Reverse micellar extraction of bromelain from pineapple peel – Effect of surfactant structure

Food Chemistry 197 (2016) 450–456

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Reverse micellar extraction of bromelain from pineapple peel – Effect of surfactant structure Jing Wan, Jingjing Guo, Zhitong Miao, Xia Guo ⇑ School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, Jiangsu 225002, PR China

a r t i c l e

i n f o

Article history: Received 26 March 2015 Received in revised form 27 October 2015 Accepted 31 October 2015 Available online 2 November 2015 Keywords: Surfactant Reverse micelle Protein extraction Pineapple peel Bromelain

a b s t r a c t Pineapple peel is generally disposed or used as compost. This study was focused on extracting bromelain from pineapple peel by using reverse micelles. It was found that gemini surfactant C12-8-C12
2Br (octame thylene-a,x-bis(dimethyldodecylammonium bromide)) showed distinctive advantage over its monomeric counterpart DTAB (dodecyl trimethyl ammonium bromide); under optimized condition, the bromelain extracted with C12-8-C12
2Br reverse micelle had an activity recovery of 163% and a purification fold of 3.3, while when using DTAB reverse micelle, the activity recovery was 95% and the purification fold was 1.7. Therefore, the spacer of gemini surfactant should play a positive role in bromelain extraction and may suggest the potential of gemini surfactant in protein separation since it has been so far rarely used in relative experiments or technologies. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Reverse micelles are aggregates of surfactant molecules with the head groups oriented toward the polar core and the hydrophobic tails into the nonpolar medium. They are characterized by the formation of water pools, located inside the micelles. Proteins can be solubilized into the water core, and hence be shielded from organic medium without losing significant biological activity (Martinek, Klyachko, Kabanov, Khmelnitsky, & Levashov, 1989; Stamatis, Xenakis, & Kolisis, 1999; Tonova & Lazarova, 2008). Now, reverse micellar extraction has been considered as an alternative to conventional separation and purification procedures for bioactive proteins since it has potential for continuous operation and is easy to scale up with no loss of native function/activity and high capacity of proteins (Gaikaiwari, Wagh, & Kulkarni, 2012a, 2012b; Harikrishna, Srinivas, Raghavarao, & Karanth, 2002; Hatton, 1989; Imm & Kim, 2009; Kadam, 1986; Kumar, Hemavathi, & Hebbar, 2011). Two steps are included in liquid–liquid reverse micellar extraction process; a target protein is selectively solubilized into the organic phase (forward extraction) and subsequently stripped into the aqueous phase (backward extraction) by addition of fresh aqueous buffer, also called stripping solution (Dungan, Bausch, Hatton, Plucinski, & Nitsch, 1991; Harikrishna et al., 2002; Kumar et al., 2011; Nandini & Rastogi, 2009). Factors affecting the performance ⇑ Corresponding author. E-mail address: [email protected] (X. Guo). http://dx.doi.org/10.1016/j.foodchem.2015.10.145 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

of reverse micelle system are rather complicated, including the nature and concentration of target protein, pH, the concentration and species of ions, type and concentration of surfactant, the composition of reverse micelles, and so on (Chen, Su, & Chiang, 2006; Ding, Cai, & Guo, 2015; Dong, Cai, Guo, & Xiao, 2013; Harikrishna et al., 2002; Hebbar & Raghavarao, 2007; Hebbar, Sumana, & Raghavarao, 2008; Imm & Kim, 2009; Noritomi, Kowata, Kojima, Kato, & Nagahama, 2006; Tonova & Lazarova, 2008; Xiao, Cai, & Guo, 2013). It has been generally considered that reverse micelle size should fit the target protein size and the transfer of protein between the two phases is primarily governed by electrostatic interaction between protein and surfactant; when using cationic surfactant, the forward extraction is generally carried out at pH higher than the isoelectric point (pI) of protein, while the backward extraction is always done at lower pH (Arshad et al., 2014; Chen et al., 2006; Harikrishna et al., 2002; Imm & Kim, 2009; Noritomi et al., 2006; Tonova & Lazarova, 2008). The hydrophobic interaction between the nonpolar domain of protein and surfactant also affects the reverse micelle extraction efficiency. It has been found that the recovery of ovalbumin from reverse micelle is more difficult than that of BSA (bovine serum albumin) due to the higher hydrophobicity of ovalbumin than BSA (Ding et al., 2015). The application of reverse micelle in enzyme extraction from real biological system should be interesting and has been expected promising potential. Bromelain is a proteolytic enzyme and widely used in food, cosmetics and pharmaceutics. It is found in the tissues of plant family Bromeliaceae. Pineapple is the best known source of bromelain, present in its pulp, core, stem and peel, with

J. Wan et al. / Food Chemistry 197 (2016) 450–456

the molecular weight being ca. 23–36 kDa (Arshad et al., 2014). Bromelain is soluble in water but insoluble in organic solvent. Its isoelectric point is 4.6 when extracted from pulp or core, while 9.5 when obtained from stem or peel (Arshad et al., 2014; Hebbar et al., 2008). CTAB (cetyl trimethyl ammonium bromide) and AOT (di-2-ethylhexyl sodium sulfosuccinate) reverse micelles were used to extract bromelain from pulp, core and stem of pineapple, and CTAB was found more suitable (Hebbar et al., 2008; Hemavathi, Hebbar, & Raghavarao, 2007). When using CTAB, the extraction efficiency was ca. 20% and the activity recovery was 97% from pineapple pulp (Hemavathi et al., 2007) or 102–106% from core and stem (Hebbar et al., 2008), while the efficiency of AOT reverse micelle was very low (Hebbar et al., 2008; Hemavathi et al., 2007). The separated protein could be analyzed by SDS–PAGE electrophoresis (Gao, Liu, & Xiao, 2011; Li et al., 2012), based on which, the purity of the extracted bromelain seemed satisfactory (Hebbar et al., 2008). Fileti, Fischer, Santana, and Tambourgi (2009) carried out bromelain extraction from pineapple pulp by using BDBAC (benzil dodecyl bis(hydroxylethyl) ammonium chloride) reverse micelle and found the maximum purification factor could be ca. 3 (Fileti et al., 2009). However, how to get bromelain from pineapple peel with high activity has been still a problem. Gemini surfactant is made up of two hydrophilic head groups, two hydrophobic chains, and a spacer linking the two head groups via covalent bonds. It has been reported that the spacer shows an obvious effect on the interaction between protein and gemini surfactant in aqueous solution (Amiri et al., 2012; Mir, Khan, Khan, Rather, & Dar, 2010). However, gemini surfactant has been rarely used in protein extraction (Dong et al., 2013). In the present paper, we will use reverse micelles from gemini surfactant C12-8-C12
2Br (shown in Scheme 1A) and its counterpart monomer – conventional surfactant DTAB (dodecyl trimethyl ammonium bromide, shown in Scheme 1B) to extract bromelain from pineapple peel, with a purpose to study whether and how the spacer of gemini surfactant works in bromelain extraction. Since pineapple peel is generally disposed or used as compost, this study should be interesting and expected to lead to value addition to the peel. 2. Materials and methods 2.1. Materials Gemini surfactant C12-8-C12
2Br (Scheme 1A, Molecular Weight: 698.86) was prepared according to the reference (Zana,

CH3

CH3

.

+ + C12H25N − (CH2)8 − NC12H25 2BrCH3

CH3

A

Br + N B Scheme 1. Structures of C12-8-C12
2Br (A) and DTAB (B).

451

Benrraou, & Rueff, 1991). Its purity (at least 98%) was checked by NMR and elemental analysis (Zana et al., 1991). DTAB (Scheme 1B, Molecular Weight: 308.34) was bought from Amresco Co. Solon, OH (99% purity). n-Hexane and 1-hexanol were bought from Chinese Chemicals (analytical grade, Sinopharm chemical reagent Co. Ltd, Shanghai, China). Matured pineapple fruits (Ananas comosus L. Merryl, Produced in Hainan, P.R. China) used in this study were purchased from the local market. The water used was deionized. The buffer used in the forward extraction was prepared by disodium hydrogen phosphate (10 mM)/citric acid (pH 6 8.0) and glycine (10 mM)/sodium hydroxide (pH > 8.0). The stripping solution in the backward extraction was prepared by acetic acid/sodium acetate (10 mM, pH 6 5.7) and disodium hydrogen phosphate/sodium dihydrogen phosphate (10 mM, pH: 5.8–8.0). 2.2. Methods 2.2.1. Preparation of reverse micelle The reverse micelle was prepared by known quantities of nhexane, 1-hexanol, surfactant and water. The volume ratio of 1hexanol to n-hexane is 1:9. 2.2.2. Preparation of crude extract The peel was manually separated from the fruit. For the preparation of crude extract, a known quantity of peel was crushed along with extraction buffer (0.04 M sodium phosphate buffer of pH 6.0, containing 5 mM EDTA) at 1:1 ratio for 5 min and then filtered through a cheese cloth. The filtrate was centrifuged at 10,000 rpm (Allegra 64R centrifuge, Beckman Coulter, USA) for 25 min and the supernatant (i.e. crude enzyme extract) obtained was used for reverse micellar extraction experiments. 2.2.3. Reverse micellar extraction There are two steps in the liquid–liquid reverse micellar extraction process: forward extraction and backward extraction. Aqueous (crude enzyme extract/buffer) and organic (reverse micelle) phases were mixed with a volume ratio of 1:1 and the mixture was vortexed for 25 min at room temperature. Phase separation was done by centrifuging at 14,000 rpm (Allegra 64R centrifuge, Beckman Coulter, USA) for 30–50 min. Backward extraction was carried out by mixing the organic phase obtained from forward extraction with an equal volume of a fresh aqueous phase, also termed as stripping phase (buffer of known pH). The mixture was then centrifuged at 14,000 rpm (Allegra 64R centrifuge, Beckman Coulter, USA) for 30–40 min, followed by phase separation. The aqueous phase obtained after backward extraction was analyzed for bromelain activity and total protein content. 2.2.4. Protein activity determination Bromelain activity was determined according to the casein digestion unit (CDU) method using casein (0.6%) as substrate in the presence of cysteine and EDTA (Murachi, 1976). The assay was based on proteolytic hydrolysis of casein substrate. The absorbance of the clear filtrate (solubilized casein) at 275 nm was measured using UV–vis spectrophotometer (Shimadzu UV-160). One unit of bromelain activity was defined as 1 lg of tyrosine released in 1 min per ml of sample when casein was hydrolyzed under the standard conditions of 37 °C and pH 7.0 for 10 min. 2.2.5. Protein content measurement Protein content in aqueous phase was determined by the dye binding method (Bradford method, using BSA as standard) (Bradford, 1976). The sample analyses were performed against respective blank solutions. The overall extraction efficiency (OEF, %), activity recovery (AR, %) and purification fold (PF) were

J. Wan et al. / Food Chemistry 197 (2016) 450–456

OEF ð%Þ ¼ ½Proteinr =½Proteinf  100

ð1Þ

AR ð%Þ ¼ ðBromelain activityÞr =ðBromelain activityÞf  100

ð2Þ

120

2.0 OEF AR PF

90

PF ¼ ðSpecific activity of bromelainÞr =ðSpecific activity of bromelainÞf

1.5

60

1.0

30

0.5

0

PF

calculated according to Eqs. (1)–(3), where the subscripts r and f represented recovery and feed, respectively (for example, [Protein]r expressed the recovered protein content (i.e. the protein content in backward aqueous phase) and [Protein]f represented the protein content in the feed). The units of protein content, bromelain activity and specific activity of bromelain were mg/ml, CDU/ ml and CDU/mg, respectively.

OEF (%) AR (%)

452

0.0 8

ð3Þ

9

10

11

12

pH

3. Results and discussion

When studying the effect of forward aqueous phase pH, the backward extraction was carried out at aqueous phase pH 4.2 and 0.5 M KBr. Fig. 1 showed the activity recovery (AR), purification fold (PF) and overall extraction efficiency (OEF) with DTAB (Panel A) and gemini surfactant C12-8-C12
2Br (Panel B) reverse micelles at different forward aqueous phase pH. From Fig. 1, it could be seen that with the increase of pH, OEF was increased gradually until pH reached 10.5 with DTAB (Panel A) or 9.5 with gemini surfactant (Panel B), and the maximum values of OEF were ca. 56%. Fig. 1 also showed AR became higher with increasing pH until pH reached 10.5 with DTAB (Panel A) or 8.0 with gemini surfactant (Panel B), and the highest values of AR were 95% when using DTAB and 163% when using gemini surfactant. Since activity recovery should be the primary criterion in enzyme extraction and purification, the optimum forward aqueous phase pH should be 10.5 when DTAB was used or 8.0 when gemini surfactant was used. At the optimum pH, AR, PF and OEF were 95%, 1.7 and 56%, respectively, with DTAB reverse micelle, or 163%, 3.3 and 50%, respectively, with gemini surfactant reverse micelle. In other words, the optimum specific activity of the recovered bromelain was 11,097 CDU/mg when gemini surfactant was used or 5774 CDU/mg when DTAB was used. Therefore, it should be concluded gemini surfactant was more efficient than monomeric surfactant. The backward aqueous phase was dialyzed and concentrated and then, loaded to 12.5% polyacrylamide gel and analyzed by SDS–PAGE electrophoresis. The SDS–PAGE pattern (Fig. 1 of Supplementary material) indicated the recovered bromelain had a satisfactory purity, with the molecular weight being around 23 kDa, close to the reported range of the molecular weight of bromelain (i.e. 23– 36 kDa, Arshad et al., 2014). The net protein charge is determined by its isoelectric point and pH of the aqueous phase. The isoelectric point of peel bromelain is 9.5 (Hebbar et al., 2008). With the increase of forward aqueous phase pH, bromelain molecules may have more negative charges, which in turn increases the electrostatic attraction of bromelain with positively charged surfactant molecules and facilitates its transfer into reverse micelles. Gemini surfactants exhibit superior surface activity if comparing to the corresponding conventional (monomeric) surfactant; the spacer of gemini surfactant can obvi-

OEF AR PF

150

3.2

120 2.4

90 60

PF

3.1. Effect of forward aqueous phase pH

4.0

180

OEF (%) AR (%)

In this study, we prepared crude extract solution first. The activity and the specific activity of the enzyme in the crude extract were 406 CDU/ml and 3404 CDU/mg, respectively. Then, we made the forward aqueous phase by adding adequate amount of crude extract solution into buffer. To illustrate the effect of surfactant structure on the bromelain extraction from pineapple peel, the forward and backward extraction parameters were both studied.

A

1.6

30 0.8 0 7

8

9

10

11

12

pH

B Fig. 1. Dependences of overall extraction efficiency (OEF), activity recovery (AR) and purification fold (PF) of bromelain on pH of forward aqueous phase (having 0.1 M of NaCl). pH of backward aqueous phase (containing 0.5 M of KBr): 4.2. Surfactant: DTAB (0.025 g/ml, A) and C12-8-C12
2Br (0.030 g/ml, B).

ously restrain the repulsive force between the two head groups and hence, the two head groups in gemini surfactant are closer and the apparent area of the head group becomes smaller and the density of the head group charge becomes higher (Menger & Keiper, 2000; Zana, 2002; Zana & Xia, 2004). Therefore, the attractive interaction between the head groups of gemini surfactant and the negatively charged center of bromelain should be stronger than that between monomeric surfactant and bromelain. As a result, the optimum forward aqueous phase pH is lower when using gemini surfactant than that when using monomeric surfactant. Bromelain extracted with gemini surfactant reverse micelle exhibited much higher AR than that with monomeric surfactant (163% vs. 95%, Fig. 1). On the one hand, this might be due to the optimum activity of bromelain extracted with gemini surfactant reverse micelle was obtained at pH 8.0, while that with monomeric surfactant reverse micelle was obtained at pH 10.5. On the other hand, this data might suggest that the gemini surfactant could retain the activity/function of bromelain much better than the monomeric surfactant. Comparative studies on the interactions of protein with gemini surfactant and its counterpart monomer have indicated that (1) gemini surfactant has much stronger binding ability with protein, and (2) although the monomeric surfactant only results in protein unfolding, refolding of protein could be observed in the presence of gemini surfactant (Gull, Sen, Khan, & Kabir-ud-Din, 2009; Li, Wang, & Wang, 2006; Mir, Khan, Khan, & Dar, 2012).

J. Wan et al. / Food Chemistry 197 (2016) 450–456

3.2. Effect of backward aqueous phase pH

100

2.0

80

1.8

60

1.6

40

1.4 O EF AR PF

20 0

PF

OEF (%) AR (%)

To make protein back into water (stripping solution) from reverse micelles, the attraction between surfactant and protein must be broken. So the backward extraction is generally carried out at pH of stripping solution lower than pI of protein. Fig. 2A is the effect of backward aqueous phase pH on OEF, AR and PF with DTAB reverse micelle. At pH 3.6, a thin film was observed between the two phases in the backward extraction process, which made OEF and AR low. When the backward aqueous phase pH was increased to 4.2, the interface between the two phases became clear and AR and OEF became much higher, which were 95% and 56%, respectively, with PF being 1.7. However, when the backward aqueous phase pH was increased to 5.0, AR and OEF was decreased, and then, they remained almost unchanged with increasing pH. According to Fig. 2A, the optimum backward aqueous phase pH with DTAB reverse micelle should be 4.2 since under this condition, maximum values of AR (95%), OEF (56%) and PF (1.7) could be obtained. When using gemini surfactant (Fig. 2B), the backward aqueous phase pH showed very little effect on OEF, which was around 50%, while the values of AR and PF were the highest at pH 4.2, which were 163% and 3.3, respectively. Therefore, the optimum backward

1.2 1.0

3

4

5

6

7

8

pH

A 160

O EF

4.0

AR

140

3.2

PF

2.4

100

PF

OEF (%) AR (%)

120

80

1.6

60

0.8

40 0.0 3.6

4.0

4.4

4.8

5.2

5.6

pH

B Fig. 2. Dependences of OEF, AR and PF of bromelain on pH of backward aqueous phase (having 0.5 M of KBr). Surfactant: DTAB (0.025 g/ml, A) and C12-8-C12
2Br (0.030 g/ml, B). 0.10 M NaCl was present in forward aqueous phase. Forward aqueous phase pH: 10.5 when using DTAB and 8.0 when using C12-8-C12
2Br.

453

aqueous phase pH for bromelain recovery with gemini surfactant reverse micelle was the same as that with DTAB reverse micelle. However, the much higher AR and PF with gemini surfactant reverse micelle should suggest that gemini surfactant should be much better than DTAB for bromelain extraction, consistent with the conclusion from Fig. 1. The isoelectric point of peel bromelain is 9.5. With the decrease of backward aqueous phase pH, on the one hand, the repulsive force between surfactant and bromelain should be increased due to higher positive charge density on bromelain, on the other hand, the protein may become unfolded and hydrophobic moiety may be exposed. The former effect should be helpful for protein recovery while the latter one would not due to the hydrophobic interaction between surfactant and protein. A combination of the two effects may make bromelain recovered most efficiently at pH 4.2. Moreover, compared to the case in Fig. 2A, the very little changed OEF with backward aqueous phase pH in Fig. 2B may suggest that the repulsion between gemini surfactant and bromelain should be stronger than that between monomeric surfactant and bromelain, probably due to the higher positive charge density on gemini surfactant. Recently, we (Xiao et al., 2013) studied the extraction efficiency of gemini surfactant for model protein BSA (bovine serum albumin) and found that the attraction between gemini surfactant and protein seemed much easier to be shielded (than that between conventional monomeric surfactant and protein) and as a result, BSA could be recovered efficiently under acidic, neutral and basic conditions (while BSA was recovered from monomeric surfactant reverse micelle only under acidic condition). 3.3. Effect of ionic strength The water structure forming salt such as NaCl has been generally used during forward extraction, while the water structure breaking salt such as KBr has been found better during backward extraction (Hebbar et al., 2008; Kinugasa et al., 2003; Lu, Chen, Li, & Shi, 1998). Hence, in the present study, NaCl was used in the forward extraction while KBr was used in the backward extraction. Fig. 3 showed the effects of NaCl (Panels A and B) and KBr (Panels C and D) contents on the extraction performances of DTAB (Panels A and C) and gemini surfactant (Panels B and D) reverse micelles. According to this figure, it could be seen that when NaCl concentration in forward aqueous phase was 0.1 M and KBr concentration in backward aqueous phase was 0.5 M, both reverse micelles could exhibit the best performances; AR, PF and OEF were 95%, 1.7 and 56% with DTAB reverse micelle and 163%, 3.3 and 50% with gemini surfactant reverse micelle. The increase of ionic strength can (1) decrease the repulsive force between the surfactant head groups, (2) decrease the size of reverse micelles and the thickness of the electric double layer, and (3) shield the attractive interaction between protein and surfactant (Hebbar et al., 2008; Lu et al., 1998; Tonova & Lazarova, 2008). The first effect is beneficial to the attractive interaction between protein and surfactant and positive for forward extraction while the latter two effects are negative for protein located in reverse micelle (Hebbar et al., 2008; Lu et al., 1998). Fig. 3 suggests that the first effect should be dominant when salt content is low, while the latter two are important at high salt content, similar to the conclusion in the literatures (Hebbar et al., 2008; Hemavathi et al., 2007; Lu et al., 1998). What should be mentioned here is that when the ionic strength in stripping solution is too high, the ‘‘salting out” effect may become obvious and decrease the solubility of protein in water and then the protein may be retained in reverse micelle phase due to its hydrophobic patches.

J. Wan et al. / Food Chemistry 197 (2016) 450–456

100

4.8 OEF AR PF

150

1.5

1.0

25

0.5

4.0

120

3.2

90

2.4

60

1.6

OEF (%) AR (%)

50

PF

75

0.8

30

0 0.0

0.1

0.2

0 0.0

0.0 0.4

0.3

0.0 0.1

0.2

[NaCl] (M)

0.3

0.5

[NaCl] (M)

A

B 180

2.0

100 OEF AR PF

7 OEF AR PF

150

1.0

25

0.5

OEF(%) AR(%)

50

PF

1.5

75 OEF (%) AR (%)

0.4

6

120

5

90

4

PF

OEF (%) AR (%)

180

2.0 OEF AR PF

PF

454

3

60

2 30 1

0 0.0

0.0 0.5

1.0

1.5

2.0

[KBr] (M)

C

0 0.25

0.50

0.75

1.00

[KBr] (M)

D

Fig. 3. Effects of NaCl content in forward aqueous phase (Panels A–B) and KBr content in backward aqueous phase (Panels C–D) on OEF, AR, and PF of bromelain extracted with DTAB (0.025 g/ml, Panels A and C) and gemini surfactant C12-8-C12
2Br (0.030 g/ml, Panels B and D) reverse micelles. Forward aqueous phase pH: 10.5 when using DTAB and 8.0 when using C12-8-C12
2Br. Backward aqueous phase pH: 4.2. In Panels A and B, 0.5 M of KBr was present in backward aqueous phase. In Panels C and D, 0.1 M of NaCl was present in forward aqueous phase.

3.4. Effect of surfactant content Fig. 4 shows the effect of surfactant concentration on extraction behavior of reverse micelle. When using DTAB reverse micelle, OEF and AR are increased with increasing surfactant content, fast first and then slowly, until DTAB content reaches 0.025 g/ml, and then, further increasing surfactant content makes OEF and AR decreased gradually (Panel A); in other words, the highest AR is 95% and the highest OEF 56% at DTAB content being 0.025 g/ml, where PF is 1.7. The gemini surfactant content affects the extraction behavior, too, and the highest AR (163%) and OEF (50%) are obtained at gemini surfactant content being 0.030 g/ml. The critical micelle concentration (cmc) for gemini surfactant is 0.16 mM (i.e. 0.11 mg/ml) in organic solvent, while that for DTAB 0.41 mM (i.e. 0.13 mg/ml) (Zheng, Zhao, Chen, & Fu, 2006). The surfactant concentrations in Fig. 4 range from 3 to 35 mg/ml for DTAB and from 2 to 50 mg/ml for gemini surfactant, much higher than cmc. An increase in surfactant concentration may increase the number of reverse micelles, which in turn enhances the transfer of protein into reverse micelle. On the other hand, with the increase of reverse micelle number, the inter-micellar interactions may lead to percolation and interfacial deformation with a change in micellar shape and micellar clustering (Regalado, Asenjo, & Pyle, 1996), which may make OEF decreased at high surfactant content. When using DTAB, the percentage of water in organic phase (obtained from forward extraction, Fig. 2A of Supplementary material) is changed little at DTAB content lower than 0.010 g/ml, then increased obviously until DTAB content reaches 0.015 g/ml and remained little changed at DTAB content between 0.015 and

0.020 g/ml, followed by a decrease at DTAB content being 0.020– 0.030 g/ml. Furthermore, Wo (i.e. the molar ratio of water to surfactant in reverse micelle) is decreased continuously, fast at DTAB content lower than 0.01 g/ml and slow at higher DTAB content. These data suggest that increasing DTAB concentration should result in (1) the increase of reverse micelle number and (2) the collapse of reverse micelle. Under the influence of these two effects, 0.015–0.025 g/ml of DTAB seems suitable for bromelain extraction (Fig. 4A). When using gemini surfactant C12-8-C12
2Br, the water percentage in organic phase (Fig. 2B of Supplementary material) is increased with C12-8-C12
2Br content, and the increase becomes faster when surfactant content reaches 0.025–0.030 g/ml. Furthermore, Wo remains little changed at C12-8-C12
2Br content lower than 0.03 g/ml, while it is increased obviously at C12-8-C12
2Br content being around 0.03 g/ml. This should suggest the percolation of reverse micelle at C12-8-C12
2Br content being ca. 0.03 g/ml. Lang and Zana found ammonium surfactant reverse micelles showed percolation for increasing water content (Lang, Lalem, & Zana, 1991; Zana & Lang, 1991). Therefore, 0.03 g/ml of C12-8-C12
2Br exhibits the optimum extraction efficiency (Fig. 4B). Besides, high content of surfactant may make protein conformation changed obviously (Li et al., 2006) and hence, show a negative effect on protein activity, resulting in a decreased AR. From Fig. 4, AR is dropped to 45% when DTAB content reaches 0.035 g/ml (Fig. 4A), however, when using gemini surfactant reverse micelle, the recovered bromelain still shows 109% activity even when the gemini surfactant content reaches 0.05 g/ml (Fig. 4B). This result should indicate again that gemini surfactant reverse micelle extraction is beneficial to get high active protein.

455

J. Wan et al. / Food Chemistry 197 (2016) 450–456

2.5

60

1.5

30

1.0

0 0.00

0.01

0.02

1.5

80

1.0

60 0.5 40

0.5 0.04

0.03

2.0 OEF AR PF

100

2.0 OEF (%) AR (%)

OEF (%) AR (%)

90

120

PF

OEF AR PF

PF

120

0.0 10

[DTAB] (g/mL)

20

30

40

50

Dilution multiple

A

A 180

7.2

180

7.5

6.0

OEF AR PF

150

120 90

3.6 60

OEF (%) AR (%)

4.8 PF

OEF (%) AR (%)

150

6.0

120

4.5

90 3.0

2.4

PF

OEF AR PF

60

30 1.2 0 0.00

0.01

0.02

0.03

0.04

1.5 30

0.05

[C12-8-C12.2Br] (g/ml)

B Fig. 4. Effects of surfactant content on OEF, AR, and PF of bromelain extracted with DTAB (Panel A) and C12-8-C12
2Br (Panel B) reverse micelles. Forward aqueous phase pH: 10.5 when using DTAB and 8.0 when using C12-8-C12
2Br. Backward aqueous phase pH: 4.2. Salt: NaCl (0.1 M) in forward aqueous phase and KBr (0.5 M) in backward aqueous phase.

3.5. Effect of the content of crude enzyme in feed Fig. 5 showed that the dilution multiple of crude enzyme solution affected the extraction behavior of reverse micelle. When using DTAB reverse micelle, the crude enzyme had to be diluted at least 10 times, otherwise, emulsification occurred. While when using gemini surfactant reverse micelle, a clear interface could be obtained and no emulsification was observed even when the crude enzyme solution was just 2 times diluted. Therefore, gemini surfactant reverse micelle could load more enzyme than DTAB reverse micelle. Critical micelle concentration (cmc) for the gemini surfactant is 0.16 mM (i.e. 0.11 mg/ml) in organic solvent, and that for DTAB 0.41 mM (i.e. 0.13 mg/ml) in organic solvent (Zheng et al., 2006), and the aggregation numbers for these two surfactants are similar, corresponding to 40–50 dodecyl chains per micelle (Danino, Talmon, & Zana, 1995; Lianos, Lang, & Zana, 1983), indicating the number of gemini surfactant reverse micelles may be similar to that of DTAB reverse micelles when the weights of the two surfactants are similar. Thus, the higher loading capacity of gemini surfactant reverse micelle should be due to (1) the stronger electrostatic attraction of gemini surfactant with bromelain (discussed in Section 3.1), and (2) the higher water contents in gemini surfactant reverse micelles (Fig. 2 of Supplementary material). Moreover, from Fig. 5A, with the increase of dilution multiple, the best performance of DTAB reverse micelle for extracting bromelain was observed when the crude enzyme solution was

0.0 0 0

5

10

15

20

25

30

35

40

Dilution multiple

B Fig. 5. Effects of crude enzyme content in feed on OEF, AR, and PF of bromelain extracted with DTAB (0.025 g/ml, Panel A) and C12-8-C12
2Br (0.030 g/ml, Panel B) reverse micelles. Forward aqueous phase pH: 10.5 when using DTAB and 8.0 when using C12-8-C12
2Br. Backward aqueous phase pH: 4.2. Salt: NaCl (0.1 M) in forward aqueous phase and KBr (0.5 M) in backward aqueous phase.

diluted 25 times or higher, where AR, OEF and PF were ca. 95%, 56% and 1.7. When using gemini surfactant reverse micelle (Fig. 5B), OEF was increased from ca. 16% to 40% when the dilution multiple was increased from 2 to 5, then changed very little (from ca. 40% to 55% with the dilution multiple increased from 5 to 35), and AR and PF were the highest when the crude enzyme solution was diluted 25 times. Since AR should be the primary criterion for enzyme extraction, the best dilution times for the crude enzyme solution should be 25, which was used in Figs. 1–4. Fig. 5 also showed that AR with gemini surfactant reverse micelle was much higher than that with DTAB reverse micelle, given the dilution multiple was the same. This also suggested the advantage of gemini surfactant over conventional monomeric surfactant in reverse micellar extraction. 4. Conclusion In summary, this study was focused on getting high active bromelain from pineapple peel by reverse micelle extraction. Gemini surfactant C12-8-C12
2Br was found more suitable than its counterpart monomer DTAB. The activity recovery and purification fold of bromelain were 163% and 3.3 with C12-8-C12
2Br reverse micelle, much higher than those with DTAB reverse micelle (which were 95% and 1.7). This should be due to the spacer of C12-8-C12
2Br

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