The limited lipolysis that occurs during the ripening of Cheddar cheese manufactured from pasteurized milk is probably effected by the lipolytic enzymes of the starter lactococci and nonstarter lactobacilli, although LAB are generally only weakly lipolytic. Screening studies using natural substrates, triglycerides, and synthetic chromogenic substrates (58) have confirmed the presence of lipase (121-123) and esterase activities in mesophilic (67,123-125) and thermophilic (126,127) dairy lactobacilli. In general the enzymes were located intracellularly, and activities were strain specific but tended to be higher in thermophilic than mesophilic strains. In the majority of strains examined, activities increased as the carbon chain length of the fatty acid decreased. The only lipase purified is a 65 kDa molecular mass intracellular enzyme produced by Lb. plantarum (128). The enzyme hydrolyzed the triglycerides trilaurin and tripalmitin but was most active against tributyrin. The purified enzyme expressed optimal activity at pH 7.5 and 35°C but retained 45% of its maximum activity at pH 5 and 15°C.
LAB esterases have also been purified and characterized. The serine tributyrin esterase of both Lc. lactis subspecies has a subunit molecular mass of 29 kDa and optimal activity at pH 7-8 (129,130). The enzyme was active on chromogenic ester substrates having a fatty acid chain length of 2-12 carbon atoms. The gene encoding the major intracellular tributyrin esterase of Lc. lactis has been cloned and characterized (131). Intracellular esterases formed by Lb. plantarum (132,133), Lb. casei (134), Lb. fermentum (135), and Lb. helveticus (136) have also been purified and characterized. The estimated molecular masses ranged from 70 to 105 kDa (subunit mass 25-40 kDa), but many of the enzyme properties were similar. They are all serine-dependent enzymes that are inhibited by PMSF. Activities were maximal with butyrate ester substrates at a temperature of 34-40 °C and at a pH of 7-8. Reduced activity was retained at pH5, at lower ripening temperatures, and in the presence of NaCl. The response of esterase activity to the effects of salt, temperature, and pH is strain-dependent (137), but retention of this activity during ripening may have important consequences for flavor as a result of both lipolysis and ester formation.
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