Purification Of Bromelain Research Articles

Use of Artificial Neural Networks to predict Aqueous Two- Phases System Optimal Conditions on Bromelain's Purification

Bromelain is the denomination chosen to the group of endoproteases obtained from pineapple and from most of plants belonging to Bromeliaceae family. These enzymes have being widely studied in researches across the world due its physiological activity and biotechnological potential. While Brazil still cultivating over 60,000 hectares of pineapple, there is a optimistic trend that aim bromelain's recovery from agriculture residues (stalk and leaves) and fruit processing residues (stem and bark) leading to a fully integrated process which aggregate value to vegetal residues. Our previous studies applied Aqueous Two-Phases Systems and Fractional Precipitation to purify bromelain and achieve purification factor and yield of 11.80 and 87.36, respectively. However, such studies were designed and analysed using Design Of Experiments (DOEs), which lead to an optimal condition but cannot predict with accuracy the complex phenomena of partitioning using ATPS. This work is part of an initiative that aims establish a protocol to calculate more accurate partitioning data through the use of Artificial Neural Networks (ANNs) over a dataset that has being improved continuously. The ANN will determine the relationship between five input parameters (temperature, PEG's molar mass, concentration of PEG, concentration of ammonium sulphate and dilution factor of sample) with three output parameters (protein partition coefficient, Activity partition coefficient and purification factor). The method applied a feed-forward neural network trained with Levenberg-Marquardt algorithm and the Bayesian regularization over the normalized experimental data. The network generated proved the reliability of the method which combined datasets from different DOEs and obtained regression coefficient (~.99) and error (MSE ~0.02) satisfactory for such amount of data used so far.

Characteristic properties of crude pineapple waste extract for bromelain purification by membrane processing

Bromelain enzyme can be extracted from pineapple fruit including its waste parts. It is a valuable ingredient for various markets especially in the food and pharmaceutical areas. This enzyme can be extracted by membrane-based technology, but there is a lack of understanding of the properties of the crude pineapple extracts to link to functional membrane properties for efficient purification operation. This study focused on establishing characteristic properties of the crude pineapple waste mixture (CWM) extract, constituting of 57 % peel, 28 % crown and 15 % core, as a source of the enzyme and relevant to membr'ane processing. Rheological properties at different temperatures and pH levels were also determined in order to propose appropriate processing conditions for a practical and an efficient membrane operation. The CWM extract contained appreciable specific enzyme activity of 394.9 CDU/mg protein that would need to be purified at least 2–4 fold to achieve a required market standard. Existence of polysaccharides, particularly pectin, in the extract would be expected to cause fouling issues in membrane process, thus pre-treatment may be required to remove this compound from the mixture. The protein molecular weight of the CWM extract ranged between 11.7 and 26.9 kDa. The selection of membrane molecular weight cut-off (MWCO) should be above or below this range to isolate the enzyme. The evaluation on rheological properties of the CWM extract showed that it demonstrated lower viscosity at higher temperature and at neutral pH. Therefore, selection on temperature of 20–25 °C and pH 7 throughout the membrane purification operation has been recommended.

Efficacy of reverse micellar extracted fruit bromelain in meat tenderization.

Reverse micellar extraction (RME) was used for the separation and purification of bromelain from pineapple core and efficacy of RME purified bromelain (RMEB) in tenderization of beef meat was compared with that of commercial stem bromelain (CSB). RME resulted in reasonably high bromelain activity recovery (85.0%) and purification fold (4.0). Reduction in meat toughness was higher in RMEB treated meat (52.1%) compared to raw (control) and CSB treated (26.7%). Significant increase in water holding capacity (WHC) was observed in RMEB treated meat (91.1%) as against CSB treated (55.6%) and control (56.6%). No change in cooking loss was observed in RMEB treated meat, whereas the loss increased by nearly 14.0% in case of CSB treated. While the meat color was retained, trichloroacetic acid (TCA) soluble protein content increased due to hydrolysis of protein in RMEB treated meat. Scanning electron microscopy (SEM) analysis revealed that RMEB treatment completely ruptures myofibril tissues, indicating a higher degree of tenderization.

Bromelain: an overview of industrial application and purification strategies.

This review highlights the use of bromelain in various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially in many applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant bromelain for novel applications in the future.

A new procedure based on column chromatography to purify bromelain by ion exchange plus gel filtration chromatographies

Bioproducts separation and purification processes are an important segment of the biotechnical industry. Bromelain is an enzyme which has great commercial value and is of wide interest in the pharmaceutical, alimentary, and textile industries, among others. The goal of this study was to develop a new method for bromelain purification from the stem residues resulting from agricultural processing of the pineapple plant. Bromelain was purified using two liquid chromatography steps, ion exchange plus gel filtration chromatography. Use of the methodology which was developed produced an enzyme with a molecular weight of 30kDa (confirmed by SDS-PAGE), high recovery of enzymatic activity (89%), and with a purification factor of 16.93, a result superior to the methodologies described in the literature. HPLC showed the presence of two peaks in the ion exchange chromatogram and only one protein in the gel filtration chromatogram. Results indicate that, depending on the destination of the bromelain, the process can be stopped after the first purification step. The MALDI-TOF MS provided the peptide mass fingerprint of bromelain and MALDI-MS/MS the fragmentation profile and sequencing of the ions of m/z 951 and 1584. Thus, the connectivity and chemical structure of bromelain was confirmed. Moreover, besides its superiority to other methodologies, it can be applied to take advantage of the agricultural and industrial pineapple plant residues.

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.

The use of papain inhibitor immobilized onto polyaniline for bioaffinity chromatography of cysteine proteases

In this work, a papain inhibitor extracted from seeds of Adenanthera pavonina was covalently immobilized onto glutaraldehyde modified polyaniline (PANIG). The immobilization was very efficient, presenting 54.24% inhibitor retention in the following conditions: immobilization time of 30min, at 4°C, pH 7.0, inhibitor concentration of 20mg and 5mg of PANIG. The resultant material (PANIG-I) was tested as stationary phase for the cysteine proteases papain, ficin and bromelain purification through bioaffinity chromatography. A commercial preparation of papain was efficiently purified, resulting in a single band with 25kDa. SDS–PAGE of purified bromelain showed a characteristic band around 28kDa and this procedure resulted in 0.89-fold purification and 32.4% yield. The purification process was more effective for ficin reaching 2.6-fold purification and a single band of 25kDa in the SDS–PAGE. These results showed that the stationary phase containing A. pavonina inhibitors immobilized onto PANIG is a very promising material for a single step purification of cysteine proteases through bioaffinity chromatography.

Extraction of bromelain from pineapple core and purification by RME and precipitation methods

Demand for bromelain is increasing because of its application in various fields like medical, food and cosmetics. While bromelain extracted from pineapple stem is commercially known, presence of bromelain is reported also in core, peel and crown of pineapple fruit. Core, which is slightly harder in texture, is one of the major wastes generated (15% of the fruit) by the pineapple processing industry. The present work mainly focuses on optimization of conditions for extraction of bromelain from pineapple (Ananas comosus L.) core and purification of bromelain by reverse micellar extraction (RME) and precipitation techniques. Extraction conditions like type of buffer, pH, temperature and strength of buffer on bromelain activity and protein content were standardized. Storage stability study of the extract under refrigerated condition (5–8°C) showed decrease in bromelain activity with duration. Mineral analysis of extract showed the presence of calcium, sodium, potassium, and magnesium in the extract. RME, one of the promising liquid–liquid extraction techniques was employed for the primary purification of bromelain from pineapple core. Selective separation of bromelain (excluding polyphenol oxidase (PPO) and peroxidase (POD)) with 3.96-fold purification and activity recovery of 78.90% was achieved with this method. Acetone and ammonium salt precipitation methods resulted in 4.90 and 3.07-fold purification under optimized conditions. The studies indicate that extraction followed by RME or precipitation method could be employed for separation and purification of bromelain from extract obtained from pineapple core.

Separation of Bromelain by Aqueous Two Phase Flotation

Aqueous two phase flotation (ATPF) system of polyethylene glycol (PEG) and potassium phosphate is studied for the separation and partial purification of bromelain from the pineapple fruit (Annanus comosus L. Merryl). The effect of PEG molecular weight (1500–20000), concentration of phase forming components (PEG 12–18% w/w and potassium phosphate 14–20% w/w), system pH, nitrogen flow rate, and flotation time were studied and optimum conditions for ATPF were obtained. At optimum conditions of the system, i.e., 14% w/w PEG 1500, 18% w/w potassium phosphate, 80 mL/min of nitrogen flow rate and pH 7, maximum yield of 91.47% and purification fold of 4.26 were obtained. ATPF was found to be an effective technique for the purification of bromelain from pineapple fruit with higher extraction yield and purification fold as compared to aqueous two phase extraction (ATPE).

Purification of bromelain from pineapple wastes by ethanol precipitation

Bromelain is an aqueous extract of pineapple that contains a complex mixture of proteases and non-protease components. These enzymes perform an important role in proteolytic modulation of the cellular matrix in numerous physiologic processes, including anti-inflammatory, anti-thrombotic and fibrinolytic functions. Due to the scale of global production of pineapple (Ananas comosus L.), and the high percentage of waste generated in their cultivation and processing, several studies have been conducted on the recovery of bromelain. The aim of this study was to purify bromelain from pineapple wastes using an easy-to-scale-up process of precipitation by ethanol. The results showed that bromelain was recovered by using ethanol at concentrations of 30% and 70%, in which a purification factor of 2.28 fold was achieved, and yielded more than 98% of the total enzymatic activity. This enzyme proved to be susceptible to denaturation after the lyophilization process. However, by using 10% (w/v) glucose as a cryoprotector, it was possible to preserve 90% of the original enzymatic activity. The efficiency of the purification process was confirmed by SDS–PAGE, and native-PAGE electrophoresis, fluorimetry, circular dichroism and FTIR analyzes, showing that this method could be used to obtain highly purified and structurally stable bromelain.

Bromelain purification through unconventional aqueous two-phase system (PEG/ammonium sulphate)

This paper focuses on the feasibility of unconventional aqueous two-phase systems for bromelain purification from pineapple processing waste. The main difference in comparison with conventional systems is the integration of the liquid-liquid extraction technique with fractional precipitation, which can decrease the protein content with no loss of biological activity by removing of unwanted molecules. The analysis of the results was based on the response surface methodology and revealed that the use of the desirability optimisation methodology (DOM) was necessary to achieve higher purification factor values and greater bromelain recovery. The use of DOM yielded an 11.80-fold purification factor and 66.38 % biological activity recovery using poly(ethylene glycol) (PEG) with a molar mass of 4,000, 10.86 % PEG concentration (m/m) and 36.21 % saturation of ammonium sulphate.

Reverse Micellar Extraction of Bromelain from Pineapple (Ananas comosus L. Merryl) Waste: Scale-up, Reverse Micelles Characterization and Mass Transfer Studies

Scale-up studies for phase transfer mode of reverse micellar extraction are attempted for the separation and primary purification of bromelain (EC 3.4.22.33) from pineapple (Ananas comosus L. Merryl) waste. Characterization of reverse micelles and mass transfer studies for the real system has been attempted for the first time. Scale-up of the extraction process employing commercial grade surfactant cetyltrimethylammonium bromide (CTAB) and solvent isooctane resulted in purification of 2.43 fold with an activity recovery 81.3%. The reverse micellar size estimated using empirical and geometrical models indicated that the reverse micelles are large enough (Rm = 7.2–9.6 nm) to host bromelain molecules that are relatively smaller in size (∼1.67 nm). The studies on the kinetics of mass transfer indicated a relatively slower rate (by ∼34%) of mass transfer in case of back extraction compared to forward extraction. Process scale-up did not significantly affect the extraction efficiency whereas purity of phase components played a major role. The mass transfer across the phases was high in the initial period of mixing for both forward and back extractions.

Preparative purification of bromelain (EC 3.4.22.33) from pineapple fruit by high-speed counter-current chromatography using a reverse-micelle solvent system

The crude protein of pineapple fruit was purified by high-speed counter-current chromatography (HSCCC). Excellent separation was achieved after careful investigation as follows: cetyltrimethylammonium bromide (CTAB) was selected to prepare the reverse micelle phase at the concentration of 0.10g/mL. The mobile phase was 0.05M sodium phosphate buffer solution, including mobile phase A (pH 9.5, containing 0.2M KCl) used for equilibration and mobile phase B (pH 7.0, containing 0.4M KCl) used for elution. The flow rate of the mobile phase was set at 1.5mL/min and the effluent was monitored at 280nm. Under these conditions, 127.3mg bromelain (EC 3.4.22.33) was produced from 200mg crude sample. A large-scale procedure was then carried out, and 3.01g bromelain was obtained from 5.00g crude extract in a 200min run. The separated protein was analysed by SDS–PAGE, compared with the standard and then identified by TOF/TOF-MS.

Affinity based reverse micellar extraction and purification of bromelain from pineapple (Ananas comosus L. Merryl) waste

Affinity based reverse micellar extraction and separation (ARMES) technique is employed for the first time to extract and purify bromelain from pineapple (Ananas comosus L. Merryl) waste. The reverse micellar system of cetyltrimethylammonium bromide (CTAB)/isooctane/butanol/hexanol and sodium bis(2-ethyl-1-hexyl) sulfosuccinate (AOT)/isooctane is used for forward and reverse extractions respectively, with concanavalin A (Con A) as an affinity ligand. The effect of various process parameters like concentration of Con A during forward extraction, type of counter ligand and its concentration during back extraction, type of surfactants and its concentration, aqueous phase pH for reverse extraction on the purification and activity recovery of bromelain has been studied in detail. The optimized conditions of extraction resulted in purification of 12.32 fold with an activity recovery of 185.6%, which is higher than that reported for conventional reverse micellar extraction (RME). The forward, back, reverse and overall extraction efficiencies were found to be 49%, 44%, 48% and 14% respectively under optimized conditions.

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% PEG and 13% salt and other at 12.6% PEG and 12.2% 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.

Separation and Purification of Bromelain by Reverse Micellar Extraction Coupled Ultrafiltration and Comparative Studies with Other Methods

Reverse micellar extraction (RME) is a promising liquid-liquid extraction technique for downstream processing of biomolecules from dilute solutions. An integrated approach of coupling RME with ultrafiltration is attempted to improve the overall efficiency of extraction and purification of bromelain from aqueous extract of pineapple core. The performance of RME is compared with aqueous two-phase extraction (ATPE), another potential liquid-liquid extraction technique and conventional ammonium sulphate precipitation technique. The reverse micellar system of cationic surfactant cetyltrimethylammoniumbromide/isooctane/hexanol/butanol used for RME resulted in an activity recovery of 95.8% and purification of 5.9-fold. The purification of bromelain increased to 8.9-fold after ultrafiltration. Alteration of aqueous phase pH during RME facilitated the differential partitioning of bromelain and polyphenoloxidase. Comparison of RME results with ATPE (activity recovery of 93.1% and purification of 3.2-fold) and the conventional ammonium sulphate precipitation (activity recovery of 82.1% and purification of 2.5-fold) indicated the improved performance of RME.

Expanded bed adsorption of bromelain (E.C. 3.4.22.33) from Ananas comosus crude extract

This work focuses on the adsorption of Bromelain in expanded bed conditions, such as the adsorption kinetics parameters. The adsorption kinetics parameters showed that after 40 minutes equilibrium was achieved and maximum adsorption capacity was 6.11 U per resin mL. However, the maximum adsorption capacity was only determined by measuring the adsorption isotherm. Only by the Langmuir model the maximum adsorption capacity, Qm, and dissociation constant, kd, values could be estimated as 9.18 U/mL and 0.591, respectively, at 25 ºC and 0.1 mol/L phosphate buffer pH 7.5. A column made of glass with an inner diameter of 1 cm was used for the expanded bed adsorption (EBA). The residence time was reduced 10 fold by increasing the expansion degree 2.5 times; nonetheless, the plate number (N) value was reduced only 2 fold. After adsorption, the bromelain was eluted in packed bed mode, with a downward flow. The purification factor was about 13 fold and the total protein was reduced 4 fold. EBA showed to be feasible for purification of bromelain.

Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes

Reverse micellar systems of CTAB/isooctane/hexanol/butanol and AOT/isooctane are used for the extraction and primary purification of bromelain from crude aqueous extract of pineapple wastes (core, peel, crown and extended stem). The effect of forward as well as back extraction process parameters on the extraction efficiency, activity recovery and purification fold is studied in detail for the pineapple core extract. The optimized conditions for the extraction from core resulted in forward and back extraction efficiencies of 45% and 62%, respectively, using reverse micellar system of cationic surfactant CTAB. A fairly good activity recovery (106%) and purification (5.2-fold) of bromelain is obtained under these conditions. Reverse micellar extraction from peel, extended stem and crown using CTAB system resulted in purification folds of 2.1, 3.5, and 1.7, respectively. Extraction from extended stem using anionic surfactant AOT in isooctane did not yield good results under the operating conditions employed.

Sedimentation properties of [formula omitted][formula omitted] RNA polymerase and its complexes with polyuridylic acid

Bioproducts separation and purification processes are an important segment of the biotechnical industry. Bromelain is an enzyme which has great commercial value and is of wide interest in the pharmaceutical, alimentary, and textile industries, among others. The goal of this study was to develop a new method for bromelain purification from the stem residues resulting from agricultural processing of the pineapple plant. Bromelain was purified using two liquid chromatography steps, ion exchange plus gel filtration chromatography. Use of the methodology which was developed produced an enzyme with a molecular weight of 30 kDa (confirmed by SDS-PAGE), high recovery of enzymatic activity (89%), and with a purification factor of 16.93, a result superior to the methodologies described in the literature. HPLC showed the presence of two peaks in the ion exchange chromatogram and only one protein in the gel filtration chromatogram. Results indicate that, depending on the destination of the bromelain, the process can be stopped after the first purification step. The MALDI-TOF MS provided the peptide mass fingerprint of bromelain and MALDI-MS/MS the fragmentation profile and sequencing of the ions of m/z 951 and 1584. Thus, the connectivity and chemical structure of bromelain was confirmed. Moreover, besides its superiority to other methodologies, it can be applied to take advantage of the agricultural and industrial pineapple plant residues.