# Recent Advances and Insights into Bromelain Processing, Pharmacokinetics and Therapeutic Uses
## 4 Bromelain Enzymatic Activity Measurement  
The detection of the enzymatic activity of the various bromelain extracts and preparations is fundamental to assessing their bromelain content, which reflects the yield efficiency of the extraction and purification techniques, as well as the stability of the proteolytic activity under operational and storage conditions (pH, temperature). A number of different substrates can be used to measure bromelain activity, ranging from casein, gelatin and hemoglobin to the more sensitive casein and albumin azo-derivatives and various artificial model peptides. Casein, which can also be used in the form of sodium caseinate, is the most frequently used substrate for proteolytic activity measurements [65], due to its high availability and low cost. Proteolytic-activity measurements are usually performed on 0.5-1% casein solutions in buffers at neutral pH over various time periods, ranging from 5 to 30 min, after which the reaction is stopped via the addition of trichloroacetic acid (TCA) [9; 35; 40; 48]. The amount of soluble peptides released by bromelain is detected at 275-280 nm and usually compared with a standard curve obtained using tyrosine solutions at increasing concentrations to calculate the enzymatic activity. It is worth noting that thiol-based reducing agents and EDTA are required for the optimal measurement of enzymatic activity [66]. The reducing agents prevent the oxidation of catalytic cysteine, whereas the EDTA chelates metals that may interfere with the enzymatic activity [67; 68]. The proteolysis of azocasein (1% solution in buffers at pH 6.5-7.5) results in the formation of peptides with histidine and tyrosine diazo sulphonic acid analogues that remain in solution after the reaction mixture is treated with 5-10% TCA. Azo-derived soluble peptides can be detected spectrophotometrically at 440 nm after the addition of an alkali solution with no significant interference from other colored compounds [8; 19; 55]. Activity measurements are usually carried out at 37 \({}^{\circ}\)C, both in the case of casein and \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Separation Method**} & **Purification** & **Activity** & **Advantages** & **Limitations** & **Reference** \\  & **Fold** & **Recovery (\%)** & & & \\ \hline \multirow{2}{*}{\
\begin{tabular}{c} Aqueous two-phase \\ extraction \\ \end{tabular} } & \multirow{2}{*}{16.3} & \multirow{2}{*}{55.6} & Aqueous medium & Poor knowledge on & \multirow{2}{*}{[62]} \\  & & & Low cost & mechanisms; High salt & \\  & & & & content & \\ \hline \multirow{2}{*}{
\begin{tabular}{c} Ion exchange \\ chromatography \\ \end{tabular} } & \multirow{2}{*}{10} & \multirow{2}{*}{84.5} & \multirow{2}{*}{Mild operation condition} & High cost; Difficult & \multirow{2}{*}{[35]} \\  & & & optimization due to the complexity & \\ \hline \multirow{2}{*}{Precipitation} & \multirow{2}{*}{4.9} & \multirow{2}{*}{85.97} & Low energy needed & High precipitant (salt) & \multirow{2}{*}{[20]} \\  & & & Many alternative as & content, hardly & \\  & & & precipitants & reciclable & \\ \hline \multirow{2}{*}{Ultrafiltration} & \multirow{2}{*}{10} & \multirow{2}{*}{90} & High product throughput & \multirow{2}{*}{Membrane fouling} & \multirow{2}{*}{[32]} \\  & & & Environmentally friendly & & \\  & & & Easy scaling up & & \\ \hline \multirow{2}{*}{Affinity membranes} & \multirow{2}{*}{2.5} & \multirow{2}{*}{-} & High selectivity & High costs and difficult & \multirow{2}{*}{[63]} \\  & & & monitoring & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Comparison of the different bromelain separation techniques. azo-casein, although a number of studies have reported that the optimum temperature for stem and fruit bromelain lies in the 50-60 \({}^{\circ}\)C range [55]. Bromelain proteolytic activity can be conveniently measured using a number of synthetic substrates that simulate model peptides, which release molecules that are easily detectable by fluorescence and/or spectrophotometry.