**2. Milk indigenous enzymes**

Numerous enzymes have been indigenously identified in milk from 1924 to 1970 [7]. A large number of enzymes with multiple functionalities are present in milk. Additional enzymes contribute in quality of milk products and also perform an antibacterial action (LP). In bovine milk more than 70 enzymes are detected [8, 9]. A 50–60 substantial number of milk enzymes with multiple functions are present in abundance in milk and are concerned with processing stability and general customer safety [10] and additionally processing suitability (ALP). Some enzymes (LP) having antibacterial characteristics are with significant importance in preservation of milk and milk products and some, e.g., plasmin (PL) and lipoprotein lipase (LPL) connected with the serum, plasma, fat globules, casein, or leukocytes are important in maintaining of quality of milk and milk products. More than 40 enzymes have been recognized in cow milk [9, 11].

Lactation period in animals involves colostrum, developed milk, peak, and production with compositional variations. Numerous indigenous enzymes present in milk are secreted by epithelial cells and their composition changes with the lactation stages.

In already recognized indigenous enzymes in milk [7], almost 20 enzymes have been well characterized and the rest of the 40 enzymes are of little significance but can be identified through their activity. These enzymes indicate the efficient process of milk pasteurization (ALP, γ-glutamyl transferase GGT) or of mastitis (phosphatases, CAT). Additional enzymes can be of significance in processing and ultimately providing safety to human beings. They play an antibacterial activity (LP) and contribute quality to milk products (e.g., LPL, PL) associated with the serum, plasma, fat globule, casein, or leukocytes.

## **2.1. Lipases and esterases**

Lipolytic enzymes have capability to hydrolyze triacylglycerols are considered as carboxylesterases [12, 13]. Those enzymes that can hydrolyze acyl glycerol having <10 carbon atom fatty acids are known as esterases or carboxylases (Enzyme Commission, EC 3.1.1.1) while those can hydrolyze ≥10 carbon atom fatty acids are considered as lipases, or triacylglycerol acyl hydrolases (EC 3.1.1.3) [14, 15].

Esterases are different from lipases due to their functions for being relatively soluble compared to emulsified ester substrates. Several esterases are present in milk [15, 16], the most prominent are carboxylesterase (EC 3.1.1.1), acetylcholinesterase (EC 3.1.1.7), and cholinesterase (EC 3.1.1.8). In bovine colostrum, lipase is not connected with casein and not activated by blood serum, therefore exhibited low lipase activity and showed slight lipolysis in early lactation. However, after few days of calving, normal milk from early lactation exhibited higher lipase activity [17, 18].

Lipases are naturally a critical group of enzymes since they are connected with the fat digestion system. Lipases are more dynamic at pH 8–9 and catalyze the advancement of hydrolytic rancidity in milk. Investigation of lipases is more alluring in the light of the fact that it would add to our comprehension about the properties and modes of these enzymes [19, 20].

The phenomenon of lipolysis is correlated with the lactation days. Higher activity is associated with its presence in fat fraction of milk. Activity of lipase in milk fat increases with the advancement in lactation stages [21]. The lipolysis process is of major apprehension in the dairy industry, as rancid off flavors are produced in milk and milk products during this phenomenon [22].

Earlier research has well established that milk lipase is sensitive to heavy metals. Copper, cobalt, and nickel have been shown to be more powerful inhibitors of lipase than iron, chromium, manganese, and silver. Enzyme activity is stimulated by blood serum albumin, ammonium, calcium ions, and mercaptoethanol. The buffer solutions, citrate, acetate, and phosphate buffers damage the enzyme activity, whereas borate and barbiturate buffers do not [23, 24].

LPL in cow milk is altered due to the breed, lactation phase, feed and fodder, season, and milk yield [22, 25]. Lipase activity increased from 0.32 to 2.98 U/mL of milk. At the point when milk fat globule membrane (MFGM) is damaged, lipolysis takes place rapidly and leads to hydrolytic rancidity and ultimately may cause variations in functionality and flavor of dairy products throughout storage period [15]. LPL found in goat milk is of low concentration in the early and late lactation stage [26].

The membrane lipase is available in higher concentration in milk from dairy animals in late lactation [27]. They additionally reported that lipase action in milk showed inclined pattern with reference to lactation stages. Hameed et al. [28] reported the expanding pattern of lipase activity with lactation stages in bovine milk. Lipase action (1.55 U/mL) was recorded higher (*p* < 0.01) in milk, examined at the last of lactation, followed by other lactations (1.29 and 1.16 U/mL, respectively).
