# Assessment of Seasonal and Diurnal Variations of Alkaline Phosphatase Activity in Pasteurized Milk
## 4 Discussion  
Pasteurization is the method of choice to significantly reduce the microbial load of raw milk. Raw, unprocessed milk is an excellent substrate for various foodborne pathogens to grow, causing serious illnesses upon consumption. Through this perspective, pasteurization is more than necessary. So far, the most popular and legally mandatory method is the inactivation of ALP. This enzyme is a hydrolase that removes one phosphoric group from various compounds, a crucial step in phosphorus metabolism, and hence is abundant in nature in procaryotic and eukaryotic cells. In milk, ALP originates from two sources [1; 2]. The first is the cellular membranes of various cells of the udder. In general, ALP shows a strong affinity for membranes and thus is also located on the membrane of the lipids of the milk [28]. The other source of ALP in milk is of bacterial origin. Bacterial cells harbor ALP, and the bacterial ALP is usually more heat resistant than the other one, causing false positive results in ALP testing after pasteurization. To tackle this problem, it is advisable--after checking the pasteurization equipment for various mechanical and technical flaws--to repeat pasteurization and then check again for ALP. If the ALP test is still positive, then the enzyme is probably from bacterial origin. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Test** & **Test A \({}^{*}\)** & **Test B \({}^{**}\)** & **“AP Test”** \\ \hline Limit of detection & 350 mU/L & 210 mU/L & 3.5 mU/L \\ \hline \multirow{3}{*}{Sample material} & Pasteurized milk, & Pasteurized milk, & Pasteurized milk, HTST \\  & Whey, cream. butter & HTST milk & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \cline{1-1}  & & & \\ \hline \hline \end{tabular} For reasons of simplification: \({}^{*}\) Test A: HEYL LACTOGNOST III kit, \({}^{**}\) Test B: SENSObiz® test.  
\end{table} 
Table 4: _Cont._  
Figure 2: Schematic representation of the seasonal variation of the mean values of the ALP activity in raw goat’s milk. ALP activity in milk is dependent on various factors such as the animal species, the health of the udder, the nutrition of the animals, the lactation period, the animal husbandry, the season, and the time of the day. The effect of the last two factors on goat's milk can be clearly seen in Table 3. Our results suggest that there are significant differences not only from month to month but also in morning and in evening milk. In Figure 2, a pattern can be observed in which ALP activity increases smoothly till August and then more rapidly until December for the morning milk, while there are two peaks for the evening milk, the higher in October and the lower in December. A sound interpretation of these findings needs of course more research but given the fact that these animals live in herds that graze all day and receive a small amount of concentrated food at night, it is the nutrition that affects to some extent the ALP concentration, since in different seasons, the biodiversity and the availability of the grazing plants varies. The steeper increases in both curves from August correspond with the beginning of the breeding season and the December peaks correspond with the beginning of the labor period, indicating a possible hormonal underlying mechanism. Since the pasteurization temperatures reduces the ALP activity of the treated milk in a logarithmic manner, it follows that if the initial content of ALP in milk is high, then the heat treatment will not eliminate the ALP, and, hence, the control will show unsuccessful pasteurization. It seems that this could be the case in caprine milk especially after August, according to our results in which the ALP activity in caprine milk is exceedingly high. The situation can become more complicated in circumstances where there is high prevalence of subclinical mastitis--very common in Greece--since unhealthy udders increase ALP activity.