Lipase heat inactivation

In addition to having the required spedfidty, lipases employed as catalysts for modification of triglycerides must be stable and active under the reaction conditions used. Lipases are usually attached to supports (ie they are immobilised). Catalyst activity and stability depend, therefore, not only on the lipase, but also the support used for its immobilisation. Interesterification reactions are generally run at temperatures up to 70°C with low water availability. Fortunately many immobilised lipases are active and resistant to heat inactivation under conditions of low water availability, but they can be susceptible to inactivation by minor components in oils and fats. If possible, lipases resistant to this type of poisoning should be selected for commercial operations. 

Thermal Inhibition, Heat treatment of milk is the most important practical means of inactivating its lipases. The temperature-time relationship necessary for partial or complete inactivation has been extensively studied, but a number of discrepancies have been apparent. These are probably due to several factors, including the sensitivity of the assay procedure, the length of the incubation period following heating, the presence and concentration of fat and solids-not-fat in the milk at the time of heating, and the type and condition of the substrate. In view of these variables, references to a number of early studies on heat inactivation have been omitted. 

Fat apparently protects the lipases to some extent from heat inactivation, 1° to 2°C higher temperatures being necessary for whole milk than for skim milk (Frankel and Tarassuk 1959 Harper and Gould 1959 Nilsson and Willart, 1960 Saito et al 1970). 

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. 

Owusu, R.K., Makhzoum, A., Knapp, J.S. 1992, Heat inactivation of lipase from psychrotrophic Pseudomonas fluorescens P38 Activation parameters and enzyme stability at low or ultra high temperature. Food Chem. 44, 261-268. 

Swaisgood, H.E., Bozoglu, F. 1984. Heat inactivation of the extracellular lipase from Pseudomonas fluorescens MC50. J. Agric. Food Chem. 32, 7—10. 

Wet heating is more effective for bran stabilization for oil extraction than is dry heating. Lipase is inactivated in 3 minutes at 100°C (37). The equipment that can be used include steam cookers, blanchers, autoclaves, and screw extruders with injected steam and water (30). Extrusion with steam injection and up to 10% added water reduces the temperature required for lipase inactivation. Temperatures are reduced to 100-120°C. Product may be held at 100°C for 1.5-3.0 minutes before drying to a stable moisture content. Bran expands as it exits the extruder, and water flashes to steam (8). Porous pellets assist in solvent percolation during oil extraction. Fines are agglomerated as well. 

A major problem with rice bran oU extraction is the high lipase activity, which results in FFA formation within a few days of milling particularly at high temperature and humidity. Free fatty acid content in rice bran increases during storage, i.e., 2-A% in a fresh crop, 5-8% in 1-year grain, and >10% in a 2-year-old crop (105). Thus, lipase is inactivated to stabihze rice bran prior to oil extraction (88). Heat-stabilized bran may be stored up to three months. However, oil extraction should be carried out within the first month to obtain better efficiency and higher quality oil. 

In our study, we conducted the enzyme-catalyzed methanolysis of rapeseed oil using Novozym 435, a well-known nonspecific lipase. Novozym 435 facilitates reactions between a wide variety of alcohols and is also a remarkably heat-tolerant enzyme [6, 8], Watanabe et al. [9] previously reported that immobilized Candida antarctica lipase was inactivated in the presence of more than half the stoichiometric amount of methanol against total fatty acids in the oil. This disadvantage was surmounted by the utilization of three-step methanolysis, in which only one third of the total amount of methanol was added in each stage [7, 9]. 

Driessen, F. M. (1989). Heat inactivation of lipases and proteinases (indigenous and bacterial). In Heat-Induced Changes in Milk, Bull. No. 238, pp. 71-93. Int. Dairy Fed., Brussels. 

Melt anhydrous milkfat or butter, and add water and emulsifiers Add lipase enzyme and react for 24 h or more at 30°C or above and then heat inactivate added enzyme Further process to obtain desired consistency and/or composition... 

Table 3.23. Heat inactivation of a lipase of Pseudomonas fluorescence dissolved in skim milk...

The release of blue dextran from the microspheres was studied in 25 mM buffer phosphate solution (pH 7.5) incubated in a rotatory shaker at 200 rpm and 37 C. Biocatalytic release of azo-BSA and sulfanilic acid from the microspheres was performed with subtilisin or lipase in 25 mM Tris-CIH buffer solution (pH=7.8) incubated at 200 rpm and 37°C. Azo-BSA and sulfanilic acid were determined spectrophotometrically at 334 nm. The presence of sulfanilic acid in the supernatant was assayed after precipitation of azo-BSA with 5% TCA for 15 minutes at 0°C, followed by centrifugation (10,000 xg, 20 minutes at 4°C). Controls without enzymes, and with protease previously inhibited with 1.0 mM diisopropyl fluorophosphate, or thermal inactivate lipase (heated at 100 C) were included. Chemical release of BSA from the microspheres was performed by incubating the microspheres in a buffer phosphate (100 mM, pH=7.4) until total microsphere disintegration. For CD analysis of BSA, samples were filtered through 100 kDa. MWCO devices (Centricon, Millipore, Billerica, MA, USA). 

To increase the stability of milk products. Lipoprotein lipase is probably the most important in this regard as its activity leads to hydrolytic rancidity. It is extensively inactivated by HTST pasteurization but heating at 78°C x 10 s is required to prevent lipolysis. Plasmin activity is actually increased by HTST pasteurization due to inactivation of inhibitors of plasmin and/or of plasminogen activators. 

The data of Nilsson and Willart (1960) indicate that heating at 80°C for 20 sec is sufficient to destroy all lipases in normal milk. Their studies included assays after 48 hr of incubation following heat treatment. At lower temperatures for 20 sec, some lipolysis was detected after the 48-hr incubation period after heating. Thus, 10% residual activity remained at 73 °C. Below the temperature of 68°C the amount of residual activity was enough to render the milk rancid in 3 hr temperatures below 60 °C had no appreciable effect on lipolysis. With holding times of 30 min, 40°C produced only slight inactivation, and at 55°C 80% inactivation was reported. 

Hetrick, J. H. and Tracy, P. H. 1948. Effect of high-temperature short-time heat treatment on some properties of milk. II. Inactivation of the lipase enzyme. J. Dairy Sci. 31, 881-887. 

Rancidity, due to free volatile latty acids liberated froni the glycerides by enzymic (lipasei hydrolysis. Lipases are normal components of raw milk, and are inactivated by the heat of pasteurization. 

Two types of enzymes in milk are important those useful as an index ol heat treatment and those responsible tor bad flavors. Phosphatase is destroyed by the heat treatments used to pasteurize milk hence its inactivation is an indication of adequate pasteurization. Lipase catalyzes the hydrolysis of milk fat which produces rancid flavors. It must be inactivated by pasteurization or more severe heat treatment to safeguard the product against off-flavor development Other enzymes reported to have been found in milk include catalase, peroxidase, protease, diastase, amylase, oleinase. reductase, aldehydrase. and lactase. 

Abrasive milling removes the outer bran layer to produce partially polished rice or, after polishing to remove the entire bran layer, white rice. Rice bran or polish may be subsequently stabilized by heat treatment to inactivate lipases. Stabilized rice bran has found use as an ingredient in human-grade processed foods. 

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. 

Krukovsky, V.N., Herrington, B.L. 1942a. Studies of lipase action. IV. The inactivation of milk lipase by heat.. / Dairy Sci. 25, 231-236. 

Vercet, A., Lopez, P., Burgos, J. 1997. Inactivation of heat-resistant lipase and protease from Pseudomonas fluorescens by manothermosonication. J. Dairy Sci. 80, 29-36. 

The less severe heat treatment of modern milk powder production can lead to problems since enzymes present in the milk are not inactivated. The enzyme in milk products, particularly milk powder, that causes problems is lipase. It should be appreciated that this is not the native... 

Lipase has a molecular weight of about 40,000 Da and an isoelectric point (pi) of 8.56 (32). It is activated by calcium and inhibited by heavy metals. The optimum pH is 7.5-8.0, and the optimum temperature is 37°C. It is inactivated by heating at 60°C for 15 minutes. Rice bran lipase preferentially hydrolyzes fatty acids from the... 

Extrusion (dry heat) cookers have been ideal for stabihzation because excess moisture is not added, eliminating the need for drying. The heating of the bran occurs through conversion of mechanical energy of the screw drive to heat the bran. Temperatures used for stabilization vary from 100° to 140°C. The bran is kept hot for 3-5 minutes after extrusion to ensure lipase inactivation. The hot bran is then cooled using ambient air. 

Parboiling of rice is also an example of wet heat stabilization. The lipase in rough rice is completely inactivated by either autoclaving for 3-20 minutes or by parboiling. 

Ideally, bran should be stabilized within a few minutes after removal from the kernel. Stabilization process inactivates enzyme lipase that causes rapid hydrolysis of TAG. Three methods developed for brown rice stabilization are (1) heat dena-turation and inactivation of lipases, (2) extraction with an organic solvent to remove... 

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