*2.3.2. Acid phosphatase*

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

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

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

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

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

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

decrease in their ratio (PL:PLG) was observed as compared to camel milk [53, 54].

*α*s1-casein and affects the quality of dairy products [32, 42].

higher activities of PL in infected glands of sheep.

period.

94 Livestock Science

**2.3. Phosphatases**

*2.3.1. Alkaline phosphatase*

has no effect on activities of ALP [61].

Acid phosphomonoesterase (ACP; EC 3.1.3.2) in milk was initially identified by Huggins and Talalay [66] and affirmed by Mullen [67], declared that ACP was ideally in the active form at 4.0 pH. It was thermally stable and for complete inactivation it required 88°C for 10 min. ACP in bovine milk hydrolyzes the phosphate group of casein particles [68]. There are some components that act as inhibitor and activator. Fluoride acts as an inhibitor for ACP activity but slightly activated by Mn2+. In milk, the ACP level is just ~2% that of the ALP level. Approximately 75% of ACP was found generally in the skim milk phase and 20% of ACP in the MFGM [68, 69]. Reducing agents, ascorbic acid and 2-mercaptoethanol increases the ACP activity by 100% in skim milk, whereas the ACP activity in MFGM is unaffected by these agents. Casein acts as a substrate for the activity of ACP and major casein fractions αs (αs1 + αs2) > β > κ also serve as competitive inhibitors as the ACP enzyme binds with the phosphate group of casein. The ability to bind calcium with κ-casein to form micelles is reduced by dephosphorylation of casein [61].

ACP in milk might be of innovative significance due to three reasons. First, ACP exhibits thermal stability and because of this property it may be used as an indicator for severe heat treatment rather than normal. Second, numerous milk items may have a pH near to that of its optimum. Third, phosphoproteins such as caseins might be dephosphorylated readily. Technological milk properties and development of dairy products depend on the integrity of casein micelles. The enhanced activity of ACP may create problem in the inactivation of ACP without affecting nutritional qualities as it is linked with gelation of ultra-high temperature (UHT) and development of cheese flavor [70, 71].

Specific activity of ACP is greater in cream; however, about 80% ACP of milk is present in skimmed milk [60]. ACP levels in milk of Sahiwal dairy animals showed a declined pattern alongside lactation stages [28]. Shakeel-ur-Rehman and Farkye [72] observed the higher activity of ACP at 5–6 days postpartum, and afterward observed declined trend up to the end of lactation stage. Nevertheless, the range of ACP levels in their study was presented from 2.6 ± 10−4 to 2.6 ± 10−3 U/mL in normal cow milk. The ACP level is 4–10 times more in mastitis milk than normal cow milk [73, 74].

## **2.4. Lysozyme**

Lysozyme (LZ; EC 3.2.1.17; muramidase) is a single polypeptide chain (14.3 KDa M.W.), crosslinked by four disulfide bonds [75, 76]. It is an important bacteriolytic protein in milk, component of the antibacterial system, that kills bacteria by cleaving the β-1,4-glycosidic bond between N-acetyl muramic acid and N-acetyl glucosamine residues in peptidoglycan of the bacterial cell wall [77, 78].

It helps in improving the human health status, especially neonate, to protect them from infections of invading pathogens with the promotion of gut microbiota until their own immune system is developed [79–81].

Basically, there are two types of LZ: hen egg-white (C-LZ) and goose egg-white (G-LZ). However, both C-LZ and G-LZ forms may be present in cow milk as these forms are present in other body fluids and in stomach tissue of the cow [82].

LZ is available at higher concentration (0.420 g/L) in human milk as compared to buffalos (3.85 μg*/*mL), cow (0.0013 g/L), and goat (0.0025 g/L) milk [83–86].

The activity of LZ was in greater extent and more stable in buffalo milk as compared to cow milk. However, colostrum possessed 5 times higher activity as compared to mature milk. It was also observed that various factors: parity of animal and lactation stage not influences the activity of LZ but it was increased during the peak summer and winter seasons [86–88]. A substantial increase of milk LZ in mastitis among different bovine species suggested that the neutrophils are the most probable source of LZ due to inflammation of mammary gland [89–91]
