Food Bioprocess Technol (2012) 5:10101018 DOI 10.

1007/s11947-010-0395-4

ORIGINAL PAPER

Separation and Purification of Bromelain by Reverse Micellar Extraction Coupled Ultrafiltration and Comparative Studies with Other Methods
Umesh H. Hebbar & B. Sumana & A. B. Hemavathi & K. S. M. S. Raghavarao

Received: 2 February 2010 / Accepted: 15 June 2010 / Published online: 2 July 2010 # Springer Science+Business Media, LLC 2010

Abstract 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. Keywords Activity recovery . ATPE . Bromelain . Partition coefficient . Reverse micelles . Ultrafiltration
U. H. Hebbar : B. Sumana : A. B. Hemavathi : K. S. M. S. Raghavarao (*) Department of Food Engineering, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore 570020, India e-mail: fed@cftri.res.in

Introduction Bromelain is a type of proteolytic enzyme, present in the plant family Bromeliaceae of which pineapple (Ananas comosus L. Merryl) is the best known source. The stem bromelain (EC 3.4.22.32) and fruit bromelain (EC 3.4.22.33) obtained from the pineapple stem and fruit, respectively, are finding wide applications in pharmaceutical and food industry (Rowan et al. 1990). Bromelain is widely used as a digestive aid, meat tenderizer to improve the texture of the skin and to promote the healing of wounds. It is used commercially in certain cosmetics and dietary supplements. Bromelain is reported to be present also in pineapple wastes such as core, peel, and leaves relatively in smaller quantities as compared to stem (Sriwatanapongse et al. 2000). Liquid-liquid extraction (LLE) using reverse micellar systems and aqueous two-phase systems has been recognized as superior and versatile techniques for downstream processing of biomolecules. High capacity, biocompatible environment, low interfacial tension, high yields, lower processing time and energy, ease of scale-up, and scope for continuous operation are some of the advantages of reverse micellar extraction (RME) and aqueous two-phase extraction (ATPE; Harikrishna et al. 2002). Several reports on the application of RME for the separation and purification of biomolecules are available for model systems (commercial samples of proteins and enzymes) (Dekker et al. 1989; Ichikawa et al. 1992; Regalado et al. 1996; Lee and Dungan 1998; Zhang et al. 2002; Rairkar et al. 2007; Hebbar and Raghavarao 2007). However, in the recent

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past, a few studies on the application of RME for real systems have been reported (Liu et al. 2004; Chen et al. 2006; Hasmann et al. 2007; Hemavathi et al. 2007, 2008; Hebbar et al. 2008). As process integration improves the overall efficiency, an attempt has been made in the present study to couple RME with ultrafiltration. Although, selective separation of biomolecules based on their charge or size is considered to be one of the important features of RME, not many reports are available on the above. Hence, the present study attempts to selectively separate bromelain from the crude, which also contains polyphenoloxidase (PPO), an enzyme that is prominently present in pineapple. Use of ATPE for downstream processing of many enzymes such as amylases (Andersson et al. 1985), xylanase (Gaikaiwari et al. 1996), plant peroxidase (Srinivas et al. 1999), amyloglucosidase (Tanuja et al. 2000), potato polyphenoloxidase (Vaidya et al. 2006), lipase (Nandini and Rastogi 2008), and lipoxygenase (Lakshmi et al. 2009) has been reported. In some of the applications, ATPE has been used as a primary purification technique to reduce the bulk of the processing stream to be followed by more selective steps such as chromatography, electrophoresis, etc. Since, reports on the application of ATPE for the extraction of bromelain from pineapple core (which is considered to be the waste in juice processing industry) and comparative studies of these two methods of LLE techniques are not available, the present study is focused on these issues. The objectives of the present study are (1) to couple RME with ultrafiltration for the separation and purification of bromelain from the extract of pineapple core, (2) to study the selective partitioning/separation of bromelain and polyphenoloxidase using RME, and (3) to compare the performance of RME with other downstream processes such as ATPE and ammonium salt precipitation.

purchased from Merck, Mumbai, India. Hexane, hexanol, polyethylene Glycol (PEG) 4000 and PEG 1500 were from SRL, Mumbai, India. Casein (Hammerstein grade), catechol, and n-butanol from Loba chemicals, India were used. All other chemicals of analytical grade were used for the experiments and analyses. Preparation of Aqueous Bromelain Extract The core of the pineapple fruit was manually separated and a known quantity of this was crushed along with extraction buffer (0.01 M sodium phosphate buffer of pH 6.5, containing 1% PVP) at 1:1 ratio for 10 min and then filtered through a cheese cloth. The filtrate was centrifuged (MP 400 R, Eltek, India) at 10,000 g for 15 min and the supernatant (crude enzyme extract) obtained was used for the experiments. Reverse Micellar Extraction Forward extraction was carried out by mixing 250 ml of organic phase consisting of 150 mM CTAB/iso-octane/ hexanol/butanol (80%/5%/15% v/v) with an equal volume of aqueous phase (crude enzyme extract of pH 8.0 with 0.1 M NaCl). Back extraction was carried out by mixing the reverse micellar phase obtained from forward extraction with fresh aqueous phase (acetate buffer of pH 4.2 and 0.5 MKBr; Hebbar et al. 2008). The phases were mixed thoroughly for 1.5 h and centrifuged at 4,000g for 10 min during both forward and back extractions. The aqueous phases after forward and back extractions were analyzed for bromelain activity and total protein content. The phase mixing and separation were carried out at room temperature (252 C). Ultrafiltration Two hundred fifty milliliter of the aqueous phase containing bromelain obtained after RME was subjected to ultrafiltration using tangential flow filtration system (TFF 50, Millipore, USA) with cellulose acetate membrane (MWCO-5 kDa) module having surface area of 50 cm 2 . The transmembrane pressure and average permeate flow rate was maintained at 1 bar and 3 ml/ min, respectively. The experiment was continued till a reduction in volume of fivefold was achieved that is up to 50 ml of retentate volume. Aqueous Two-phase Extraction

Materials and Methods Materials Pineapple Fruit Mature pineapple fruits (Ananas comosus L. Merryl. cv. Kew, 7-9Brix) available in the local market were used for the extraction. Chemicals Cetyltrimethylammonium bromide (CTAB) was obtained from Merck, Germany. Iso-octane (HPLC grade) was

The phase systems were prepared on a percent w/w basis by mixing the required quantities of phase forming solutes in dilute enzyme extract. The phases were mixed thor-

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oughly for 30 min using a magnetic stirrer (Cintex, Mumbai) and allowed (5 h) to separate. The top and bottom phases were analyzed for bromelain activity and protein content. The pH of the phase was adjusted using either 0.1 N NaOH or 0.1 N HCl whenever required. The phase diagrams of PEG 4000 and PEG 1500 with K2HPO4/KH2PO4 system reported by Zaslavsky (1995) was used for the selection of phase composition. Ammonium Sulphate Precipitation Known quantity of ammonium sulphate was added to the enzyme extract and the mixture was stirred gently for 1 h at 4-6 C. Then it was centrifuged to separate out precipitated pellets. The precipitate was dissolved in known quantity of 0.01 M phosphate buffer and dialyzed against deionized water before analyzing for protein content and bromelain activity. Bromelain Assay Bromelain activity was determined according to the casein digestion unit (CDU) method using Hammerstein grade casein (0.6% w/v) as substrate in the presence of cysteine and EDTA (Murachi 1976). The assays were based on proteolytic hydrolysis of the casein substrate. The absorbance of the clear filtrate (solubilized casein) was measured at 275 nm using spectrophotometer (Shimadzu UV-160, Japan). One unit of bromelain activity is defined as 1 g of tyrosine released in 1 min/ml of sample when casein is hydrolyzed under the standard conditions of 37 C and pH 7.0 for 10 min. Polyphenoloxidase Assay PPO was assayed according to the spectrophotometric method described by Das et al. (1997). The assay mixture consisted of 2.6 ml of sodium phosphate buffer (0.01 M, pH 6.5), 0.3 ml of catechol (0.5 M), and 0.1 ml of enzyme extract. The increase in absorbance at 420 nm was measured. One unit of enzyme activity is defined as the amount of the enzyme that causes an increase in absorbance of 0.001/min at 25 C. Protein Content Protein content in aqueous phase was determined by measuring absorbance at 280 nm in case of RME system. The dye binding method of Bradford (1976) was used for ATPE systems. The sample analyses were performed against respective blank solutions. Protein concentration readings were taken in triplicate and an average value is used for the calculation of specific

activity. The bromelain activity recovery (%) and purification (fold) were estimated as shown below. Activity recovery% 1

bromelain activtity in the aqueous phase after RME bromelain activity in the feed 100

Purificationfold specific activity of bromelain after RME specific activity of bromelain in the feed 2

Partition Coefficient The partition coefficient (K) of the enzyme during ATPE was calculated by the following equation K CT =CB 3

where, CT and CB is the equilibrium concentration of enzyme in the top and bottom phases, respectively. The bromelain activity recovery in the top phase is estimated using the following equation Activity recovery % volume of top phase bromelain activity 100 volume of crude extract bromelain activity 4

SDS-PAGE Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out according to the method of Lammeli (1970) using a resolving gel of 10% acrylamide and a 5% stacking gel (1 mm thick). The staining of gel was performed using coomassie brilliant blue R-250. Statistical Analysis Significant differences between means were determined by t test (paired two samples for mean) using Microsoft Excel. Significance of differences was defined at p <0.05. The standard deviation is provided for the measured values.

Results and Discussion Reverse Micellar Extraction Studies on the optimization of processing conditions for the extraction and purification of bromelain from crude extract of pineapple core using reverse micellar system reported

Food Bioprocess Technol (2012) 5:10101018 Table 1 RME coupled ultrafiltration for extraction and purification of bromelain Sample Volume (ml) Activity (CDU/ml) 306.2 1,414.5 34.9 Protein concentration (mg/ml) 1.17 3.57 0.49 Activity recovery (%) Specific activity (CDU/mg) 261.71.9a 396.22.5b 71.21.8c

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Purification (fold)

Aqueous phase after RME UF Retentate UF Permeate

250 50 200

95.81.6a 92.42.2a 9.10.3b

5.90.10a 8.90.15b 1.60.10c

Specific activity of crude extract is 44.4 CDU/mg of protein. Means of the same column with different superscripts differ significantly (p <0.05)

by Hebbar et al. (2008) is used in the present study. The optimized conditions of extraction (forward extraction: 150 mM CTAB, 0.10 M NaCl, feed pH 8.0; back extraction: acetate buffer pH 4.2, 0.5 M KBr) resulted in an activity recovery of 106% and a purification of 5.2fold in 10-ml scale. In the present study, RME was carried out at a slightly higher scale (phase volume of 250 ml) and all the processing conditions except duration of phase mixing were maintained the same as that of previous (10 ml scale) study. The mixing duration was increased from 60 to 90 min for both forward and back extractions in order to have an efficient phase mixing. The activity recovery and purification fold measured at the end of back extraction is presented in Table 1. Marginal reduction in activity recovery (by 10%) was observed as compared to 10 ml studies while the purification increased from 5.2- to 5.9-fold.

Ultrafiltration Reverse micellar extraction was followed by ultrafiltration (UF) to improve the overall efficiency. The flow diagram for the combined process of RME and UF is shown in Fig. 1. The aqueous phase (250 ml) obtained from RME was subjected to UF in a tangential flow membrane system. The molecular weight cut-off of the membrane (5 kDa) was selected so as to retain bromelain (23-28 kDa) and separate out the other solutes having lower molecular weight. The extraction was continued (100 min) till the volume of the retentate reduced to 50 ml (i.e., reduction by fivefolds). The concentration of bromelain in the retentate increased to 1,414.5 CDU/ml against 319.7 CDU/ml in the feed. Purification of 1.5-fold was achieved during the UF step, leading to a reasonably high purification of 8.9-fold for the overall process (Table 1). Concentrated bromelain preparation is more stable than diluted one in terms of proteolytic

Fig. 1 Flow diagram for RME coupled ultrafiltration process for the extraction and purification of bromelain from pineapple core

Feed (Crude extract), 250 ml + 0.1 M NaCl

Activity: 319.7 CDU/ml Purification: 1.0 fold

Organic phase, 250 ml (150 mM CTAB + 80 % isooctane + 5% hexanol + 15% butanol)

Forward Extraction Reverse micellar phase, 250 ml

Left over aqueous phase Spent organic phase

Back Extraction

Acetate buffer, 250 ml + 0.5 M KBr

Activity: 306.2 CDU/ml Purification: 5.9 fold

Aqueous phase with bromelain, 250 ml

Permeate, 200 ml

Ultrafiltration, 250 ml

Activity: 1414.5 CDU/ml Purification: 8.9 fold

Bromelain concentrate (Retentate), 50 ml

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scale. The activity recovery obtained in the present study is more than that reported by Lopes et al. (2009; 85%) using membrane processes and Devakate et al. (2009; 84.5%) using ion exchange chromatography. SDS-PAGE Analysis The aqueous phase containing bromelain obtained from back extraction was dialysed overnight at 4 C and lyophilized. The concentrated sample was loaded to 10% gel along with marker. The SDS lane pattern of reverse micellar extracted bromelain and UF sample is shown in Fig. 2. The purified sample gave a single band for bromelain indicating reasonably good purification of the enzyme. Differential Partitioning of Bromelain and PPO in RME Bromelain and PPO are the two important enzymes present in the pineapple. Babu et al. (2008) studied the differential partitioning of these two enzymes using ATPE. In the present study, attempts were made to use the RME technique to selectively extract bromelain (23-28 kDa, pI 4.6) from the dilute extract (feed), leaving behind the large molecular weight PPO (104 kDa, pI 6.5). During the forward extraction the aqueous phase pH was maintained between the isoelectric points of bromelain (4.6) and PPO (6.5) in order to have the selective extraction of negatively charged bromelain by the cationic surfactant CTAB. pH values of 5.0 and 5.5 were used in the study and the other conditions of forward and back extractions were maintained

45 36 29 24 20 kDa 1 2 3 4

Fig. 2 SDS-PAGE pattern of reverse micellar extracted sample 1 marker, 2 crude extract, 3 RME sample, 4 UF retentate

activity (Lopes et al. 2009). UF used in the present study thus facilitated both purification and concentration of bromelain which is more stable than the crude bromelain extract. It may be expected that any traces of CTAB, a low molecular weight (364.46) surfactant and salt present in the aqueous phase would be removed during UF. With the developments in membrane processing technology, the feasibility of ultrafiltration technique for commercial production is not a constraint. Hence, the combined process of RME and UF might well be used as an effective process for the extraction and purification of bromelain at higher

Table 2 Differential partitioning of bromelain and PPO at different feed pH during RME Sample Protein concentration (mg/ml) Bromelain Activity recovery (%) Specific activity (U/mg) Purification (fold) Polyphenoloxidase Activity recovery (%) Specific activity (U/mg) Purification (fold)

FE at pH 5.0 Leftover aqueous phase after forward extraction Aqueous phase after RME FE at pH 5.5 Leftover aqueous phase after forward extraction Aqueous phase after RME FE at pH 8.0 Leftover aqueous phase after forward extraction Aqueous phase after RME

4.14 1.98 4.46 2.05 4.56 1.29

50.43.1a 69.60.9d 39.81.4b 77.11.7e 31.82.6c 98.11.1f

44.562.11a 104.112.08d 32.712.28b 117.632.30e 18.731.10c 216.660.73f

1.00.15a 2.40.15b 0.80.11a 2.70.11c 0.40.15a 5.00.15d

94.10.6a 11.40.7d 82.30.9b 23.60.6e 56.71.0c 44.41.2f

62.701.08a 12.460.70d 54.160.70b 25.410.72e 43.271.22c 30.191.09f

2.10.1a 0.40.1d 1.80.1b 0.80.05e 1.50.1c 1.00.05f

Specific activity of crude extract is 29.3 CDU/mg of protein. Means of the same column with different superscripts differ significantly (p <0.05) FE forward extraction

Food Bioprocess Technol (2012) 5:10101018 Table 3 Effect of phase composition and PEG molecular weight on bromelain extraction using aqueous two phase systems S. No. Sample Partition coefficient Purification (fold)

1015 Activity recovery (%)

Means of the same column with different letters differ significantly (p <0.05)

Phase composition variation [PEG 4000/(K2HPO4/KH2PO4)] (% w/w) 1 13.5/15.0 0.600.02a 1.40.15a 2 12.0/14.0 0.680.01a 2.70.10b 3 11.5/11.0 0.890.01b 1.40.15a PEG molecular weight variation 1 PEG 4000 0.540.02 2.60.13 2 PEG 1500 1.450.03 1.70.11

68.40.80a 93.20.80b 81.31.75c 95.11.04 90.01.88

similar to that employed in the above study (described in Reverse Micellar Extraction section). The bromelain and PPO activity was estimated in both the aqueous phases (forward and back extraction) and results are presented in Table 2. Higher activity of bromelain and PPO in the back extracted and leftover aqueous phases, respectively, indicated the selective extraction of bromelain during RME. Both the conditions resulted in higher PPO activity recovery (94-82%) and purification (2.11.8-fold) in the aqueous phase leftover after forward extraction. The bromelain purification (2.42.7-fold) was fairly good in the above conditions. At pH 8.0, bromelain activity recovery (98%) and purification (5.0) were maximum but the selective extraction efficiency decreased marginally, as PPO (pI 6.5) was also coextracted due to electrostatic interaction. However, higher difference between aqueous phase pH and isoelectric point (pH-pI) might have resulted in better extraction of bromelain at pH 8.0. Thus process parameters control during RME enhances its selectivity for the targeted biomolecule. Aqueous Two-phase Extraction of Bromelain In ATPE, the mechanism governing the partition of biological materials is not well understood; the observed

solute partition is reported to be the result of van der Waals, hydrophobic, hydrogen bond, and ionic interactions of the solutes with the surrounding phase (Gunduz and Korkmaz 2000). ATPE, in many cases, offers a better alternative to existing methods, especially in the early processing stages, with regard to scale of operation and purification of desired enzymes/proteins from a complex mixture. In aqueous twophase extraction, the purification of most of the proteins is mainly due to the differential partitioning of the target protein to one phase and the contaminant proteins to the other phase. The partitioning of biomolecules is influenced by many factors such as phase composition, molecular weight of phase forming polymer, phase volume ratio, phase system pH, etc. In the present work, the above parameters were altered to study the effect on activity recovery, purification, and partition coefficient of bromelain and the performance was compared with that of RME. Effect of PEG Molecular Weight Polymer molecular weight influences the protein partitioning as a direct result of available free volume. It has been found that the increase in PEG molecular weight decreases the partition coefficient as the free volume in the top phase decreases (Raghavarao et al. 1995). However, the effect of PEG molecular weight on partitioning of lower molecular weight proteins was reported to be very less (Silva and Franco 2000). It has been reported that the molecular mass of PEG contributes significantly to the partitioning behavior of proteins having a molecular mass greater than 50 kDa (Vaidya et al. 2006). It was also observed that with a decrease in the molecular mass of phase forming polymers, the interfacial tension decreases (Wu et al. 1996), which would facilitate the partitioning of solutes. In the present study, PEG 1500 and 4000 were used and the results are shown in Table 3. Although, the activity recovery of enzyme remained almost the same, the purification increased with the higher PEG molecular weight. However, the partition coefficient was higher with PEG 1500 as compared to that of PEG 4000. It is attributed that with the decrease in polymer molecular weight, the solutes (bromelain as well as others proteins) partitioned more towards the

Fig. 3 Effect of system pH on activity recovery, partition coefficient, and purification of bromelain

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Effect of System pH The pH of the system influences the ionizable groups of a protein and alters the protein surface charges. The pH range of 7-8 was selected as RME studies of bromelain gave good results in this pH range. A change in pH is often used to alter the partitioning of a biomolecule and the partition coefficient varied exponentially with the electrochemical potential difference between the phases and the net charge of the partitioned biomolecule (Gunduz and Korkmaz 2000). Partition coefficient increased with the increase in pH (Fig. 3) with a maximum value of 1.41 at pH 8.0. The activity recovery and purification fold decrease with the increase in pH. Higher values of activity recovery (93.1%) and purification (3.2-fold) were obtained at pH 7.0. System pH decides the surface charge of biomolecules and has direct influence on their partitioning. In general, negatively charged proteins prefer the top phase. At the pH range studied, bromelain will be negatively charged as a result it was partitioned to the top phase. Effect of Phase Volume Ratio It is reported that as the phase volume ratio (ratio of volume of top phase to bottom phase) increases, the free volume in the bottom phase decreases, leading to partitioning of proteins to top phase (Raghavarao et al. 1995). The effect of phase volume ratio in PEG 4000/K2HPO4/KH2PO4 system was studied. When the phase volume ratio increased from 0.31 to 1.43 the partition coefficient increased almost by twofold, indicating a higher protein transfer to the top phase. Increase in protein transfer with phase volume ratio decreased the purification but activity recovery of bromelain remained almost constant (Fig. 4). The primary importance of any purification process depends on its suitability for achieving high purification, concentration, and yield of the target biomolecule. In ATPE, this is achieved by changing the volume ratio. At higher volume ratio, even proteins with relatively low partition coefficients come to the top phase. The activity recovery and purification fold obtained in the present study were found to be higher as compared to the bromelain partitioning in aqueous two phase system reported by Rabelo et al. (2004) using thermoseparating polymers

Fig. 4 Effect of phase volume ratio on activity recovery, partition coefficient, and purification of bromelain

top phase resulting in higher partition coefficient. The lower interfacial tension and increased free volume caused by PEG of lower molecular mass might have contributed to the higher partitioning of proteins other than bromelain also, which in turn resulted in lower purification. Effect of Phase Composition Variation in phase composition changes the physical properties such as viscosity, density, and interfacial tension of the system (Nagaraj et al. 2002), which in turn affect the solute partitioning. Asenjo et al. 2002 reported the increase in interfacial tension with the phase composition and attributed it to the higher difference between the top and bottom phase composition. The change in free volume was reported to be the reason for variation in solute partitioning (Grossman and Gainer 1988). The concentrations of PEG and phosphate were selected (Table 3) such that the system would lie above the binodal curve to ensure phase separation. Decrease in PEG concentration increased the partition coefficient. The purification (2.7-fold) and activity recovery (93.2%) were maximum at 12% (w/w) PEG concentration and it decreases above and below this concentration. Recovery of bromelain in top phase decreases above 12% PEG concentration; this may be due to volume exclusion effect as reported by Babu et al. (2008).

Table 4 Ammonium sulphate precipitation for bromelain purification Sample Activity (CDU/ml) 398.52.6 743.81.8 Protein concentration (mg/ml) 7.180.22 5.270.14 Specific activity (CDU/mg) 55.501.14 141.142.36 Purification (fold) 1.0 2.50.2 Activity recovery (%) 100.0 82.12.4

Feed Ammonium sulphate precipitate

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which resulted in an activity recovery of 79.5% and purification of 1.2-fold. Ammonium Sulphate Precipitation Ammonium sulphate precipitation is conventionally used for the primary purification of bromelain followed by other purification methods such as chromatography, electrophoresis, etc. Although the above method is simple, it is tedious and time consuming. The present study was undertaken to compare the performance of the above method with RME. Precipitation was carried out by adding predetermined weighed quantity of ammonium sulphate to cold crude extract with continuous stirring. The mixture was allowed to stand undisturbed for 2 h for complete precipitation. The precipitate obtained at 50% ammonium sulphate concentration (this concentration is selected based on maximum recovery of bromelain) was dissolved in phosphate buffer of pH 7.0 and dialyzed overnight at 4-6 C using 10 kDa dialysis membrane. The activity recovery and purification obtained was 82.1% and 2.5-fold, respectively (Table 4). Although the purification of bromelain increased after ammonium sulphate precipitation it is lower than that observed for RME. The experimental findings suggests that RME coupled ultrafiltration and ATPE can be used effectively for separation and purification of bromelain with higher yield, since these liquidliquid extraction methods are gentle enough to preserve biological activity compared to conventional method of ammonium sulphate precipitation. Practically liquidliquid extractions can be easily scaled up and can be operated in continuous mode compared to precipitation.

References
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Conclusions Reverse micellar extraction resulted in good extraction efficiency of bromelain both in terms of activity recovery and purification compared to ATPE and ammonium sulphate precipitation. RME coupled ultrafiltration process was successfully used for obtaining higher bromelain activity recovery and purification. By varying the aqueous phase pH during RME, it is possible to selectively separate and purify bromelain and PPO simultaneously in two aqueous phases. Application of ATPE technique for bromelain extraction was encouraging, although the efficiency was not as high as that observed in case of RME.
Acknowledgments The authors thank Dr. V Prakash, Director, CFTRI, for the encouragement and keen interest in the area of downstream processing. Authors wish to thank Department of Biotechnology, New Delhi for funding (No.BT/PR-6418/PID/20/259/ 2005) the project.

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