# Recent Advances and Insights into Bromelain Processing, Pharmacokinetics and Therapeutic Uses
## 3 Extraction and Purification Methods  
The National Library of Medicine (MEDILINE; January 1970 to June 2021) and the Cochrane Register of Controlled Trials were used in the preparation of this review. The terms used for the electronic search strategy were: 'bromelain', 'pineapple', 'bromelain extract', 'bromelain purification', 'bromelain extraction', 'human' and 'clinical trial'. The selected references were screened, including those regarding clinical applications from clinical trials, and bromelain extraction and purification methods. The work includes a general introduction, an explanation of the main bromelain extraction and purification methods, a brief description of the mechanisms of action, the pharmacokinetics and safety of this enzyme complex and bromelain properties, followed by an exploration of the effects observed in clinical studies divided by therapeutic area. In general, plant proteases are produced in the early stages of fruit development, as has been demonstrated for papayas [22]. However, bromelain is extracted from pineapple  
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when the fruit is mature, with the highest concentration of proteases only being reached at that point [23]. As highlighted in the introduction, the production of bromelain is expensive (price for extract: 2400 USD/Kg) [12], as it entails several manufacturing processes, including extraction, purification, drying and packing. Purification is the most significant of all these processes, with it accounting for at least two-thirds of the total cost of bromelain production [5]. Furthermore, no guidelines for bromelain purification are currently available. The starting product, which can include the fruit, stem and other parts of the pineapple, is usually ground, without adding water, to obtain a raw crude extract. Subsequently, the extract (cooled pineapple juice) is processed further using conventional methods, such as centrifugation, ultrafiltration and lyophilisation, giving a yellowish powder (Figure 1) [24]. However, several unconventional methods have been developed in recent years to improve both enzymatic yield and purity (Table 2) [25]. The conventional approach to bromelain extraction is currently the most widely used. It consists of a first phase in which the raw extract is homogenized to disrupt pineapple cells and it is then centrifuged to remove the particulate material without denaturing the proteases [26]. The enzymatic concentration of stem bromelain has been reported to be higher than that of fruit bromelain [24]. Although centrifugation produces a homogenous extract, subsequent processing is required to eliminate or decrease the quantities of impurities [27]. ultrafiltration (-100 min) is followed by centrifugation, which is the gold standard for protein concentration, using membranes with molecular weight cut-offs from 3 to 100 kDa, leading to an -8.9-fold increase in bromelain concentration. Convection (driven by difference in pressure applied to the membrane) and diffusion (driven by concentration gradient across the membrane) are the two main mechanisms that influence mass transfer during membrane filtration [28]. Other effects that can influence mass transfer include dielectric exclusion, steric effects and the Donnan equilibrium [29]. Membrane filtration, used in combination with other processes, has been proven to improve bromelain purification. A study by [PERSON] et al. has reported a yield of 98% for combined microfiltration, ultrafiltration, ammonium sulphate precipitation and ultracentrifugation, although proteolytic activity was found to be lower by the end of the processes [30]. [PERSON] et al. have obtained 64.7% activity recovery and a 5.3-fold concentration using nano-TiO\({}_{2}\) and ultrafilters [31]. The simultaneous use of both microfiltration (plain membrane) and ultrafiltration (10 kDa Millipore kit) resulted in activity recovery of 90% and bromelain that was concentrated 10-fold [32]. [PERSON] et al.