Reverse Micellar Extraction of Bromelain from ...

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

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H. Umesh Hebbar , A. B. Hemavathi , B. Sumana & K. S. M. S. Raghavarao

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Department of Food Engineering, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore, India Available online: 12 Apr 2011

To cite this article: H. Umesh Hebbar, A. B. Hemavathi, B. Sumana & K. S. M. S. Raghavarao (2011): Reverse Micellar Extraction of Bromelain from Pineapple (Ananas comosus L. Merryl) Waste: Scale-up, Reverse Micelles Characterization and Mass Transfer Studies, Separation Science and Technology, 46:10, 1656-1664 To link to this article: http://dx.doi.org/10.1080/01496395.2011.572110

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Separation Science and Technology, 46: 1656–1664, 2011 Copyright # Taylor & Francis Group, LLC ISSN: 0149-6395 print=1520-5754 online DOI: 10.1080/01496395.2011.572110

Reverse Micellar Extraction of Bromelain from Pineapple (Ananas comosus L. Merryl) Waste: Scale-up, Reverse Micelles Characterization and Mass Transfer Studies H. Umesh Hebbar, A. B. Hemavathi, B. Sumana, and K. S. M. S. Raghavarao

Downloaded by [Umesh H. Hebbar] at 21:26 15 July 2011

Department of Food Engineering, Central Food Technological Research Institute, Council of Scientific and Industrial Research, Mysore, India

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 ¼ 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. Keywords bromelain; CTAB; mass transfer; reverse micelles; scale-up

INTRODUCTION Reverse micelles are thermodynamically stable, optically transparent, nanometer sized droplets of an aqueous solution stabilized in an apolar environment by the surfactant present at the interface (1). Reverse micelles provide simple, energy-efficient and mild separation conditions for enzyme recovery in active form. Low interfacial tension, ease of scale-up, and potential for continuous operation are some of the advantages of reverse micellar extraction (RME) (2,3). Several reports on the application of RME for separation and purification of biomolecules are available for model systems (4–8). In recent years, a few studies Received 22 October 2010; accepted 11 March 2011. Address correspondence to K. S. M. S. Raghavarao, Department of Food Engineering, Central Food Technological Research Institute (CFTRI), Mysore 570 020, India. Tel.: 91-0821-2513910; Fax: 91-0821-2517233. E-mail: [email protected]

on RME of biomolecules from natural=real systems have been reported (9–15). Bromelain, a proteolytic enzyme found in the tissues of 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 the pharmaceutical and food industry. Bromelain is reported to be also present in pineapple wastes such as core, peel, and crown although relatively in smaller quantities as compared to stem (16). Bromelain is widely used as a digestive aid, meat tenderizing agent, cleansing agent, anti-inflammatory agent, antibiotic potentiating agent, wound debridement agent, and in cosmetics (17). Pineapple processing industries generate nearly 35% of wastes (represent the mass of waste compared to the total mass of pineapple processed) which are either disposed off or used in composting (18). So, there is a need to use the waste for value addition and to decrease the load on the environment. Hebbar et al. (13) reported the laboratoryscale extraction of bromelain from pineapple wastes (peel, crown, core, and extended stem). The present study attempts the scale-up of RME technique for the extraction and primary purification of bromelain from core, which is one of the main wastes generated in pineapple processing (juice, canned slices) industry. Studies at higher scale are essential as the data provides valuable information that will facilitate further scale up of the process to industrial scale. Although, on the laboratory scale (10–25 mL phase volume) RME for downstream processing of biomolecules is well established, only a few reports on the large scale extraction (maximum reported being 2 L scale) using this technique are available (19,20). The cost of the phase components could significantly influence the process economics and there is a need to investigate this aspect during scale-up. Hence, in the present work, extraction at higher scale was attempted using commercial grade phase components to study the

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SCALE-UP STUDIES FOR REVERSE MICELLAR EXTRACTION OF BROMELAIN

effect of purity of phase components as well as scale-up on the extraction efficiency of bromelain. Models proposed in the literature for reverse micelles characterization range from simple geometric models (22– 26) to more rigorous molecular thermodynamic models (27–29). Several experimental methods and empirical models have been employed to determine the size and shape of reverse micellar aggregates. Dynamic light scattering (DLS), small angle neutron scattering (SANS), quasi-elastic light scattering (QELS), fluorescence recovery after fringe pattern photobleaching, small angle X-ray scattering (SAXS), or ultracentrifugation are some of the well known techniques employed for size measurement. Several empirical models, which show a linear relationship between water content (W0) and the hydraulic core radius (Rm) have been reported (30–33). In this study, some of these size estimation models (geometrical as well as empirical) derived for model systems, have been tested for the real system. Diffusion of solute and solute containing reverse micelles in aqueous and organic phases, and the formation of solute containing reverse micelles are considered to be the major steps involved in RME (34). Determination of mass transfer rates are important not only for designing an extraction process but also to gain a more fundamental understanding of the physical processes occurring during interfacial solubilization of biomolecules. Most of the mass transfer studies are on model solutes employing sodium-bis-2-ethylhexyl sulfosuccinate (AOT)=isooctane system (35,36). Hence, the present work focuses on the above aspects with respect to the real system using the cationic surfactant CTAB. The objectives of the present study are

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other chemicals used for the experiments and analyses were of analytical grade. Methods Preparation of Crude Extract The core of the pineapple fruit was manually separated and a known quantity was crushed in a fruit mill (B Sen Berry, New Delhi) along with buffer (0.01 mol=L sodium phosphate buffer of pH 6.5 and containing 1% polyvinylpyrrolidone) at 1:1 ratio, followed by expelling of juice in a screw press (B Sen Berry, New Delhi). The extract was centrifuged at 12,000 g using a continuous disk centrifuge (Westfalia Separator AG, Germany). The supernatant (crude enzyme extract) thus obtained was used for the experiments. The flow diagram and material balance for the preparation of crude extract for 5 L (phase volume) scale is shown in Fig. 1. Reverse Micellar Extraction of Bromelain The optimized RME conditions reported by the authors (13) are used in the present study. Forward extraction was carried out by contacting known volume (10 mL, 250 mL,

i. to study effect of scale-up and purity of phase components on extraction efficiency of bromelain ii. to estimate the size of reverse micelles (filled and unfilled) and study its relationship with water content iii. to estimate the mass transfer coefficient for forward and back extractions.

MATERIALS AND METHODS Materials Pineapple Fruit Mature pineapple fruits (Ananas comosus L. Merryl. cv. Kew) available in the local market were used for the extraction of bromelain. Chemicals CTAB (ultrapure grade) and CTAB (commercial AR grade) were obtained from Merck, Darmstadt, Germany and Himedia, Mumbai, India, respectively. Isooctane (HPLC and AR grade) was purchased from Merck, Mumbai. Hexanol from SRL, Mumbai, casein (Hammerstein grade) and n-butanol from Loba chemicals, India were used. All

FIG. 1. Process flow diagram and material balance for the preparation of crude extract.


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H. U. HEBBAR ET AL.

or 5 L) of organic phase consisting of 150 mmol=L CTAB= 80% (v=v) iso-octane=5% (v=v) n-hexanol=15% (v=v) n-butanol with an equal volume of aqueous phase (crude enzyme extract of pH 8.0 with 0.1 mol=L 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 mol=L KBr). Commercial grade CTAB and isooctane were used for experiments at 10 mL and 5 L, whereas for extraction at 250 mL, ultrapure components were used. For both forward and back extractions at 10 and 250 mL scale the phases were mixed thoroughly using a magnetic stirrer in a glass container for 30 min and centrifuged at 4000 g for 10 min. In case of 5 L phase volume, mixing was carried out for 1.5 h using pitched blade agitator (250 rpm) in a stainless steel (AISI 304) container (20 L capacity) fitted with baffles. In this case, gravity separation (2 h, determined by visual appearance of two distinct clear phases) was employed in place of centrifugal separation. 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 (25 2 C) during RME. Estimation of Water Content and Size of the Reverse Micelle The amount of water present in the organic phase after forward extraction was measured using Karl Fischer (KF) auto titrator (DL 32, Mettler Toledo, Germany). The water percent value was converted into molar value and used for the estimation of water content (W0, which is the molar ratio of water to surfactant per reverse micelle). The W0 value obtained was used for the estimation of the radius of reverse micellar core using the empirical=geometrical models reported in the literature (Table 1) and values were expressed in nanometers. Bromelain Assay Bromelain activity is determined by the casein digestion unit (CDU) method using Hammerstein grade casein (0.6% w=v) as substrate in the presence of cysteine and ethylenediaminetetraacetic acid (EDTA) (37). 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 mg of tyrosine released in 1 min per mL of sample when casein is hydrolyzed under the standard conditions of 37 C and pH 7.0 for 10 min. Protein Content The protein content was determined by measuring the absorbance at 280 nm using BSA as standard. The sample analysis was performed against respective blank solutions. The protein concentration readings were taken in triplicate and an average value is used for the calculation. The enzyme

TABLE 1 Models used for the estimation of reverse micellar radius (Rm) Geometrical models Levashov et al. (22) Sheu et al. (23)

n o1=3 Rm ¼ 43p ðN0ag W0 Vw Þ For empty 3 micelle 1=3 s Rem ¼ 4p Nw e V w þ Ne V S For filled 3 micelle 1=3 s Rfm ¼ 4p Nw f Vw þ Nf VS þ Vp

Regaldo et al. (24)

W0 Rm ¼ 3VAtSm

Krei and Hustedt (25) Jolivalt et al. (26) Empirical models Bru et al. (30) Gaikar and Kulkarni (31) Motlekar and Bhagwat (32) Kinugasa et al. (33)

o Mw Rm ¼ 3W As Nq w

Rm ¼ 3VAwSW0 Rm ¼ 0.175 W0 Rm ¼ 0.164 W0 Rm ¼ 0.15 W0 Rm ¼ 0.145 W0 þ 0.57

activity recovery (%) and purification (fold) were estimated as shown below Activity recovery ð%Þ ¼ enzyme activity in aqueous phase after RME volume of aqueous phase enzyme activity in feed volume of feed

ð1Þ 100

Specific activity ðU=mgÞ ¼ enzyme activity in aqueous phase protein concentration in aqueous phase

ð2Þ

Purification ðfoldÞ ¼ specific activity of enzyme in aqueous phase after RME specific activity of enzyme in feed ð3Þ

Overall Mass Transfer Coefficient The overall mass transfer coefficient, ka (min1) for forward and back extraction was estimated using the equations proposed by Dungan et al. (35) n o 0 ¼ A V kf 0 t ¼ kfo at ¼ Kf t In 1 ðCorg =Caq Þ ð4Þ

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In

n o 0 1 ðCaq =Corg Þ ¼ A V kb0 t ¼ kbo at ¼ Kb t ð5Þ


where, Corg and Caq are the concentrations of solute in the organic and aqueous phases, superscript ‘0’ indicates the initial concentration, ‘A’ is the interfacial area, ‘V’ is the volume of phase, and ‘t’ is the phase mixing time. Subscripts ‘f’ and ‘b’ indicates forward and back extraction respectively. ‘K’ is the overall mass transfer coefficient. RESULTS AND DISCUSSION Effect of Purity of Phase Components Laboratory scale (10 mL) studies on the extraction of bromelain from pineapple core using CTAB=isooctane= n-hexanol=n-butanol system reported by the authors (13) using pure grade CTAB and HPLC grade isooctane had resulted in an activity recovery of 106% and purification of 5.2 fold. In order to study the effect of purity of phase components (surfactant and solvent), laboratory scale studies (10 mL) were carried out using commercial grade components. Lower activity recovery (90.07%) and purification (2.86 fold) with commercial grade components indicated the significant influence of purity of phase components on overall extraction efficiency. Impurities present might have hindered the extraction and also reduced the purification of bromelain extracted. Since, cost of phase components has a direct effect on process economics, it is required to have a trade off to decide on the purity of components to be used. Scale-up Studies To study the effect of process scale-up on extraction efficiency, the studies were carried out in two levels; i. 250 mL phase components,

volume

using

pure

grade

phase

ii. 5 L phase volume with commercial grade phase components.

The scale-up of the process to 250 mL resulted in an activity recovery of 97.15% and purification of 5.28 fold. The results are almost the same as that obtained at 10 mL phase volume with same phase components. An activity recovery of 81.32% and purification of 2.43 fold was obtained at 5 L scale (Table 2). The above result is similar to that obtained with laboratory scale (10 mL) extraction using commercial grade phase components. The above-mentioned studies indicated that scale-up of the process does not significantly affect the performance of RME. Although, the type of mixing and method of phase separation were different at a higher scale when compared to laboratory scale studies, the efficiency of extraction did not change appreciably. Process scale-up studies (upto 2 L, i.e., scale-up by 200 times) have reported reduction in activity recovery upto 10–30% (19,20), which was attributed to a lower degree of phase contact at higher volume, enhanced shear forces generated, longer phase separation time under gravity rather than centrifugation, and also due to extended complexation of the solute with the surfactant in the aqueous phase. However, in the present study only 9% decrease in recovery was observed for scale-up of 500 times. This indicated the adequacy of processing conditions as well as systems employed in scale-up experiments. In addition the results indicated the scope for further scale-up. In order to confirm the stability of bromelain during the processing period, control sample was kept at same condition (25 2 C). Activity of bromelain found to be quite stable indicates that reduction in activity recovery was not due to the long time of gravity separation at higher scale. Characterization of Reverse Micelles Determination of Water Content The amount of water present in reverse micelles at different concentrations of CTAB was estimated using the KF titration method. The amount of water present in the reverse micellar phase increased linearly with the surfactant concentration for unfilled reverse micelles. In case of filled reverse micelles, a similar trend was observed

TABLE 2 Scale-up studies for reverse micellar extraction of bromelain Sample Crude extract (feed) RME (10 mL) RME (250 mL) RME (10 mL) RME (5000 mL)

Specific activity (CDU=mg)

Activity recovery (%)

68.36 100 With pure grade phase components 356.15 1.65 106.04 3.1 361.29 1.29 97.15 2.4 With commercial grade phase components 195.51 1.15 90.07 2.5 166.15 1.36 81.32 2.1

Purification (fold) 1.00 5.21 0.2 5.28 0.1 2.86 0.1 2.43 0.1


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up to CTAB concentration of 150 mmol=L (Fig. 2a). The increase in the amount of water is mainly due to an increase in the number of reverse micelles formed. At constant salt concentration, increasing surfactant concentration yields an increase in the number of reverse micelles of constant size. Whereas, at constant surfactant concentration varying the salt concentration varies the reverse micelles size rather than number (38). When both the surfactant and the salt concentration are constant, the number of reverse micelles formed are fixed but the size will vary based on the water content and=or biomolecule extraction. The study by Hebbar et al. (13) on the effect of surfactant concentration has shown a reduction in forward extraction efficiency at higher CTAB concentration (above 150 mmol=L) and it was attributed to the inter-micellar collision and collapse of reverse micellar structure. Decrease in water content in the organic phase above 150 mmol=L CTAB concentration substantiated the observation made earlier. Figure 2b shows the change in W0 values with CTAB concentration. For unfilled micelles, the W0 value remained almost constant at different CTAB concentrations. In case of filled micelles, the concentration did not change

FIG. 2. (a). Effect of CTAB concentration on percentage of water in reverse micellar phase; (b). Effect of CTAB concentration on water content.

appreciably, except for concentration above 150 mmol=L. This indicates that with the increase in the surfactant concentration, the size of reverse micelles does not change and will remain almost the same but additional molecules of the surfactant added would contribute to the formation of new reverse micelles of the same size (as salt concentration remains constant at 0.1 mol=L). Except at CTAB concentration of 200 mmol=L, the W0 values for the filled reverse micelles (64–68) were higher as compared to that of the unfilled ones (51–55). Ichikawa et al. (3) also reported that the amount of solubilizing water in the proteincontaining system to be more than in the protein-free system. Rabie and Vera (39) had reported the effect of surfactant (AOT) concentration on W0, wherein with an increase in AOT concentration, the water uptake increased, although the increase was not linear. Estimation of Size of Reverse Micelles Reverse micelle size is a very important parameter that determines protein selectivity, enzyme activity, and water content. Size measurement of reverse micelles formed by other surfactants (i.e., other than AOT) has been sparingly reported (40). In the present study, the water content values obtained at different concentrations of CTAB were used for the estimation of the size of the reverse micelles employing the equations listed in Table 1. The size of the reverse micelles estimated for unfilled and filled micelles using empirical and geometrical models are shown in Tables 3a and 3b, respectively. All the models resulted in bigger size of filled reverse micelles compared to that of the unfilled ones, except at concentration of 200 mmol=L. An increase in size of nearly 15–21% was observed for filled reverse micelles. Among the empirical models, the model proposed by Bru et al. (30) resulted in relatively higher values for both unfilled (8.9–9.6 nm) and filled (8.4–11.9 nm) reverse micelles, which is obviously due to the higher multiplication factor of W0. The change in surfactant concentration did not significantly change the size of the unfilled reverse micelle, which indicated that increase in surfactant concentration only increases the number of micelles while the size of the micelle is not affected. The same trend was observed with filled reverse micelles, except for concentration above 150 mmol=L. Like in the case of empirical models, the size of the filled reverse micelles was bigger when compared to the unfilled reverse micelles for geometrical models. The increase in size was in the range of 15–20%. The geometrical models proposed by Leavashov et al. (22) and Sheu et al. (23) resulted in relatively higher Rm values (7.9–10.9 nm) compared to other geometrical models (7.1–9.5 nm) and were closer to the values obtained from empirical models. The values were almost similar for the other three geometrical models for both filled and unfilled reverse micelles. It has been reported (21,23,29,30) that incorporation of solute into micelles increases the size of the microstructure

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TABLE 3a Size of the reverse micelles estimated using empirical models Radius of the reverse micelle (nm) Gaikar & Kulkarni (31)

W0

Kinugasa et al. (33)

Motlekar & Bhagwat (32)

Bru et al. (30)

CTAB (mmol=L)

UF

F

UF

F

UF

F

UF

F

UF

F

50 100 150 200

53 55 52 51

68 66 64 48

8.7 9.0 8.5 8.4

11.1 10.8 10.5 7.9

8.3 8.5 8.1 8.0

10.4 10.1 9.8 7.5

7.9 8.2 7.8 7.6

10.2 9.9 9.6 7.2

9.3 9.6 9.1 8.9

11.9 11.5 11.2 8.4


F-filled, UF-unfilled.

to some extent. The results obtained in the present study have also indicated the increase in size of reverse micelle with the inclusion of enzyme. However, a few studies (22,41) have reported that reverse micelles adjust the size in such a manner that it remains more or less the same for both filled and unfilled reverse micelles. To derive at an empirical relationship between W0 and size of the reverse micelle (Rm), a plot of Rm at different W0 values was drawn (Fig. 3) for both filled and unfilled reverse micelles. The geometrical model reported by Krie and Hustdet (25) was used for the estimation of Rm as this model was tested for the CTAB system. The data has given a good fit with high coefficient of regression (R2) value of 0.92 and 0.99, respectively for unfilled and filled reverse micelles. The correlation obtained is given below and is found to be almost the same for filled and unfilled reverse micelles. Rm ¼ 0:138 W0 ; for unfilled reverse micelles

ð6Þ

Rm ¼ 0:136 W0 ; for filled reverse micelles

ð7Þ

Validation of the Model To check the goodness of the empirical equation obtained, the experiments were carried out as detailed earlier at different CTAB concentrations (20, 40, 60, and 80 mmol=L). The W0 and reverse micellar size were estimated as detailed earlier. A parity plot of estimated and predicted values of reverse micellar size showed an excellent fit (R2 ¼ 0.99) for both unfilled and filled reverse micelles (Fig. 4). Estimation of Mass Transfer Coefficients To study the mass transfer kinetics during forward extraction, phase mixing was carried out for different time intervals and protein concentrations were estimated for studies at 250 ml phase volume. The transfer of protein from aqueous phase to reverse micellar phase was high in the first 20 min of phase mixing with nearly 60% (of total 65%) of the extraction taking place during the above period. To estimate thenforward extraction o mass transfer coefficient, a plot of In

0 1 ðCorg =Caq Þ

against mixing

time ‘t’ was drawn for the initial period of mixing (30 min), during which maximum extraction took place (Fig. 5a). The mass transfer coefficient Kf was estimated from the

The equation obtained above was found to be closer to that (Rm ¼ 0.15 W0) reported by Motlekar and Bhagwat (32).

TABLE 3b Size of the reverse micelles estimated using geometrical models Radius of the reverse micelle (nm) Levashov et al. (22)

W0

Sheu et al. (23)

Krei & Hustedt (25)

Jolivalt et al. (26)

Regaldo et al. (24)

CTAB (mmol=L)

UF

F

UF

F

UF

F

UF

F

UF

F

UF

F

50 100 150 200

53 55 52 51

68 66 64 48

8.6 8.9 8.5 8.6

10.9 10.6 10.3 7.9

8.0 8.2 7.8 7.7

9.8 9.2 9.3 6.8

7.3 7.7 7.1 7.1

9.2 9.1 8.7 6.7

7.4 7.6 7.2 7.1

9.5 9.2 8.9 6.7

7.3 7.6 7.2 7.1

9.4 9.1 8.9 6.6

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FIG. 3.

Variation of reverse micelles size with water content.

slope of the line (trend line) drawn. Since, it is difficult to estimate the interfacial area or contact area under mixing conditions, the mass transfer coefficient was estimated in terms of kfoa (min1). The estimated mass transfer coefficient was found to be 0.038 min1. Back transfer experiments were carried out using the reverse micellar phase obtained from forward extraction. For all the experiments, forward extraction time was maintained the same (1 h). Back extraction was carried out for different time intervals and the corresponding protein concentration in the aqueous phase was measured. Like in the case of forward extraction, nearly 43% (of total 49%) of the extraction took place in the first 20 min. Practically, there was no extraction of solute observed after 30 min. The mass transfer coefficient was estimated from n for back extraction o the plot of In

0 1 ðCaq =Corg Þ

(Fig. 5b) against time ‘t’

and it was found to be 0.025 min1, which is lower than that obtained for forward extraction. Dungan et al. (35) reported the back transfer rate to be three orders of magnitude slower than the forward transfer

FIG. 4.

Parity plot for reverse micelles size estimation.

FIG. 5. (a) Estimation of mass transfer coefficient for forward extraction of bromelain; (b) Estimation of mass transfer coefficient for back extraction of bromelain.

(for model systems). Coalescence of the protein filled reverse micelles with the bulk interface dominates the back transfer kinetics indicating interfacial resistance to be the limiting factor. They reported kf in the range of 0.06– 0.0006 cm=min and kb in the range of 0.0012–0.00036 cm= min. In the present study we found the back transfer rate to be 66% of forward transfer rate. Dovyap et al. (7) predicted kf to be in the range of 0.078–0.3612 cm=min under optimized conditions for amino acid extraction (model system). Liu et al. (42) obtained Kf in the range of 0.063–0.093 min1 and Kb in the range 0.00114– 0.00096 min1 for the model system, wherein Kb found to be very less than the Kf indicating high resistance to mass transfer during back extraction. CONCLUSIONS Studies at higher scale (5 L phase volume) with commercial grade CTAB and isooctane resulted in an activity recovery of 81.32%, which is only 9% lower than that obtained



with laboratory scale studies (10 mL phase volume). Purity of phase components significantly affected the extraction efficiency of bromelain. All the models predicted a bigger size of the filled reverse micelles as compared to that of the unfilled ones. The empirical model obtained showed a good correlation (R2 ¼ 0.99) between the estimated and the predicted values of Rm for both filled and unfilled reverse micelles. The protein transfer across the phases was high in the initial period of mixing (20 min) for both forward and back extractions. The studies on kinetics of mass transfer indicated a relatively slower rate (by 34%) of mass transfer in case of back extraction compared to forward extraction. ACKNOWLEDGEMENTS The authors thank Dr. V. Prakash, Director, CFTRI, for the encouragement. Authors wish to thank Department of Biotechnology, New Delhi for funding (#BT=PR-6418= PID=20=259=2005) the work. REFERENCES 1. Harikrishna, S.; Srinivas, N.D.; Raghavarao, K.S.M.S.; Karanth, N.G. (2001) Reverse micellar extraction for downstream processing of proteins=enzymes. Adv. Biochem. Eng. Biotechnol., 75: 119. 2. Goklen, K.E.; Hatton, T.A. (1987) Liquid–liquid extraction of low molecular weight proteins by solubilization in reversed micelles. Sep. Sci. Technol., 22: 831. 3. Ichikawa, S.; Imai, M.; Shimizu, M. (1992) Solubilizing water involved in protein extraction using reversed micelles. Biotechnol. Bioeng., 39: 20. 4. Poppenborg, L.H.; Brillis, A.A.; Stuckey, D.C. (2000) The kinetic separation of protein mixtures using reverse micelles. Sep. Sci. Technol., 35: 843. 5. Shin, Y.; Weber, M.E.; Vera, J.H. (2003) Reverse micellar extraction and precipitation of lysozyme using sodium di(2-ethylhexyl) sulfosuccinate. Biotechnol. Prog., 19: 928. 6. Chun, B.S.; Park, S.Y.; Kang, K.Y.; Wilkinson, G.T. (2005) Extraction of lysozyme using reverse micelles and pressurized carbon dioxide. Sep. Sci. Technol., 40: 2497. 7. Dovyap, Z.; Bayraktar, E.; Mehmetoglu, U. (2006) Amino acid extraction and mass transfer rate in the reverse micelle system. Enz. Microb. Technol., 38: 557. 8. Hebbar, H.U.; Raghavarao, K.S.M.S. (2007) Extraction of bovine serum albumin using nanoparticulate reverse micelles. Process Biochem., 42: 1602. 9. Jarudilokkul, S.; Stuckey, D.C. (2001) Continuous forward and back extraction of lysozyme from egg white using reverse micelles. Sep. Sci. Technol., 36: 657. 10. Manocha, B.; Gaikar, V.G. (2006) Permeabilization of Aspergillus niger by reverse micellar solutions and simultaneous purification of catalase. Sep. Sci. Technol., 41: 3279. 11. Hemavathi, A.B.; Hebbar, H.U.; Raghavarao, K.S.M.S. (2007) Reverse micellar extraction of bromelain from Ananas comosus L. Merryl. J. Chem. Tech. Biotechnol., 82: 985. 12. Hemavathi, A.B.; Hebbar, H.U.; Raghavarao, K.S.M.S. (2008) Reverse micellar extraction of b-galactosidase from barley (Hordeum vulgare). Appl. Biochem. Biotechnol., 151: 522. 13. Hebbar, H.U.; Sumana, B.; Raghavarao, K.S.M.S. (2008) Use of reverse micellar systems for the extraction and purification of bromelain from pineapple wastes. Bioresour. Technol., 99: 4896.

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