Polymer-based alternative method to extract

bromelain from pineapple peel waste

Letıcia Celia de Lencastre

Novaes1∗

Valeria de Carvalho Santos

Ebinuma1

Priscila Gava Mazzola2

Adalberto Pessoa Junior1

1Department of Biochemical and Pharmaceutical Technology, School ofPharmaceutical Science, University of Sao Paulo, Sao Paulo, Brazil2Department of Clinical Pathology, Faculty of Medical Sciences, Universityof Campinas, Campinas, Brazil

Abstract

Bromelain is a mixture of proteolytic enzymes present in alltissues of the pineapple (Ananas comosus Merr.), and it isknown for its clinical therapeutic applications, foodprocessing, and as a dietary supplement. The use of pineapplewaste for bromelain extraction is interesting from both anenvironmental and a commercial point of view, because theprotease has relevant clinical potential. We aimed to study theoptimization of bromelain extraction from pineapple waste,

using the aqueous two-phase system formed by polyethyleneglycol (PEG) and poly(acrylic acid). In this work, bromelainpartitioned preferentially to the top/PEG-rich phase and, in thebest condition, achieved a yield of 335.27% with a purificationfactor of 25.78. The statistical analysis showed that allvariables analyzed were significant to the process. C© 2013International Union of Biochemistry and Molecular Biology, Inc. Volume 60,Number 5, Pages 527–535, 2013

Keywords: aqueous two-phase system, bromelain, pineapple waste,polyethylene glycol, poly(acrylic acid)

1. IntroductionIn the biotechnology industry, aqueous two-phase systems(ATPS) find extensive application for the separation and pu-rification of biomolecules such as proteins, enzymes, viruses,and nucleic acids [1]. ATPS can be obtained by the addition oftwo chemically different water-soluble polymers to water ora combination of a water-soluble polymer and salt [2]. Afterachieving phase equilibrium, which is rapid, these systems con-tain up to 80%–90% of water (w/w), and each phase containsmainly one of the compounds [2,3]. Because of the high water

Abbreviations: PEG, polyethylene glycol; ATPS, aqueous two-phasesystems; PAA, poly(acrylic acid); EC, enzyme commission number;Na2SO4, sodium sulfate; Ytop, yield in the top phase; PF, purification factor;K, partition coefficient.∗Address for correspondence: Letıcia Celia de Lencastre Novaes, MSc,Universidade de Sao Paulo, Departamento de TecnologiaBioquımico-Farmaceutica – Faculdade de Ciencias Farmaceuticas. Av. Prof.Lineu Prestes, 580, Bloco 16 Cidade Universitaria 05508–000 – SaoPaulo/SP, Brazil. Tel.: +55 11 30913862; Fax: +55 11 38156386;e-mail: lclnovaes@usp.br.Received 13 March 2013; accepted 23 April 2013DOI: 10.1002/bab.1121Published online 6 September 2013 in Wiley Online Library(wileyonlinelibrary.com)

content, ATPS have several advantages when compared withcommonly used separation and purification techniques. Theyhave a low interfacial tension, in addition to being nontoxic,nonflammable, biocompatible, and being easily scaled-up with-out considerable changes in the efficiency of the process [2–5].

Most common polymer-polymer-based ATPS are thoseformed with polyethylene glycol (PEG) and dextran. The indus-trial usage of the PEG + dextran system is limited because of thehigher costs of dextran ($100–200/kg) [4, 6]. Saravanan et al.[2, 4] showed that poly(acrylic acid) (PAA; $1–10/kg) is a goodalternative to dextran to be used as one of the phase-formingpolymers in ATPS with PEG. Johansson et al. [7–9] studied thepartition of several proteins and pDNA, through ATPS formedby PEG + PAA, and found that proteins could be partitionedwith almost 100% retained activity [8].

Bromelain is a mixture of proteolytic enzymes presentin stem and fruit of the pineapple (Ananas comosus Merr.,Bromeliaceae family), constituting an unusually complex mix-ture of different thiol-endopeptidases. Stem-bromelain (EC3.4.22.32) is distinguished from fruit-bromelain (EC 3.4.22.33),as they have different proteolytic activities, molecular masses(23.8 and 23 kDa), and isoelectric points (>10 and 4.6, respec-tively) [10,11].

Bromelain has been known for its clinical and therapeuticapplications, particularly for modulation of tumor growth,

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selective burn debridement, improvement of antibiotic action,and anti-inflammatory, antithrombotic, and fibrinolytic ac-tivities [10, 12, 13]. Recently, it has been shown to modulatethe cellular responses of lymphocyte after oral use [14]. It isalso used in food processing for meat tenderization and as adietary supplement [11]. Bromelain is used in the United Statesand Europe as an alternative or complementary medicationto glucocorticoids and antirheumatics, as well as nonsteroidaland immunomodulatory agents [10]. Isolation of enzymes frompineapple has been investigated since 1893, when Chittendenisolated bromelain, formerly called bromelin, by salt precipita-tion [15]. The continuous focus on bromelain, for its numerousapplications in the food industry, medicine, and pharmacology,makes the search for new alternatives for its purification veryinteresting. The preparation in pure form of a proteolytic en-zyme has always proved difficult, and bromelain appears to beno exception [16].

Pineapple waste (peel, core, crown, and leaves) usuallyaccounts for 50% (w/w) of its total weight [17]. Sriwatanapongseet al. [18] reported that bromelain is present in pineapple wastein smaller quantities when compared with stem. The use ofpineapple waste for bromelain extraction is interesting fromboth environmental and commercial viewpoints, as commercialbromelain costs up to $2,400/kg [15].

Experimental design in ATPS has been used in severalstudies to optimize the extraction of biomolecules [19–26].It reduces the number of experiments and also can identifythe independent variables with statistical significance for theprocess, as well as their significant interactions [25]. In thepresent study, an experimental design was used to analyzebromelain extraction from pineapple waste by polymer-basedATPS for potential pharmaceutical application. This workaimed to evaluate a new extractive/purification techniqueas an alternative to other methodologies used for bromelainextraction.

2. Materials and Methods2.1. MaterialsPineapple peel residues were donated by a juice industry fromSao Paulo. Peels were sliced and blended. The extract obtainedwas centrifuged at 10,000g, for 20 Min, for removal of insolublematerials. Supernatant aliquot was stored at −20 ◦C until themoment of its use. Poly(acrylic acid) and PEG were purchasedfrom Sigma (St. Louis, MO, USA). All the other chemicals usedwere of analytical grade.

2.2. Bromelain activity determinationBromelain activity was measured by the methodology describedby Kunitz [27] and modified by Walter [28], using 2% (w/v)casein as the substrate and tyrosine as the standard. In short,the methodology consists of allowing bromelain casein cleavageat 37 ◦C for 10 Min, following the addition of trichloroaceticacid. The casein cleavage releases tyrosine residues, which aredetected by spectroscopy.

2.3. Protein determinationProtein determination was made by the Sigma bicinchoninicacid kit [29]. Absorbance measurements were made in amicroplate reader at 562 nm (SpectraMax Plus 384; MolecularDevices, Sunnyvale, CA, USA).

2.4. Bromelain extraction by ATPSThe systems were prepared in 15 mL graduated glass tubes byadding PAA, PEG, saline solution (Na2SO4), McIlvaine buffer(pH 5.0), and the pineapple waste extraction, resulting in a5 g total mass system. The amount of pineapple extract ineach system was 10% of the total weight, the initial bromelainactivity was 2.1 ± 0.2 U/mL, and the concentration of totalproteins was 10.3 ± 0.7 mg/mL. The system components wereadded by weighing, and further homogenized in an orbitalshaker (Barnstead/Thermolyne, model 400110) at 8 rpm for5 Min at room temperature.

Afterward, the system was transferred to a controlledtemperature bath and kept at rest for 30 Min for phaseseparation and balance. After resting, the samples of the topand bottom phases were carefully collected using Pasteurpipettes, and the concentration of proteins and bromelainactivity were determined in both phases.

The partitioning behavior of bromelain in ATPS wasquantified in terms of partition coefficient, purification factor,and yield. The partition coefficient (K) is defined as

K = [bromelain]t/[bromelain]b

where [bromelain]t represents bromelain activity in the topphase and [bromelain]b represents bromelain activity in thebottom phase. The partition coefficient, K, is often used toevaluate the extension of biomolecule separation in the aque-ous two-phase polymeric systems. When K is significantlydistinct for the target biomolecule and for other biomoleculespresent in the system, the extraction is more effective. In otherwords, K values greater than unity indicate the effectiveness ofpartitioning in the ATPS [3].

The purification factor is defined as

PF = SAphase/SAinitial

where SAphase is the bromelain specific activity in one phase andSAinitial is the bromelain specific activity before the partition.

Yield is defined as

Y= (Actphase × Volphase)/(Actinitial × Volinitial)

where Actphase is the bromelain activity in one phase, Volphase isthe volume of one phase, Actinitial is the bromelain activity beforethe partition, and Volinitial is the initial volume of bromelain.

2.5. Experiment design and statistical analysisThe bromelain extraction in this study was evaluated usingtwo successive experimental designs. First, a 26–2 fractionalstatistical design was performed to verify the effects andinteractions of PEG and PAA concentrations, PEG and PAAmolar mass, temperature, and salt (Na2SO4) concentration.

528 Bromelain Extraction from Pineapple Peel Waste

TABLE 1Factor levels used in the 26–2 design

Levels

Factor (−1) (0) (+1)

PEG concentration (%, w/w) 6 7 8

PAA concentration (%, w/w) 6 7 8

PEG molar mass 2,000 4,000 6,000

PAA molar mass 1,200 8,000 15,000

Na2SO4 (%, w/w) 6 6.5 7

Temperature (◦C) 20 25 30

TABLE 2Factor levels used in the 25–1 design

Levels

Factor (−1) (0) (+1)

PEG concentration (%, w/w) 8.5 9 9.5

PAA concentration (%, w/w) 8.5 9 9.5

PEG molar mass 1,000 1,500 2,000

PAA molar mass 1,200 8,000 15,000

Temperature (◦C) 20 25 30

The screening design used was a resolution IV; the analysisof the results used contrast, where the main effects wereconfounded with the three-factor interactions, while the two-factor interactions were confounded with each other. A factorialmodel can be interpreted as modeling of a given response bya special type of potency series, where higher-order termsare assumed to be less important than those of a minororder; otherwise, the series would not converge and the modelobtained would be useless. It is natural, therefore, to assumethat third-order terms are less important than first-order,and, in a first approximation, they could be neglected [30,31];therefore, the third-order interactions are negligible in thiswork.

Next, a 25–1 fractional statistical design was used to studyPEG and PAA concentrations, PEG and PAA molar mass, andtemperature. In both designs, a set of 19 experiments with threereplicates at the central point was performed, and yield in thetop phase and purification factor were treated as responses.The range and levels of the components under study are givenin Tables 1 and 2 for the first and second experimental designs,respectively.

For statistical elaboration, the actual values of each in-dependent variable were coded to give −1, 0, and +1 coded

levels, according to the equation

xi=Xi − Xo/�Xi (1)

where xi represents the corresponding coded values, Xo theactual values at the central point, and �Xi the step changevalue.

To identify the best conditions for bromelain extraction,linear models expressed by the following equation were used:

yi = b0 +∑

bixi +∑

bij xixj (2)

where yi are the predicted values for each response, b0 and bi

are the intercept and linear coefficients, respectively, and bij

are the interaction coefficients.Statistica Software Version 10 (StatSoft, Tulsa, OK, USA)

was used for regression and graphical analysis of data. Thestatistical significance of the regression coefficients was deter-mined by the Fischer’s test for analysis of variance (ANOVA)at a significance level (P) ≤0.05, and the extent of varianceexplained by each model was given by the coefficient of de-termination, R2. To minimize the error of ANOVA, the assayscorresponding to the central point were repeated three times.The experimental and predicted values were compared todetermine the model validity. Once ANOVA had evidenced themost significant effects and interactions on each response, onlythose were maintained in Eq. (2), thus providing the final modeldescribing the conditions able to maximize each of them.

3. Results and Discussion3.1. Variables that influence bromelain extraction byATPS (PEG/PAA)To minimize the number of experiments needed to identifythe best bromelain extraction conditions, a 26–2 fractionalfactorial design was performed. The independent variables an-alyzed were PEG and PAA concentrations, PEG and PAA molarmass, salt (Na2SO4) concentration, and temperature, whereasthe responses were yield in the top phase (Ytop) and purifi-cation factor (PF). Experimental results are summarized inTable 3. It is well known that polyelectrolytes, such aspoly(acrylic acid), can precipitate proteins. Aiming to avoidbromelain precipitation, we used a 6% or higher concentrationof Na2SO4. High salt concentration causes in general a decreasein the electrostatic interaction by the Debye-screening effect[32]. The polymer concentration was based on the binodalcurve, and the temperature chosen was based on the previousstability studies of bromelain, as well the binodal curves.

According to Table 3, the highest value not only for yield inthe top phase (335.27%) but also for purification factor (25.78)was obtained at run 14. The yield was calculated based onthe bromelain activity; therefore, the yield, above 100%, couldbe due to migration of other proteins to the bottom phase,which probably were inhibiting bromelain activity. In addition,no bromelain activity was observed in the bottom phase, andany influence that the components of the top phase could haveon bromelain activity determination was taken into account.

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TABLE 3Results of bromelain extraction by ATPS (PEG/PAA) on response yield in the top phase (Ytop) and purification factor (PF)

according to the 26–2 fractional factorial statistical design analysis

Independent variable

PEG PAA PEG PAA Salt Temperature Response

Run (%, w/w) (%, w/w) (g/mol) (g/mol) (%) (◦C) Ytop (%) PF

1 6 6 2,000 1,200 6 20 37.36 1.98

2 6 6 2,000 15,000 6 30 20.05 0.59

3 6 6 6,000 1,200 7 20 16.87 1.86

4 6 6 6,000 15,000 7 30 18.03 1.07

5 6 8 2,000 1,200 7 30 46.40 4.25

6 6 8 2,000 15,000 7 20 n.c.a n.c.a

7 6 8 6,000 1,200 6 30 10.86 1.29

8 6 8 6,000 15,000 6 20 1.44 1.17

9 8 6 2,000 1,200 7 30 50.43 8.17

10 8 6 2,000 15,000 7 20 132.22 6.57

11 8 6 6,000 1,200 6 30 16.47 0.98

12 8 6 6,000 15,000 6 20 n.c.a n.c.a

13 8 8 2,000 1,200 6 20 84.75 8.82

14 8 8 2,000 15,000 6 30 335.27 25.78

15 8 8 6,000 1,200 7 20 19.83 7.59

16 8 8 6,000 15,000 7 30 6.34 0.68

17b(C) 7 7 4,000 8,000 6.5 25 13.27 0.92

18b(C) 7 7 4,000 8,000 6.5 25 13.13 0.88

19b(C) 7 7 4,000 8,000 6.5 25 13.17 0.89

an.c., not calculated.bCentral point.

In the literature, several studies presented a yield higherthan 100% for enzyme extraction using ATPS [20, 26, 30, 33].Cavalcanti et al. [30] in extraction studies on phospholipase Cusing PEG/phosphate ATPS obtained a yield of 230%.

At runs 6 and 12, it was not possible to calculate bothparameters, once no bromelain activity was observed. Resultsof ANOVA are illustrated in Fig. 1 in the Pareto chart forboth responses, omitting the less significant interactions.In this chart, the length of each bar is proportional to thestandardized effect of the related variable or interaction, andthe bars extending beyond the vertical line correspond to thestatistically significant effects at a confidence level of 95% [31].

It can be seen from Fig. 1A that all variables evaluatedshowed a significant effect on the response yield in the topphase. Conversely, PAA molar mass had no significant effect

on purification factor (Fig. 1B), and salt concentration wasvery close to the significance line. According to Fig. 1, thevariables PEG and PAA concentrations and temperaturesshowed a significant positive effect, suggesting that an increasein these parameters would improve bromelain extraction byATPS (PEG/PAA). PEG molar mass had a negative effect, whichmeans that work with lower PEG molar mass could improveboth responses (yield and purification factor).

Although PAA molar mass did not cause a significant effecton purification factor, it showed a positive effect on yield. Underthe highest PAA molar mass, the effect of exclusion volumecould take place, rejecting bromelain in the PEG (top) phase;in addition, the hydrophobic and electrostatic forces could actin order to make bromelain migrate to the PEG phase. As PAAis a negatively charged polymer, it will have a strong repulsive

530 Bromelain Extraction from Pineapple Peel Waste

FIG. 1Pareto chart for the effects of variables PEG (1) andPAA (2) concentration, PEG (3) and PAA (4) molarmass, salt concentration (5), and temperature(6) on yield in the top phase (A) and purificationfactor (B) of bromelain extraction by ATPS(PEG/PAA).

electrostatic interaction with anionic biomolecules. Thus, thepartition of bromelain in PEG/PAA ATPS can be driven byenthalpic and entropic forces.

The salt concentration showed a negative effect for bothresponses evaluated, that is, a decrease in the salt concentra-tion could improve the results for these extraction parameters,but result in bromelain precipitation, hindering the extraction,as previously stated.

Although the partition coefficient (K) has not been analyzedas a response in the fractional factorial design, it is presentedin Fig. 2.

In many experimental conditions, it was not possible tocalculate the partition coefficient (K) due to the null bromelainactivity in the bottom phase. The highest K (69.10) was obtained

FIG. 2Partition coefficient (log K) of bromelain extractionby ATPS (PEG/PAA).

at run 3, threefold higher than the one obtained in the secondbest condition (23.72, at run 13).

Babu et al. [12] studied the partitioning of bromelain bythe PEG/potassium phosphate system, and this enzyme pref-erentially partitioned to the top (PEG) phase, which suggestsa hydrophobic interaction between bromelain and PEG. Thiskind of interaction can be also responsible for the partition ofbromelain in the PEG phase of the PEG/PAA system.

Taking the above into consideration, new assays werecarried out according to a 25–1 fractional factorial design, withNa2SO4 concentration fixed at 6% (w/w) and new levels for thevariables PEG and PAA concentrations and PEG molar mass.Temperature and PAA molar mass were also evaluated, but atthe same level of the first design.

3.2. Second statistical analysisOnce the first experimental design showed that salt concen-tration must be as low as possible, the second factorial design(25–1) was performed with a fixed salt concentration (6%),and PEG and PAA concentrations, PEG and PAA molar mass,and temperature were varied. Table 4 shows the results ofbromelain extraction by ATPS.

The highest top phase yield (51.45%) was observed at run34, with the highest levels of PEG and PAA concentrations(9.5%) and PEG molar mass (2,000 g/mol) and the minor PAAmolar mass (1,200 g/mol) at 20 ◦C. The purification factorshowed values from 0.01 (run 27) to 31.63 (run 24). Comparingthe runs with the highest yield and purification factor, thefollowing parameters were in common: PAA concentration(9.5), PAA molar mass (2,000 g/mol), and temperature (20 ◦C).

Ketnawa et al. [34] found that the lower molar weight ofPEG was preferable for bromelain partitioning in the systemformed by PEG and MgSO4. Navapara et al. [35] observed thatthe partition coefficient and yield decreased with an increasein molecular weight of PEG, in an ATPS formed by PEG andpotassium phosphate. This was probably due to the effect ofsteric exclusion and the hydrophobic nature of the polymer

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TABLE 4Results of bromelain extraction by ATPS (PEG/PAA) on response yield in the top phase (Ytop) and purification factor (PF)

according to the 25–1 fractional factorial statistical design analysis

Independent variable

PEG PAA PEG PAA Temperature Response

Run (%, w/w) (%, w/w) (g/mol) (g/mol) (◦C) Ytop (%) PF

20 8.5 8.5 1,000 1,200 30 36.71 1.05

21 8.5 8.5 1,000 15,000 20 55.57 1.64

22 8.5 8.5 2,000 1,200 20 47.88 2.95

23 8.5 8.5 2,000 15,000 30 8.79 0.22

24 8.5 9.5 1,000 1,200 20 39.87 31.63

25 8.5 9.5 1,000 15,000 30 n.c.a n.c.a

26 8.5 9.5 2,000 1,200 30 27.98 1.06

27 8.5 9.5 2,000 15,000 20 0.23 0.01

28 9.5 8.5 1,000 1,200 20 50.08 1.92

29 9.5 8.5 1,000 15,000 30 n.c.a n.c.a

30 9.5 8.5 2,000 1,200 30 42.92 1.63

31 9.5 8.5 2,000 15,000 20 3.85 0.17

32 9.5 9.5 1,000 1,200 30 39.76 1.10

33 9.5 9.5 1,000 15,000 20 3.62 0.13

34 9.5 9.5 2,000 1,200 20 51.45 2.25

35 9.5 9.5 2,000 15,000 30 n.c.a n.c.a

36b (C) 9 9 1,500 8,000 25 45.56 1.52

37b (C) 9 9 1,500 8,000 25 34.82 1.16

38b (C) 9 9 1,500 8,000 25 38.68 1.24

an.c., not calculated.bCentral point.

in the top phase, which increases enzyme partitioning in thebottom phase. In the same work, the authors found a maximumenzyme partition coefficient of 12.62 with 90.33% yield and2.4-fold, the maximum bromelain partition was predicted fromthe ATPS composed of 14% (w = w) PEG, 17.66% (w = w)potassium phosphate, 1 mM NaCl, and pH 7.5.

All variables had a significant effect on the purificationfactor, as shown in the Pareto chart (Fig. 3). Although PAAconcentration had a positive effect, the other variables hada negative one. As all variables were significant, it was notpossible to exclude any independent variable. Table 5 showsthe ANOVA results for the response yield in the top phase.

It can be seen that the strongest effect on Ytop at 95%confidence level (P ≤ 0.05) was that exerted by PAA molar mass

(0.007) and temperature (0.047). Both effects were negative,which means that a decrease in both parameters could generatebetter results in bromelain extraction by ATPS (PEG/PAA). Theobtained model did not exhibit a lack of adjustment, and thedetermination coefficient (R2 = 93%) showed that 93% ofvariations in the Ytop results can be explained by the model.According to Silva et al. [19], values of R2 > 90% are very goodin the experimental design of bioprocess. Figure 4 illustratesthe interaction between PAA molar mass and temperature tothis response.

Figure 4 clearly shows that the systems with a PAA molarmass of 1,200 g/mol and opposite level combinations (−1 and+1) of temperature (20 and 30 ◦C, with prominence to thefirst one) generated the highest results. As the temperature

532 Bromelain Extraction from Pineapple Peel Waste

FIG. 3Pareto chart for the effects of variables PEG (1) andPAA (2) concentrations, PEG (3) and PAA (4) molarmass, and temperature (5) on purification factor ofbromelain extraction by ATPS (PEG/PAA).

increased, the PAA structure may extend. It is important to bearin mind that temperature can influence the phase diagrams andprotein partitioning. Thus, any change in the system, such as anincrease in the temperature, could influence the concentrationof polymers in the phases. Furthermore, increased temperaturecould also extend the PEG structure [4]. The regression analysisof Ytop data, taking into account the linear simple effects aswell as the above most significant interactions, provided thefollowing first-order model:

Ytop = 27.7394 − 16.5686 × PAAmolarmass

− 6.0250 × Temperature (3)

The results showed that the two studied responses requiredifferent conditions to be optimized. Concerning purificationfactor, more assays should be carried out, because all variablesanalyzed showed a significant effect. The condition: PEG1,000 g/mol a 8.5%, PAA 1,200 g/mol a 9.5% at 20 ◦C showedthe highest result for purification factor (31.63). Regarding

TABLE 5ANOVA applied to the regression models used for yield in top phase according to the 25–1 fractional factorial design

Factor SS df MS F P value

(1) PEG concentration 40.167 1 40.167 1.3575 0.364139

(2) PAA concentration 429.552 1 429.552 14.5175 0.062495

(3) PEG molar mass 112.873 1 112.873 3.8148 0.190032

(4) PAA molar mass 4,392.452 1 4,392.452 148.4511 0.006669a

(5) Temperature (◦C) 580.804 1 580.804 19.6294 0.047355a

1 by 2 388.609 1 388.609 13.1338 0.068418

1 by 3 169.342 1 169.342 5.7232 0.139163

1 by 4 493.778 1 493.778 16.6881 0.055024

1 by 5 119.674 1 119.674 4.0446 0.181999

2 by 3 78.006 1 78.006 2.6364 0.245927

2 by 4 131.257 1 131.257 4.4361 0.169788

2 by 5 107.787 1 107.787 3.6429 0.196526

3 by 4 157.140 1 157.140 5.3108 0.147689

3 by 5 149.664 1 149.664 5.0582 0.153453

4 by 5 9.860 1 9.860 0.3332 0.622078

Lack of fit 488.606 1 488.606 16.5133 0.055558

Pure error 59.177 2 29.589

Total SS 7,908.750 18

aSignificant at a level of 95%.

SS, sum of squares; df, degrees of freedom; MS, mean of square. R2, 0.93; pure error, 29.59.

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FIG. 4Yield in the top phase, Ytop, as a function oftemperature and PAA molar mass.

yield in the top phase, only the factors PAA molar mass andtemperature showed significant effects.

3.3. Comparison of the two statistical analysesAlthough the first statistical analysis has shown some condi-tions to improve both response yields in the top phase andpurification factor, the second analysis did not promote thebest results. It was possible to obtain a great yield (335.27%)with a good purification factor (25.78) at run 14, of the firstexperimental design, which had the following conditions: 8%(w/w) of PEG and PAA, PEG 2,000 g/mol, PAA 15,000 g/mol,and 6% (w/w) of Na2SO4 at 30 ◦C.

Depending on the pharmaceutical formulation, one canuse bromelain direct from the partition, in a rich PEG solution.In addition, if necessary, bromelain can be removed fromthe PEG phase using a simple PEG/salt ATPS, as pointed outby Johansson et al. [8] to back-extraction of lysozyme fromPEG/PAA systems.

Babu et al. [12] reported 228% activity recovery and a 4.0-fold increase in purity of bromelain in PEG/phosphate ATPS.Hebbar et al. [36] studied bromelain extraction by reversemicellar systems and obtained the following best results:106% of activity recovery and purification factor of 5.2. Soareset al. [37] achieved a purification factor of 2.28 and yieldedapproximately 98% in the extraction of bromelain using theethanol precipitation process. Thus, PEG/PAA ATPS promotedhigher values for yield and purification factor when comparedwith other extraction techniques. Further investigation must becarried out to increase both results. However, these results arealready interesting as a new bromelain purification method.

4. ConclusionPEG/PAA systems provided good results of both parameters—yield in the top phase and purification factor—as compared withother methods available in the literature, which demonstratesthe potential of this system as a new and an alternative methodfor bromelain extraction from pineapple waste. Consideringthe yields obtained, extracted bromelain has the potential to beincorporated in pharmaceutical or cosmetic formulations, afterthe purification process.

5. AcknowledgementsThis research was supported by grants from the Coordinationfor the Improvement of Higher Level Personnel (Capes), Na-tional Council for Scientific and Technological Development(CNPq), and State of Sao Paulo Research Foundation (Fapesp),Brazil.

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