Major antimicrobial proteins in milk
Written by MM
Milk provides the newborn (neonate) with nutrients and an array of antimicrobial factors. These are believed to help protect neonates from infection until their own immune system has developed.This section of the dairy science website reviews the properties and potential nutritional and industrial significance of the major antimicrobial systems of milk, with particular reference to the lactoperoxidase system.
The major antimicrobial proteins of milk are lysozyme, lactoferrin (Lf), lactoperoxidase (LP), and the immunoglobulins. These have been discussed in summary form in the section on Inhibitors in Milk. In some species minor proteins including a folate binding protein have been characterised. While these ‘minor’ factors are not discussed here they can be important in certain situations. Goat milk can have significant levels of folate binding protein and it is advisable to supplement goat milk with folic acid in addition to the normal ‘humanising’ compositional adjustments when used to feed babies.
Lysozyme (E.C. 18.104.22.168) is a relatively small basic protein and is classified as a 1,4-ß -N-acetylmuramidase.
Lysozyme is present in secretions such as saliva, egg white, milk and blood. Egg white lysozyme and human milk lysozyme are similar proteins (Dubois et al., 1982). Lysozyme cleaves the glycosidic bond between N-acetylmuramic acid and N-acetyglucosamine in bacterial peptidoglycans, which constitute the major part of the bacterial cell wall of gram-positive bacteria.
The susceptibility of different bacteria to lysozyme depends on a number of factors including the accessibility of the substrate and the ionic environment. Gram-positive bacteria are generally more susceptible because they have a much simpler cell wall consisting to a major extent (90%) of peptidoglycan. Staphylococci, on the other hand, are more resistant. This is probably due to the presence of teichoic acid in their cell walls. In gram-negative bacteria the peptidoglycan concentration in the cell wall is much lower (5-10%) and it is protected by an outer layer of lipopolysaccharide, which prevents lysozyme from reaching its substrate. If this barrier is disrupted, which in vitro can be achieved by various chemical and physical treatments, many gram-negative bacteria become sensitive to lysozyme.
Because egg-white lysozyme will lyse Clostridium tyrobutyricum, lysozyme is used to prevent blowing in cheeses of low lactic acid content e.g. brined-cheeses such as Gouda and Grana Padano.
Lysozymes are generally heat-stable in an acid medium. Bovine milk lysozyme losses only 43% of its activity after heating for 20 min at 100°C at pH 4.0 (Eitenmiller et al., 1975). Human milk lysozyme, under the same conditions, has been reported to loose 85% of its activity (Parry et al., 1969). At an alkaline pH, i.e. pH 9.0, bovine milk lysozyme is inactivated completely within a few minutes at 100°C.
The biological role of lysozyme is still not understood completely. Lysozyme may have an antibacterial role in milk, serum and avian eggs.
While bovine milk normally contains very low levels of lysozyme, i.e. 0.1 µg/ml, mastitic milk, however, contains higher concentrations (1-2 µg/ml). Human milk is much richer in lysozyme and contains on average 100 µg/ml.
It has also been suggested that lysozyme may have an indirect effect on the defence systems of hosts as an immunomodulator through the stimulation of the immune system by break down products from the hydrolysis of peptidoglycan (Jolles, 1976). This hypothesis has been supported by the findings that feeding infants lysozyme-enriched formulas results in an increased level of secretory IgA in faeces (Lodinové and Jouja, 1977).
Lysozyme may also be responsible for some false positive results for antibiotics in milk. However, such milk is likely to be of high somatic cell count.
There are several closely related iron binding proteins in animals and biological secretions. An iron chelating protein, Lf occurs in many exocrine secretions of mammals. It is closely related to transferrin, a soluble glycoprotein that binds two iron ions (III) per molecule. Lf is also related to an iron chelating protein found in hen egg white, ovotransferrin or conalbumin.
All three iron-containing proteins have similar structures and biological properties. They consist of a single polypeptide chain of molecular weight 80,000 daltons to which one or two carbohydrate chains are attached. The molecules have two symmetrical parts, which each has one site for binding of a ferric iron ion. For each iron ion that is bound one bicarbonate ion is also bound. Lf has been found in milk of some 12 species (Masson, 1970). In humans it also has been shown to be present in most exocrine secretions, including bronchial mucus, tears, saliva, nasal secretions, cervical mucus and gastric juice (Masson, 1970).
In human milk Lf is one of the major whey proteins, constituting 10-30% of the total protein content. Nagasawa et al. (1972) have reported levels of Lf in human colostrums and milk of 4.9 and 1.6 mg/ml respectively. In bovine milk the levels are substantially lower, i.e. about 1 mg/ml and 0.2 mg/ml in colostrums and milk, respectively. However, the concentration of lactoferrin in bovine milk increases significantly with inflammatory conditions of the mammary glands, e.g. mastitis.
In the native state Lf in only partially saturated with iron. In human milk, which contains 0.3 to 0.5 µg/ml Fe, only 2-4% of this is bound to Lf. A major part of the Fe in milk is found in the fat phase, mainly as a component of xanthine oxidase. The rest is present in the form of low molecular complexes.
A number of investigations have been carried out to elucidate the biological role of Lf. Although a number of activities have been demonstrated in vitro, the function of Lf in vivo is not well understood. However, two main roles for LF in vivo have been postulated, its antibacterial effect and role in the iron metabolism.
The antibacterial effect of Lf in vitro is well established (Bullen & Armstrong, 1979). The antibacterial mechanism has been assigned to the ability of capacity to chelate iron and thereby depriving bacteria of the iron that is essential for growth. This is supported by the fact that Lf saturated with iron does not demonstrate an antibacterial effect. To what extent the antibacterial effect of Lf also is active in vivo is less clear. While there is limited evidence of antibacterial effect in humans there is indirect evidence, which suggests that Lf has an important role in vivo. Iron is an essential nutrient for both mammalian cells as well as many bacteria. It has been postulated that Lf supplies a source of necessary iron that can be utilized by the host but is not available for bacteria. Growth of the latter are therefore inhibited due to lack of essential iron. However, some bacteria have evolved mechanisms for obtaining iron that has been ‘bound’ in this way.
Lf may also be responsible for some false positive results for antibiotics in milk. However, such milk is likely to be of high somatic cell count.
LP the protein has been discussed previously in the introductory section on inhibitors in milk.
LP (EC 22.214.171.124) is a basic (it is negatively charged below its isoelectric point) glycoprotein that has a molecular weight of 78,000 and contains one heme group. The enzyme has an iron content between 0.068-0.071% and a carbohydrate content ranging from 9.9 - 10.2% (Carlström, 1969). The identity and type of binding of the heme moiety has been investigated. Initially it was proposed that the heme group was covalently bound to the protein moiety via an ester or amide bond (Hultqvist & Morrison, 1963). Slevers (1979) has however shown that no covalent heme-protein bond exists and that the prosthetic group is protoheme IX. Peroxidases catalyse reactions in which hydrogen peroxide is reduced and an appropriate electron donor is subsequently oxidised. A wide variety of organic and inorganic substances can serve as electron donors, but substrate specificity varies between various peroxidases.
LP itself has no antibacterial effect but in combination with certain co-factors, thiocyanate (SCN- ) and hydrogen peroxide (H2O2) ,forms a potent antimicrobial system. The mechanism of this inhibition is largely understood as a result of studies of the inhibition of acid production by lactococci in cheese manufacture.
The antibacterial mechanism is caused by oxidation of vital SH-groups by OSCN-/ O2SCN- (Thomas & Aune, 1978) in vital metabolic enzymes, e.g. hexokinase and/or depletion of reduced nicotinamide adenine nucleotides. A wide variety of bacteria are influenced by the LP-system. With some bacteria the effect is reversible, i.e. the bacteria are inhibited for a certain period of time, for other bacteria the effect is bactericidal. Many Gram-positive bacteria such as lactococci and lactobacilli are inhibited while many gram-negative bacteria such as Escherichia coli, Pseudomonas spp, Salmonella spp are killed (Reiter et al., 1976, Björck et al., 1975).
Mammalian cells do not appear to be adversely affected by the LP-system. The LP-system did not have any mutagenic effects when tested against several mutagen-sensitive strains of Salmonella typhimurium (White et al., 1983).
The activity of the LP system in vivo is not fully known. In bovine milk, LP is always present in excess (Björck et al., 1975). Buffalo's milk (Härnulv & Kandasamy, 1982), ewe's milk (Medina et al., (1989) and goat's milk (Zapico et al., 1990) generally also contain high levels of LP. The factors limiting the activity of the LP system in milk from these species are the low concentrations of thiocyanate and hydrogen peroxide present. Some thiocyanate is normally present in milk although the level is variable, i.e. between 1 to 15 mg/l. There is some evidence of in vivo production of hydrogen peroxide by lactic acid bacteria (Marschall, 1983; Mullan, Extrand and Waterhouse, unpublished information).
Most investigations of the antibacterial activity of the LP system have been carried out in vitro. In these experiments LP, thiocyanate and hydrogen peroxide are added to a suitable medium. In milk, the system can be "activated" by addition of thiocyanate and hydrogen peroxide or an appropriate hydrogen peroxide source. There are several methods for determining the concentration and antimicrobial activity of the LP system.
How to cite this article
Mullan, W.M.A. (2003) .
[On-line]. Available from: https://www.dairyscience.info/index.php/exploitation-of-anti-microbial-proteins/52-antimicrobial-proteins.html . Accessed: 26 May, 2019.