## 4 Bromelain Use as an Exfoliant Agent in Cosmetic Applications
### Methods of Extraction and Purification of Bromelain  
Bromelain extraction and purification includes many methods, varying from conventional techniques to modern, sustainable approaches, all aiming to isolate bromelain with maintained enzymatic activity and high purity [35; 43; 44]. The extraction and purification procedure involves various previous steps, including pre-treatment (cleaning, peeling, and size reduction), cell disruption, and debris removal, including filtration. Each method has advantages and limitations, and thus, selecting the appropriate method depends on the intended application [23; 37; 44; 45]. Conventional methods often include homogenization and crushing, followed by ultrafiltration and centrifugation to create a crude extract and determine bromelain concentration. For example, precipitation with ammonium sulfate or ethanol separates bromelain, and is followed by dialysis to remove salts and lyophilization in order to produce a stable powder (Figure 3) [23; 37; 44; 45].  
With a view to increasing the efficacy, sustainability, extraction yield, and purity of the extracts, several unconventional methods have emerged. Firstly, aqueous two-phase systems (ATPS), which use two immiscible aqueous phases (i.e., polyethylene glycol (PEG) and a salt or block copolymers) to separate bromelain, often act as a cost-effective, eco-friendly alternative [45; 46]. Reverse micellar extraction (RME) is another effective technique where bromelain is solubilized in surfactant-stabilized water droplets within an organic solvent, yielding a high level of enzyme activity [46; 47]. In addition, chromatographic methods, like ion exchange chromatography, are frequently used for their precision and efficiency, with cationic exchange chromatography, specifically, resulting in up to 10-fold purification [44; 46]. Other chromatographic procedures like gel filtration, affinity, and high-speed counter-current chromatography (HSCCC) also provided very promising results [45; 46]. Furthermore, environmentally friendly methods like polysaccharide precipitation using carrageenan, as well as membrane filtration optimized by polysaccharide precipitation, are gaining vast popularity [35; 48]. Precipitation processes by adding salts, non-ionic polymers, or organic solvents like ethanol, and microwave- and ultrasound-assisted extraction (MAE and UAE, respectively) provided high enzyme recovery, while the newly emerged recombinant DNA technology showed high bromelain thermal stability and anti-microbial potential (Figure 3) [44; 49; 50; 51].  
The selection of an extraction or purification method is based on the intended application of bromelain, often involving a blend of low- and high-resolution techniques to optimize cost, yield, selectivity, and enzyme activity [44; 45]. However, limitations surrounding such methods, including concentration variations, high salt content, cost and complexity, membrane fouling, and the possible loss of the enzymatic activity during pu  
Figure 3: Schematic representation of bromelain extraction and purification. (Parts of this figure were obtained from the free database [[https://smart.servier.com](https://smart.servier.com)]([https://smart.servier.com](https://smart.servier.com)) (accessed on 29 January 2025)).  
rification because of pH or heat alterations, highlight the practical issues that formulators face, which require the urgent development of future methods (Figure 3) [44; 45].