Proteolytic Enzyme Activities of Bromelain, Ficin, and Papain from Fruit By-Products and Potential Applications in Sustainable and Functional Cosmetics for Skincare

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The distinct characteristics and efficacies of the natural proteolytic enzymes bromelain, ficin, and papain in cosmetic applications for skincare, skin renewal, and against hyperpigmentation.

Abstract

Enzyme peels are an emerging and effective cosmetic technique for controlled skin exfoliation. Naturally occurring proteolytic enzymes such as bromelain, ficin, and papain have gained increasing attention as promising cosmetic and cosmeceutical ingredients due to their exfoliating and skin resurfacing properties. These enzymes catalyze the hydrolysis of keratin protein bonds, facilitate the removal of dead skin cells from the outermost layer of the epidermis, and promote cell turnover. The role of these enzymes in skin care is particularly noteworthy due to their gentle, yet effective, exfoliating action, their ability to improve the penetration of active ingredients, and their contribution to skin renewal and regeneration. While proteolytic enzymes are traditionally extracted from fruit pulp, recent research highlights fruit by-products such as pineapple peels, fig latex, and papaya peels, as sustainable and environmentally friendly sources. These by-products, which are often discarded in the food and agricultural industries, are rich in enzymatic activity and bioactive compounds, making them valuable alternatives for cosmetic applications. Their use is in line with the principles of the circular economy. They contribute to waste prevention while improving the availability of effective enzymatic exfoliants. This review provides a comparative analysis of bromelain, ficin, and papain, highlighting their different biochemical properties, their efficacy in cosmetic formulations, and their common mechanisms of action. In addition, the extraction processes from fruit by-products, their incorporation into skin care formulations, and their potential for sustainable cosmetic applications are examined. The results underline the growing importance of proteolytic enzymes, not only as exfoliating agents, but also as multifunctional bioactive components in next-generation cosmetic products.

1. Introduction

The quest for healthy, radiant skin has driven significant advancements in cosmetic science, leading to the emergence of various groundbreaking cosmetics. Exfoliation is an essential part of skincare, as it promotes skin renewal. Natural exfoliants made from fruits and dairy products have always been used in skincare, implying that bioactives play an important role in skin health. Ancient civilizations such as the Egyptians, Mesopotamians, Chinese, and Greeks, used natural substances like milk, honey, and fruits for skin rejuvenation. For example, Cleopatra bathed in sour milk, which was known to be acidic, an action which foreshadowed more targeted enzyme treatments [1,2]. Modern commercial exfoliants appeared during the 19th century and contained ground nutshells, pumice, or other abrasives, while chemical exfoliants started a new era, offering a gentler alternative to traditional exfoliation. Nowadays, enzyme-based exfoliants are prominent in the cosmetic industry, and exfoliation is an effective method in skincare routine, globally [1,3,4,5].

Skin exfoliation is a process that involves removing dead (non-vital-keratinized) cells and impurities from the surface of the skin, aiding in skin cell renewal. Eliminating this layer from the skin makes the stratum corneum, namely the outermost skin layer, more uniform, improves skin texture and homogeneity, prevents clogged pores and dullness, and improves the skin’s overall appearance. The active ingredients of other cosmetic or dermatological products are also more capable of penetrating skin barriers [5,6,7,8,9].

Three possible depths exist concerning exfoliation: superficial (60 μm from the stratum corneum, until the papillary dermis, impacting the epidermis and plexus superficialis), medium (450 μm from the papillary dermis up to the reticular dermis, also interacting with plexus superficialis) and deep (from mid-reticular dermis and up to 600 μm, including the plexus profundus above the subcutaneous fat) (Figure 1) [1,5,6,10,11]. Superficial exfoliation removes the outermost layer of dead skin cells improving overall texture, while preparing the skin for an efficacious absorption of skincare products. Medium-depth exfoliation is performed to reduce fine lines, hyperpigmentation, acne scars, and uneven tone. Finally, deep exfoliation treats severe scarring, deep wrinkles, and sun damage, while promoting collagen production. The depth of exfoliation caters to a wide range of skincare needs, from everyday maintenance to intensive corrective treatments [1,5,6,12].

All exfoliation methods used can be broadly categorized into physical and chemical, each serving different skin needs. Physical exfoliation (or mechanical) is the most traditional exfoliation way, and relies on physical abrasion to slough off dead skin cells. Such products have abrasive particles, like microbeads, sugar, coffee grounds, salt, or fruit kernels. Specifically, for physical exfoliation, certain tools such as brushes, special sponges, and dermaplaning razors may also be used [5,8,13,14,15,16]. Chemical exfoliation relies on many chemical agents (i.e., α/β hydroxy acids or enzymes), which exfoliate the skin due to their acidic character that dissolves bonds between dead skin cells [5,8,13,14,15,16].

Enzymatic-based exfoliation represents a gentler alternative to traditional physical and chemical exfoliation, providing effective skin renewal without compromising the skin barrier. Enzyme peels are a relatively new category of exfoliating treatment employing naturally occurring enzymes, like bromelain, ficin, and papain. Enzymatic reactions take place in order to hydrolyze proteins like keratin, leading to the removal of corneocytes from the stratum corneum and the shedding of dead skin cells. This method provides effective exfoliation, resulting in a smoother, toned, more radiant complexion and an enhanced skin texture and appearance [8,11,17,18]. Unlike chemical exfoliants which dissolve the intercellular matrix via pH-dependent desquamation, enzymatic exfoliation is less aggressive. Since they minimize the risk of irritation, they are suitable for several skin types, i.e., sensitive, dry, oily, problematic, or combined ones, and for individuals with acne-prone skin, rosacea, or inflammatory conditions [8,11,17,18]. Additionally, enzymatic exfoliants do not require extreme pH levels to function, and thus, they are easily incorporated into mild, skin-friendly cosmetics; however, they are easily influenced by several factors, like temperature, enzyme concentration, and formulation stability. When they are extracted from fruit by-products, they may require additional stabilization techniques to maintain their bioactivity. Advances in eco-friendly extraction methods, nanoencapsulation, and product stabilization techniques have notably improved the longevity, bioactivity, and possibility of integrating these alternative sources in cosmetic formulations (i.e., peels, masks, and cleansers) [19,20,21].

Such natural peeling factors are not new; the concept of using natural enzymes regarding traditional skincare practices dates back to many centuries ago. During the late 20th century, as cosmetic industries began to prioritize natural alternatives to traditional exfoliants, enzyme peels gained increasing attention. For the past 50 years, the scientific understanding of enzymes has grown remarkably due to their ability to offer effective exfoliation without causing irritation [1,5,15]. Scientists and cosmetic formulators recognized their potential not only for their exfoliating properties, but also for their anti-inflammatory, anti-melanoma, antioxidant, and soothing effects [18,22,23,24,25,26]. Initially, enzyme peels were a luxury, found primarily in high-end spas and professional skincare treatment. Over time, their growing popularity created a demand for more accessible formulations, leading to the introduction of enzyme peels in over-the-counter products. Modern formulations provide the benefits of enzyme peels in innovative cosmetic products [1,8,15,27].

Despite the increasing use of enzyme-based exfoliants in cosmetics, the current literature remains limited in several key aspects, including the lack of comparative analyses of bromelain, ficin, and papain’s cosmetic benefits, their fruit by-products’ potential as alternative sources in skincare formulations, and the consolidated literature regarding enzyme formulation techniques, innovations, and cost-effectiveness in the cosmetic science domain. This review aims to bridge such knowledge gaps by integrating an all-inclusive comparison and the latest research findings considering these proteolytic enzymes [1,5,15].

The principal aim of this research is to investigate the proteolytic function of these enzymes used in cosmetic formulations, namely bromelain, ficin, and papain, which are widely known for partaking in skin exfoliation. A comprehensive evaluation and comparative analysis of each enzyme’s proteolytic activity, biochemical mechanisms of action, optimal conditions, effectiveness in skin exfoliation and rejuvenation, and their extraction from fruit by-products as a sustainable alternative will be provided. Particular attention is paid to their cosmetic applications, economic value, and role in promoting the principles of the circular economy. A guide for future development in the field of enzyme-based skincare will also be provided. The main highlights of our work are depicted in Figure 1.

2. Materials and Methods

A systematic method was employed in this review in order to compile and synthesize the relevant scientific literature on the proteolytic activity of bromelain, ficin, and papain and their applications in cosmetics. A detailed review of the relevant scientific literature was conducted, focusing on the biochemical properties, efficacy in exfoliation, and comparative performance of such useful and efficacious enzymes. The review specifically focuses on the biochemical properties, exfoliation efficacy, and comparative analysis of bromelain, ficin, and papain, highlighting their relevance in cosmetic applications.

During the literature review, all references were publicly accessible through valid scientific databases including MDPI, Google Scholar, Scopus, PubMed, Science Direct, Google Scholar, and Research Gate. The following combination of keywords was used during the data collection phase: “proteolytic enzymes”, “bromelain”, “ficin”, “papain”, “enzyme peels”, “exfoliants”, “skin exfoliation”, “physical exfoliation”, “chemical exfoliation”, “natural products”, “fig”, “pineapple”, “papaya”, “extraction”, “purification”, “molecular structure”, “chemical properties”, “stability”, “chemical activity”, “inhibitors”, “stabilizers”, “mechanism of action”, “biochemical enzyme properties”, “anti-inflammatory”, anti-melanoma”, “antimicrobial”, “antioxidant”, “soothing effect”, “wound healing”, “tissue repair”, “cosmetics”, “cosmetic applications”, “cosmetic formulations”, “incorporation methods”, “sustainability”, and “circular economy”, employing different keyword combinations with the Boolean operators AND/OR. The selection criteria ensured that only high-quality, peer-reviewed studies related to enzymatic exfoliation, enzyme stabilization techniques, and cosmetic formulations were included.

To ensure the incorporation of up-to-date information, only publications from 2015 onwards were considered. The literature review process was carried out between February 2024 and January 2025, with a view to reflecting the latest advancements and trends in the field of cosmetic enzyme research and to compare them with past relevant information, considering all limitations and suggesting future development aspects.

All total of 207 references were included in this study, which were meticulously selected using pre-established criteria, such as being exclusively research-based articles, peer-reviewed in the English language, and published between 2015 and 2025. A few important articles published prior to 2015 with relevant information, which have not been included in more recent publications, were also included. All articles cited in our manuscript were credible research papers, reviews, peer-reviewed journals, scientific books, academic publications, reports from research institutes and universities, valid clinical trials, or even meta-analyses, and they were all evaluated for their quality according to their title, abstract, keywords, and included data. The references were thoroughly screened with Zotero “6.0.36”, a reference management tool, to identify and remove duplicate entries, verifying the uniqueness and reliability of the cited sources.

Studies with no relevant experimental/review data, articles retrieved from non-valid databases, duplications, and non-English publications were excluded. Conference papers, book chapters, theses, short surveys, old reviews, blog articles, etc., were also excluded from our review, along with low impact factor/relevance score publications.

The references for each enzyme are distributed as follows:

• Bromelain: approximately 31% of the references, focusing on its source (pineapple), its biochemical properties, purification techniques, and cosmetic applications.
• Ficin: approximately 20% of the references, focusing on the extraction of ficin from the fig latex, the unique substrate specificity, and its gentle exfoliating properties.
• Papain: approximately 24% of the references, focusing on the derivation of papain from the papaya latex, its strong proteolytic activity, and its role in deep exfoliation.

The remaining references provided general information about enzyme mechanisms, proteolytic activity, relevant applications in cosmetic formulations, methods of incorporation, sustainability, and circular economy aspects, as well as the supporting data for comparison and analysis. No animal or human intervention studies were conducted in this study. The ethical considerations pertained to citing data sources responsibly and ensuring transparency in discussing enzyme properties and applications. Although, in this study, the existing literature was analyzed comprehensively, it did not involve new experimental work, and this may insert restrictions on data availability. Recent clinical data are limited, and there are, as yet, no standardized data on the optimal experimental conditions in which all enzymes are addressed. Additionally, the reliance on publicly accessible databases may have excluded studies published in less accessible journals.

3. Brief Overview of Proteolytic Enzymes: Bromelain, Ficin, and Papain’s Classification

Proteolytic enzymes, also referred to as proteases or peptidases, form a crucial class of enzymes that catalyze the hydrolysis of peptide bonds in proteins. This process leads to the breakdown of proteins into their smaller building blocks, namely monopeptides or even amino acids. More than 4000 peptidases are known, with around 700 found in humans, and they are extensively used across many industries, including food (i.e., in cheese making, where rennet induces milk proteins’ coagulation and transformation into curd), dairy, meat, bakery, brewing, feed, detergent, leather, and pharmaceutical ones [28,29,30].

Proteolytic enzymes belong to the class of hydrolases and can be classified in a variety of ways. Considering their chemical mechanism of catalysis, proteolytic enzymes are divided into serine proteinases, aspartic proteinases, threonine peptidases, metalloproteinases, and cysteine proteinases—the latter category includes bromelain, ficin, and papain, our enzymes of focus [28,30,31,32]. Furthermore, proteolytic enzymes are categorized by their mode of action into exopeptidases and endopeptidases (proteinases). Exopeptidases cleave peptide bonds near the amino or carboxyl termini of the substrate, generating multiple peptides, depending on the cleavage site. If the exopeptidases act on the N-terminus, they produce a single amino acid (aminopeptidases), or a di- or tripeptide, while exopeptidases targeting the C-terminus increase the production of a tripeptide or dipeptide (carboxypeptidases). Dipeptidase and omega-peptidase exopeptidases may occur as well. Endopeptidases, on the other hand, hydrolyze chemical bonds located within the peptide chain. Combining these two categories, an enzyme can be, for example, a serine endopeptidase or a serine exopeptidase, etc., [28,30,31,32]. Bromelain, ficin, and papain are cysteine endopeptidases. More specifically, bromelain cleaves peptide bonds between aromatic, basic, and hydrophobic residues [33], ficin cleaves bonds in proteins like casein or gelatin [34], and papain owns a broad specificity [34]. Moreover, the protease source, which is mainly plant (where bromelain, ficin, and papain belong), animal, or microbial, and their distinct biological purposes, such as wound healing, defense mechanism, anti-diabetic, anti-inflammatory, anti-melanoma, antioxidant, skin renewal, and skin soothing action, are secondary ways of proteolytic enzyme classification that further highlight their importance in cosmetic products and formulations [31]. All the aforementioned categories are illustrated in Figure 2.

The following analysis focuses on the biochemical properties, enzymatic activity, and mechanisms of action of these enzymes. Further investigations concern their sources, extraction, and purification methods, as well as factors influencing their stability and efficacy. Their applications in cosmetic formulations are also explored, highlighting their role in exfoliation, skin renewal, and general skin care, along with sustainability considerations and their potential for use in environmentally friendly cosmetic products.

4. Bromelain Use as an Exfoliant Agent in Cosmetic Applications

4.1. General Profile and Source of Bromelain

Bromelain is a complex blend of proteolytic enzymes and isoforms, primarily derived from the Bromeliaceae plant family, with the pineapple (Ananas comosus) being its most abundant natural source. This enzyme complex encloses several thiol endopeptidases, phosphatases, glucosidases, peroxidases, cellulases, and glycoproteins [35,36].

Although bromelain is present in all pineapple plant parts like the fruit, stem, peels, leaves, core, crown, and root, the concentration varies considerably depending on the specific plant part [23,35]. Historically, stems were the main origin of bromelain, with high concentrations being found in stems rather than fruit parts [35,37]. However, researchers have explored other pineapple plant parts, including waste portions, for efficiently extracting bromelain. For instance, pineapple waste like peel (30–42% w/w), core (9–10% w/w), stems (2–5% w/w), crowns (2–4% w/w), and leaves contain bromelain, though in lesser quantities compared to the fruit (~51%), while other studies have claimed that the crown may own the highest proteolytic activity (173,000 and 322,000 units for Nang Lae and Phu Lae, respectively) [23,38,39]. Hence, such cost-effective sources reduce environmental waste and align with green chemistry and circular economy principles [40,41]. The inconsistency in commercially produced products, influenced by concentration variability in plant parts, geographic origin, and processing techniques, has posed significant challenges in pharmaceutical development [42].

4.2. 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 purification 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].

4.3. Chemical Properties and Structure of Bromelain

4.3.1. Molecular Structure

Bromelain is primarily recognized as a cysteine protease, also known as thiol endopeptidase, and it has a very low toxicity [36]. Its molecular structure is glycoprotein-based, containing an oligosaccharide chain with sugars such as xylose, fucose, mannose, and N-acetyl glucosamine [52]. Bromelain’s molecular weight (MW) varies slightly depending on the source and extraction method. For stem bromelain, the MW is approximately 23.8 kDa, with some studies reporting values around 28.5–37 kDa when analyzed through different techniques [36]. Fruit bromelain, on the other hand, varies in molecular weight from 24.5 to 32 kDa (acidic protein) [35], while crown-derived bromelain revealed a MW~28 kDa [38]. Its enzymatic activity is facilitated by specific catalytic residues, such as cysteine, histidine, and asparagine that are crucial for its proteolytic function, as they are involved in the enzyme’s ability to cleave peptide bonds. Bromelain’s distinct binding sites allow it to interact with several substrates, contributing to its capability of catalyzing different reactions and enhancing various cosmetic formulas [52].

4.3.2. Stability and Activity Under Different pH and Temperature Conditions

Bromelain’s stability and proteolytic activity are significantly influenced by both pH and temperature conditions. The optimal pH values for bromelain’s activity are generally between 3 and 7, with some sources citing a slightly narrower range of 5.5–8.0, or even 6.5–7.5. Beyond this range, its activity declines, although both acidic and basic forms preserve substantial enzymatic activity, down to pH 4.0 [35,36,46]. Stem bromelain operates within a pH range of 4–10, although its activity decreases above pH 10 (optimal values of 4–8), whereas fruit bromelain functions properly at pH values of 3–8 [36]. Its stability is also pH-dependent; it remains stable at pH 5.0 over extended time periods and across all temperatures, likely due to the proximity to the pH of fresh pineapple fruit. However, its activity and specific activity continue to rise up to pH 7.0 [35,36,46].

Temperature is a major destabilizing factor for bromelain, with higher temperatures leading to enzyme degradation and decreased activity, while lower temperatures are believed to improve its stability. Bromelain is inactivated at pasteurization temperatures and its thermal denaturation is irreversible. However, it does exhibit some heat stability, maintaining proteolytic activity between 40 and 60 °C, with an optimal temperature of around 50 °C [35,36,38]. Nonetheless, the specific values may vary considerably among different clinical trials. Stem bromelain, for instance, functions properly at an optimum temperature of 40–60 °C, in contrast to fruit-derived bromelain’s optimal temperature range of 37–70 °C [36]. The glycosylated bromelain form reaches maximum activity at 30 °C and pH 7.0, with its activity decreasing by 17% when the temperature increases to 40–60 °C. Bromelain retains more than half of its original action after 30 min at 60 °C, which is completely lost when heated to 100 °C for 10 min. Regarding its storage, it is mainly stable at 4 °C (lowest activity loss) and for extended periods at −20 °C. Bromelain auto-degrades at 37 °C (optimal), where it exhibits peak proteolytic activity. The impact of temperature on bromelain activity is, therefore, pH-dependent [44].

4.4. Bromelain Stabilizers and Inhibitors in Cosmetic Formulations

In cosmetic formulations, maintaining the activity of bromelain, a thiol endopeptidase, requires a careful balance of stabilizers and the avoidance of inhibitors. Stabilizers play a crucial role in enhancing bromelain’s longevity and efficacy and include mostly non-ionic, but otherwise soft, surfactants, which are preferred over ionic ones for causing less activity loss [44,53]; however, recent studies claim that the binding of ionic surfactants is greater than that of non-ionic ones. Plus, among ionic and anionic surfactants, anionic ones, like sodium dodecyl sulfate (SDS), may vastly alter protein conformation [44,53,54]. Polyol stabilizers such as PEG, and particularly a mix of PEG (8% w/w) and ammonium sulfate (15% w/w), enhanced both the activity (stem and fruit had 4.0 and 3.6 units/mg, respectively) and stability. Meanwhile, an aqueous mixture of 15% w/w PEG-3000 and 20% magnesium sulfate (20%) results in an enzyme activity of 5.23 units/mg in ATPS [44,55]. Additionally, stabilizers, like soluble polymers (i.e., Shizophyllum commune-derived (SC) glucan) [56] and chitosan-based nanoparticles [57], enhance enzyme activity and improve stability via encapsulation. In general, certain salts (i.e., sodium sulfate (Na₂SO₄) [58] and calcium chloride (CaCl₂) [59]) facilitate enzymatic activity, while lyoprotectors, like maltose, maintain enzyme activity in freeze-dried formulations [57,60]. Finally, cream–gel formulations have shown notable promise in providing a suitable base for bromelain, especially when stored at 4 °C [61]. Chemical additives, like EDTA and non-ionic surfactants, may enhance bromelain’s stability and activity, while, in order to prevent its degradation, incorporating antioxidants (i.e., Vitamin C, Vitamin E) and maintaining appropriate storage conditions are also recommended [20,42,54,62,63,64,65].

Conversely, inhibitors may compromise bromelain’s activity. Heavy metals (Ag⁺, Hg²⁺, and Cu²⁺), for example, can directly inhibit its proteolytic function [66,67], iodoacetate may obstruct its active site [46], and anhydrous gels may create an incompatible environment for this enzyme [42,43]. High temperatures and oxidation are also detrimental as potent inhibitors, leading to enzymatic degradation and activity loss [44]. Exposure to light and the presence of various oils and surfactants in complex cosmetic products can also affect bromelain’s stability and structure [45]. To summarize, the main challenge in incorporating bromelain into cosmetic formulations is to maintain its enzymatic activity over a longer period of time. Factors such as pH fluctuations, temperature variations, and interactions with other ingredients can lead to the degradation of the enzyme. Strategies such as enzyme encapsulation, particularly for nanoemulsions, pH buffering systems, and stabilizers are currently being explored to improve the long-term efficacy of bromelain in skin care products [45,62].

5. Ficin Use as an Exfoliant Agent in Cosmetic Applications

5.1. General Profile and Ficin Source

Similarly to bromelain, ficin, belongs to a complex family of cysteine proteases and their isoforms, mainly originating from the Moraceae plant family [68,69]. Ficin, from the cysteine protease enzyme family, is obtained from fig trees’ latex and the Ficus genus, and is characterized by laticifer cells that contain and secrete this milky fluid in cases of tree injury, serving as a defense mechanism [68,69]. While ficin can be found in various Ficus species, its most common sources are Ficus carica and Ficus glabrata [69] or commercially available Ficus carica cell cultures [70]. Ficin complex encloses multiple thiol-based endopeptidases, along with peroxidases, phosphatases, amylases, and glycoproteins [68,69].

The composition of ficin extracts is influenced by the species used and the environmental conditions under which they are obtained [68,69]. Ficin is present in the unripe fruit, stem, milky fig latex fluid, root, leaves, and bark, and is extracted in concentrations ranging depending on the plant part and environmental factors [68,69,71]. Interestingly, unripe fig latex has been claimed to be the primary source of ficin extraction as it contains higher protease activity [72]. However, more qualitative information about ficin’s concentrations in distinct fig plant parts is required, in order to create valid comparative analyses. Ficin crude extract from ficin latex, produced by Ficus carica, has been shown to have high proteolytic activity of 209,000 units/mg [73]. However, alternative sources have been explored, like fig by-products and agricultural waste (i.e., leaves, unripe fruit residues), to develop cost-effective, sustainable extraction techniques. The variability of ficin content due to factors like fig variety, climate, and collection methods has posed challenges in standardizing its commercial cosmetic, medicinal, and food processing applications [68,69,71].

5.2. Methods of Extraction and Purification of Ficin

Ficin extraction and purification typically start with latex collection of a complex mix of ficin and other metabolites [74], which is usually collected from immature green fruits or via incisions on the plant [69,75]. Following latex collection, several methods are employed to isolate, extract, and purify ficin, each offering distinct advantages. The extraction and purification procedure involves various previous steps, similarly to bromelain, including pre-treatment (cleaning, peeling, and size reduction), cell disruption, and debris removal, including filtration. Conventional ficin extraction begins with latex collection, as mentioned, and the latex is then diluted in a buffer solution in order to prevent enzyme inactivation. Mechanical homogenization, sonication, or enzyme-assisted extraction (EAE) may be employed to enhance yield and preserve enzymatic activity. The crude extract undergoes ultrafiltration and centrifugation to remove insoluble debris and determine the ficin concentration. Precipitation methods, using ammonium sulfate and t-butanol, separate ficin from other proteins, and then dialysis is performed to remove salts and lyophilization or spray drying in order to produce a stable powder [44,69,76,77]. MAE, EAE, and UAE are believed to provide high enzyme recovery, as well (Figure 4) [78,79,80].

Three-phase partitioning (TPP) is an efficacious method using ammonium sulfate and tert-butanol to separate ficin from other substances in the crude extract. In TPP, ficin is selectively partitioned into the interfacial precipitate, while contaminants like pigments and lipids move into other phases. TPP is favored for its simplicity, cost-effectiveness, and scalability, and also for providing a high recovery rate of ficin while preventing autolysis [69]. Other less commonly employed techniques, like ATPS [81], RME [82], and supercritical fluid extraction (SFE) [83], have also been confirmed as efficient for ficin extraction, but more recent trials need to be conducted in this context (Figure 4).

Chromatographic techniques are also widely used for ficin extraction and purification, and separate proteins based on their physical and chemical properties. Gel filtration-associated chromatography separates proteins according to their specific size, whereas ion exchange chromatography distinguishes them by charge. One example of an effective ion exchange technique is sulphopropyl (SP)-Sepharose [76]. Affinity chromatography is yet another method using specific binding ligands to isolate ficin with high yields [84,85]. Chromatographic methods may achieve high purity, but are often more expensive and time-consuming than TPP; hence, they are a less preferred option in industry (Figure 4) [68].

Precipitation methods, such as using ammonium sulfate and ethanol or t-butanol, are also employed to selectively precipitate ficin from a crude extract [70]. Ultrafiltration, which uses membranes with specific molecular weight cut-offs, can concentrate ficin by separating it from smaller molecules and other peptides [86,87]. Finally, ficin can also be produced from Ficus carica cell cultures, in a more controlled and reproducible way than direct latex extraction. Cell cultures are optimized for enzyme formation and micropropagation techniques, and may be used for large-scale plant production (Figure 4) [88].

The choice of extraction and purification method is dependent on the desired purity, yield, scale, and cost. TPP is particularly useful for its simplicity and cost-effectiveness, while chromatography is valuable for achieving high purity; however, it requires more resources and time. Moreover, challenges like the variability in ficin concentration depend on fig maturity, harvesting time, sensitivity to oxidation, and non-standard environmental conditions. Relative, existent clinical trials regarding ficin extraction and purification are now outdated, and updated data are needed for valid comparisons of methods [68,70,88].

5.3. Chemical Properties and Structure of Ficin

5.3.1. Molecular Structure

Ficin is a cysteine protease, also known as a sulfhydryl enzyme, which is characterized by its essential catalytic cysteine residue within its active site [25,68]. This protease consists of a single polypeptide chain, and has a molecular mass of approximately 23–27 kDa, although this can slightly vary based on the source and determination method used [68,89,90]. Ficin is stabilized by three disulfide bonds which contribute to its overall structural stability [25,90]. The N-terminal sequences of different ficin isoforms show high identity, with a core sequence that is homologous to other plant cysteine proteases [90]. Ficin’s active site is characterized by the presence of a catalytically active cysteine residue and a histidine residue, which are both essential for its proteolytic function [25,90]. The secondary structure of ficin consists of approximately 22% α-helices and 26% β-sheets [89]. While ficin can interact with a variety of substrates, it demonstrates a preference for more basic amino acids at the P1 position, such as arginine or lysine, and aromatic residues at the P2 position [91]. Ficin’s structural and functional properties align with other cysteine proteases that belong in the C1 papain family, and this enzyme shares many similarities with papain, mainly considering their substrate specificity and catalytic mechanism [89,90].

5.3.2. Stability and Activity Under Different pH and Temperature Conditions

Ficin as a cysteine protease, is strongly dependent on pH and temperature values for its activity and stability [68,75,91]. The optimal temperature for ficin activity is generally around 60 °C, with a functional range between 40 and 70 °C, where it retains more than 50% of its initial activity [68,75,91]. Temperatures exceeding 70 °C, however, lead to its thermal denaturation and decreased activity [75,90]. The optimal pH for ficin activity falls within a neutral range, typically between 6.0 and 7.5, with the highest activity observed at pH 6.5 [68,75,90,91]. Ficin is in fact significantly less active under acidic conditions, displaying instability at low pH [75,90]. Ficin’s stability is also maximized around pH 7.0, with deviations towards both acidic and alkaline conditions reducing its thermal stability and causing a decrease in its apparent thermal denaturation temperature [75,90]. Therefore, ficin’s activity is influenced by its environment with extreme pH values causing denaturation, while the optimal pH varies depending on the specific substrate [75,90].

5.4. Ficin Stabilizers and Inhibitors in Cosmetic Formulations

The preservation of ficin’s structural integrity and activity necessitates precise formulation strategies that incorporate stabilizing agents while mitigating the impact of potential inhibitors. Stabilizing compounds, including established co-solvents such as trehalose, sucrose, sorbitol, and xylitol, have been shown to enhance the thermal stability of ficin by increasing its denaturation temperature. Notably, trehalose demonstrated the most significant stabilizing effect, thereby effectively maintaining the enzyme’s native conformation [90]. The microencapsulation of proteolytic enzymes like ficin, has also been confirmed as an alternative stabilizing method [92]. Plus, TPP with tertiary butanol (t-butanol) and ammonium sulfate not only purifies ficin, but also concentrates it, enhancing its stability by partitioning it away from contaminants that could destabilize it. Additionally, it has been shown that ficin immobilization, chemical additives, and non-ionic surfactants in cream formulations enhance their stability and activity [27,75,90,93].

Conversely, certain substances act as inhibitors and must be avoided in ficin-based formulations. Ficin is highly susceptible to inhibition by compounds that target its active site. For instance, E-64 is a known cysteine protease inhibitor that is able to block ficin’s enzymatic activity [89]. Moreover, metal ions, specifically zinc ions (Zn²⁺), have been shown to completely suppress ficin activity at a concentration of 1 mM. This inhibitory effect of Zn²⁺ is relevant when considering formulation ingredients, as certain compounds may contain trace amounts of metal ions [89]. In addition to specific inhibitors, surfactants can also denature ficin, with non-ionic surfactants being a better choice to minimize this effect [27]. The pH of the formulation is critical as well, as ficin demonstrates its optimal activity in pH values ranging from 6.5 to 8.5, indicating that slightly acidic or alkaline conditions can compromise its enzymatic activity. Therefore, the formulation’s pH should be controlled to match ficin’s optimum pH range of around 6.5, while also taking the skin’s pH conditions of approximately 5 into consideration [27,89,94]. Furthermore, the temperature must be controlled, since temperatures over 40 °C can lead to the thermal degradation of the enzyme. Usually, its storage requires moderate temperatures (maintaining its stability up to 37 °C). In addition, ficin’s autolytic ability may lead to concerns, considering the fact that the autolysis process increases with storage time, regardless of the storage temperature [86]. Finally, the concentration of ficin can influence its stability; higher concentrations may increase its susceptibility to changes, while lower concentrations are deemed as more stable [27,89,94]. Thus, a holistic approach that combines the use of appropriate stabilizers and the avoidance of inhibitors, as well as controlling parameters like pH, temperature, and concentration, is essential for ensuring that ficin remains stable and effective in cosmetic formulations [25,27,89,94,95].

6. Papain Use as an Exfoliant Agent in Cosmetic Applications

6.1. General Profile and Source of Papain

The cysteine protease enzyme of papain is mainly sourced from Carica papaya latex, a plant originated from the Caricaceae plant family [96,97,98]. Unlike bromelain and ficin, which exist as enzyme mixtures, papain is a single major proteolytic enzyme, even though papaya latex also contains chymopapain, caricain, and glycyl endopeptidases [98]. Papain latex refers to a milky fluid harvested by making incisions on the surface of unripe fruits; nonetheless, papain can also be extracted from other plant parts, like peels and leaves [74,99]. Although papain is an endopeptidase, it also functions as an amidase, esterase, transamidase, transesterase, and thiolesterase [74,100,101]. Papaya production is highly prominent in tropical and subtropical areas, like India, Brazil, and Nigeria [102,103].

Papain is distributed throughout different papaya plant parts like latex (16.76% w/w) [96], fruit, peel, seeds, and leaves, and its concentration highly differentiates among ripe and unripe fruit [104]. Traditionally, unripe papaya fruit latex has been the primary commercial source of papain, as it contains high proteolytic activity (1834–4293 units/mg), varying across different studies [105]. However, with increasing interest in sustainability and waste utilization, studies now focus on extracting papain from papaya by-products, including peels or seeds, to minimize environmental waste and align with green chemistry principles. The enzymatic activity of papain is challenging and varies depending on fruit maturity, processing techniques, geographic origin, etc., [74,100,101].

6.2. Methods of Extraction and Purification of Papain

Comparable to ficin, 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 [74,96,98,107]. 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].

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].

6.3. Chemical Properties and Structure of Papain

6.3.1. Molecular Structure

Papain’s molecular weight is found to be approximately between 23 and 25 kDa [94,96], and it is a globular cysteine protein composed of a single polypeptide chain containing 212 amino acid residues [100,113]. Its chain is stabilized by three disulfide bridges, contributing to its structural stability [91]. Papain also features a sulfhydryl group, which is essential for its proteolytic character [91,98], while the enzyme’s three-dimensional structure is characterized by two distinct structural domains separated by a cleft, within which the active site is located. This active site contains a catalytic dyad consisting of cysteine-25 (Cys-25) and histidine-159 (His-159), which are both critical for its catalytic activity [91,98,107,113]. The papain molecule has both α-helices and anti-parallel β-sheet domains, contributing to its complex structure. The polypeptide chain is folded along the three disulfide bridges, leading to strong interactions between side chains, enhancing the enzyme’s stability [74,91,98].

6.3.2. Stability and Activity Under Different pH and Temperature Conditions

Papain’s stability and activity are significantly influenced by both temperature and pH, with optimal conditions varying based on the context. Generally, papain exhibits stability over a broad temperature range, with some studies indicating activity from 10 to 90 °C [101], while others suggest an optimal temperature between 40 and 80 °C [107]. However, the optimal temperature for papain activity is often reported at around 60 °C [91,97,107], with the enzyme retaining its activity at higher temperatures, but experiencing decreased stability over time [91]. Papain retains activity at higher temperatures, but its stability decreases over time [101]. For example, at 80 °C, the enzyme functions properly, but may rapidly lose its activity within an hour at 90 °C. Papain’s stability is reportedly enhanced when immobilized in different chitosan- or alginate-based membranes or graft copolymers [97].

Papain’s activity is also highly pH-dependent, with optimal performance often reported between pH 6 and 7, although a range from 3.0 to 9.0 is also cited, depending on the substrate [91,94]. Some studies specify optimal pH ranges around 5.0–5.5 [74,98], while others report it to be around 7.5 [113]. Optimal pH values may shift to more alkaline ones when the enzyme is immobilized. For instance, immobilization can shift the optimal pH values from 7 to 8 [97]. Papain’s stability significantly decreases under strong acidic conditions and extreme pH levels, with better stability at neutral pH values [98]. Immobilized papain can retain more activity at pH 12 than free papain [97].

6.4. Papain Stabilizers and Inhibitors in Cosmetic Formulations

In cosmetic formulations, stabilizing agents are essential for preserving the activity of papain, as environmental factors, such as ultraviolet radiation and humidity, can adversely affect the enzyme’s stability. Such substances work by preventing denaturation and preserving the enzyme’s structure and function. Key stabilization methods include conjugation with soluble polymers, such as dextran, PEG, and SC-glucan, which shield the enzyme from harsh conditions [97,114]. Another powerful approach to stabilizing papain is immobilization, which involves attaching the enzyme to a solid support. These supports include cellulose nanocrystals coated with polydopamine, magnetic nanoparticles coated with chitosan, and incorporation into polymeric film matrices [93,115,116].

The microencapsulation of proteolytic enzymes has also been confirmed as an alternative stabilizing method [92,117,118,119]. Additionally, chemical additives like EDTA, cysteine, and dimercaptopropanol, as well as non-ionic surfactants in cream formulations, enhance stability and activity [27,74]. Furthermore, formulation components such as glycerol, sodium carboxymethyl cellulose, propylene glycol, and triethanolamine are utilized in papain gel creation [113,116].

While the primary goal in cosmetics is to harness papain’s enzymatic action, the role of inhibitors must be considered for a comprehensive understanding of its behavior. Though not typically used directly in cosmetic products, certain substances can inhibit papain’s activity. Cysteine protease inhibitors can bind to its active site, preventing protein hydrolysis [120]. Heavy metals (i.e., Zn²⁺) and oxidizing agents may also inhibit it, necessitating care to avoid metallic materials, with a view to preventing oxidation [94]. Alpha-1-antitrypsin (AAT), as a plasma antiprotease, inhibits papain’s activity in healthy tissues, ensuring that its action is limited to damaged areas, which is highly important during wound debridement [100]. Papain’s activity is also critically influenced by pH, and its stability is significantly improved by proper formulation and delivery systems, such as liposomes, hydrogels, and lyophilized powders [27,94,121].

7. Bromelain, Ficin, and Papain Exfoliants in Cosmetic Formulations: Mechanisms of Action and Biochemical Pathways

7.1. Bromelain, Ficin, and Papain Mechanisms of Action

Bromelain, ficin, and papain, all examined in this study, are classified as plant-derived, cysteine proteinases, and belong specifically to the peptidase family C1 (papain family). The latex of papayas has been understood since 1880, and includes a large family of peptidases which are homologous to papain [122,123,124]. The dyad catalytic residues of the C1 family are the amino acids cysteine (Cys, C₃H₇NO₂S) and histidine (His, C₆H₉N₃O₂). The two residues located at the active site are glutamine (Gln), which is positioned before Cys, and asparagine (Asn), located after His. His, in fact, is a semi-essential amino acid, while Cys is a non-essential, sulfuric residue. Cys, especially, comprises a thiol group, and thus is a unique amino acid residue (R-SH; sulfur takes the place of the oxygen in the hydroxyl). Gln is claimed to assist in “oxyanion holes” creation, whereas Asn helps to position the imidazolium ring of His (Gln and Asn residues are not illustrated in Figure 6) [122,123,124].

Therefore, regarding their mechanism of action, even though they follow the same catalytic strategy, they have slight differences according to their optimal function conditions, stability, and substrate specificity, as reported. Bromelain exhibits broad substrate specificity, favoring peptide bonds adjacent to lysine alanine, tyrosine, and glycine basic amino acid residues [42], while it can hydrolyze proteins including gelatin, casein, and fibrinogen [125,126]. Stem bromelain, for instance, preferentially cleaves substrates with arginine at the P1 and P2 positions [127]. Ficin, on the contrary, has a higher affinity for hydrophobic residues (i.e., phenylalanine (Phe)) located at the P1 position [128]. Similarly, papain has a broad specificity, cleaving basic, hydrophobic, and often polar residues, while working well on bulky amino acids like Phe [129]. All enzymes share high proteolytic activity and peptide bond hydrolysis patterns due to their structural similarity, having the same active sites and target proteins, and similar biochemical skin effects, as they are highly effective for skin exfoliation and renewal [22,32,91].

The peptide bond hydrolysis process of papain family enzymes is stepwise, consisting of three stages: (1) the nucleophilic attack by the sulfur (thiol) group of cysteines on the peptide bond’s carbonyl carbon, (2) the formation of a tetrahedral intermediate which is stabilized by Gln, His, and Asn, and (3) peptide bond cleavage, resulting in the release of smaller peptides or amino acids [123]. More specifically, after a peptide manages to bind to the active site, His-159 deprotonates Cys-25, allowing it to attack the carbonyl carbon in order to, again, create a tetrahedral intermediate, stabilized by an oxyanion hole (Figure 6(1)). Non-covalent-based interactions, such as hydrogen or ionic bonds, van der Walls forces, or hydrophobic interactions, along with Cln-19, aid in stabilizing the complex at its high-energy state. His-159, by acting as an acid, then protonates the nitrogen of the peptide bond, allowing it to act as a leaving group. This action concludes in carbonyl reformation and the release of the C-terminal peptide (Figure 6(2)). Water can subsequently access the active site and attack the carbonyl carbon once again, which is deprotonated again by His-159 in order to form another tetrahedral intermediate, stabilized by the oxyanion hole (Figure 6(3)). Ultimately, carbonyl is reformed, and Cys-25’s sulfur behaves as the leaving group, releasing the N-terminal peptide portion and regenerating the enzyme (Figure 6(4,5)). The enzyme, as a catalyst, returns to its original conformation, ready to mediate another reaction cycle with a new substrate molecule (Figure 6(6)). The general mechanism of bromelain, ficin, and papain cysteine proteases catalysis is presented in Figure 6 [29,122,124,130].

7.2. Bromelain, Ficin, and Papain Biochemical Pathways of Action and Relative Properties

The outermost stratum corneum skin layer comprises dead skin cells held together by keratin, a tough, key-target protein, in cosmetic applications. Keratin forms a protective barrier, but can also trap many dead cells, leading to a dull and uneven skin appearance. Another interesting mechanism of proteolytic enzymes is that they specifically target and hydrolyze keratin peptide bonds, weakening dead skin cells in the located bonds and boosting their removal [27,63]. Keratin is a substrate that enables proteolytic enzyme binding in the active site, in order to interact with peptide bonds. By breaking down keratin or desmosome proteins, proteolytic enzymes weaken the intercellular adhesion between dead skin cells, namely corneocytes, which leads to their easier detachment and removal from the skin’s surface [27,42,63,131,132,133].

Keratin and desmosome proteins are cleaved onto peptides and amino acids, allowing corneocyte detachment and the removal of dead skin cells, thus enhancing skin hydration, firmness, elasticity, and barrier repair. Such gentle exfoliation results in keratinocyte turnover and in fresher, smoother skin, without damaging the underlying skin layers. It also stimulates the underlying epidermal cells to proliferate and migrate to the surface, which, overall, promotes the regeneration of new, healthy skin cells. Regular enzymatic exfoliation helps maintain a healthy skin barrier, preventing the buildup of dead cells that clog pores and promote a rough texture while reducing hyperpigmentation, improving the skin tone, and increasing product efficacy by providing an easier penetration ability (Figure 7) [27,42,63,131,132,133].

Considering the anti-inflammatory actions of proteolytic enzymes, bromelain exhibits significant anti-inflammatory properties, which are valuable in managing skin-associated conditions. More specifically, bromelain modulates the inflammatory response by selectively restraining the biosynthesis of pro-inflammatory prostaglandins and influencing the arachidonate cascade of thromboxane (TxA₂) synthetase, all while shifting the balance towards anti-inflammatory prostaglandins [46]. Moreover, bromelain effectively reduces pro-inflammatory cytokines levels, including interleukins 1β and 6 (IL-1β and Il-6, respectively), interferon γ (IFN-γ), and the tumor necrosis factor α (TNF-α), a crucial mediator in the inflammatory response [35,42,134]. Bromelain also modulates immune cell activity by altering surface adhesion components on T cells, macrophages, dendritic cells, (DCs), phagocytosis, etc., and by also stimulating cytokine formation from peripheral blood mononuclear cells (PBMCs), often increasing IL-2 secretion. Additionally, it has been shown to interfere with platelet activation factor PAF signaling, as it regulates PAF/PAF receptor (PAF-R) and mitogen-activated protein kinase (MAPK) cascades, and thus inhibits PAF-cholinetransferase (PAF-CPT) and lysophosphatidylcholine (LPC) acyltransferase thrombo-inflammation [24]. Bromelain inhibits T cell signaling by targeting the Raf-1/extracellular-regulated kinase (ERK)-2 pathway, leading to reduced activation of CD4+ and CD8+T lymphocytes [134]. Cyclooxygenase (COX)-2 expression, implicated in tumor-inducing prostaglandin E2 (PGE2) synthesis, is also inhibited, as it modulates tumor growth factor β’s (TGF-β) expression, potentially influencing tumor growth and inflammatory signaling cascades [35,42,134]. Thrombin, collagen, adenosine diphosphate (ADP), and TxA₂ synthetase are finally restrained; hence, skin inflammation and pigmentation is reduced, by suppressing the nuclear factor-kappa B (NF-κΒ) cascade and halting the inflammatory gene’s expression (Figure 8) [42].

Additionally, bromelain has displayed vast therapeutic potential against pre-aging and inflammaging induced by advanced glycation end (AGE) products [135]. Similarly to bromelain, ficin reduces pro-inflammatory cytokines activity, aiding in inhibiting post-inflammatory pigmentation, redness and irritation, and soothing the skin, working best for the milder exfoliation of sensitive skin [68,70,85]. While being the most aggressive towards keratin bonds and playing a stronger role in deep exfoliation, papain has a weaker, but also notable, anti-inflammatory impact. Specifically, papain reduces inflammation by modulating cytokine expression and decreasing inflammation-associated mediators, helping with decreasing skin irritation (Figure 8) [26,74,101,107].

Bromelain and ficin possess significant antioxidant properties, effectively scavenging reactive oxygen species (ROS) and free radicals, in order to safeguard cells and tissues against oxidative damage and related disorders. Papain exhibited higher antioxidant action than those of bromelain and ficin, both in free radical scavenging and oxidized polar lipids (ox-PLs) peroxidation inhibition [35,42,70,96,136,137,138]. Plus, bromelain peel, stem and crown crude, and purified extracts have been shown to display important antimicrobial activity against S. aureus, followed by the P. acne, S. epidermidis, C. diphtheria, and E. coli bacteria, while P. aeruginosa displayed the highest resistance. In the same clinical trial, a facewash prepared using the above extracts showed high activity against S. aureus, followed by P. acne, C. diphtheria, and E. coli bacteria, in contrast to commercial acne-reducing formulations [23]. Such antimicrobial activity is induced by bromelain’s interaction with intestinal secretory signaling cascades, e.g., the adenosine 3:5′-cyclic monophosphatase pathway [35,46]. Ficin exhibits strong antimicrobial properties, particularly against S. aureus, S. epidermidis, S. gordonii, and S. mutans [139,140], by disrupting bacterial cell walls as a potent alternative to antibiotics in wound care. Ficin also effectively inhibited C. albicans biofilm generation [141]. Papain also had a notable anti-biofilm impact against S. aureus, S. epidermidis, and S. jejuni [142,143], a vast anti-bacterial impact, along with bromelain, against A. acidoterrestris [144], and mitochondria dysfunction-related C. albicans inhibition, induced by a papaya seed extract (Figure 8) [145].

In terms of tissue repair and subsequent wound healing, bromelain promotes both via several mechanisms. As of debridement, by hydrolyzing the devitalized tissue and breaking down necrotic tissues, bromelain facilitates the removal of damaged tissue from the wound site in cases of burn treatment and other skin injuries [35,42,133,146]. Moreover, it enhances cell proliferation by facilitating the replacement of impaired tissue with new cells [42,46,57]. Bromelain’s proteolytic activity aids in breaking down the proteins involved in edema formation, reducing the accumulation of fluid in tissues, and stimulating fibroblast. This activity leads to decreased bruising and swelling, improved circulation, and boosted collagen deposition and epithelialization [42,57]. Ficin is also known for its similar potent proteolytic, wound healing, and skin regeneration actions, making it useful for reducing post-injury edema and swelling, as well as infection risk [140,147]. Similarly, papain accelerates wound healing and skin repair, removes necrotic tissue, stimulates collagen synthesis, and reduces edema and swelling; thus, it is beneficial against burn treatment, scar healing, and pressure ulcers (Figure 8) [148,149,150].

Bromelain, ficin, and papain proteolytic enzymes have also exhibited antithrombotic, anti-skin cancer, and anti-tumor potential. As with bromelain, properly inhibiting the action of NF-κB, COX-2, and PGE2, may represent a potent anti-skin carcinoma and anti-tumorigenesis targeted cure [35]. In the same context, bromelain led to the suppression of proliferation of cell lines like those of the human epidermoid carcinoma-A431 and melanoma-A375, induced basal cell carcinoma destruction, and halted their anchorage-independent growth [151]. F. carica latex-derived ficin possesses polyphenolic constituents acting as potent antioxidants, and is employed to treat skin aberrations based on its anti-proliferative impact [152], while it has also a notable capability of inhibiting tumor formation [153]. Finally, papain has displayed significant antitumor properties. Papain suppresses atopic skin inflammation in atopic dermatitis (AD)-suffering mice and human keratinocytes (HaCaT cell lines) [26]. Also, papain has shown anti-proliferative effects on tight junction proteins, as observed in human primary keratinocytes (C57BL/6 cell lines) and toll-like receptor (TLR) 4-deficient mice, where it affected skin barrier function by elevating transepidermal water loss (TEWL), degrading tight junction proteins, and promoting vasodilation [154]. All biochemical cascades and effects of bromelain, ficin, and papain are depicted in Figure 8.

8. Bromelain, Ficin, and Papain Peels By-Products-Based Cosmetic Applications

Proteolytic enzymes like bromelain, ficin, and papain are widely used in cosmetics due to being able to break down proteins, facilitate exfoliation, and enhance skin renewal. They provide a gentler, yet equally effective, alternative to traditional mechanical and chemical exfoliants, rendering them ideal for a diverse range of skincare, haircare, and generally dermatological applications. Such enzymes have been or may be integrated in enzyme peels, masks, exfoliating cleansers, scrubs, anti-wrinkle creams and serums, acne spot treatments, oil control masks and toners, dandruff or scalp exfoliating shampoos, scalp serums treatment, foot peels, and hand and cuticle treatments, as well as many other cosmetic products and cosmeceutical formulations. Bromelain is commonly used in exfoliating masks, cleansers, and anti-inflammatory creams, ficin is preferred in mild exfoliants for sensitive skin, and papain features in deep exfoliators [18,45,52,155,156].

Proteolytic enzyme-based formulations have provided great benefits and efficacy in skincare, such as gentle exfoliation. Bromelain, ficin, and papain effectively degrade keratin stratum corneum-located proteins, facilitating the removal of dead skin cells without causing significant irritation. They are non-abrasive and gentle on the skin, and are thus ideal for acne- or rosacea-prone skin and do not cause over-exfoliation. Therefore, they are suitable for sensitive skin types that may react negatively to harsh exfoliants [22,27,157]. Also, such enzymes exhibit anti-inflammatory effects by inhibiting pro-inflammatory cytokine activity, reducing excessive immune activation. By breaking down swelling-inducing proteins, damaged peptides, and other skin inflammatory mediators, they can reduce inflammation and fluid accumulation. Proteolytic enzymes promote collagen formation and enhance the skin barrier without interfering with its natural defense system [26,42].

The systemic usage of products containing bromelain, ficin, or papain can lead to a more even skin tone and an improved texture. Such proteolytic enzymes remove melanin-rich keratinocytes (pigmented dead skin cells), but they also slow melanin production, preventing new dark spots and, therefore, targeting hyperpigmentation. By inhibiting enzymes involved in melanin production, they lighten age spots, acne scars, and even sun damage, leading to a more uniform complexion. They also help smooth out rough areas of the skin caused from clogged pores, dead skin accumulation, or uneven collagen structure, leading to smoother, softer skin with fewer breakouts [18,156]. The enhanced absorption of active ingredients is also observed. By exfoliating the outer layer of dead skin cells, proteolytic enzymes modulate the skin’s permeability, allowing larger or complex molecules penetration. Enhanced penetration leads to the better absorption of other active constituents in skincare-based products, and since the bioavailability of key ingredients is improved, skincare and haircare treatments show better efficacy [27,46,94].

Enzyme-based hair and scalp care has also been investigated. Since they dissolve excess keratin buildup, bromelain, ficin, and papain can reduce scalp flakiness, dandruff, and/or scalp irritation. Such enzymes may gently exfoliate the scalp, preventing clogged hair follicles, boosting hair growth, strengthening hair strands, and promoting overall hair follicle health [27,46,94]. Moreover, considering wound healing and scar reduction, the proteolytic activity of these enzymes is beneficial by debriding necrotic tissue; decreasing redness, irritation and pain; stimulating collagen formation; and consequently, promoting the formation of new, healthy tissue. They can also help in reducing scar appearance, accelerating the healing process, and improving overall skin health [147,158,159,160,161], as observed in post-surgery healing ointments and creams for after-burn treatment [162]. Ultimately, proteolytic enzymes can be found in several cosmetic products, each serving a different purpose. Enzyme-based formulations could be a key ingredient in future cosmetic and cosmeceutical treatments [17,22,27,94,163].

9. Methods of Incorporation of Bromelain, Ficin, and Papain Peel By-Products-Based Cosmetic Applications

In order to discuss, bromelain-, ficin-, and papain-related methods of incorporation in cosmetic products, their solubility must be discussed. Bromelain and papain are highly water-soluble (

Table 1. Physicochemical properties of proteolytic enzymes.

Enzymes	Water Solubility (mg/L, 25 °C) *	logKow *	pKa *
Bromelain	1000	−11.6	6.4; 7.1
Ficin	10,000	2	8–9
Papain	10,000	−4.5	8.3

* Physicochemical parameters were collected from PubChem (https://pubchem.ncbi.nlm.nih.gov) (accessed on 24 January 2025).

) [46,117], while ficin is less soluble, but is still viable in buffered or aqueous solutions. Thus, organic solvents reduce the solubility of all three enzymes [70]. Furthermore, it must be noted that α or β-hydroxy acids (AHAs and BHAs, respectively) and some surfactants in cleansers or emulsifiers may denature enzymes and reduce their activity [5,8,13,14,15,16]. Therefore, pH buffering, encapsulation techniques, the use of stabilizers, and formulation type selection (leave-on products like masks or serums, wash-off products like cleansers and scrubs or dry formulations, i.e., powder exfoliants), may be the key answer to preventing enzyme deactivation and enhancing the product’s overall efficacy [17].

The creation of lyophilized enzyme powders follows a process that involves freeze-drying the enzyme to preserve its activity and stability. A lyophilized powder can easily be incorporated into dry formulations or rehydrated for use in liquid cosmetics [129]. Lyophilized enzymes reportedly have a longer shelf-life and are less prone to degradation during storage, while they can be activated by being mixed with a liquid component, ensuring maximum activity at the time of application [44,57,161,164]. Moreover, enzyme powders can be incorporated into anhydrous (water-free) formulations like oils, balms, and powders, which minimize the risk of enzyme degradation due to hydrolysis. On the other hand, for water-based products like gels, creams, and lotions, enzymes are often added in the final stages of formulation, and pH adjusters are used to maintain optimal conditions for preserving efficacious enzyme activity [165,166].

The utilization of encapsulation with a view to effectively protecting enzymes from harsh formulation conditions and ensuring controlled release, as aforementioned, is of great importance. Encapsulation involves encapsulating the enzyme in a protective coating to boost its stability and control its release, while common encapsulating agents include liposomes, polymers, silica particles, etc. Encapsulation shields the enzyme from external factors like pH, temperature, and UV radiation [167,168]. Liposomes are phospholipid vesicles that encapsulate enzymes, ensure their gradual release, enhance their penetration capability, and promote product efficiency during skin delivery [33,169,170,171,172]. Similarly, polymeric nanoparticles, with materials like chitosan or alginate-enclosing proteolytic enzymes, also enhance prolonged cosmetic product shelf-life [93,116,148,161,165,167,173,174,175]. Microencapsulation, for instance using microspheres, is ideal for powdered cosmetics that prevent premature activation [176,177,178]. There is great importance in controlling release formulations to prevent over-exfoliation and ensure prolonged enzymatic product activity; thus, delivery systems that enhance enzyme skin penetration have been examined. Nanoencapsulation by using nanocarriers (i.e., nanoemulsions, nanomotors) which improve enzymatic bioavailability and enhance product efficacy [57,97,119,157,167,170,173,174,179,180,181,182,183] has resulted in several beneficial skin outcomes. Finally, hydrogel formulation may provide a moisture-rich medium for preserving enzyme stability and enabling skin hydration [184,185,186,187,188].

For the maximum efficacy in the incorporation of proteolytic enzymes in cosmetic products, incorporating stabilizing agents such as sugars, polyols, complexes, and proteins can help maintain enzyme activity during formulation and storage. Such agents (i.e., trehalose, glycerol, or EDTA) or antioxidants, such as ascorbic acid, can create a protective environment around enzyme molecules in cosmetic formulations [90,114,173].

10. Comparative Analysis of Several Aspects Regarding Bromelain, Ficin, and Papain Enzyme Peel By-Products Utilized in Cosmetic Applications

Bromelain, ficin, and papain are all highly valued for their proteolytic properties, with each enzyme offering unique benefits in skincare applications. Several factors influence their cosmetic and cosmeceutical efficacy; thus, a comparison among these cosmetic agents is essential for comprehending their role, overcoming the current limitations, and developing advanced methods to exploit their full potential [2,17].

Although they share a similarly high proteolytic activity, due to their structural similarity and same active sites, peptide bond hydrolysis patterns demonstrate that many distinct characteristics and approaches exist among these enzymes regarding their source, methods of extraction and purification, molecular structure, optimal function pH and temperature, substrate specificity, stability, biochemical profile, exfoliation efficacy, skin type suitability, methods for incorporation in cosmetic applications, potential side effects, stabilizers and inhibitors, and finally, sustainability aspects [2,17].

All of the similar and different general profiles, characteristics, properties, and cosmetic efficacies of bromelain, ficin, and papain that have been examined, cited, and thoroughly analyzed in our study are synopsized in

Table 2. Detailed comparison of the source and structural characteristics and the extraction and purification profiles of bromelain, ficin, and papain.

Aspect/Feature *	Proteolytic Enzyme			References
	Bromelain	Ficin	Papain
Source, Origin, and General Profile	Pineapple plant (Ananas comosus) Found in stem, fruit, leaves, core, crown, root, and peel Stem bromelain is more abundant The crown has the highest proteolytic activity Complex of several thiol endopeptidases, phosphatases, glucosidases, peroxidases, cellulases, and glycoproteins	Fig (Ficus carica L.) latex Found in milky latex, unripe fruit, leaves, stem, bark, root, and peels Fig latex is more abundant The latex has the highest proteolytic activity Complex of several thiol endopeptidases, phosphatases, amylases, peroxidases, and glucoproteins	Papaya (Carica papaya) latex Found in unripe fruit, but also peels and leaves Unripe papaya latex is more abundant and has the highest proteolytic activity Can be produced from Ficus carica cell cultures Not a complex It may function as an amidase, esterase, transamidase, transesterase, and thiolesterase	[23,35,69,70,74,91,98,100,121]
Extraction and Purification	Precipitation using ammonium sulfate ethanol or acetone ATPS (often using PEG and a salt) Ultrafiltration RME Chromatography including ion exchange, gel filtration, and HSCCC MAE and UAE Recombinant DNA technology	Precipitation using ammonium sulfate ethanol/t-butanol TPP ATPS RME and SFE (less preferred) Chromatographyincluding ion exchange with SP-sepharose, affinity, and gel filtration Ultrafiltration Cell cultivation MAE, UAE, and EAE	Precipitation with ethanol and saturated ammonium sulfate ATPS TPP Chromatography including ion exchange, affinity, and gel filtration RME SFE MAE, UAE, and EAE	[43,45,47,49,50,51,68,70,74,78,79,81,98,106,107,108,111,161,189,190]
Molecular Structure	Cysteine protease MW of 24.5–32 kDa for the fruit, 23.8–37 kDa for the stem and ~28 kDa for the crown Catalytically active Cys and a His residue	Cysteine protease belonging to the papain family MW 23–27 kDa ~22% α-helices and 26% β-sheets Catalytically active Cys and a His residue	Cysteine protease, part of the papain family MW 23–25 kDa ~Both α-helices and anti-parallel β-sheets Catalytically active Cys and a His residue	[69,89,90,94,96,127]
Optimal pH	Stem bromelain: 6–7, 5.5–8.0 Fruit bromelain: 3–8 Can range from 4.5 to 9.5–10 Depends on the study—non-standardized	Optimal activity near pH 6–7.5, but can be active in a broad range pH 5–8 Depends on the study—non-standardized	Optimal activity near pH 6–7, but active across a broader range of 3–9 Optimal activity may vary slightly with different substrates Depends on the study-non-standardized	[69,74,89,90,91,94]
Optimal Temperature	Optimal temperature of 60 °C Stem bromelain: 50–60 °C Fruit bromelain: 37–70 °C Depends on the study—non-standardized	Optimal temperature of 60 °C Can range from 40 to 70 °C Depends on the study—non-standardized	Optimal temperature of 60 °C Can range from 10 to 90 °C Depends on the study—non-standardized	[35,44,69,75,91,97,101,107]

* General profile, characteristics, properties, cosmetic efficacy, and sustainability.

,

Table 3. Detailed comparison of the substrate specificity, mechanisms, and biochemical cascades of action, properties, and relative exfoliation efficacy of bromelain, ficin, and papain.

Aspect/Feature *	Proteolytic Enzyme			References
	Bromelain	Ficin	Papain
Substrate Specificity	Bromelain exhibits broad substrate specificity It has preference for peptide bonds adjacent to Lys, Ala, Tyr, and Gly basic residues Stem bromelain cleaves substrates with Arg at P1 and P2 positions Hydrolyzes various proteins, including casein, gelatin, and fibrinogen Similar specificity to ficin and papain, hydrolyzing bradykinin and angiotensin Possible affinity to elastin	Ficin, on the contrary, has a higher affinity for hydrophobic residues (i.e., Phe) located at the P1 position Similarly to bromelain and papain, hydrolyzes bradykinin and angiotensin Affinity to gelatin and casein with optimum pH of 7.5 and 6.7, respectively Possible affinity to elastin	Papain has a broad specificity It can cleave basic, hydrophobic, and often polar residues It works well on bulky amino acids, such as Phe Hydrolyzes a broad range of proteins, including casein and gelatin Possible affinity to elastin	[22,42,77,125,126,127,128,129,131,137,176,191]
Exfoliation Efficacy	Strong keratin breakdown, smoothens skin	Gentle exfoliation, suitable for sensitive skin	Strong protein breakdown, deep exfoliation	[13,22,27,116,156]
Mechanism of Action	Same peptide bond hydrolysis patterns The catalytic dyads of the C1 family are Cys and His amino acids Structural similarity, same active sites, same target proteins	Same peptide bond hydrolysis patterns The catalytic dyads of the C1 family are Cys and His amino acids Structural similarity, same active sites, same target proteins	Same peptide bond hydrolysis patterns The catalytic dyads of the C1 family are Cys and His amino-acids Structural similarity, same active sites, same target proteins	[22,29,32,74,91,122,124,125,126,127,128,129,130]
Biochemical Pathways and Properties	All hydrolyze keratin via same peptide bond hydrolysis patterns High anti-inflammatory activity High antioxidant activity High antimicrobial action against S. aureus followed by P. acne, S. epidermidis, C. diphtheria, and E. coli P. aeruginosa displayed the highest resistance Higher antimicrobial action, in contrast to known commercial acne-related products High wound healing, anti-swelling, and anti-edema activity Proper antithrombotic impact against skin carcinoma and skin melanoma	All hydrolyze keratin via same peptide bond hydrolysis patterns High anti-inflammatory activity High antioxidant activity Strong antimicrobial properties, particularly against S. aureus, S. epidermidis, S. gordonii, and S. mutans High wound healing, anti-swelling, and anti-edema activity Notable skin anti-tumor formation activity	All hydrolyze keratin via same peptide bond hydrolysis patterns Weaker but high anti-inflammatory activity The highest antioxidant activity Notable anti-biofilm impact towards S. aureus, S. epidermidis, and S. jejuni Vast anti-bacterial impact, along with bromelain, against A. acidoterrestris Mitochondria dysfunction-related, C. albicans inhibition High wound healing, anti-swelling, and anti-edema activity Suppressive activity against atopic skin inflammation in atopic dermatitis (AD)	[25,26,35,42,68,70,74,85,96,101,106,107,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154]

* General profile, characteristics, properties, cosmetic efficacy, and sustainability.

and

Table 4. Detailed comparison of the skin type suitability, common cosmetic applications, incorporation techniques, potential side effects, formulation stability, and major stabilizers and inhibitors of bromelain, ficin, and papain.

Aspect/Feature *	Proteolytic Enzyme			References
	Bromelain	Ficin	Papain
Skin Type Suitability and Corresponding Cosmetic Applications	Best for normal-to-oily and inflamed skin Versatile and suitable for most skin types Exfoliating masks, anti-inflammatory creams, and post-inflammatory pigmentation treatments	Ideal for dry and irritation-prone skin Suitable for sensitive skin types Suitable against inflammatory skin conditions (rosacea or acne) Enzyme, low-irritation masks	Effective for thick, rough skin textures, clogged pores, and hyperpigmentation Recommended for more resilient skin types Deep-cleansing peels and enzyme-based serum formulations	[23,26,35,42,46,88,119,165,192,193,194]
Incorporation Methods in Cosmetic Applications	Incorporated into liposomes, creams, gels, and nanoemulsions Microencapsulation and nanoencapsulation Powdered enzyme formulations Hydrocolloid-based systems Hydrogel systems	Ficin-based cosmetics usually rely on nanoparticles like chitosan or alginate, gels, nanomotors, and biofilms Microencapsulation and nanoencapsulation Dry-powdered products Hydrogel systems	Can be incorporated into liposomes for delivery Incorporated into topical creams and gels Due to being prone to fast degradation, it is often integrated into microparticle-based cosmetics, polymeric nanoparticles, and natural encapsulators Enzyme-infused masks and deep cleansers Hydrogel systems	[33,44,57,93,97,116,119,129,148,157,161,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188]
Potential Side Effects	Skin rashes, itching, swelling, and more severe reactions are possible, especially for those with pineapple allergies It can also cause false-positive latex skin prick tests May be cross-reactive with other allergens such as latex, papain, or wheat flour	Can cause irritation and allergic reactions, particularly in those sensitive to fig latex High prevalence of cross-sensitization with fig byproducts and birch pollen allergens	May cause rashes, itching, contact dermatitis, and hives Keratinolytic activity degrades tight junction proteins, increasing skin permeability People with sensitive skin may be more prone to reactions	[42,46,195,196,197]
Stability	Stable at pH 5.0 for extended periods, and activity is able to increase up to pH 7.0 Inactivated at pasteurization temperature, retains activity at 40–60 °C Loses activity when heated at 100 °C for 10 min Stable at 4 °C, retains stability for long periods at −20 °C Influenced by pH, temperature, and exposure to surfactants	Less active under acidic conditions, unstable at low pH Retains >50% activity between 40 and 70 °C Denatures above 70 °C Moderate stability at lower temperatures Influenced by autolysis, metal ions, and pH variation	Stable at a broad range of temperatures, better at a neutral pH Active at 10–90 °C but loses stability over time Rapid loss of activity at 90 °C Stability increases when immobilized Influenced by oxidation, inhibitors, and formulation conditions	[35,44,57,69,90,91,97,98,101,107,133]
Major Stabilizers and Inhibitors	Stabilizers: polyols (PEG), SC-glucan, soluble polymers, chitosan nanoparticles, Na2SO4, CaCl2, lyoprotectants like maltose and cream–gel formulations Inhibitors: heavy metals (Ag+, Hg2+, Cu2+), iodoacetate, anhydrous gels, oxidation Various surfactants Thermal stability is affected by pH and vice versa	Stabilizers: trehalose, sorbitol, sucrose, xylitol, TPP, immobilization Inhibitors: E-64, Zn2+, non-optimal pH, autolysis over time Various surfactants Thermal stability is affected by pH and vice versa	Stabilizers: PEG, SC-glucan, EDTA, cysteine, encapsulation in cream or gel formulations Inhibitors: heavy metals, oxidation, alpha-1-antitrypsin (AAT) Various surfactants Thermal stability is affected by pH and vice versa	[46,69,89,94,100,114,115,116]

* General profile, characteristics, properties, cosmetic efficacy, and sustainability. Overall, some significant data concerning the proteolytic activity of bromelain, ficin, and papain from fruit peel by-products and their potential applications in functional cosmetics destined for skincare must be examined. Bromelain, ficin, and papain are cysteine proteases with distinct specificities, impacting their dermatological applications, effects on skin types, allergic reactions, and their potential within a circular economy.

.

Papain, derived from papaya latex, exhibits a strong preference for cleaving peptide bonds adjacent to lysine and arginine residues, making it effective in exfoliating the stratum corneum and removing dead skin cells, which can improve skin texture and appearance. However, papain’s potent keratinolytic activity and its ability to degrade tight junction proteins may lead to irritation and sensitization, especially regarding sensitive skin types [154]. Pineapple-derived bromelain possesses broader substrate specificity and targets peptide bonds predominantly located near lysine and tyrosine, with stem bromelain preferring arginine at both P1 and P2 positions, and fruit bromelain favoring hydrophobic residues at P2. Bromelain offers anti-inflammatory and antimicrobial benefits, making it useful for acne-prone skin, wound healing, and reducing post-procedure bruising [35,46,125,126,127]. Ficin, which is obtained from fig latex, targets basic residues at the P1 position, and hydrolyzes proteins like casein, gelatin, and collagen [68,70,85,128]. Ficin displays both protease and peroxidase activity, and offers antioxidant, anti-inflammatory, and whitening effects, reducing melanin synthesis and improving skin tone [70,147,152,183]. All proteolytic enzymes displayed vast anti-inflammatory, antioxidant, antimicrobial, anti-thrombotic, and wound healing properties, displaying both great similarities and differences in their specific activity.

Enzymatic exfoliants provide a milder option compared to chemical ones, making them ideal for sensitive skin; however, individual sensitivities may still arise. Oily and acne-prone skin can benefit from bromelain’s antimicrobial and anti-inflammatory properties [23,133], as well as ficin’s soothing effects [70]. For dry and aging skin, papain’s ability to break down collagen and ficin’s influence on the extracellular matrix contribute to improved hydration and skin renewal [198,199]. Additionally, hyperpigmented skin may come across enhancement through the exfoliating and anti-melanogenic effects of both papain and ficin, promoting a more even skin tone [25,121].

At this point, it is interesting to add that although allergic reactions to bromelain, ficin, and papain are rare, they should still be considered, especially for individuals with sensitivities to certain plant-based allergens. Bromelain, which is derived from pineapple, may cause skin rashes, itching, or swelling reactions in individuals with pineapple allergies, and can also lead to false-positive latex skin prick tests, while cross-reactivity with allergens such as latex, papain, or wheat flour is also possible [42,46,196]. Ficin may trigger irritation and allergic reactions, particularly in those sensitive to fig by-products, with a notable link to cross-sensitization with birch pollen allergens [136,165,197]. Finally, papain, while widely used in skincare, may cause rashes, itching, contact dermatitis, or hives, and its keratinolytic activity can degrade tight junction proteins, increasing skin permeability. Individuals with sensitive skin are more prone to reactions, making it important to patch-test enzyme-based products before use [154,197].

Bromelain, ficin, and papain each offer unique benefits for cosmetic applications, with varying degrees of exfoliation, anti-inflammatory properties, and whitening effects. However, bromelain’s strong exfoliation and calming properties, ficin’s gentle exfoliation profile, and papain’s deep cleansing and acne treatment characteristics serve as promising cosmetic agents [18,22,44,158]. The choice of enzyme depends on the desired formulation outcomes, skin type compatibility, and stability considerations. Future research and development may focus on optimizing enzyme delivery, and enhancing stability for more effective skincare solutions [18,22,44,158].

11. Sustainability and Circular Economy of Bromelain, Ficin, and Papain Enzyme Peel By-Products-Based Cosmetic Applications

Incorporating enzymes like bromelain, ficin, and papain into cosmetic formulations requires an understanding of the economic aspects of sustainability and market availability. Bromelain is relatively cost-effective and widely available due to the extensive cultivation of pineapples and the use of by-products like stems. Bromelain’s strong exfoliating properties and anti-inflammatory benefits are utilized in various skincare products [27]. Ficin is slightly more expensive and less readily available because of figs’ limited cultivation and the labor-intensive extraction process. However, ficin is valued in niche markets for its gentle exfoliating action towards sensitive skin types in specialized skincare [200]. Finally, papain is cost-effective and widely used across multiple industries as it is abundant and easily extracted. Papain’s exfoliating value, robust market presence, and cost-efficiency contribute to its broad appeal in cosmetic formulations [99].

The use of bromelain, ficin, and papain in cosmetics exemplifies a commitment to circular economy principles and sustainability, via the innovative use of by-products and green methods of extraction. Such plant-derived enzymes that are often discarded represent a strategic shift towards waste reduction and resource efficiency [68,201]. Bromelain from pineapple waste, such as stems and peels, ficin from fig tree latex, and papain from unripe papaya fruit latex, highlight how agricultural by-products may be repurposed into valuable cosmetic ingredients [41,68,181,201]. Such an approach not only limits waste from agricultural processing, but also lessens industry’s reliance on synthetic materials, aligning with a growing demand for eco-conscious products. To some extent, dietary fiber from pineapple by-products can also serve as a food additive to enhance texture and consistency as a substrate for microbial enzyme production [40,202].

Utilizing plant-based enzymes often involves green methods of extraction that minimize the environmental impact as compared to the production of synthetic alternatives. For instance, bromelain powder with potent enzymatic activity was successfully generated by pineapple by-products via organic, solvent-free extraction, ultrafiltration, and freeze-drying [125], while pectin and furanocoumarins from fig leaves and peels were easily recovered by ionic liquids and deep eutectic solvents [68]. Similarly, papain from various papaya parts was extracted using distilled water and a citric phosphate buffer, and purified in a two-step salt precipitation method [98]. Additionally, green extraction methods, like ATPS, MAE and UAE, are energy- and time-efficient techniques able to reduce the use of harmful solvents and enhance extraction yield, and have provided great results in bromelain, ficin, and papain-based experiments [49,50,51,68,81,99,108,189]. However, additional relevant clinical trials are needed to demonstrate their sustainability, effectiveness, and applicability in cosmetic formulations [189].

Integrating biodegradable enzymes contributes to sustainability and environmental impact reduction in cosmetic industries. Using plant-based by-products as raw materials decreases an industry’s reliance on non-biodegradable newly extracted or synthesized ones (sustainable production cycle) [41,181]. Such enzymes serve as renewable alternatives to harsh chemical exfoliants, and are ideal for sensitive skin types [42]. Beyond their skincare advantages, they also aid in reducing environmental pollution caused by waste disposal. The shift towards using bromelain, ficin, or papain enzymes supports biodiversity by promoting the cultivation of plants that are able to offer useful by-products, like fiber-rich fruit peels capable of being converted into composts and residual biomass repurposed for animal feed or energy biogas formation [203,204]. Additionally, utilizing such enzymes effectively aids in minimizing complications and enhancing skin health via their anti-inflammatory and wound-healing properties; thus, a growing demand for sustainable skincare solutions has emerged [41,181].

The utilization of bromelain, ficin, and papain also offers significant economic advantages, driving innovation and product development [205]. This approach adds value to what would otherwise be discarded, making the production process more cost-effective. Moreover, the use of natural ingredients caters to consumer demand for sustainable and ethically sourced products capable of boosting a company’s profitability. Technological advancements in nanocarrier systems (i.e., liposomes, nanoemulsions) facilitate the effective delivery of these enzymes, further enhancing their efficacy and promoting their use in innovative cosmetic formulations. Hence, integrating natural enzymes into nanotechnology creates a more sustainable and efficacious model for the cosmetic industry. Lately, cosmetic industries have prioritized eco-conscious practices, such as using enzymes in “green beauty” cosmetic or cosmeceutical formulations [41,181,205,206].

12. Current Research Gaps, Future Directions, and Perspectives

Although enzymes have been used in skincare formulations for several years, significant gaps and limitations still remain. Most studies and clinical trials focus on short-term exfoliation effects, but the long-term skin health impact remains largely unexplored [6]. Key challenges include stability [114], the risk of over-exfoliation and skin irritation [1,5,15], variability in enzyme activity due to different extraction and purification methods, and non-standardized pH, temperature, and concentration conditions across plant sources [163]. Additionally, cost and accessibility [44,45], regulatory hurdles, consumer acceptance [6], and environmental and sustainability concerns [68,201] must be addressed in order to create efficacious, enzyme-based cosmetic products. In this context, the following section explores future perspectives and potential solutions.

The use of proteolytic enzymes in cosmetics is evolving rapidly, driven by advances in science, sustainability, and a deeper understanding of skin biology. The cosmetic industry is continuously growing, and although proteolytic enzymes are already used in various cosmetic products, the field is still expanding with new applications, improved formulations, and cutting-edge technologies. With the rise in biotechnology, bioengineering, and artificial intelligence (AI), proteolytic enzymes in cosmetic products will become “smarter”, more sustainable, and highly customized. Bromelain’s strong exfoliation and calming properties, ficin’s gentle exfoliation profile, and papain’s deep cleansing and acne treatment characteristics serve as promising cosmetic agents [18,22,44,158]

One of the most significant trends is the move towards personalized skincare solutions. Advances in biotechnology and genomics enable the creation of skincare products tailored to individual skin types and conditions. Proteolytic enzymes can be customized to provide bespoke exfoliation and treatment options based on a person’s unique genetic predispositions to certain skin conditions, such as acne, hyperpigmentation, and issues like sensitivity [163,207]. Smart enzyme-based cosmetics, enzyme diagnostic tools (i.e., biosensors) [208], and AI-powered skin analysis [209], can adapt to environmental changes like temperature or pollution level to optimize skin renewal. Companies are currently shifting their focus towards DNA-based skincare recommendations, where specific enzymes facilitate unique skin needs [163,207]. Recombinant DNA technology, where genetically engineered microorganisms generate bromelain, ficin, and papain enzymes under controlled, low-impact conditions, has offered high proteolytic enzyme thermal stability, high enzyme yield, and significant proteolytic activity [3,17]. Enzyme immobilization is also promising in extending usability and reducing waste in cosmetic products [93,115,116].

Sustainability and ethical sourcing are also becoming increasingly important in the cosmetic industry. Biotechnology offers innovative methods for genetically producing enzymes via microbial fermentation (i.e., engineered bacteria and fungi), reducing environmental impact compared to traditional extraction methods [210]. Additionally, the shift towards cruelty-free and vegan products is fueling the use of plant-based proteolytic enzymes like bromelain, ficin, and papain, which aligns with such ethical considerations [121]. Their production parameters can be optimized to minimize waste; for instance, by using agricultural by-products (like pineapple stems for bromelain, or papaya or fig peels for papain and ficin) as raw materials, the industry can reduce waste and utilize materials that would otherwise be discarded, enhancing the concept of the circular economy [91,101,190]. Many companies in the cosmetic industry currently integrate circular economy principles, including this, by using by-products [158].

Another strategy to align with the circular economy is developing stable enzyme formulations that maintain their activity over longer periods and are able to reduce the need for frequent repurchases, at the same time minimizing packaging waste. Advanced encapsulation techniques can shield enzymes from degradation, extending the shelf-life of cosmetic products. Also, proteolytic enzymes generate biodegradable, non-toxic waste (i.e., amino acids, peptides, and simple sugars), which does not accumulate in the environment, and offers a promising alternative to conventional chemical exfoliants, like microplastics, thereby reducing long-term pollution [22]. Future skincare products are likely to combine multiple proteolytic enzymes to harness their synergistic effects, providing enhanced exfoliation and further benefits like anti-inflammatory, antioxidant, and moisturizing properties [22]. Plus, a combination of proteolytic enzymes with antioxidants, peptides, and probiotics could enhance skin barrier function and hydration, as they could be integrated into hybrid products, like enzyme-infused moisturizers, serums, and masks, offering multifunctional skincare solutions, all in a single product [211].

Moreover, each enzyme’s stability and activity may be optimized through pH, buffering, encapsulation, or pairing with stabilizers, as pH-responsive enzyme activation and time-release enzyme formulations may revolutionize skincare applications and offer adaptation to several skin types and conditions [92,118,163,212]. Enzyme stability is one of the biggest challenges, since their effectiveness heavily depends on this. Future developments should additionally focus on delivery systems to enhance enzyme performance, extend their shelf-life, and optimize their activity during application. Nanotechnology enables the creation of advanced delivery systems that protect enzymes from degradation and enhance their skin penetration ability. Encapsulation methods, such as liposomes, microencapsulation, and polymeric nanoparticles, stabilize enzymes, regulate their release, and increase their cosmetic efficacy [57,92,118,172,175,177,212].

Regenerative skincare is another very promising area for proteolytic enzymes. Such enzymes are being explored for their potential in anti-aging applications, since they promote collagen production and aid in the acceleration cell turnover for a firmer, more youthful complexion with reduced wrinkles [213]. Additionally, the wound healing properties of certain proteolytic enzymes, like bromelain and papain, are harnessed in medical skincare therapies, aiming at repairing and rejuvenating damaged skin, concurrently preventing infections and healing wounds [147,159,161]. Proteolytic enzymes skincare is not still only limited to existing enzymes. Advances in biotechnology and bioinformatics are facilitating the discovery of novel enzymes from diverse natural sources, such as extreme environments including deep-sea vents and hot springs as well as unexplored plant species to identify enzymes with unique properties that are suitable for skincare products. CRISPR-Cas9 technology allows for modifications in enzyme genes and directed evolution could evolve enzymes with desired traits [214].

The future of proteolytic enzymes in cosmetics is bright, with emerging trends focusing on personalization, sustainability, multifunctionality, and advanced delivery. By integrating biotechnology, nanotechnology, and dermatological research, proteolytic enzymes are set to redefine skincare and offer solutions that are not only more effective, but also eco-friendly, gentle, and tailored to individual needs, meeting the evolving demands of consumers and cosmetic industries. However, more toxicological studies and long-term safety assessments, regulatory approval, and consumer education must be conducted to develop clear guidelines on enzyme-based exfoliation and skincare [17,94,163].

13. Conclusions

The study investigated the proteolytic activity of three important enzymes in cosmetics: bromelain (pineapple), ficin (fig), and papain (papaya). Bromelain and papain are highly effective exfoliants, while ficin offers a gentler approach, suitable for sensitive skin. Papain is slightly stronger in some formulations, while ficin offers a milder exfoliation. The properties of each enzyme allow for targeted skincare solutions to combat issues such as dullness, hyperpigmentation, and uneven skin texture. Skin safety is critical, especially for sensitive skin, where over-exfoliation and irritation must be avoided. Stabilizers, pH buffers, and soothing agents help to minimize the risks. Sustainability is also important. The use of fruit by-products such as pineapple peel and fig and papaya latex is in line with the circular economy principles.

Compared to chemical exfoliants such as AHAs and BHAs, proteolytic enzymes provide controlled exfoliation by breaking down keratin proteins without causing irritation or post-inflammatory hyperpigmentation. While proteolytic enzymes are useful, they may cause allergic reactions in individuals who are sensitive to pineapple, fig latex, or papaya extracts. Patch testing and controlled formulations are necessary to ensure skin tolerance and avoid adverse reactions. Improving the stability of enzymes in skin care products requires advanced techniques such as enzyme immobilization, cross-linking, and coating with hybrid polymers to extend shelf life, while maintaining efficacy. Future research should focus on improving enzyme stability, exploring synergies with bioactivity, and evaluating the long-term effects on skin health. Clinical studies are essential to validate their safety for different skin types and ensure sustainable integration into modern cosmetic formulations. Advances in biotechnology and personalized skincare will further establish these enzymes as versatile, eco-friendly solutions that meet a wide range of skincare needs, while meeting the consumer demand for natural, gentle, and effective products.