Milk Lipase

Early research on lipolytic enzymes in cows milk suggested that at least two major lipases were present a plasma lipase in the skim portion and a membrane lipase associated with the milk fat globule membrane (Tarassuk and Frankel, 1957) while later research indicated that there might be up to six different molecular species with lipase activity (Downey and Andrews, 1969). However, work by Korn (1962) showed that milk contained a lipoprotein lipase (EC 3.1.1.34) (LPL) with properties very similar to those of post-heparin plasma, adipose tissue and heart LPLs, particularly the enhancement of its activity on emulsified triglycerides by blood serum lipoproteins. It is now accepted that LPL is the major, if not the only, lipase in cows milk. Its properties have been reviewed by Olivecrona et al. (2003). 

LPL is synthesized in the mammary gland secretory cells and most is transported to the capillary endothelium where it hydrolyzes triglycerides in circulating lipoproteins to FFAs and 2-monoglycerides. These products are absorbed by the mammary gland and used for the synthesis of milk fat. The LPL in milk appears to be identical with the enzyme in the mammary gland (Askew et al., 1970) and to be the result of a spillover. Its level in milk is low at parturition but increases rapidly during the first few days of lactation and remains almost constant for the remainder of the lactation (Saito and Kim, 1995). 

LPL is a glycoprotein (8% by weight carbohydrate) with a native molecular weight of around 100 000 Da and a monomer subunit of about 50 000 Da (Kinnunen et al., 1976). Senda et al. (1987) calculated the molecular weight of the unglycosylated form as 50 548 Da based on the cDNA encoding it. LPL has a serine at the active site, located in a beta turn in the enzyme, similar to that at the active site of other serine hydrolases (Reddy et al., 1986). 

High-temperature short-time (HTST) treatment (72°C x 15 s) of milk almost completely inactivates the enzyme (Luhtala and Antila, 1968 Andrews et al., 1987 Farkye et al., 1995) so that little if any lipolysis caused by milk lipase occurs in pasteurised milk (Downey, 1974). Somewhat higher temperatures are required for cream pasteurization because of the protective effect of the fat (Nilsson and Willart, 1961 Downey and Andrews, 1966). However, some workers have reported that a more severe heat treatment, [e.g., 79°C x 20 s, (Shipe and Senyk, 1981) or 85°C x 10 s (Driessen, 1987)] is required to inactivate completely milk lipase. 

Phospholipids also have a role in the LPL-catalyzed hydrolysis of triglycerides. The activator apo-LPs exhibit enhanced activation in the presence of phospholipids such as phosphatidyl choline (La Rosa et al., 1970 Blaton et al., 1974) and in milk there is evidence that apo-LPs in the absence of phospholipids are unable to initiate lipolysis of intact milk fat globules by the indigenous LPL (Driessen and Stadhouders, 1974 Clegg, 1980). The phospholipids are believed to be involved in the reaction through their interaction with the substrate rather than with the enzyme (Blaton et al., 1974). 

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Consumer acceptance of milk is strongly determined by its sensory characteristics. The development of off-flavor in milk as a result of lipolysis can reduce the quality of milk. The enzymatic release, by milk lipase, of free fatty acids (FFA) from triglycerides causes a flavor defect in milk described as rancid . Triglycerides in milk contain both long chain and short chain fatty acids, which are released at random by milk lipase. The short chains FFA, like butyric acid, are responsible for the off-flavor. 

Olivecrona, T., Vilaro, S. and Bengtsson-Olivecrona, G. (1992) Indigenous enzymes in milk Lipases, in Advanced Dairy Chemistry, Vol. 1 Proteins, 2nd edn (ed. P.F. Fox), Elsevier Applied Science. London, pp. 292-310. 

Lipolysis. Some lipolysis occurs in all cheeses the resulting fatty acids contribute to cheese flavour. In most varieties, lipolysis is rather limited (Table 10.5) and is caused mainly by the limited lipolytic activity of the starter and non-starter lactic acid bacteria, perhaps with a contribution from indigenous milk lipase, especially in cheese made from raw milk. 

Blue cheeses undergo very extensive lipolysis during ripening up to 25% of all fatty acids may be released. The principal lipase in Blue cheese is that produced by Penicillium roqueforti, with minor contributions from indigenous milk lipase and the lipases of starter and non-starter lactic acid bacteria. The free fatty acids contribute directly to the flavour of Blue cheeses but, more importantly, they undergo partial /J-oxidation to alkan-2-ones (methyl O... 

The literature on the subject is quite large. The present review has been limited to milk lipases, but good reviews on this, other dairy products, milk esterases, and microorganisms are available (International Dairy Federation 1974, 1975, 1980 Shipe et al 1978 Deeth and FitzGerald 1976 Downey 1980A Jensen and Pitas 1976 Shahani et al 1980 Lawrence 1967 Kitchen 1971). 

Enzymes are produced and elaborated by living cells—a fact that has prompted some investigations into the origin of milk lipases. It is only relatively recently that the synthesis of glycerides by milk lipases has been demonstrated (Koskinen et al. 1969 Luhtala 1969 Luhtala... 

Bovine blood serum is lipolytically active, but cows producing milk which goes rancid quickly do not have sera that are more lipolytically active than those producing normal milk. Leukocytes, which are present in large numbers in milk, are especially high in mastitic milk they are the source of milk catalase but are apparently not the source of milk lipases (Nelson and Jezeski 1955). 

The effect of the estrous period on rancidity has also been investigated. According to Wells et al. (1969), who studied lipase activity in the milk and blood of cows throughout their lactation period, the peak blood plasma lipase values occur about 24 hr before the onset of observed estrous. Changes in blood lipase activity were reflected and magnified in the milk, although it was noted that the increase in milk lipase level occurred 9 to 15 hr after it was observed in the blood. Bachmann (1961) also has indicated that hormonal disturbances are linked to rancidity. He differentiates between rancidity produced by cows in late lactation and rancidity due to hormonal disturbances on the basis of an increased in lipase concentration in the latter. 

The binding of milk lipases to casein micelles apparently imparts some stability to the enzyme, for as purification progresses, the milk lipase becomes less stable, and more so as the concentration of casein decreases (Downey and Andrews 1966 Egelrud and Olivecrona 1972). 

Human Milk Lipases. Two lipases have been identified in human milk by Hernell and Olivecrona (1974A,B). One of these, lipoprotein... 

Downey and Andrews (1966) experiments indicate that there is a bivalent cation requirement for full milk lipase activity. Dunkley and Smith (1951) had previously stated that small amounts of CaCl2 accelerate lipolysis. These observations are in keeping with those made on lipases from other sources where Ca2+ was found to stimulate activity (Wills 1965 Egelrud and Olivecrona (1973). 

The milk lipase system is reported to be activated by mercuric chloride. Raw milk preserved with corrosive sublimate sometimes contains a much larger concentration of free fatty acids that do unpreserved samples. Pasteurized milk preserved in a similar fashion does not show an increase in free fatty acids (Manus and Bendixen 1956). 

Irradiation by ionizing radiation and its effect on milk lipase activity have also been studied (Tsugo and Hayashi 1962). Irradiation doses of 6.6 x 104 rads destroyed 70% of the activity. The udders of lactating cows, when exposed to 60 Co gamma rays, gave milk with decreased lipase and esterase activity (Luick and Mazrimas 1966). 

Heavy metals usually affect enzymes adversely, and milk lipases are... 

Other chemicals which inhibit milk lipase include hydrogen peroxide, animal cephalin, sodium arsenite, diisopropyl fluorophosphate, 2,4 din-itro-l-fluorobenzene, p-hydroxymercuribenzoate, potassium dichromate, lauryl dimethyl benzyl ammonium chloride, aureomycin, penicillin, streptomycin, and terramycin (Schwartz 1974). 

An extensive study of the effects of formaldehyde in milk lipase inhibition showed that formaldehyde acts as a competitive inhibitor and, under the proper conditions, selectively inhibits the lipases of raw skim milk (Schwartz et al. 1956A). This study showed that the inhibitory effect of formaldehyde was dependent on such factors as pH, time of addition of the inhibitor, length of the incubation period, concentration... 

The incubation of raw skim milk at pH 6.0 and at pH 8.9 for 1 hr at 37 °C in the absence of substrate was subsequently shown to cause a 47% and 40% decrease, respectively, in lipase activity when the milk was later incubated with milk fat. When tributyrin was the substrate the inhibition was even more marked. Although some of the inactivation was due to temperature, the majority of it was attributable to pH exposure. Stadhouders and Mulder (1964) have also demonstrated that milk lipase subjected to incubation at pH 5.0 is almost completely destroyed. 

The point on the acid side of the pH curve where milk lipase activity ceases is of considerable practical importance, but there is still controversy regarding it. Willart and Sjostrom (1962) found that milk lipase is active in the range pH 4.1 to 5.7, whereas Schwartz et al. (1956B) could detect no activity at pH 5.2 on butterfat. Although Peterson et al. (1948) found no milk lipase activity on tributyrin at pH 7.0, activity was reported on this substrate at pH 5.0 and even at pH 4.7 when 24-hr incubation periods were used (Stadhouders and Mulder 1964). 

Stability. Some discussion regarding stability of milk lipases was presented in the preceding section. Egelrud and Olivecrona (1973) found that the enzyme fractions from heparin-Sepharose can be stored frozen at -20°C with less than 10% loss of activity in 2 weeks. The purified enzyme had only moderate stability at 4°C high concentrations of salt or a pH below 6.5 or above 8.5 increases the rate of inactivation. 

Titration. Titration of the fatty acids formed by the action of the milk lipase system has been the most widely used procedure. Titration has... 

Radioactive Substrates. Koskinen et al (1969), Luhtala et al. (1970-A,B), and Scott (1965) have used labeled triglycerides as substrates for milk lipases. This method, which is extremely sensitive, requires that the acids released by lipase action be isolated uncontaminated with any tagged glycerides. It also requires the preparation of labeled substrate and, of course, counting equipment. 

Clegg, R. A. 1980. Activation of milk lipase by serum proteins Possible role in the occurrence of lipolysis in raw bovine milk. J. Dairy Res. 47, 61-70. 

Deeth, H. C. and Fitz-Gerald, C. H. 1977. Some factors involved in milk lipase activation by agitation. J. Dairy Res. 44, 569-583. 

Fitz-Gerald, C. H. 1974. Milk lipase activation by agitation—influence of temperature. Aust. J. Dairy Technol. 29, 28-32. 

Frankel, E. N. and Tarassuk, N. P. 1956A. The specificity of milk lipase. I. Determination of the lipolytic activity in milk toward milk fat and simpler esters. J. Dairy Sci. 39, 1506-1516. 

Harper, W. J. and Gould, I. A. 1959. Some factors affecting the heat-inactivation of the milk lipase enzyme system. 15th Int. Dairy Congr. Proc. 6, 455-462. 

Harper, W. J., Gould, I. A. and Badami, M. 1956A. Separation of the major components of the milk lipase system by supercentrifugation. J. Dairy Sci. 39, 910. 

Hyasawa, H., Kiyosawa, I. and Nagasawa, T. 1974. Some observations on human milk lipase. Proc. XIXth Int. Dairy Congr. IE, 559. 

Hernell, O. 1975. Human milk lipases. III. Physiological implications of the bile-salt stimulated lipase. Eur. J. Clin. Invest. 5, 267. 

Hernell, O. and Olivecrona, T. 1974A. Human milk lipases. I. Serum-stimulated lipase. J. Lipid Res. 15, 367-374. 

Luick, J. R. and Mazrimas, J, A. 1966. Biological effects of ionizing radiation on milk synthesis. III. Effects on milk lipase, esterase, alkaline phosphatase, and lactoper-oxidase activities. J. Dairy Sci. 49, 1500-1504. 

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