Extraction Of Bromelain Research Articles

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.

Reverse Zymography Alone does not Confirm Presence of a Protease Inhibitor

Reverse zymography is applied for identification and semi-quantification of protease inhibitors that are of protein in nature. However, a protein that shows band in reverse zymography against a protease used for digestion of the gel need not be an inhibitor; it might be resistant to degradation by the protease. We demonstrate that in reverse zymography, avidin, streptavidin and the leaf extract of Catharanthus roseus behave like inhibitors of proteases like papain, ficin, bromelain extracts from pineapple leaf, stem and fruit and trypsin. Still, they do not act as inhibitors of those proteases when enzyme assays were done in solution. In reverse zymography, the extract of pineapple crown leaf shows two major inhibitor bands against its own proteases. Identification of these proteins from sequences derived from MALDI TOF MS analysis indicated that they are fruit and stem bromelains. Avidin, streptavidin and bromelains are 'kinetically stable proteins' that are usually resistant to proteolysis. Thus, it is recommended that identification of an inhibitor of a protease by reverse zymography should be supported by independent assay methods for confirmation.

Efficacy of Extrinsic Stain Removal by Novel Dentifrice Containing Papain and Bromelain Extracts

To evaluate the stain removal efficacy of a novel dentifrice containing papain and bromelain extracts (Glodent) in comparison with a control dentifrice (Colgate regular). This was a randomized, positive-controlled, double-blinded, clinical study. Subjects were randomly divided into one of the two study groups. Pre-treatment and post treatment photographs of the 4 anterior teeth were recorded under standardized conditions and analyzed for lightness or luminosity values using Adobe Photoshop. The difference between the mean pre-treatment luminosity of test and control groups was not statistically significant. In both test and control groups, the post-treatment luminosity was significantly higher than pre- treatment luminosity (P < 0.001 and P = 0.003 respectively). The mean post-treatment luminosity for test group was found to be significantly higher than control group. The mean percentage removal of stains for test group was significantly higher than control group. The test dentifrice showed significant stain removal when compared to control which could be attributed to the role of proteolytic enzymes in the test dentifrice.

Study of Enzyme Activity and Stability to pH and Temperature of Bromelain Extracted from Curaua (Ananas erectifolius L. B. SMITH) Purple and White

Ananas erectifolius (curaua) is a fibrous vegetable that can be found in North region of Brazil. It has physico-chemical features that provide great potential in the automobile industry as a source of fibers. In addition, from the same plant, the development of new extraction and purification processes for bromelain have been studied. To optimise this process, it is necessary to investigate enzyme stability, so that bromelain retains its biological activity when stored, isolated, purified or subjected to any other manipulation, such as autodigestion, proteolytic enzymes and heating. This study aimed to evaluate the activity of the enzyme bromelain extracted from the leaves of curaua, white and purple, and study its stability. The proteolytic activity was measured for each of the tests by the azocasein method. The stability study to pH and temperature was accomplished by the enzyme activity after storage at 4°C and -18°C, compared with the lyophilized crude extract, over a period of 30 days. The samples were subjected to different conditions for a period of 60 minutes and then the remaining bromelain activity was measured. Results showed that bromelain extracted from lyophilized curaua provided better stability and a higher enzymatic activity, and was also more stable at 20°C, losing only 5% of its activity

Pineapple wastes: A potential source for bromelain extraction

This study investigates the isolation and characterization of bromelain extract from the wastes of Nang Lae and Phu Lae pineapple cultivars (economical fruits of Chiang Rai province, Thailand). The waste portions such as the peel, core, stem and crown were 29–40%, 9–10%, 2–5% and 2–4% (w/w), respectively. The extract of crown from both cultivars gave the highest proteolytic activity and protein contents, while the extract from the stem exhibited the lowest values. SDS–PAGE showed that the major protein band in the extracts was ∼28kDa. Activity staining of the crown extracts from both cultivars confirmed that the major protein band showed caseinolytic activity on the casein substrate-gel. All of the crude extracts from both cultivars gave high caseinolytic activity (>80% relative) in a board pH range (3–9). The optimum temperatures for all crude extracts were about 50–60°C. This study founded that there is much added value into local Thailand pineapple wastes because of bromelain extraction.

Antioxidant, Antimicrobial, 15‐LOX, and AGEs Inhibitions by Pineapple Stem Waste

Pineapple stem has been extensively used for bromelain extraction; however, almost no attention has been given to the waste obtained during bromelain manufacturing. In this regard, antioxidant, antimicrobial, and inhibitions against 15-lipoxygenase and advanced glycation end product formations by pineapple stem waste (PSW) obtained during bromelain manufacturing process were studied. The PSW had moderate bioactivities in all the performed assays. It also showed a considerable inhibition against fungal growth, probably due to high amounts of the benzoic acid present in the sample. These results indicate that PSW could be utilized as an economic source of preventive or therapeutic agent in disease and in different functional food industries. A large amount of wastes are generated during bromelain manufacturing from pineapple stem. So far, these wastes are not utilized and are often considered as a burden while disposing them. However, we found some important phytochemicals with considerable bioactivities in these wastes. We believe that these wastes may have a promising usage as a cheap source of one of the ingredients in functional food based industries.

Aqueous two-phase extraction of bromelain from pineapple peels (‘Phu Lae’ cultv.) and its biochemical properties

This study examines the extraction of bromelain from pineapple peels by using an aqueous two-phase system (ATPS). Bromelain from the crude extract predominantly partitioned to the polyethylene glycol rich phase. The best condition for bromelain partitioning was found to be 18% PEG6000-17% MgSO4, which increased purity 3.44-fold with an activity recovery of 206%. Protein patterns and activity staining showed the molecular weight of bromelain to be about 29 kDa. The bromelain showed the highest relative activity at pH 8.0 and at 60°C. When increasing NaCl concentrations (up to 1.5%, w/v), its activity continuously decreased. The bromelain (0–0.3 units) was applied to hydrolyze collagen. The β, α1, α2 components of collagen extensively degraded into small peptides when treated with the bromelain. This study showed that the ATPS can be employed to isolate the bromelain from pineapple peels and the bromelain extract could be used as a meat tenderizing agent.

Extraction of bromelain from pineapple peels

Large amount of pineapple peels (by-products) is left over after processing and they are a potential source for bromelain extraction. Distilled water (DI), DI containing cysteine and ethylenediaminetetraacetic acid (EDTA) (DI-CE), sodium phosphate buffer pH 7.0 (PB) and PB containing cysteine and EDTA (PB-CE) were used as extractants for bromelain from the pineapple peels. The highest bromelain activity was obtained when it was extracted with PB-CE (867 and 1032 units for Nang Lae and Phu Lae cultv, respectively). The PB could maintain the pH of the extract (pH 5.1-5.7) when compared with others. Under sodium dodecyl sulfate polyacrylamide gel electrophoresis, the extract showed protein bands in the range 24-28 kDa. The protein band with a molecular weight of ∼28 kDa exposed the clear zone on blue background under the casein-substrate gel electrophoresis. The effects of the bromelain extract on the protein patterns of beef, chicken and squid muscles were also determined. Trichloroacetic acid soluble peptide content of all the treated muscles increased when the amount of bromelain extract increased. Decrease in myosin heavy chains and actin was observed in all the muscle types when bromelain extract was used. The best extractant for bromelain from pineapple peels was PB-CE. Moreover, bromelain extract could be used as a muscle food tenderizing agent in food industries.

Application of Response Surface Methodology for Optimization of Bromelain Extraction in Aqueous Two-Phase System

Extraction of bromelain from pineapple fruit in an aqueous two phase system (ATPS) composed of polyethylene glycol (PEG) 1500 and potassium phosphate has been studied using response surface methodology. The various process variables such as PEG, potassium phosphate and NaCl concentration, and pH were optimized using a central composite rotatable design (CCRD) of response surface methodology (RSM) based on the partition coefficient, % yield, and purification factor of an enzyme. An optimized ATPS composed of 14% (w/w) PEG 1500, 17.66% (w/w) potassium phosphate and 1 mM sodium chloride at pH 7.5 was used to purify bromelain from a pineapple fruit. With this system, a maximum enzyme partition coefficient of 12.62 and %yield of 90.33 in the top PEG-rich phase with a purification factor of 2.4 was predicted. The enzyme partition coefficient, % yield, and purification factor obtained from experimentation are 12.22, 89.65, and 2.8, respectively, in the top PEG phase. The response model is validated by the closeness between the predicted and experimental results.

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.

Application of Bromelain Extract for Muscle Foods Tenderization

This study investigates the tenderizing effects of bromelain extract (BE) obtained from the top phase of an aqueous two-phase system comprising of 18% PEG 6000–17% MgSO4 in muscle foods. Uniform-sized chunks of beef, chicken, and squid were marinated with different concentrations of BE (0, 3, 7%, and 20%, w/w) for 1 h at room temperature. The physico-chemical and quality characteristics of the marinated samples were determined. Decreases in pH and moisture content were observed in the samples BE treated, but the TCA-soluble peptides content significantly increased (p < 0.05). Water holding capacity and cooking yields were observed in all of the tested samples, especially when the concentration of BE increased. In addition, a reduction of meat firmness and toughness were also observed in all of the samples when compared to the control (p < 0.05). Electrophoretic patterns also revealed extensive proteolysis and a reduction in number and intensity of the protein bands in all of the treated samples. At the microstructural level, tissue fibers were broken, cell membranes were more strongly degraded, the connections between the sarcolemma and the myofibrils were disappeared, and the generation of numerous gaps could be clearly observed when 20% (w/w) BE was added. The results show that the bromelain extract derived from the two-phase extraction of a pineapple peel could be used as an effective meat tenderizer.

Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar

Extraction of bromelain from pineapple peel ( Nang Lae cultv.) using aqueous two phase system (ATPS) was optimized. Some biochemical properties including collagen hydrolysis were also investigated. Bromelain predominantly partitioned to the polyethylene glycol (PEG)-rich phase. The highest enzyme activity recovery (113.54%) and purification fold (2.23) were presented in the top phase of 15% PEG2000–14% MgSO 4. Protein pattern and activity staining showed the molecular weight (MW) of bromelain to be about 29 kDa. The extracted bromelain showed the highest relative activity at pH 7.0 and 55 °C. Its activity was decreased continuously by increasing NaCl concentration (up to 1.5% (w/v)). The bromelain extract was applied to hydrolyze the skin collagen of beef and giant catfish (0–0.3 units). The β, α 1, α 2 of giant catfish skin collagen extensively degraded into small peptides when treated with 0.02 units of the bromelain extract. Bovine collagen was hydrolyzed using higher bromelain up to 0.18 units. This study showed the ATPS can be employed to partially purify bromelain from Nang Lae pineapple peel and the enzyme effectively hydrolyzed the collagens.

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.

Stain removal efficacy of a novel dentifrice containing papain and Bromelain extracts - an in vitro study

To assess the in vitro stain removal efficacy of a novel commercially available dentifrice. Twenty four human molar teeth (12 in test and 12 in control group) were cut to obtain enamel specimens approximately 9 mm(2) and placed on a microscopic slide using mounting adhesive. They were cycled in human saliva, staining model and dried for brushing regimen using a customized brushing apparatus. Lightness values were recorded at baseline, after staining and after brushing regimen using Adobe Photoshop software. Stain removal efficacy was tested using the either of the two dentifrice slurries for 2 one minute brushing cycles. The mean lightness increment for test and control after brushing regimen was 13.7 ± 6.35 and 3.16 ± 1.29 respectively. There was statistically significant difference in percentage removal of stains for test and control groups (66.99 ± 9.57 and 25.89 ± 16.2 P < 0.001). There was significant stain removal with new whitening dentifrice when compared with control. Further clinical trials are recommended to evaluate this significant difference.

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.

Comparative study of extraction, purification and estimation of bromelain from stem and fruit of pineapple plant

Comparative study of extraction, purification and estimation of bromelain from stem and fruit of pineapple plant

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% 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.

Safety & efficacy of bromelain extract in debriding deep burns: Recent summary of all available data

Safety & efficacy of bromelain extract in debriding deep burns: Recent summary of all available data

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% of bromelain activity was recovered. Ultra-filtration retained 100% 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.

Preparation and Some Functional Properties of Rice Bran Protein Concentrate at Different Degree of Hydrolysis using Bromelain and Alkaline Extraction

Rice bran protein was extracted by using defatted rice bran and water at 1:6 (w/w) and 6% of bromelain at pH 9.0, 50°C, 500 rpm for 15 and 30 mins. The degree of hydrolysis (DH) of rice bran protein extract (RBPE) was 19 and 36.5%, respectively, and their nitrogen solubility was higher than the controls. Rice bran protein concentrate (RBPC) was prepared by spray drying. Emulsion activity of RBPC produced from 19% DH RBPE was increased while emulsion stability index was not significantly different from the control. Foam capacity and rehydration ability of RBPC were greater than the control.