Enhancement of caudal fin regeneration in diabetic zebrafish: An insight into proangiogenic potential of bromelain

Objectives: Diabetes mellitus is a chronic metabolic disease that is characterised by hyperglycaemia, altered lipids, carbohydrates and protein metabolism, and in the long-term, with eye, kidney, cardiovascular and neurological complications. Poor wound healing is one of the major complications faced by diabetes mellitus patients. Angiogenesis is critical for tissue regeneration and wound healing. Impaired angiogenesis may lead to poor blood flow to the wound and hence delayed wound healing. Hence, it is important to find an antidote to speed up wound healing. Hence, this study focuses on the proangiogenic potential of bromelain in the caudal fin regeneration of diabetic zebrafish. Materials and Methods: Zebrafish were divided into control, Diabetic and Diabetic + Bromelain treated groups. Diabetes mellitus was induced using multiple doses of streptozotocin (350 mg/kg b.w./i.p.) on days 1, 3, 5, 12 and 19. Glucose levels were estimated on day 21 to confirm the induction of diabetes mellitus, and then, the caudal fin was amputated. After the amputation of the caudal fin, bromelain was administered orally at a dosage of 40 mg/kg b.w. on every alternate day for 15 days. Body weight, blood glucose level, total area and percentage of fin regeneration were observed on day 36. Images were compared and areas of regeneration were analysed with the help of Image J software. One-way analysis of variance followed by Tukey’s multiple comparison tests was used to analyse the data. Results: A significant increase in the blood glucose level was observed in the diabetic group compared with control. A significant decrease in the percentage of tail regeneration and area of regeneration was observed in diabetic fishes compared to the control. Bromelain treatment has significantly increased the percentage and area of regeneration and significantly decreased the blood glucose level in the treatment group compared with the control. Conclusion: The study confirms that bromelain can promote tissue regeneration; hence, it can be used to improve wound healing, which is one of the most common complications in diabetes mellitus.

Systemic therapy with bromelain-trypsin-rutoside combination in inflammation: A narrative review of the pharmacodynamics

Inflammation and wound healing are attended by interactions of numerous cell types, mediators, cytokines, and the vascular system. Any imbalance in these interactions can set off a cascade of processes, often working in different directions, and which continue till homeostasis is restored. Systemic enzyme therapy with trypsin-bromelain-rutoside combination has long been used as dietary supplements or pharmaceuticals commercially to counter various inflammatory conditions. The clinical benefits are believed to be due to the multipronged action of the combination, which include anti-kinin, antioxidant, antiplatelet, anti-chemokine, pro-fibrinolytic, and pro-fibrotic activities; with more recent evidence indicating inhibition of pro-inflammatory signal transduction. The primary modes of action of the individual agents on different components and processes involved in inflammation have been evaluated and elucidated, using various in vitro, animal model, and clinical studies. A narrative review of the evidence collected over the years has been provided.

Action of Bromelain and Ficin on horse anti Bothrops sp venom Antibodies

Abstract The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)'2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)'2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

In vitro enzyme activity and in vivo healing activity of the protein extract from pineapple peel

The pineapple is a fruit that has wide acceptance worldwide both in natural form, as industrialized. Your peel is a residue generated by food industries and from this residue can obtain a protein extract which is a good source of bromelain. This study aimed to obtain a protein extract from pineapple peel, evaluate its enzyme activity and its healing properties in skin lesions in rats. Seven animal groups were used: control, treated with 5% of protein extract, 10% of protein extract and pure protein extract; 5% of commercial bromelain, 10% of commercial bromelain and pure commercial bromelain. The animals were subjected to a tissue incision and treated for 21 days. Proteolytic and specific activities of the protein extract were 1.30 U mg-1 and 45 x 10-3 U μg-1 and, for commercial bromelain, 1.04 U mg-1 and 6 x 10-3 U μg-1, respectively. In the histology of the lesion, there was no significant difference between the control and treated groups; however, macroscopically, the prepared topical formulations assisted in the recovery of skin lesions, providing a significant reduction in their width, in the groups treated with pure protein extract, 5 and 10% commercial bromelain, and pure bromelain.

Effect of proteolytic agents on microleakage of etch and rinse adhesive systems

Objective: this study aimed to assess the effect of treatment of phosphoric acid etched dentin surface with 5% bromelain enzyme and Nd:YAG laser prior to the use of etch and rinse adhesive systems on microleakage margins of class V composite restorations. Materials and Methods: sixty sound premolar teeth were selected. Standard class V cavities were prepared in the buccal and lingual surfaces of each teeth. Preparation in that way 1 mm of the cavity was above and 1 mm of it was below the cementoenamel junction. The teeth were then randomly divided into three groups including 20 teeth(40cavities in each gorup). Two ethanol base etch and rinse system (Adper Single Bond and OptiBond Solo) was applied in each group. In the control group, cavities were etched with 37% phosphoric acid. Adper Single Bond was applied to 20 and OptiBond Solo was applied to the remaining 20 cavities and they were restored with Z250 composite (n=40). The same procedures were performed in bromelain and Nd:YAG laser groups with the difference that prior to the application of adhesive, aqueous 5% bromelain was applied in bromelain group while Nd:YAG laser was irradiated in laser group. All teeth were thermocycled and their apices were sealed with sticky wax. The tooth surfaces were coated with nail varnish except for 1 mm around the restoration margins, and the teeth were then immersed in fuchsine and sectioned by a diamond disc into mesial and distal halves. Marginal microleakage at both sides was determined and scored under a stereomicroscope at x40 magnification. Then data were analyzed using the Kruskal Wallis and Mann Whitney tests (α=0.05). Results: according the result of Mann Whitney test, Cervical margins showed significantly higher microleakage than the occlusal margins (P=0.000) but no significant difference was noted in microleakage of occlusal (P=0.362) or gingival (P=0.147) margins among the three groups by Kruskal Wallis test. Conclusion: in conclusion, application of proteolytic agents(5% bromelain solution and Nd:YAG laser) on acid-etched dentin surface prior to the application of adhesive has no significant effect on marginal microleakage of class V composite restorations (AU)

Enzyme Therapy: Current Perspectives.

Enzymes control all metabolic processes in human system from simple digestion of food to highly complex immune response. Physiological reactions occuring in healthy individuals are disturbed when enzymes are deficient or absent. Enzymes are administered for normalizing biological function in certain pathologies. Initially, crude proteolytic enzymes were used for the treatment of gastrointestinal disorders. Recent advances have enabled enzyme therapy as a promising tool in the treatment of cardiovascular, oncological and hereditary diseases. Now, a spectrum of other diseases are also covered under enzyme therapy. But, the available information on the use of enzymes as therapeutic agents for different diseases is scanty. This review details the enzymes which have been used to treat various diseases/disorders.

Bromelain and cardiovascular risk factors in diabetes: An exploratory randomized, placebo controlled, double blind clinical trial / 中国结合医学杂志

<p><b>OBJECTIVE</b>To assess whether the dietary supplement (bromelain) has the potential to reduce plasma fibrinogen and other cardiovascular disease (CVD) risk factors in patients with diabetes.</p><p><b>METHODS</b>This randomized placebo controlled, double blind, parallel design, efficacy study was carried out in China and investigated the effect of 12 weeks of bromelain (1,050 mg/day) on plasma fibrinogen. This randomized controlled trial (RCT) recruited 68 Chinese diabetic patients [32 males and 36 females; Han origin, mean age of 61.26 years (standard deviation (SD), 12.62 years)] with at least one CVD risk factor. Patients were randomized into either bromelain or placebo group. While bromelain group received bromelain capsule, the placebo group received placebo capsule which consisted inert ingredient and has no treatment effect. Subjects were required to take 1,050 mg (3×350 mg) of either bromelain or starch-filled placebo capsules, two to be taken (2×350 mg) after breakfast and another (350 mg) after dinner, daily for 12 weeks. Plasma fibrinogen, CVD risk factors and anthropometric indicators were determined at baseline and at 12 weeks.</p><p><b>RESULTS</b>The change in the fibrinogen level in the bromelain group at the end of the study showed a mean reduction of 0.13 g/L (standard deviation (SD) 0.86g/L) compared with the mean reduction of 0.36 g/L (SD 0.96 g/L) for the placebo group. However, there was no significant difference in the mean change in fibrinogen between the placebo and bromelain groups (mean difference=0.23g/L (SD 0.22 g/L), =0.291). Similarly, the difference in mean change in other CVD risk factors (blood lipids, blood pressure), blood glucose, C-reactive protein and anthropometric measures between the bromelain and placebo groups was also not statistically significant. Statistical differences in fibrinogen between bromelain and placebo groups before the trial despite randomization may have influenced the results of this study.</p><p><b>CONCLUSION</b>This RCT failed to show a beneficial effect in reducing fibrinogen or influencing other selected CVD risk factors but suggests other avenues for subsequent research on bromelain.</p>

Diseño asistido por computadora de la inmovilización covalente de bromelina y papaína / Computer-aided design of bromelain and papain covalent immobilization

Enzymes as immobilized derivatives have been widely used in Food, Agrochemical, Pharmaceutical and Biotechnological industries. Protein immobilization is probably the most used technology to improve the operational stability of these molecules. Bromelain (Ananas comosus) and papain (Carica papaya) are cystein proteases extensively used as immobilized biocatalyst with several applications in therapeutics, racemic mixtures resolution, affinity chromatography and others industrial scenarios. The aim of this work was to optimize the covalent immobilization of bromelain and papain via rational design of immobilized derivatives strategy (RDID) and RDID1.0 program. Were determined the maximum protein quantity to immobilize, the optimum immobilization pH (in terms of functional activity retention), was predicted the most probable configuration of the immobilized derivative and the probabilities of multipoint covalent attachment. As support material was used Glyoxyl-Sepharose CL 4B. The accuracy of RDID1.0 program´s prediction was demonstrated comparing with experimental results. Bromelain and papain immobilized derivatives showed desired characteristics for industrial biocatalysis, such as: elevate pH stability retaining 95% and 100% residual activity at pH 7.0 and 8.0, for bromelain and papain, respectively; high thermal stability at 30 °C retaining 90% residual activity for both immobilized enzymes; a catalytic configuration bonded by immobilization at optimal pH; and the ligand load achieve ensure the minimization of diffusional restrictions.

Las enzimas inmovilizadas han sido ampliamente utilizadas en las industrias Alimentaria, Agroquímica, Farmacéutica y Biotecnológica. La inmovilización de proteínas es, probablemente, la tecnología más empleada para elevar la estabilidad operacional de estas moléculas. La bromelina (Ananas comosus) y la papaína (Carica papaya) son cisteín proteasas extensamente usadas como biocatalizadores inmovilizados con disímiles aplicaciones en la terapéutica, resolución de mezclas racémicas, cromatografía de afinidad, entre otros escenarios industriales. El objetivo del presente trabajo fue optimizar la inmovilización covalente de las enzimas bromelina y papaína a través de la estrategia de diseño racional de derivados inmovilizados (RDID) y el programa RDID1.0. Se predijo la cantidad máxima de proteína a inmovilizar, el pH óptimo de inmovilización (en términos de retención de la actividad funcional), la configuración más probable del derivado inmovilizado y la probabilidad de enlazamiento covalente multipuntual. Como soporte de inmovilización de empleó Glioxil-Sepharose CL 4B. La precisión de las predicciones llevadas a cabo con el programa RDID1.0 fue validada comparando con los resultados experimentales obtenidos. Los derivados inmovilizados de bromelina y papaína mostraron características deseadas para la biocatálisis a nivel industrial, tales como: elevada estabilidad al pH reteniendo el 95% y 100% de actividad residual a pH 7.0 y 8.0, para la bromelina y la papaína, respectivamente; una elevada estabilidad térmica con la retención del 90% de actividad residual a 30 °C para ambas enzimas; al pH de inmovilización óptimo la configuración obtenida es catalíticamente competente; y la carga de ligando alcanzada asegura la disminución de las restricciones difusionales.

The influence of pH, polyethylene glycol and polyacrylic acid on the stability of stem bromelain

Enzyme stability is critical in biotechnology, pharmaceutical and cosmetic industries. Investigations on this subject have drawn attention because of its practical application. Bromelain is a thiol-endopeptidase, obtained from pineapple (Ananas comosus), known for its clinical and therapeutic applications, particularly to selective burn debridement and improvement of antibiotic action and anti-inflammatory activities. To date, the use of bromelain in pharmacological or industrial applications is limited, due to commercial availability, costs, and sensitivity to pH and temperature. Therefore, a better understanding of enzyme stability would be of great interest. The aim of this study was to evaluate bromelain activity and stability in several pH (2.0 to 8.0) and in polyethylene glycol and polyacrylic acid solutions. We observed that bromelain was able to maintain its stability at pH 5.0 for the temperatures studied. PEG solutions increased bromelain stability, but PAA solutions had the opposite effect.

Estabilidade de enzimas é uma questão fundamental em indústrias biotecnológicas, farmacêuticas e cosméticas. As investigações sobre o assunto têm chamado a atenção por sua aplicação prática. A bromelina é uma tiol-endopeptidase, obtida a partir do abacaxi (Ananas comosus). É conhecida por suas aplicações clínicas e terapêuticas, especialmente para desbridamento seletivo de queimaduras, melhoria de ações antibiótica e de atividades anti-inflamatórias. Até o momento, a utilização da bromelina em aplicações farmacológicas industriais é limitada, devido à disponibilidade comercial, os custos, a sensibilidade ao pH e temperatura. Portanto, a maior compreensão da estabilidade desta enzima seria de grande interesse. O objetivo deste estudo foi avaliar a estabilidade da atividade da bromelina em vários pH (2,0 a 8,0) e em soluções de polietilenoglicol e de ácido poliacrílico. Observamos que a bromelina foi capaz de manter a sua estabilidade em pH 5.0, em todas as temperaturas estudadas. Soluções de PEG aumentaram a estabilidade da bromelina, enquanto que soluções de PAA obtiveram efeito oposto.

Isolation and purification of bromelain from waste peel of pineapple for therapeutic application

The aim of this work was to isolate and purify bromelain extracted from the pineapple peel by ammonium sulfate precipitation (40-80%), followed by desalting and freeze-drying with a 75% activity recovery and 2.2 fold increased specific activity. Ion exchange chromatography on DEAE-Sepharose was able to separate the polysaccharides from the enzyme, which was recovered in the elution step, maintaining its enzymatic activity. The batch adsorption of bromelain was evaluated in terms of total protein and enzymatic activity using Langmuir and Langmuir-Freundlich models. Results showed that the process could be suitable for the recovery and purification of the enzyme, maintaining its specific activity.

Extração de bromelina dos resíduos de abacaxi ( Ananas comosus ) por sistemas de duas fases aquosas e sua aplicação em hidrogel polimérico / Extraction of bromelain from pineapple waste ( Ananas comosus ) by aqueous two-phase systems and its application in polymeric hydrogels

Bromelina é um nome coletivo para enzimas proteolíticas encontradas no talo, fruto e folhas do abacaxi (Ananas comosus Merr). A bromelina possui propriedades anti-inflamatórias, de debridamento, entre outras. Para a produção da bromelina deve-se, preferencialmente, usar resíduos do abacaxi, visto que os produtos do fruto têm aplicação comercial. Este trabalho teve como objetivo a extração de bromelina a partir de cascas de abacaxi através de sistema de duas fases aquosas (SDFA), e sua aplicação em hidrogel polimérico. Foram realizados estudos de estabilidade da bromelina comercial, em que se observou maior estabilidade em pH 5,0 com menor perda da atividade relativa em todas as temperaturas estudadas (20, 30, 40 e 50°C). O estudo da extração da bromelina em SDFA formado por polietileno glicol (PEG) e ácido poliacrílico (PAA) (com auxílio da análise de variância de parâmetros como rendimento, fator de purificação e coeficiente de partição) proporcionou rendimento de 335% e fator de purificação de 25,8. Os hidrogéis poliméricos à base de PEG estudados apresentaram-se flexíveis, com pouca elasticidade e taxa de absorção superior a 1000%. Hidrogel carreado de bromelina pelo método de turgescência proporcionou a maior liberação da enzima, assim como a maior atividade (80% da bromelina liberada em 24 h e 278 ± 89 U/mL)

Bromelain is a collective name for the proteolytic enzymes found in the stem, fruit and leaves of pineapple (Ananas comosus Merr.). Bromelain possesses anti-inflammatory properties, debridement, among others. For bromelain production one should preferably use the waste materials, whereas pineapple fruit products have commercial application. This study aimed to extract bromelain from pineapple peels using aqueous two-phase system (ATPS), and its application in polymeric hydrogels. Stability studies of commercial bromelain were performed, which found greater stability at pH 5.0 with minor loss of relative activity at all temperatures studied. The study of bromelain extraction in ATPS composed by polyethylene glycol (PEG) and poly acrylic acid (PAA) (with assistance of variance analysis of parameters such as yield, purification factor and partition coefficient) showed yield 335% and purification factor of 25.8. The PEG-based hydrogels studied presented flexibility, low elasticity and swelling ratio higher than 1000%. Hydrogel containing bromelain, loading by embedding (solvent sorption) method, yielded the highest enzyme release, as well as the highest activity (80% bromelain released over 24 h and 278 ± 89 U / mL)

Quantity and quality of bromelain in some Indonesian pineapple fruits.

This research aimed to extract bromelain from all parts of some variant of Indonesian pineapple fruits in order to evaluate their amount and characteristics as well. Each part of pineapple (crown, flesh, core, and peel) was separately blended with sodium phosphate buffer 0.01 M pH 7.0 at ratio of 1:1 (w/v) then filtered using cheese cloth. The filtrate was centrifuged at 1250 g for 10 minutes. The supernatant was collected and then centrifuged at 10000 g for 15 minutes. All extracts from each stage (blended mixture, pulp, filtrate, supernatant 1, pellet 1, supernatant 2, and pellet 2) were collected and characterized. The concentration of protein was measured by using BSA as standard and the proteolytic activity was determined using casein 1% (w/v) as substrate under the standard conditions (37 oC, pH 8.00, 10 min). The optimum temperature (ranged 27-80°C) and pH (ranged 3-10) was also evaluated. The result showed that three stepwise extraction process (blending with buffer – filtration – centrifugation) could be applied to obtain bromelain from all parts of pineapple, but some amount of bromelain was still left in the residue. Due to its price and weight, Subang pineapple was the most potential Indonesian pineapple as the source of bromelain. The maximum protein content and bromelain activity in one of Subang pineapple were 236.29 mg and 787.92 unit, respectively, while those in Bogor and Palembang pineapples were 168.27-190.10 mg and 464.97-510.37 unit, respectively.

Determination of optimal condition to obtain the bromelain from pineapple plants produced by micropropagation

This study aimed to obtain the condition of maximum bromalein activity in different parts of pineapple plants produced in vitro, by micropropagation. The sStems and leaves of Pérola and Imperial cultivar plants were evaluated after three and eight months of in vitro cultivation in Murashige and Skoog medium without growth phytoregulator, macerated in potassium phosphate buffer at different pH values (5.7, 6.7 and 7.7). Total protein and proteolytic activity were determined in the samples after three- and eight-month cultivation periods. For both the cultivars, the best results were obtained at pH 5.7 in extraction media. Pérola cultivar, showed higher bromelain activity in the leaves cultivated in vitro for three months (0.0194U/mL) while in the Imperial cultivar, it was higher in the stem after eight months (0.0179 U/mL). Imperial cultivar showed higher bromelain activity than the Pérola's.

Viability in the production of a drug extracted from Ananas comosus by a flat membrane system

The aim of this work was to study the production of e bromelain from the Ananas comosus L. Merril, by determining the process conditions using flat membranes. The production system modeling generated a hyperbolical curve and the optimization by response surfaces showed an influence of the transmembrane pressure higher than the pH influence. The cost of the production of bromelain from A. comosus was estimated 9 to 13 times lower than Sigma's retail sales price and 6.5 to 8.5 times lower than when this enzyme was obtained through a liquid-liquid extraction, which showed the economical feasibility of the process.

The effect of pH on bromelain partition from Ananas comosus by PEG4000/phosphate ATPS

In this work, the thermodynamic equilibrium and applying of PEG4000/Phosphate ATPS on the purification of bromelain extracted from pineapple was studied. A rigorous study of the equilibrium curves and tie-line length from PEG4000/phosphate ATPS were done for the pH 6-11 at 25ºC. Results showed that there was augment in the PEG and salt contents with the high pH value from PEG4000/Phosphate ATPS and two-phase formation needed only increasing the PEG content. Two tie-line length at pH 11 from PEG4000/Phosphate ATPS were optimal condition for bromelain purification, one on composition of 14 percent PEG and 13 percent salt and other at 12.6 percent PEG and 12.2 percent salt, while a 25-62 folds of enzyme was found. SDS-PAGE electrophoreses had one band only, which showed that bromelain was purified. Optimum conditions of bromelain use were found at pH 7 and between 30-40ºC.

Caracterización cinética de un preparado semipurificado de bromelina para uso antitumoral / Kinetic characterization of a semipurified preparation from bromelain for antitumor use

INTRODUCCIÓN: la mayor parte de la producción mundial de enzimas está destinada a la obtención de proteasas. Las aplicaciones de estas biomoléculas con fines terapéuticos son muy variadas. Algunas se conocen como remedios naturales y otras son fármacos modificadores de la respuesta biológica. Las plantas de piña contienen varias cisteíno-proteasas, el componente mayoritario aislado del tallo es la bromelina de tallo (EC 3.4.22.32). En el campo de la salud se le atribuyen varias acciones entre las que se destaca la de inducir la diferenciación de células tumorales y atenuar el crecimiento del tumor. OBJETIVOS: caracterizar cinéticamente un preparado semipurificado de bromelina obtenida por un procedimiento desarrollado en Cuba para ser evaluado en el campo de la salud. MÉTODOS: el extracto crudo de bromelina, obtenido a partir de tallos de plantas adultas de Ananas comosus (L.) Merr cv Española roja (piña) se purificó por cromatografía de intercambio iónico en carboximetilcelulosa-52 en un sistema semibach. RESULTADOS: la caracterización cinética del extracto purificado permitió comprobar la existencia de un preparado activo y estable con una actividad específica de 0,94 U/mg de proteínas (2,34 veces superior a la del extracto crudo original), con una fracción proteica mayoritaria de masa molar 24 500 Da y un punto isoeléctrico, localizado en la zona de pH básico (9,55). Tiene pH óptimo 6,8 al utilizar hemoglobina 2 por ciento como sustrato a 37 ºC y una estabilidad en función del pH y de la temperatura aceptable. Si se conserva liofilizado a - 20 ºC mantiene 80 por ciento de la actividad durante 1 año. CONCLUSIONES: la cromatografía de intercambio iónico en carboximetilcelulosa-52 posibilitó la obtención de un preparado semipurificado de bromelina, con una actividad específica superior a la del extracto original que puede ser utilizado en la medicina como antitumoral

INTRODUCTION: most of the world production of enzymes is devoted to proteases obtaining. Applications of these biomolecules for therapeutical objectives are very varied. Some are known as natural remedies and other are drugs modifying the biological response. Pineapple plants contain some cysteine-proteases, the majority component isolated from stem is the stem Bromelain (EC 3.4.22.32). In the health field it has attributed some actions including the differentiation induction of tumor cells and attenuation of tumor growth. OBJECTIVES: to characterize kinetically a semipurified preparation from Bromelain obtained by a procedure developed in Cuba to be assessed in the health field. METHODS: the crude extract from Bromelain obtained from the adult plant stems of Ananas comosus (L.) Merr Cv Spanish red (pineapple) was purified by ion-exchange chromatography in carboxylmethylcelulose-52 in semibatch system. RESULTS: kinetic characterization of purified extract allowed us to verify the existence of a stable and active preparation with a specific activity of 0,94 U/mg of proteins (2,34 times superior to that of original crude extract) with a protein fraction in the main of molar mass 24 500 Da and a isoelectric point, located in the basic pH zone (9,55). It has a 6,8optimal pH using the 2 percent hemoglobin as substrate at 37ºC and stability depending on pH and on acceptable temperature. If it is preserved lyophilized at 20 ºC it maintains the 80 percent of activity for a year. CONCLUSIONS: the ion-exchange chromatography in carboxyl methylcellulose-52 allowed the achievement of semipurified preparation from Bromelain with a specific activity higher to that of original extract that may to be used in medicine as antitumor agent

Neural modeling of bromelain extraction by reversed micelles

A pulsed-cap microcolumn was used for bromelain extraction from pineapple juice by reversed micelles. The cationic micellar solution used BDBAC as the surfactant, isooctane as the solvent and hexanol as the co-solvent. In order to capture the dynamic behavior and the nonlinearities of the column, the operating conditions were modified in accordance with the central composite design for the experiment, using the ratio between the light phase flow rate and the total flow rate, and the time interval between pulses. The effects on the purification factor and on total protein yield were modeled via neural networks. The best topology was defined as 16-9-2, and the input layer was a moving window of the independent variables. The neural model successfully predicted both the purification factor and the total protein yield from historical data. At the optimal operating point, a purification factor of 4.96 and a productivity of 1.29 mL/min were obtained.

Uma micro-coluna com campânulas pulsantes foi utilizada para a extração de bromelina a partir de suco de abacaxi, usando micelas reversas. A solução catiônica micelar foi composta do surfactante BDBAC, do solvente iso-octano e do co-solvente hexanol. Seguindo um planejamento experimental, perturbações foram impostas à coluna de extração com o objetivo de capturar seu comportamento dinâmico e suas não-linearidades, usando a razão entre a vazão da fase leve e vazão total, e o intervalo de tempo entre os pulsos. Os efeitos das variáveis independentes sobre o fator de purificação e sobre o rendimento em proteínas totais foram modelados via redes neurais artificiais. A melhor topologia de rede obtida foi definida como 16-9-2, usando um esquema de janela móvel no tempo das variáveis independentes. O modelo neural obtido do histórico do processo se mostrou adequado para predizer simultaneamente o fator de purificação e o rendimento do processo em proteínas totais. No ponto ótimo de operação, foi encontrado um fator de purificação de 4.96, com produtividade de 1.29 mL/min.

Batch and continuous extraction of bromelain enzyme by reversed micelles

The main aim of this study was to optimize the conditions for bromelain extraction by reversed micelles from pineapple juice (Ananas comosus). The purification was carried out in batch extraction and a micro-column with pulsed caps for continuous extraction. The cationic micellar solution was made of BDBAC as a surfactant, isooctane as a solvent and hexanol as a co-solvent. For the batch process, a purification factor of 3 times at the best values of surfactant agent, co-solvent and salt concentrations, pH of the back and forward extractions were, 100 mM, 10 percent v/v, 1 M, 3.5 and 8, respectively. For the continuous operation, independent variables optimal point was determined: ratio between light phase flow rate and total flow rate equal to 0.67 and 1 second for the time interval between the pulses. This optimal point led to a productivity of 1.29 mL/min and a purification factor of 4.96.

Este trabalho teve como objetivo principal otimizar as condições para extração da bromelina do suco do abacaxi (Ananas comosus) por micelas reversas. A purificação foi feita usando o processo de extração em batelada e contínuo, este último em uma micro-coluna de campânulas pulsantes. A solução micelar catiônica foi preparada com o surfactante BDBAC, i-octano como solvente e hexanol como co-solvente. Na extração em batelada encontrou-se um fator de purificação de 3 vezes, e seus melhores valores de concentração do agente surfactante, co-solvente e sal, de pH da re-extração e extração, foram respectivamente iguais a: 100 mM, 10 por cento v/v, 1 M, 3,5 e 8. Para a operação contínua, as variáveis independentes ótimas foram: 0,67 para a razão entre as taxas de fluxos da fase leve e a total e 1 s para o intervalo de tempo entre pulsos das campânulas. Este ponto ótimo leva a uma produtividade de 1,29 mL/min e a um fator de purificação igual a 4,96.

Concentration by membrane separation processes of a medicinal product obtained from pineapple pulp

The concentration of pineapple juice is needed to retain the bromelain activity and to standardize the composition and proteolytic activity. Thus, this work aimed to obtain a pure bromelain extract from the Ananas comosus L. Merril juice by membrane separation process. A 2² experimental planning was used to study the influence of pH and transmembrane pressure on the activity recovery by micro-filtration using a plain membrane. In second step, this enzyme was purified by the ultra-filtration using a 10 kDa millipore kit. The best operation condition to bromelain concentration using the plain membrane was at pH 7.5 and transmembrane pressure of 0.05 bar, while 85 percent of bromelain activity was recovered. Ultra-filtration retained 100 percent of proteolytic activity and concentrated in 10 fold the bromelain extract. SDS-PAGE electrophoresis showed that the ultra-filtrated had high purity and the bromelain from A. comosus pulp had a molecular weight of 24.5 kDa.

A concentração do suco de abacaxi é necessária para manter a atividade da bromelina e padronizar a composição e atividade proteolítica. Assim, este trabalho objetivou a obter um extrato de bromelina pura do suco do Ananas comosus L. Merril por processos de separação por membranas. Um planejamento experimental do tipo 2² foi feito para estudar a influência do pH a da pressão transmembranar sobre a recuperação da atividade por micro-filtração usando uma membrana plana. Em uma segunda etapa, purificou-se a enzima alvo por ultra-filtração usando um "kit millipore" de 10 kDa. A melhor condição para a concentração da bromelina foi a pH 7,5 e pressão transmembranar de 0,05 bar, onde 85 por cento da atividade da bromelina foi recuperado. A ultra-filtração manteve 100 por cento da atividade proteolítica e concentrou em 10 vezes o extrato de bromelina. A eletroforese via SDS-PAGE mostrou que o ultra-filtrado teve alta pureza e a bromelina da polpa do Ananas comosus tem um peso molecular de 24.5 kDa.