Starch- and carboxymethyl cellulose-based films as active beauty masks with papain incorporation.

This work aimed to evaluate the effects of the incorporation of papain in biopolymeric-based beauty face masks with exfoliation activity of the skin. The masks were produced by casting with starch and carboxymethyl cellulose blend (50:50 weight percentages). The macro and microstructure, protein distribution, thickness, moisture content, water contact angle, solubility matter, and mechanical properties were evaluated. Moreover, the in vitro proteolytic and exfoliation activity and storage stability were also evaluated. The films with papain had a more concise matrix which provided higher mechanical properties and lower water solubility when compared to the control film (without papain). Films with 1, 2, and 5 % of papain had enzymatic activity for casein and porcine skin substrates. The micrographs of porcine skin treated with 2 and 5 % of papain showed more difference when compared to the control sample, indicating the enzymatic exfoliation. Differently from the solution of papain, the enzyme that was immobilized in the films maintained its activity for up 90 days during the storage stability assay. Based on the physicochemical properties and proteolytic activities, the films preserved the exfoliation activity of papain and have interesting characteristics to act as beauty masks in the cosmetic field.

In vitro effect on the proteolytic activity of papain with proteins of the skin as substrate.

OBJECTIVE: This work aims to evaluate the effects of enzyme concentration, pH, temperature and time course degree of hydrolysis (DH) of papain regarding further development of pharmaceutical and cosmetic formulations. METHODS: The hydrolysis of casein, collagen, keratin and porcine skin at pH and temperature ranges of the human skin was evaluated. Also, low contact times of enzyme-substrate were studied. The incorporation of 3mM of cysteine improved the caseinolytic (PU), collagenolytic (CU) and keratinolytic (KU) activities of papain. RESULTS: In general, the increase from 0.1 to 1.0 or 2.0 mgmL-1 of papain improved PU, CU and KU. When 2.0 mgmL-1 of papain was used, the highest DH of casein, collagen and keratin was obtained at 240min (14%, 35% and 6% respectively). The decrease in pH and temperature reduced all proteolytic activities, but papain maintained at least 50% and 40% of its activity at 26°C and pH  4.5 respectively. Scanning electron micrographs of the surface of the skin showed that papain application had exfoliating activity. CONCLUSION: This preformulation study demonstrated that papain concentration, time of application and pH of the product should be evaluated when developing a product to promote the hydrolysis of the proteins of the skin.

OBJECTIF: Ce travail vise à évaluer les effets de la concentration d'enzyme, du pH, de la température et du degré d'hydrolyse (DH) de la papaïne concernant le développement ultérieur de formulations pharmaceutiques et cosmétiques. MÉTHODES: L'hydrolyse de la caséine, du collagène, de la kératine et de la peau porcine à des plages de pH et de température de la peau humaine a été évaluée. De plus, des faibles temps de contact enzyme-substrat ont été étudiés. L'incorporation de 3mM de cystéine a amélioré les activités caséinolytiques (PU), collagénolytiques (CU) et kératinolytiques (KU) de la papaïne. RÉSULTATS: En général, l'augmentation de 0,1 à 1,0 ou 2,0 mg.mL−1 de papaïne a amélioré la PU, la CU et la KU. Lorsque 2,0 mg mL−1 de papaïne ont été utilisés, les DH les plus élevées de caséine, de collagène et de kératine ont été obtenues à 240 min (14, 35 et 6 %, respectivement). La diminution du pH et de la température a réduit toutes les activités protéolytiques, mais la papaïne a maintenu au moins 50 et 40 % de son activité à 26 °C et pH 4,5, respectivement. Des micrographies obtenues par microscopie électronique à balayage de la surface de la peau ont montré que l'application de la papaïne avait une activité exfoliante. CONCLUSION: Cette étude de pré-formulation a démontré que la concentration de papaïne, le temps d'application et le pH du produit doivent être évalués lors du développement d'un produit pour favoriser l'hydrolyse des protéines de la peau.

An overview of the use of proteolytic enzymes as exfoliating agents.

BACKGROUND: Proteolytic enzymes are biological catalysts that can compose cosmetic formulations: These enzymes are capable of mimicking the desquamation process of the skin, acting as exfoliants. Although enzymatic exfoliation is not new and commercial products were easily found, there is a lack of scientific literature about this topic. METHODS: A search was carried out until 2021 in different scientific databases (Web of Science, Scopus, Scielo, PubMed, etc.). In vitro and in vivo studies that evaluated the application of enzymes aiming to exfoliate the skin or with a similar cosmetic or dermatological application were selected. RESULTS: Only 11 articles were found, and, among them, few studies applied enzymes as exfoliants in clinical trials. Nevertheless, the results demonstrate that the enzymes can exfoliate the skin and improve some desired characteristics of the organ. Papain, bromelain, keratinases, and microbial proteases are some enzymes already applied as exfoliants. The study of pH, temperature, and stabilization of the enzymes in cosmetic formulations were also demonstrated to be important aspects to be evaluated, principally in preventing loss of enzyme activity and possible allergens/irritations on the skin. CONCLUSION: This literature review showed the main aspects that should be evaluated before considering producing or applying proteolytic enzymes in exfoliation products/procedures. The use of enzymatic exfoliation has potential in the cosmetic industry. Hence, further robust in vivo studies are needed before the enzymatic exfoliation can be recommended with safety as a treatment modality in the current conditions.

Optimization, kinetic, and scaling-up of solvent-free lipase-catalyzed synthesis of ethylene glycol oleate emollient ester.

The use of enzymatic catalysts is an alternative to chemical catalysts as they can help to obtain products with less environmental impact, considered sustainable within the concept of green chemistry. The optimization, kinetic, lipase reuse, and scale-up of enzymatic production of ethylene glycol oleate in the batch mode were carried out using the NS 88011 lipase in a solvent-free system. For the optimization step, a 23 Central Composite Design was used and the optimized condition for the ethylene glycol oleate production, with conversions above 99%, was at 70 °C, 600 rpm, substrates molar ratio of 1:2, 1 wt% of NS 88011 in 32 H of reaction. Kinetic tests were also carried out with different amounts of enzyme, and it showed that by decreasing the amount of the enzyme, the conversion also decreases. The lipase reuse showed good conversions until the second cycle of use, after which it had a progressive reduction reaching 83% in the fourth cycle of use. The scale-up (ninefold increase) showed promising results, with conversion above 99%, achieving conversions similar to small-scale reactions. Therefore, this work proposed an environmentally safe route to produce an emollient ester using a low-cost biocatalyst in a solvent-free system.

Enzymatic pretreatment and anaerobic co-digestion as a new technology to high-methane production.

The population growth is causing an increase in the generation of effluents (mainly organic fraction of municipal solid waste (OFMSW) and agro-industrial waste), which is an old problem in agro-industrial countries such as Brazil. Contrastingly, it is possible to add value to these residual biomasses (residues) through the application of new technologies for the production of bioenergy. Anaerobic digestion (AD) of sewage sludge is being applied in many effluent treatment plants for the sustainable and economically viable production of biogas. However, the biogas produced from AD (sludge) or co-digestion (sludge with other residues) presents a concentration of methane between 60 and 70% on average, which is relatively low. This review is aimed at analyzing studies involving (i) production of lipases by solid-state fermentation (SSF) by different microorganisms for the application in enzymatic pretreatments prior to the anaerobic treatment of effluents; (ii) pretreatment followed by AD of various residues, with an emphasis on OFMSW and sewage sludge; and (iii) more recent studies on anaerobic co-digestion (AcoD) and hybrid technologies (pretreatment + AD or AcoD). There are many studies in the literature that demonstrate the enzymatic pretreatment or AcoD applied to the optimization of methane production. Nevertheless, few studies report the combination of these two technologies, which can improve the process and reduce or eliminate the costs of biogas purification, which are major challenges for the viability of this route of bioenergy production. KEY POINTS: • Municipal and agro-industrial wastes have potential as medium for lipase production. • Enzymatic pretreatment and anaerobic co-digestion are low cost for high-methane production. Graphical abstract Interactions among various factors optimization methane production from enzymatic pretreatment and AcoD.

Lipase-Catalyzed Esterification of Geraniol and Citronellol for the Synthesis of Terpenic Esters.

This article describes the synthesis of terpenic esters derived from geraniol and citronellol (geranyl and citronellyl alkanoates) through esterification reactions catalyzed by the immobilized lipases from Thermomyces lanuginosus (Lipozyme TL IM®) and Candida antarctica (Novozym 435®). Geraniol was esterified with oleic, lauric, and stearic acids; and citronellol was esterified with oleic and stearic acids. For all the synthesized flavor esters, the best conditions were 35 °C, and the molar ratio between acid and alcohol was 1:1. Geranyl and citronellyl alkanoates reached yields between 80-100% within 4 h of reaction. For the synthesis of the citronellyl and geranyl oleate, higher yields were obtained in the absence of organic solvents. For the esters from lauric and stearic acids, using solvent was indispensable to improve the miscibility between the substrates. The reuse of Novozym 435® and Lipozyme TL IM® was performed for two more cycles after the first use, with yields higher than 60%. The results demonstrated the efficiency of the reaction catalyzed by these two commercial enzymes and the feasibility of the methodology for the production of synthetic flavor esters through enzymatic catalysis. The flavor esters synthesized were not described in the literature up to the date, giving this research an innovative feature.

Toxicity of enzymatically decolored textile dyes solution by horseradish peroxidase.

The oxidative systems including enzymatic systems have been widely studied as an alternative for textile effluents treatment. However, studies have shown that some oxidative processes can produce degradation products with higher toxicity than the untreated dye. In this work, enzymatic dye decolorization was evaluated by horseradish peroxidase enzyme (HRP) and the toxicity of discoloration products was evaluate against Daphnia magna, Euglena gracilis algae, and Vibrio fischeri. Dye decolorization kinetics data were evaluated and the pseudo-second-order model showed the best-fitting to the experimental data. In addition, it was observed an increased acute and chronic toxicity associated with the decolorization efficiency. The Reactive Blue 19 and Reactive Black dye showed the highest toxicity against D. Magna (16 toxicity factor) and V. Fischeri (32 toxicity factor) after enzymatic decolorization. For the chronic toxicity against D. Magna, Reactive Red was the only dye with no fertility inhibition. In relation to toxicity tests with E. gracilis algae, it was not observed photosynthetic inhibition for all dyes. This study verified the viability of the enzyme horseradish peroxidase in the textile dyes decolorization and the importance to evaluate the decolorization products.

Study of a reactor model for enzymatic reactions in continuous mode coupled to an ultrasound bath for esters production.

The main objective of this work was to investigate the enzymatic production of ethyl esters in continuous mode coupled to an ultrasound bath. For esterification reactions soybean free fatty acids (FFA) and ethanol were used as substrates, and Novozym 435 and Lipozyme TL IM, as catalysts. The experimental system consisted of a packed bed glass reactor immersed in an ultrasound bath and the following variables were studied: ethanol to FFA molar ratio of 1:1, 1:3 and 1:6, substrates flow rate of 2.5 and 5 mL/min, at 65 °C and 132 W ultrasound power output. It was noticed that the excess alcohol favored the esterification reaction with the best conversions observed, 95%, at 6 min reaction for 1:6 FFA to ethanol molar ratio, 2.5 mL/min feeding flow rate. Increasing the substrates feeding flow rate decreased the conversion down to 87% and replacing the Novozym 435 by Lipozyme TL IM no satisfactory conversions were achieved. This type of reactor demonstrated good performance for ethyl esters production, opening promising perspectives for use in the production of other types of esters such as those aromatic and others with high value.

Immobilization of laccase from Aspergillus oryzae on graphene nanosheets.

Laccase enzymes of Aspergillus oryzae were immobilized on graphene nanosheets by physical adsorption and covalent bonding. Morphological features of the graphene sheets were characterized via microscopy techniques. The immobilization by adsorption was carried out through contact between graphene and solution of laccase enzyme dissolved in deionized water. The adsorption process followed a Freundlich model, showing no tendency to saturation within the range of values used. The process of immobilization by covalent bonding was carried out by nitration of graphene, followed by reduction of sodium borohydride and crosslinking with glutaraldehyde. The process of immobilization by both techniques increased the pH range of activity of the laccase enzyme compared to the free enzyme and increased its operating temperature. On operational stability, the enzyme quickly loses its activity after the second reaction cycle when immobilized via physical adsorption, while the technique by covalent bonding retained around 80% activity after six cycles.

Transfer and consumption of oxygen during the cultivation of the ectomycorrhizal fungus Rhizopogon nigrescens in an airlift bioreactor.

The study had the objective of examining the aspects involved in the cultivation of ectomycorrhizal fungi for the production of commercially sustainable inoculant to attend the demands of the seedling nursery industry. It focused on certain parameters, such as the oxygen consumption levels, during the cultivation of the ectomycorrhizal fungus Rhizopogon nigrescens CBMAI 1472, which was performed in a 5-L airlift bioreactor. The dynamic method was employed to determine the volumetric coefficient for the oxygen transfer (k L a) and the specific oxygen uptake rate (Q O2 ). The results indicate that specific growth rates (µ X ) and oxygen consumption decline rapidly with time, affected mainly by increases in biomass concentration (X). Increases in X are obtained primarily by increases in the size of pellets that are formed, altering, consequently, the cultivation dynamics. This is the result of natural increases in transferring resistance that are observed in these environments. Therefore, to avoid critical conditions that affect viability and the productivity of the process, particular settings are discussed.

Modeling the Growth of Filamentous Fungi at the Particle Scale in Solid-State Fermentation Systems.

Solid-state fermentation (SSF) with filamentous fungi is a promising technique for the production of a range of biotechnological products and has the potential to play an important role in future biorefineries. The performance of such processes is intimately linked with the mycelial mode of growth of these fungi: Not only is the production of extracellular enzymes related to morphological characteristics, but also the mycelium can affect bed properties and, consequently, the efficiency of heat and mass transfer within the bed. A mathematical model that describes the development of the fungal mycelium in SSF systems at the particle scale would be a useful tool for investigating these phenomena, but, as yet, a sufficiently complete model has not been proposed. This review presents the biological and mass transfer phenomena that should be included in such a model and then evaluates how these phenomena have been modeled previously in the SSF and related literature. We conclude that a discrete lattice-based model that uses differential equations to describe the mass balances of the components within the system would be most appropriate and that mathematical expressions for describing the individual phenomena are available in the literature. It remains for these phenomena to be integrated into a complete model describing the development of fungal mycelia in SSF systems.

Effects of nitrate injection on microbial enhanced oil recovery and oilfield reservoir souring.

Column experiments were utilized to investigate the effects of nitrate injection on sulfate-reducing bacteria (SRB) inhibition and microbial enhanced oil recovery (MEOR). An indigenous microbial consortium collected from the produced water of a Brazilian offshore field was used as inoculum. The presence of 150 mg/L volatile fatty acids (VFA´s) in the injection water contributed to a high biological electron acceptors demand and the establishment of anaerobic sulfate-reducing conditions. Continuous injection of nitrate (up to 25 mg/L) for 90 days did not inhibit souring. Contrariwise, in nitrogen-limiting conditions, the addition of nitrate stimulated the proliferation of δ-Proteobacteria (including SRB) and the associated sulfide concentration. Denitrification-specific nirK or nirS genes were not detected. A sharp decrease in water interfacial tension (from 20.8 to 14.5 mN/m) observed concomitantly with nitrate consumption and increased oil recovery (4.3 % v/v) demonstrated the benefits of nitrate injection on MEOR. Overall, the results support the notion that the addition of nitrate, at this particular oil reservoir, can benefit MEOR by stimulating the proliferation of fortuitous biosurfactant-producing bacteria. Higher nitrate concentrations exceeding the stoichiometric volatile fatty acid (VFA) biodegradation demands and/or the use of alternative biogenic souring control strategies may be necessary to warrant effective SRB inhibition down gradient from the injection wells.

Kinetics of ultrasound-assisted lipase-catalyzed glycerolysis of olive oil in solvent-free system.

This work reports experimental kinetic data of solvent-free glycerolysis of olive oil using a commercial immobilized lipase (Novozym 435) under the influence of ultrasound irradiation. The experiments were performed in a mechanically stirred reactor under ultrasound irradiation, evaluating the effects of temperature (50-70 °C), enzyme concentration (2.5-10 wt%) and glycerol to oil molar ratio (0.8:1-3:1). Results show that ultrasound-assisted lipase-catalyzed glycerolysis might be a potential alternative route to conventional methods, as high contents of reaction products, especially monoglycerides, were achieved at mild irradiation power supply (~130 W) and temperature, in a relatively short reaction time (2h) and low enzyme content (7.5 wt%). To completeness, two simplified kinetic modeling approaches, based on the ordered-sequential bi bi mechanism and reaction stoichiometry, were employed to represent the experimental data, thus allowing a better understanding of the reaction kinetics.

Ultrasound irradiation promoted efficient solvent-free lipase-catalyzed production of mono- and diacylglycerols from olive oil.

This work reports the enzymatic production of mono- and diacylglycerols under the influence of ultrasound irradiation, in a solvent-free system, with and without the presence of surfactants at a constant temperature of 65°C, glycerol to oil molar ratio of 2:1 and a commercial immobilized lipase (Novozym 435) as catalyst. For this purpose, two operation modes were adopted: the use of a sonotrode (ultrasonic probe), without agitation, varying reaction time, irradiation amplitude (25-45% of the total power) and type of surfactant, and a mechanically stirred reactor (600 rpm) under ultrasound irradiation in a water bath, testing different surfactants. Results show that very satisfactory MAG and DAG yields, above 50 wt.%, can be obtained without the use of surfactant, at mild irradiation power supply (∼130 W), with no important enzyme activity losses verified, in a relatively short reaction time (2h), and low enzyme content (7.5 wt.%). Also, reaction kinetic results show that contents of MAG+DAG as high as ∼65 wt.% can be achieved at longer times (6h), indicating a promising route for producing MAG and DAG using ultrasound irradiation.