724 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 thiocyanate (SCN~) and hydrogen peroxide (H202) as well as cal applications of the LP system. The effect against oral the catalytic enzyme 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 enzymes. 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 peroxidase 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, saliva 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 Oxygen 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, catalase and peroxidases. ing the first week after delivery (26,35,76,121). Contrary The first enzyme converts the superoxide radical 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 catalysis 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 myeloperoxidase derived from milk leuco­ extensively studied since the beginning of enzymology. cytes (58). In addition to SCN", bromide and iodide, 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), eosinophil peroxidase (115), is 22 units/ml which was observed in guinea pigs (97). thyroid peroxidase (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.