXIDE (26). In contrast, it was reported that the saliva of the calf does not contain any LP at birth (61) or possibly It is generally assumed that milk does not contain varying low concentrations (26). While salivary LP could H202. Although the mammary tissue is metabolically be important for the human infant, because it is continu­ very active during lactation, any H202 formed is rapidly ously secreted and swallowed to enter the stomach, the reduced by catalase or peroxidase. When these enzymes calf's saliva would normally enter the undeveloped were inactivated with azide, it was, for instance, possible rumen, except during feeding when the rumen is by­ to detect nanomolar concentrations of H202 generated by passed and the saliva enters the digestive abomasum to­ and diffused from bovine polymorphonuclear leucocytes gether with the milk which is the primary source of LP. when suspended in milk (41).

JOURNAL OF FOOD PROTFCT10N. VOL 47, SEPTEMBER 1984 726 REITER AND HARNULV

Another possible source of H202 in milk is the The cyanogenic glucosides are found in cassava, sweet metabolic activity of lactic acid bacteria either con­ potatoes, maize, millet, sugar cane, peas, beans and the taminating the milk or added deliberately as a starter cul­ kernel of various fruits. When hydrolyzed, these ture. Many lactobacilli and streptococci produce sufficient glucosides release cyanide, which in a reaction with H202 under aerobic conditions to activate the LP system thiosulfate (metabolic product of sulfur amino acids) is (19-21,34,67,84,90,107). detoxified by conversion into SCN . The latter reaction Because of the high reactivity and rapid turnover of is catalyzed by the enzyme rhodanase (EC 2.8.1.1.) H202, it is difficult to make accurate quantitative deter­ which is in the liver, kidney and thyroid (24,59,119). minations of the concentration of H202 in saliva or gas­ Cyanide from tobacco smoke is metabolized in the same tric juice. The presence of H202-producing bacteria in way. human saliva has, however, been known for a long time Bovine milk normally contains 1 to 10 ppm of SCN (42) and the recent detection of the primary oxidation (16,44,53,118), but higher concentrations have been re­ product of SCN in aseptically drawn saliva also indicates ported, particularly in milk with high somatic cell counts the production of H 0 in the salivary glands (70). 2 2 (39). On the other hand, supplementation of feed in Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/9/724/1650811/0362-028x-47_9_724.pdf by guest on 29 September 2021 Moreover, traces of H202 have been detected in samples short-term experiments with as much as 3 g of SCN per taken from the calf abomasum, which was found to be day failed to raise the concentration in the milk above colonized by large numbers of lactobacilli. Up to 60% 10 ppm (65). of these were H202-producers and they appeared to form Excessive intake of SCN" can produce goiter (or de­ enough H 0 to activate the LP system in vivo (50,81). 2 2 press hyperactivity of the thyroid gland), but only indi­ THIOCYANATE rectly through interference with iodine metabolism. In rats, high levels of SCN" in the diet (1 to 5 mg/d) in­ The thiocyanate anion (SCN") is widely distributed in creased the plasma level from about 5 up to 13 to 27 animal tissues and secretions. The concentration in ppm, but the renal clearance increased in proportion to human body fluids is shown in Table 1. The level of the quantity of SCN" ingested (24). SCN" is related to the diet and habits such as smoking. Milk was thought to have a goitrogenic effect because Thiocyanate is mainly excreted in the urine (57) and, of its SCN" content. It was clearly demonstrated, how­ with normal renal function, the half-life of elimination ever, that milk containing the highest concentration of is 2 to 5 d (15,89). Moreover, the rate of excretion in­ SCN" achievable by feeding "goitrogenic" plants to creases with increasing serum concentration (22,24). cows (chiefly Brassica), failed to prevent accumulation There are two major dietary sources of SCN", i.e., of labelled iodide by the thyroid gland in man. A reduc­ glucosinolates and cyanogenic glucosides. Vegetables be­ tion of iodide uptake could only be achieved by a dose longing to the genus Brassica (family Cruciferae), such of at least 200 to 400 mg, which would be equivalent as cabbage, kale, brussel sprouts, cauliflower, turnips and to an intake of 10 to 20 L of milk containing 20 ppm rutabaga, are particularly rich in glucosinolates which SCN". Furthermore, if the iodide intake is adequate, ten upon hydrolysis yield SCN" in addition to other reaction times the dose of SCN" would be required (112). products (24,110,119). Data compiled by Wood (119) in­ In a recent study (22), a group of people were given dicate that concentrations of SCN" up to 100 ppm are not unusual. In single cases, even higher values have an extra daily quantity of 8 mg of SCN" by way of milk been reported (52,53,113). (0.4 L of milk containing 20 ppm of SCN" per day). The results show that the serum levels of SCN" initially in­ TABLE 1. Concentration of thiocyanate (average values ex­ creased, but this effect coincided with an increased excre­ pressed as ppm of SCN~) in human body fluids. tion in the urine. From week 4 and onwards, however, Fluid Non-smoker Smoker Reference the concentration of SCN" decreased. Based on measure­ ments of levels of thyroxine, triiodothyronine and thyro­ Plasma 1.9 6.7 8 4.0 8.4 22 tropic hormone, it is concluded that this additional intake Saliva of SCN" does not have any apparent effect on thyroid Adults 37 155 10 function. Similar results have recently been obtained in 52 198 23 Sudan with a group of subjects having a low iodine status 52 133 87 (A. Bergmark, personal communication). 72 159 99 In the past, SCN" has been used as an antihypertensive Infants 17 - 26 agent (9). At a plasma level of 80 to 120 ppm, high Urine 16 23 10 14 39 23 blood pressure can be effectively reduced (89). The Gastric juice 23 64 87 therapy, however, came into disrepute because the mar­ (fasting) gin between therapeutic and toxic concentrations was too Tear fluid ca.10 111 narrow and varied widely among individuals. Thiocyanate Milk as a drug was therefore replaced by sodium nitroprusside 5.0 114 which, via cyanide, is metabolized to SCN" (109). This 4-wk post­ drug can have a good therapeutic effect without being partum 2.6 114 toxic (89).

JOURNAL OF FOOD PROTECTION. VOL. 47. SEPTEMBER 1984 LACTOPEROXIDASE ANTIBACTERIAL SYSTEM 727

In recent years, the effect of SCN" on the formation and amino acids as well as synthesis of protein, DNA, of nitrosamines has also been studied. In vitro experi­ and RNA is also inhibited (48,55,75). The leakage ments showed that SCN" increased the rate of nit- phenomenon, however, does not explain the difference rosamine formation in the presence of nitrites and between the bacteriostasis with streptococci and the bac­ amines, but only at very low pH. At pH 3.5 and above, tericidal effect on coliforms because the cytoplasmic however, the effect was negligible or non-existent (17). membranes of both types of organsims are damaged, al­ Since ingestion of milk increases the pH of gastric juice beit less so with the former organisms (49). It is possible because of its high buffering capacity (51,81), the result­ that streptococci are capable, at least partially, of "neut­ ing pH should be well above the critical level. In vivo ralizing" the oxidation product(s) of the LP system or experiments with rats fed SCN" showed that the concen­ of repairing the damage caused. It has been observed that tration of nitrosamines in the stomach was raised only the activity of an enzyme catalyzing the oxidation of re­ slightly and that animals fed additional SCN" neither gen­ duced nicotinamide adenine dinucleotide by OSCN" is erated more tumors nor did existing tumors develop faster much higher in resistant than in sensitive streptococci (91). (19,63). Alternatively, the cell wall of gram-positive or­ ganisms may be a more effective barrier than that of Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/9/724/1650811/0362-028x-47_9_724.pdf by guest on 29 September 2021 MODE OF ANTIBACTERIAL gram-negative organisms. Smooth strains of Salmonella ACTION OF THE LP SYSTEM typhimurium possessing a complete sequence of polysac­ charides are, for instance, less susceptible to the LP sys­ The LP-catalyzed reaction yields short-lived inter­ tem than are rough strains (73). mediary oxidation products of SCN" (6,31,33,63, Different groups of bacteria show a varying degree of 64,68,71,82), which may be further oxidized to innocu­ resistance to the LP system as indicated above and in ous end-products, such as sulfate, carbon dioxide and the review by Korhonen (40). Gram-negative, catalase- ammonia (64), or may be reduced back into SCN positive organisms, such as pseudomonads, coliforms, (7,104). It is the intermediary oxidation product(s) that salmonellae and shigellae, are not only inhibited by the show antibacterial activities, such as inhibition of growth, LP system but, depending on the pH of the medium, the oxygen uptake and lactic acid production. In addition, it temperature, the incubation time, the cell density and the has been shown that bacterial enzymes, including particular electron donor, may be killed provided H202 hexokinase and glyceraldehyde-3P-dehydrogenase, are in­ is supplied exogenously, either chemically, enzymatically hibited (2,19,54,63,64,82,101,107). or by H202-producing bacteria (14,73,83,85,103). The major intermediary oxidation product is Gram-positive, catalase-negative bacteria, such as , OSCN" (2,6,19,33,68,72,98,100,101, streptococci and lactobacilli, on the other hand, are gen­ 105-107,116), which has also been chemically synthe­ erally inhibited but not killed by the activated LP system sized and shown to inhibit the acid production of strep­ (63,82). These organisms can be self-inhibitory under tococci and various enzymes (2,54). Earlier results, im­ aerobic conditions in milk in the presence of LP and plicating higher oxyacids, such as cyanosulfurous acid SCN" provided they generate the third component of the (H0 SCN) and cyanosulfuric acid (H0 SCN) (31), have 2 3 LP system, namely H202. The catalase in milk does not now gained renewed credence. Recent kinetic and prevent inhibition. Under anaerobic conditions, inhibition polarographic studies (68,71) suggested that very short­ is prevented and in the presence of reducing agents it lived oxidation products, such as 02SCN and 03SCN , is reversed (82). Self-inhibition is probably the reason for may be formed when H202 is present at higher than a slight delay in the development of acidity recently ob­ equimolar concentration with SCN . It is possible that served in some cheese-making experiments (124). In ear­ such higher oxyacids are better oxidizing agents than lier work, the development of acidity in cheese-milk (79, OSCN" and have a bactericidal effect on Escherichia O. Aule, personal communication), coagulation time (//) coli, whereas the more stable OSCN" is only bacteriosta­ and cheese maturation (79) were all found to be normal tic (12,18,71). in trials with LP system-activated milk, which indicates The oxidation of sulfhydryl (SH) groups of enzymes that the starter cultures then used were either resistant to and other proteins has been considered to be the key to the LP system or did not produce enough H202. the antibacterial action of the LP system In practical experiments with milk containing a mixed (7,54,56,104,120). However, more recently (43) it was flora of varying resistance to the LP system, a small drop demonstrated that the SH-independent enzyme D-lactate in the viable count followed by an extended lag-phase dehydrogenase is also inhibited by the LP system. The is generally observed. This extension depends, among lactate dependent uptake of proline was shown to be inhi­ other things, on the temperature of the milk and on the bited in whole organisms or membrane vesicles because initial level of contamination (13,29,123). The following the proton gradient was affected by the LP system. relation between storage temperature and lag-phase has Moreover, the bacterial cytoplasmic membrane is obvi­ been reported (13): 15°C, 24 to 26 h; 20°C, 16 to 17 h; ously structurally damaged or changed because organisms 25°C, 11 to 12 h; and 30°C, 7 to 8 h. exposed to the LP system immediately leak potassium At near neutral pH (as in milk, pH 6.6 to 6.8), OSCN" ions, amino acids and polypeptides into the medium. is the main oxidation product (68,105) and the LP system Subsequently, uptake of , purines, pyrimidines is predominantly bacteriostatic for the important conta-

JOVRNAL OF FOOD PROTECTION. VOL. 47, SEPTEMBER 1984 728 REITER AND HARNULV minants of raw, uncooled milk. The bactericidal effect, activate streptococcal and coli bacterial viruses (bac­ however, increases with decreasing pH (83,107), indicat­ teriophages) had failed (unpublished). ing a stronger antibacterial effect of the uncharged The non-toxic effect of the LP system has also been hypothiocyanous acid (HOSCN, pKa = 5.3). observed with HeLa cells, Chinese hamster ovary cells, Sensitive bacteria inoculated into milk in which the LP and human gingival fibroblasts (28), although the acid system had been activated before heating at 60°C for 15 production from glucose by these cells was found to be min, were not inhibited (14). This indicates that the inter­ partially inhibited. Moreover, Hoogendoorn (32) reported mediary oxidation products of the LP system are decom­ that the LP system actively reduced lesions of the oral posed during pasteurization of milk. mucosa. It is not clear whether the primary function of the LP system is to promote healing, to prevent further inflammation, or to act by reducing the bacterial flora. COMPARISON OF THE EFFECT OF THE LP SYSTEM ON BACTERIAL EVIDENCE OF THE IN VIVO

AND MAMMALIAN CELLS ACTIVITY OF THE LP SYSTEM Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/9/724/1650811/0362-028x-47_9_724.pdf by guest on 29 September 2021

It is now generally accepted that the major evolutio­ When E. coli were fed to 2-to 3-month-old calves in nary step in development from the procaryotic cell to the heated milk (LP inactivated), practically all organisms more complex eucaryotic cell was achieved by the inter­ could be recovered in the abomasal fluid. However, when nalization of metabolic functions. These are located in the E. coli were fed in raw milk (containing active LP) with­ cytoplasmic membrane of bacteria but are in a multitude out an exogenous source of H202, only 1 to 10% of the of organized "inner" membranes (e.g., mitochondria, ingested organisms were recovered. To prove that the LP

Golgi apparatus, etc.) in mammalian cells. The cytoplas­ system, or rather its activation by H202 was involved, mic membrane of eucaryotic cells has, therefore, lost a reducing agent was added to the abomasal samples and metabolic functions retained by the bacterial cytoplasmic nearly all ingested organisms were recovered. When membrane. either lactobacilli, glucose/ or magnesium When whole liver cells were exposed to the LP sys­ peroxide was added to the raw milk as an exogenous tem, their capability to respire, as measured by their oxy­ source of H202, there was an increase in the number of gen uptake, remained unimpaired. The oxygen uptake of E. coli inactivated in the abomasum of the calves. This mitochondria isolated from the liver cells, however, was indicates that the LP system can be activated by feeding inhibited (unpublished). The cell membrane of the liver raw milk (containing LP), but that the in vivo concentra­ cells therefore seems to constitute a barrier against inter­ tion of H202 is suboptimal (78). mediary oxidation product(s) of SCN . To test this Another indirect proof of the in vivo activity of the further, erythrocytes were selected as a model for cell LP system was established in human saliva. The inter­ membrane integrity. Previously it had been shown that mediary oxidation product, OSCN , has been detected in erythrocytes were lysed by the myeloperoxidase-H202 - freshly obtained saliva (47,72,100,101,105). OSCN ap­ chloride system (36). The LP-H202-SCN" system, how­ pears to be H202, which is assumed to be provided either ever, was found to have no effect on erythrocytes, failing by the salivary glands (70) or by lactic acid bacteria re­ to hemolyze them (unpublished). The former system gen­ sistant to the LP system and colonizing the oral cavity. erates hypochlorite and singlet oxygen; both oxidation Partially resistant organisms have recently been isolated products are apparently more reactive on the mammalian (19,107). These organisms were either different H202- membrane than are the oxidation products of SCNT which producing species or strains within a species, which is failed to produce hemolysis. analogous to earlier observations on sensitive and resis­ Adamson and Carlsson (/) recently suggested that the tant strains within various serogroups of streptococci LP system is not only atoxic to human cells (e.g., the (63,82). oral mucosa), but may protect these cells against the

toxic effect of H202, which has been observed at a con­ CONCLUSIONS centration as low as 0.34 ppm (28). In a similar study,

3.4 ppm of H202 was found to reduce by over 80% The strongest arguments against any undesirable side- radiolabeled thymidine incorporation into human gingival effect resulting from the activation of the LP system in fibroblasts. This toxic effect could be totally prevented milk are based on: (a) the wide distribution of all three by the addition of LP and SCN , converting the toxic components of the system in man and animals, (b) evi­

H202 into OSCN . Concentrations of OSCN of up to dence of the in vivo activity of the LP system in calves, 22.2 ppm had no structural effect and did not reduce the (c) the detection of one of the major oxidation products, incorporation of thymidine (98), unlike in bacteria. These OSCN , in human saliva, and (d) the selective damage results substantiate the findings of White et al. (117) that to the bacterial cytoplasmic membrane but not to mam­ OSCN" failed to damage calf thymus DNA or produce malian cell membranes. The innocuous nature of the LP any mutagenic effect on mutagen-sensitive strains of 5. system is further supported by results from in vitro exper­ typhimurium or Saccharomyces cerevisiae. In retrospect, iments with animal cell cultures showing no toxic effect it is therefore not surprising that previous attempts to in­ of the system. The LP system can therefore be regarded

JOURNAL OF FOOD PROTECTION, VOL. 47. SEPTEMBER 1984 LACTOPEROXIDASE ANTIBACTERIAL SYSTEM 729 as a naturally occurring antibacterial system selected 5. Arnold, R. R., K. M. Pruitt, M. F. Cole, J. M. Adamson, and throughout the evolution of mammals to be a part of the J. R. McGhee. 1979. Salivary antibacterial mechanisms in im­ defense system against bacterial infections on mucosal munodeficiency. pp. 449-462. In I. Kleinberg, S. A. Ellison and I. D. Mandel (eds.) Saliva and dental caries. Sp. Supp. Micro­ surfaces in, for instance, the gastrointestinal tract. Fur­ biology Abstracts, Information Retrieval Inc., New York. thermore, it appears that the LP system offers protection 6. Aune, T. M., and E. L. Thomas. 1977. Accumulation of against toxic H202 evolved during the metabolism of tis­ hypothiocyanite ion during peroxidase-catalyzed oxidation of sues and commensal bacteria. thiocyanate ion. Eur. J. Biochem. 80:209-214. To preserve the quality of milk, the addition of small 7. Aune, T. M., and E. L. Thomas. 1978. Oxidation of protein sulfhydryls by products of peroxidase-catalyzed oxidation of amounts of SCN" (ca. 12 ppm) and H202 (ca. 8 ppm) thiocyanate ion. Biochemistry 17:1005-1010. has been suggested. This will result in an approximately 8. Ballantyne, B. 1977. Factors in the analysis of whole blood equimolar initial concentration (0.25 mM) of the two thiocyanate. Clin. Toxicol. 11:195-210. reactants in the milk (assuming an average natural SCN 9. Barker, M. H. 1936. The blood cyanates in the treatment of content of 0.05 mM). H202 itself at that low concentra­ hypertension. J. Am. Med. Assoc. 106:762-767. tion (50 to 100 times lower than in the "traditional" 10. Barylko-Pikielna, N., and R. M. Pangbom. 1968. Effect of method of using H 0 to preserve the quality of milk) cigarette-smoking on urinary and salivary . Arch. En­ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/9/724/1650811/0362-028x-47_9_724.pdf by guest on 29 September 2021 2 2 viron. Health 17:739-745. is unlikely to have any deleterious effect because it is 11. Bjorck, L. 1978. Antibacterial effect of the lactoperoxidase sys­ rapidly used up in the oxidation of SCN". The highly tem on psychrotrophic bacteria in milk. J. Dairy Res. 45:109-118. reactive intermediate oxidation products of SCN" are 12. Bjorck, L., and O. Claesson. 1980. Correlation between the con­ short-lived (particularly the higher oxyacids) and the centration of hypothiocyanate and antibacterial effect of the lac­ complete oxidation of SCN" results in innocuous end- toperoxidase system against Escherichia coli. J. Dairy Sci. 63:919-922. products. Any remaining unoxidized SCN" (or OSCN" 13. Bjorck, L., O. Claesson, and W. Schulthess. 1979. The lac- reduced to SCN") would not alter substantially the high toperoxidase/thiocyanate/hydrogen peroxide system as a temporary concentration of SCN" in the saliva or stomach in man preservative for raw milk in developing countries. Milchwis- (Table 1). Indeed, the consumption of milk with the senschaft 34:726-729. above addition of SCN" will actually dilute the concentra­ 14. Bjorck, L., C-G. Rosen, V. M. Marshall, and B. Reiter. 1975. Antibacterial activity of the lactoperoxidase system in milk against tion of SCN" in the gastric juice. Moreover, in medical pseudomonads and other gram-negative bacteria. Appl. Microbiol. studies on the consumption of milk with deliberately in­ 30:199-204. creased levels of SCN", no adverse effects were ob­ 15. Bbdigheimer, K., F. Nowa, and W. Schoenbom. 1979. Phar- served. makokinetik und Thyreotoxizitat des Nitroprussid-Natrium- Metaboliten Thiocyanat. Dtsch. Med. Wochenschr. 104:939-943. Finally the world-wide consumption of lactic acid cul­ 16. Boulange, M. 1959. Fluctuation saisonniere du taux des thiocyan­ tures in the form of fermented milks and starters used ates dans le lait frais de Vache. C. R. Seances Soc. Biol. in cheese-making should be considered. As long as milk 153:2019-2020. used in the manufacture of these fermented products is 17. Boyland, E., and S. A. Walker. 1974. Effect of thiocyanate on not heated to temperatures which would inactivate LP, nitrosation of amines. Nature 248:601-602. 18. Carlsson, J., M.-B. K. Edlund, and L. Hanstrom. 1984. Bacteric­ the indigenous SCN" will be oxidized by the H202 gener­ idal and cytotoxic effects of hypothiocyanite-hydrogen peroxide ated by the starter organisms and the above mentioned mixtures. Infect. Immun. 44:581-586. reaction products will be formed. Since these types of 19. Carlsson, J., Y. Iwami, and T. Yamada. 1983. Hydrogen dairy products have been consumed for thousands of peroxide excretion by oral streptococci and effect of lac- years, so often regarded to promote health, it would be toperoxidase-thiocyanate-hydrogen peroxide. Infect. Immun. surprising if the consumption of LP-treated milk would 40:70-80. 20. Collins, E. B., and K. Aramaki. 1980. Production of hydrogen turn out to have any harmful effect. peroxide by Lactobacillus acidophilus. J. Dairy Sci. 63:353-357. 21. Dahiya, R. S., and M. L. Speck. 1968. Hydrogen peroxide for­ ACKNOWLEDGMENTS mation by lactobacilli and its effect on aureus. J. Dairy Sci. 51:1568-1572. The assistance of Mrs. Gerlinde Mayntz is greatly appreciated. We 22. Dahlberg, P-A, A Bergmark, L. Bjorck, A. Bruce, L. Ham- also thank Profes sor K. M. Pruitt for helpful discussions. breaus, and O. Claesson. Intake of thiocyanate by way of milk and its possible effect on thyroid function. Am. J. Clin. Nutr. (in press). REFERENCES 23. Densen, P. M., B. Davidow, H. E. Bass, and E. W. Jones. 1967. A chemical test for smoking exposure. Arch. Environ. 1. Adamson, M., and J. Carlsson. 1982. Lactoperoxidase and Health 14:865-874. thiocyanate protect bacteria from hydrogen peroxide. Infect. 24. Ermans, A. M., F. Delange, M. van der Velden, and J. Kinth- Immun. 35:20-24. aert. 1972. Possible role of cyanide and thiocyanate in the etiol­ 2. Adamson, M., and K. M. Pruitt. 1981. Lactoperoxidase- ogy of endemic cretinism, pp. 455-486. In J. B. Stanbury and catalyzed inactivation of hexokinase. Biochim. Biophys. Acta R. L. Kroc (eds.) Human development and the thyroid gland. 658:238-247. Advances in Experimental Medicine and Biology Series, vol. 30. 3. Alexander, N. M., and B. J. Corcoran. 1962. The reversible dis­ Plenum Press, New York. sociation of thyroid iodide peroxidase into apoenzyme and 25. Fridovich, I. 1978. The biology of oxygen radicals. Science prosthetic group. J. Biol. Chem. 237:243-248. 201:875-880. 4. Anonymous. 1983. 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120. Wright, R. C, and J. Tramer. 1958. Factors influencing the activ- dar cheese. Milchwissenschaft 38:203-206. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/47/9/724/1650811/0362-028x-47_9_724.pdf by guest on 29 September 2021

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