**2.2. Plasmin**

milk properties and some of the enzyme activities [5, 6]. Solids-non-fat (SNF) content is

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

Lactation period in animals involves colostrum, developed milk, peak, and production with compositional variations. Numerous indigenous enzymes present in milk are secreted by

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

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

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

epithelial cells and their composition changes with the lactation stages.

with the serum, plasma, fat globule, casein, or leukocytes.

frequently highest throughout first 2–3 weeks of lactation.

**2. Milk indigenous enzymes**

in cow milk [9, 11].

92 Livestock Science

**2.1. Lipases and esterases**

hydrolases (EC 3.1.1.3) [14, 15].

activity [17, 18].

Plasmin (PL; EC 3.4.21.7) is an alkaline serine proteinase enzyme that proteolytically cleaves the blood clots [29]. This enzyme has affinity toward arginine (Arg) and lysine (Lys) residues, specifically breaks the Arg-X and Lys-X bonds [30, 31]. The activity of enzyme is increased with the multiple factors that include lactation stage, lactation number and severity of mastitis infection [32–34].

On the basis of origin, the PL and plasminogen (PLG) are considered to be migrated from blood to milk, and higher activity of PL in peak lactation designating more conversion of PLG into PL in bovine milk [35–37].

PL is basically released in the form of PLG in normal milk. The concentration of PLG (0.8–2.8 μg/mL) in fresh milk is varied and its concentration is 2–30 times higher than that of PL (0.1– 0.7 μg/mL). It is activated by storage or when milk is stayed in the lumen of mammary glands before milking. A considerable interest has been involved in the activation of PLG, upon which activity of PL depends [38–41]. It promptly hydrolyzes the bonding of *β*-casein, *α*s2-casein, and *α*s1-casein and affects the quality of dairy products [32, 42].

Advancing lactation stage is an essential factor that influences PL activity and percentage, suggesting that more PL activity in milk from goat and older cows is a result of increased PLG activation [43–45]. However, the relevant information about the varied concentration and activity of PL during lactation stages is controversial. Leitner et al. [46] declared significantly higher activities of PL in infected glands of sheep.

Caroprese et al. [47] and Albenzio et al. [33] found that there was decrease in PL activity in ewe's milk from the early to the late lactation stage whereas Koutsouli et al. [48] and Bianchi et al. [49] announced that PL activity significantly affected by udder health status and found an increased level of PL activity due to more somatic cell counts (SCCs) during the late lactation period.

The variation in PLG-derived activity and total PL plus PLG-derived activity is greatly influenced by lactation stage and seasonal changes. It is linked with reduction of milk yield and advancement in lactation stage [45, 50, 51]. Due to increased activity of plasminogen, more entry of PL occurred from blood to milk inside the mammary glands [52]. The PL and PLG activities were significantly increased in the advancement of lactation and a nonsignificant decrease in their ratio (PL:PLG) was observed as compared to camel milk [53, 54].

#### **2.3. Phosphatases**

## *2.3.1. Alkaline phosphatase*

In 1925, for the first time, phosphatase enzyme in milk is documented by Demuth and then considered as an alkaline phosphatase (ALP; EC 3.1.3.1) indigenous to milk by Graham and Kay [55]. It became recognizable when it was confirmed that the requirement for timetemperature relationships to inactivate the ALP required slightly higher as compared to kill *Mycobacterium tuberculosis* [56, 57]. Almost 40% activity of ALP in raw cow milk is declared to be linked with the milk fat globule membrane (MFGM) in the cream phase, though the rest is soluble or dispersed in whey membrane particles (WMP) in skim milk [58]. Between individuals and herds, higher ALP levels vary significantly and its concerned activity is correlated with lactation stages and mastitis [59, 60]. Magnesium and zinc ions are promoter of ALP while tin, copper, cobalt, and ethylenediaminetetraacetic acid (EDTA) have inhibitory action and iron has no effect on activities of ALP [61].

ALP activity is in inverse relationship with yield but the other factors, e.g., fat content, breed, and feed, have no effect. For ovine milk, the ALP content is contrarily linked with milk production and directly to the milk fat substance, while infected milk (mastitis) has higher ALP activity [62, 63]. It is reported that ALP activity is low at the mature milk production stage, increased to maximum activity during the peak production stage and again decreased at the end production stage [28]. ALP activity in cow increases as lactation stage proceeds. Immediately after parturition, there is a decrease in ALP activity with a further sharp decrease after the first milking period. ALP activity then continues to decrease and noted minimum at the first week. Then increased slowly and found maximum by the 28th week of lactation [64]. In another study, ALP activity in milk was found lowest in the early lactation stages and progressed along with advancement of lactation stages and milk yields decreased. These ALP activities were also noticed greater in milk samples from evening milk as compared with morning milk [65].
