Search Results (37)

Rapeseed meal, a byproduct of oil extraction, is increasingly recognised as a valuable source of plant protein and health-promoting peptides. This study aimed to identify key proteins in cold-pressed rapeseed meal and assess their potential to release bioactive peptides through in silico hydrolysis using plant-derived proteases, namely papain, bromelain, and ficin. Proteomic profiling via two-dimensional electrophoresis and MALDI-TOF/TOF mass spectrometry revealed cruciferin as the dominant protein, along with other metabolic and defence-related proteins. In silico digestion of these sequences using the BIOPEP database generated thousands of peptide fragments, of which over 50% were predicted to exhibit bioactivities, including ACE and DPP-IV inhibition, as well as antioxidant, neuroprotective, and anticancer effects. Among the evaluated enzymes, bromelain exhibited the highest efficacy, yielding the greatest quantity and diversity of bioactive peptides. Notably, peptides with antihypertensive and antidiabetic properties were consistently identified across all of the protein and enzyme variants. Although certain rare functions, such as anticancer and antibacterial activities, were observed only in specific hydrolysates, their presence underscores the broader functional potential of peptides derived from rapeseed. These findings highlight the potential of rapeseed meal as a sustainable source of functional ingredients while emphasising the necessity for experimental validation to confirm the predicted bioactivities. Full article

The investigation of structure–function relationships in enzyme polysaccharide complexes provides a theoretical foundation for modulating enzyme properties and expanding their industrial applications. In this study, the interaction of cysteine proteases—bromelain, ficin, and papain—with a grafted chitosan–poly(N-vinylpyrrolidone) copolymers, Cs-g-PVP, was examined, and its effect on the catalytic and stability properties of the enzymes was assessed. Molecular docking and Fourier-transform infrared spectroscopy were used to analyze the topology of the resulting complexes and identify macromolecular fragments involved in binding. Based on the obtained results, it was hypothesized that complex formation would lead to a slight reduction in the catalytic activity of cysteine proteases. In vitro studies of the complexes confirmed this hypothesis, showing that the enzymes retained more than 63% of their proteolytic activity while their half-inactivation time during storage increased by up to ~12-fold. The investigated Cs-g-PVP copolymers demonstrated high efficiency as supports for the studied enzymes, capable of retaining up to 100% of the added enzymes. Full article

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. Full article

This study explores various methods for the covalent immobilization of cysteine proteases (ficin, papain, and bromelain). Covalent immobilization involves the formation of covalent bonds between the enzyme and a carrier or between enzyme molecules themselves without a carrier using a crosslinking agent. This process enhances the stability of the enzyme and allows for the creation of preparations with specific and controlled properties. The objective of this study is to evaluate the impact of covalent immobilization under different conditions on the proteolytic activity of the enzymes. The most favorable results were achieved by immobilizing ficin and bromelain through covalent bonding to medium and high molecular weight chitosans, using 5 and 3.33% glutaraldehyde solutions, respectively. For papain, 5 and 6.67% glutaraldehyde solutions proved to be more effective as crosslinking agents. These findings indicate that covalent immobilization can enhance the performance of these enzymes as biocatalysts, with potential applications in various biotechnological fields. Full article

Lupin (Lupinus angustifolius L.) proteins are potential sources of bioactive peptides (LBPs) that can inhibit dipeptidyl peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE-I) activity. However, the capacity of different enzymes to release LBPs, the pharmacokinetic and bioactivities of the peptides released, and their binding affinities with the active sites of DPP-IV and ECA-I are topics scarcely addressed. Therefore, we used in silico hydrolysis (BIOPEP-UWM platform) with various enzymes to predict the release of LBPs. Among the bioactive peptides identified in lupin proteins (n = 4813), 2062 and 1558 had DPP-IV and ACE-I inhibitory activity, respectively. Ficin, bromelain, and papain released the highest proportion of ACE-I (n = 433, 411, and 379, respectively) and DPP-IV (n = 556, 544, and 596, respectively) inhibitory peptides. LBPs with favorable pharmacokinetics and gastrointestinal stability tightly interacted with the active sites of ACE-I (–5.6 to –8.9 kcal/mol) and DPP-IV (–5.4 to –7.6 kcal/mol). Papain generated the most bioavailable LBPs (n = 459) with ACE-I (n = 223) and DPP-IV (n = 412) inhibitory activity. These peptides were non-toxic and gastrointestinal digestion stable. Notably, papain-based hydrolysis released some LBPs (n = 270) that inhibited both ACE-I and DPP-IV. Plant protease-based hydrolysis is a promising approach for producing lupin hydrolysates with ACE-I and DPP-IV inhibitory activities. Full article

Micro- and nanoparticles of chitosan and carboxymethyl chitosan were synthesized, both with and without ascorbic acid. Methods were developed to form complexes between these micro- and nanoparticles and plant proteases—ficin, papain, and bromelain. It was demonstrated that the activity of cysteine protease complexes with carboxymethyl chitosan micro- and nanoparticles was higher compared to those with chitosan micro- and nanoparticles. Additionally, the complexes of ficin, papain, and bromelain with chitosan and carboxymethyl chitosan micro- and nanoparticles synthesized in the presence of ascorbic acid exhibited greater proteolytic activity than those formed with particles prepared without ascorbic acid. Molecular docking studies revealed that the amino acid residues of ficin, papain, and bromelain primarily interact with chitosan and carboxymethyl chitosan through hydrogen bonding and hydrophobic interactions. The amino acid residues in the active sites of these enzymes participate in a complex formation, which likely contributes to the increased activity and stability of cysteine proteases in complexes with chitosan and carboxymethyl chitosan micro- and nanoparticles. Full article

Plant-based foods offer a sustainable alternative to meet the growing protein demand. Legumes are the most promising of these, as they contain relatively high concentrations of protein, low digestible starch, and dietary fiber, as well as them possibly featuring low levels of fat. Enzymatically modified legume proteins provide us with tempting perspectives in terms of enhancing foods’ biological values. However, their bioavailability and digestibility are generally less sufficient than that of proteins of animal origin, which may be improved by well-tailored enzyme modification. In this study, the efficacy of three plant-based proteases (bromelain, ficin, and papain) were evaluated at two distinct concentrations (2.5% and 10%) and three hydrolysis durations (1, 2, and 12 h) when transforming chickpea and lentil proteins. The degree of hydrolysis (DH), peptide profiles, and free amino acid content were analyzed to determine the efficiency of each enzyme. Results showed significant variations in DH, which was influenced by enzyme type, concentration, and hydrolysis duration. Papain exhibited the highest DH, particularly at a 10% concentration, reaching 27.8% efficiency in chickpea and 34.8% in lentils after 12 h. Bromelain and ficin were proven to be less effective, with ficin showing the least hydrolytic activity. SDS-PAGE analysis revealed substantial protein degradation, especially subsequent to papain treatment, pointing out that most proteins were cleaved into smaller peptides. SEC-HPLC indicated a predominant release of peptides within the 200–1000 Da range, suggesting enhanced bioavailability. Papain and bromelain treatments resulted in a significant release of oligopeptides and dipeptides. UHPLC analysis highlighted a marked post-hydrolysis increase in total free amino acids, with arginine, leucine, and lysine being the most abundant ones. Notably, tryptophan, being undetectable in untreated samples, was released in measurable amounts post-hydrolysis. These findings demonstrate papain’s superior performance in protein hydrolysis and its potential in producing bioactive peptides, highlighting its applicability in food processing and the development of both nutraceuticals and functional foods. Full article

It has been reported that the modification of immobilized glyoxyl–ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl–ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking). Full article

The immune response of humans may be modulated by certain biopeptides. The present study aimed to determine the immunomodulatory potential of plant-derived food proteins and hydrolysates obtained from these proteins via monocatalytic in silico hydrolysis (using ficin, stem bromelainm or pepsin (pH > 2)). The scope of this study included determinations of the profiles of select bioactivities of proteins before and after hydrolysis and computations of the frequency of occurrence of selected bioactive fragments in proteins (parameter A), frequency/relative frequency of the release of biopeptides (parameters A_E, W) and the theoretical degree of hydrolysis (DH_t), by means of the resources and programs available in the BIOPEP-UWM database. The immunomodulating (ImmD)/immunostimulating (ImmS) peptides deposited in the database were characterized as well (ProtParam tool). Among the analyzed proteins of cereals and legumes, the best precursors of ImmD immunopeptides (YG, YGG, GLF, TPRK) turned out to be rice and garden pea proteins, whereas the best precursors of ImmS peptides appeared to be buckwheat (GVM, GFL, EAE) and broad bean (LLY, EAE) proteins. The highest number of YG sequences was released by stem bromelain upon the simulated hydrolysis of rice proteins (AE = 0.0010–0.0820, W = 0.1994–1.0000, DH_t = 45–82%). However, antibacterial peptides (IAK) were released by ficin only from rice, oat, and garden pea proteins (DH_t = 41–46%). Biopeptides (YG, IAK) identified in protein hydrolysates are potential immunomodulators, nutraceuticals, and components of functional food that may modulate the activity of the human immune system. Stem bromelain and ficin are also active components that are primed to release peptide immunomodulators from plant-derived food proteins. Full article

While fibrinolytic enzymes and thrombolytic agents offer assistance in treating cardiovascular diseases, the existing options are associated with a range of adverse effects. In our previous research, we successfully identified ficin, a naturally occurring cysteine protease that possesses unique fibrin and fibrinogenolytic enzymes, making it suitable for both preventing and treating cardiovascular disorders linked to thrombosis. Papain is a prominent cysteine protease derived from the latex of Carica papaya. The potential role of papain in preventing fibrino(geno)lytic, anticoagulant, and antithrombotic activities has not yet been investigated. Therefore, we examined how papain influences fibrinogen and the process of blood coagulation. Papain is highly stable at pH 4–11 and 37–60 °C via azocasein assay. In addition, SDS gel separation electrophoresis, zymography, and fibrin plate assays were used to determine fibrinogen and fibrinolysis activity. Papain has a molecular weight of around 37 kDa, and is highly effective in degrading fibrin, with a molecular weight of over 75 kDa. Furthermore, papain-based hemostatic performance was confirmed in blood coagulation tests, a blood clot lysis assay, and a κ-carrageenan rat tail thrombosis model, highlighting its strong efficacy in blood coagulation. Papain shows dose-dependent blood clot lysis activity, cleaves fibrinogen chains of Aα, Bβ, and γ-bands, and significantly extends prothrombin time (PT) and activated partial thromboplastin time (aPTT). Moreover, the mean length of the infarcted regions in the tails of Sprague–Dawley rats with κ-carrageenan was shorter in rats administered 10 U/kg of papain than in streptokinase-treated rats. Thus, papain, a cysteine protease, has distinct fibrin and fibrinogenolytic properties, suggesting its potential for preventing or treating cardiovascular issues and thrombosis-related diseases. Full article

In the last decade, Ficin, a proteolytic enzyme extracted from the latex sap of the wild fig tree, has been widely investigated as a promising tool for the treatment of microbial biofilms, wound healing, and oral care. Here we report the antibiofilm properties of the enzyme immobilized on soluble carboxymethyl chitosan (CMCh) and CMCh itself. Ficin was immobilized on CMCh with molecular weights of either 200, 350 or 600 kDa. Among them, the carrier with a molecular weight of 200 kDa bound the maximum amount of enzyme, binding up to 49% of the total protein compared to 19–32% of the total protein bound to other CMChs. Treatment with pure CMCh led to the destruction of biofilms formed by Streptococcus salivarius, Streptococcus gordonii, Streptococcus mutans, and Candida albicans, while no apparent effect on Staphylococcus aureus was observed. A soluble Ficin was less efficient in the destruction of the biofilms formed by Streptococcus sobrinus and S. gordonii. By contrast, treatment with CMCh200-immobilized Ficin led to a significant reduction of the biofilms of the primary colonizers S. gordonii and S. mutans. In model biofilms obtained by the inoculation of swabs from teeth of healthy volunteers, the destruction of the biofilm by both soluble and immobilized Ficin was observed, although the degree of the destruction varied between artificial plaque samples. Nevertheless, combined treatment of oral Streptococci biofilm by enzyme and chlorhexidine for 3 h led to a significant decrease in the viability of biofilm-embedded cells, compared to solely chlorhexidine application. This suggests that the use of either soluble or immobilized Ficin would allow decreasing the amount and/or concentration of the antiseptics required for oral care or improving the efficiency of oral cavity sanitization. Full article

Several plant papain-like cysteine proteases are exploited by the food, cosmetic, pharmaceutical and textile industries. However, some of these enzymes can cause allergic reactions. In this context, we investigated the frequency of sensitization and allergic reactions to some fruit and/or latex cysteine proteases, which are used as additives by the food industry to improve and modify the quality of their products. The FABER test was used to analyse the patients‘ sensitization towards five plants and, for comparison, two homologous mite cysteine proteases. In an Italian population of 341 allergic patients, 133 (39%) had IgE specific for at least one of the seven cysteine proteases under investigation. Most of the patients were IgE positive for Der p 1 and/or Der f 1 (96.38%) reported a clinical history suggestive of respiratory allergy to mites, whereas none of the subjects sensitized to the homologs from papaya, pineapple and fig reported allergy symptoms following ingestion of these foods. Only one patient referred symptoms from ingesting kiwifruit. Therefore, the obtained results showed that sensitization to the fruit enzymes was only rarely concomitant with allergic reactions. These observations, together with the literature reports, suggest that the allergy to plant papain-like cysteine proteases might mainly be an occupational disease. Full article

This study investigates the features of interactions between cysteine proteases (bromelain, ficin, and papain) and a graft copolymer of carboxymethyl cellulose sodium salt with N-vinylimidazole. The objective is to understand the influence of this interactions on the proteolytic activity and stability of the enzymes. The enzymes were immobilized through complexation with the carrier. The interaction mechanism was examined using Fourier-transform infrared spectroscopy and flexible molecular docking simulations. The findings reveal that the enzymes interact with the functional groups of the carrier via amino acid residues, resulting in the formation of secondary structure elements and enzyme’s active sites. These interactions induce modulation of active site of the enzymes, leading to an enhancement in their proteolytic activity. Furthermore, the immobilized enzymes demonstrate superior stability compared to their native counterparts. Notably, during a 21-day incubation period, no protein release from the conjugates was observed. These results suggest that the complexation of the enzymes with the graft copolymer has the potential to improve their performance as biocatalysts, with applications in various fields such as biomedicine, pharmaceutics, and biotechnology. Full article

Meat processing involves different transformations in the animal muscle after slaughtering, which results in changes in tenderness, aroma and colour, determining the quality of the final meat product. Enzymatic glycolysis, proteolysis and lipolysis play a key role in the conversion of muscle into meat. The accurate control of enzymatic reactions in meat muscle is complicated due to the numerous influential factors, as well as its low reaction rate. Moreover, exogenous enzymes are also used in the meat industry to produce restructured products (transglutaminase), to obtain bioactive peptides (peptides with antioxidant, antihypertensive and gastrointestinal activity) and to promote meat tenderization (papain, bromelain, ficin, zingibain, cucumisin and actinidin). Emerging technologies, such as ultrasound (US), pulsed electric fields (PEF), moderate electric fields (MEF), high-pressure processing (HPP) or supercritical CO₂ (SC-CO₂), have been used to intensify enzymatic reactions in different food applications. This review aims to provide an overview of the enzymatic reactions taking place during the processing of meat products, how they could be intensified by using emerging technologies and envisage potential applications. Full article

Papain, bromelain, and ficin are commonly used plant proteases used for meat tenderization. Other plant proteases explored for meat tenderization are actinidin, zingibain, and cucumin. The application of plant crude extracts or powders containing higher levels of compounds exerting tenderizing effects is also gaining popularity due to lower cost, improved sensory attributes of meat, and the presence of bioactive compounds exerting additional benefits in addition to tenderization, such as antioxidants and antimicrobial effects. The uncontrolled plant protease action could cause excessive tenderization (mushy texture) and poor quality due to an indiscriminate breakdown of proteins. The higher cost of separation and the purification of enzymes, unstable structure, and poor stability of these enzymes due to autolysis are some major challenges faced by the food industry. The meat industry is targeting the recycling of enzymes and improving their stability and shelf-life by immobilization, encapsulation, protein engineering, medium engineering, and stabilization during tenderization. The present review critically analyzed recent trends and the prospects of the application of plant proteases in meat tenderization. Full article