## 6 Papain Use as an Exfoliant Agent in Cosmetic Applications
### Methods of Extraction and Purification of Papain  
Comparable to fiction, the extraction and purification of papain, as well as other proteases from _Carica papaya_, involve a range of methodological approaches, with the latex serving as the principal source of enzymatic activity, while the plant leaves provide an additional, but less prominent, source. A traditional approach involves collecting latex from the unripe fruit by making incisions on its surface [100; 106], while the leaves are also an abundant source. The extraction and purification procedure involves various previous steps, like pre-treatment (cleaning, peeling, and size reduction), cell disruption, and debris removal, including filtration. After its collection, the latex is then diluted in a buffer solution in order to prevent enzyme inactivation. Then, the latex is processed by drying, followed by homogenization (in a buffer solution), sonication (ultrasound), or EAE to release papain and enhance yield [74]. The crude extracts obtained from fig latex and leaves often require a further clarification step of ultrafiltration and centrifugation to remove debris matter prior to further purification. Later, dialysis is performed to remove salts and lyophilization, or spray drying is used to produce a stable papain powder (Figure 5) [74; 107].  
Various methods are employed to purify papain extracted from papaya, including precipitation, ATPS, TPP, and different chromatographic techniques [74; 81; 98; 106; 108]. The ATPS method comprises two immiscible aqueous phases, which are usually generated by mixing polymers, like PEG, and salts, such as ammonium sulfate or sodium citrate, to separate papain from other molecules [98; 106]. Papain partitions into one of the phases depending on the conditions, while impurities partition into another. TPP employs a combination of a salt (i.e., ammonium sulfate) and a solvent (i.e., t-butanol), which can also be employed in order to purify papaya latex-derived papain [107]. MAE [51], EAE [109], and UAE [80; 110] are believed to provide high enzyme recovery [78; 79; 80]. RME [82; 111] and supercritical fluid extraction (SFE) [112] have been proven efficient for papain extraction, but more new trials need to be conducted in this context.  
Precipitation using salts, like ammonium sulfate, is a common, cost-effective technique which is performed by adjusting the salt concentration to reduce the solubility of the protein, causing it to precipitate [74; 98; 107; 96]. Organic solvents like ethanol, acetone, and methanol can additionally be used for precipitation [74; 107]. Chromatographic methods such as ion exchange- or affinity-based chromatographic procedures, as well as gel filtration, can also be used to provide high-purity papain. More specifically, the ion exchange technique, which separates molecules based on their charge, differs from the affinity method which employs specific binding interactions, while gel filtration separates molecules based on size measurements. All techniques have been used, and have demonstrated great efficacy [45; 106]. Finally, micellar systems using biodegradable surfactants also represent an effective method for papain recovery (Figure 5) [45; 106].  
Figure 5: Schematic representation of papain 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 30 January 2025)).  
Method choice depends on factors such as cost, desired purity, and the scale of production [45; 106]. Challenges like variability in papain concentrations, harvesting time, and non-standard environmental conditions, as well as the limited relative, existent clinical trial data, must be studied and overcome to enable valid method comparisons to be conducted [45; 106].