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Recovery of phenolic compounds from pequi (Caryocar brasiliense Camb.) fruit extract by membrane filtrations: Comparison of direct and sequential processes
Journal of Food Engineering 257 (2019) 26–33
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Recovery of phenolic compounds from pequi (Caryocar brasiliense Camb.) fruit extract by membrane filtrations: Comparison of direct and sequential processes
T
Flávia de Santana Magalhães, Marcelo de Souza Martins Sá, Vicelma Luiz Cardoso, Miria Hespanhol Miranda Reis∗ Faculdade de Engenharia Química, Universidade Federal de Uberlândia, Av. João Naves de Ávila, 2121, 38400-902, Uberlândia, Minas Gerais, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Fruit Pequi Polyphenols Membrane Microfiltration Ultrafiltration Nanofiltration
Pequi is a local important Brazilian fruit, which presents substantial phenolic compounds that justify the processing of this fruit. Here we investigated the clarification and concentration of pequi fruit extract by membrane processes. Microfiltration through a membrane of 0.22 μm was applied to clarify the pequi extract and total solids and lipids were reduced in 50 and 65%, respectively. However, the microfiltered extract still presented a cloudy appearance (turbidity of 649 NTU) and sequential processes (ultra and nanofiltration) were applied for a further clarification and concentration. Sequential ultra and nanofiltrations presented retention factor lower than 45% for total polyphenol compounds. Ultrafiltration was especially efficient in retaining larger molecules (ellagic acid), while the smallest molecule (p-coumaric acid) was retained by the sequential nanofiltration process. Direct ultra and nanofiltration processes presented greater retention factors and yields than the sequential processes. However, the permeate flux in the direct process were lower than in the sequential membrane filtration processes. Thus, sequential membrane filtration processes are recommended to clarify and fractionate pequi fruit extract, while direct ultra and nanofiltrations presented the greatest retention factors and yields for bioactive compounds from pequi extract.
1. Introduction Pequi (Caryocar brasiliense Camb.) is a drupe fruit with a seed in the mesocarp and covered with thin and tough thorns. This fruit is found in the Brazilian Cerrado (a tropical savanna ecoregion) and it is usually consumed as local dishes. However, pequi fruit presents a great potential to be explored as a nutrient source. Khouri et al. (2007) suggested the effectiveness of aqueous pequi fruit extract against clastogenicity due to its high concentration of antioxidant compounds. Also due to its health-promoting benefits, some studies have been reported in the literature about the processing of pequi fruit (de Santana Magalhães et al., 2018; Machado et al., 2015; Machado et al., 2016). According to Machado et al. (2015), membrane filtration processes can be successfully applied to concentrate carotenoids and phenolic acids from pequi aqueous extract. de Santana Magalhães et al. (2018) evaluated the clarification of pequi fruit extract with different bioadsorbents (chitosan and moringa seeds). However, the number of studies about clarification of pequi extract and concentration of its bioactive compounds is still scarce. Moreover, the efficiency of the membrane
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processes for the processing of fruit extracts should be deeply investigated focused on the juice clarification and on the recovery of bioactive compounds. Recent reviews are published in the literature about application of membrane processes for the treatment of fruit juices (Castro-Muñoz, 2018; Nath et al., 2018; Urošević et al., 2017). Some strategies are suggested to improve the membrane performance, such as the integration of membrane operations (Urošević et al., 2017). Ng et al. (2015) proposed the sequential fractioning of bioactive compounds from coconut products by membrane processes. Galanakis (2015) highlighted the importance of macroscopic pre-treatments prior to ultrafiltration to improve the performance of the UF membrane. Ghosh et al. (2018) compared the efficiency of centrifugation and microfiltration on the clarification of jamun juice. In addition to the concentration and clarification of fruit juices, membrane processes have been also studied for the purification of phenolic compounds from food matrices. Conidi et al. (2018) published a review about membrane-based processes applied for the recovery of polyphenols from different natural sources. More specifically, Galanakis
Corresponding author. E-mail address: [email protected] (M. Hespanhol Miranda Reis).
https://doi.org/10.1016/j.jfoodeng.2019.03.025 Received 30 October 2018; Received in revised form 15 March 2019; Accepted 30 March 2019 Available online 04 April 2019 0260-8774/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. (a) Sequential and direct membrane processes applied for pequi fruit extract filtration, (b) Membrane filtration apparatus. Table 1 Characteristics of the selected flat membranes for pequi fruit extract treatment. Membrane type
Manufacturer
Material
Characteristic
Pore size/MWCO
Water permeability (L h−1 m−2 bar−1)
Microfiltration Ultrafiltration Nanofiltration
Millipore™ Microdyn-Nadir® Microdyn-Nadir®
Mixed cellulose esters Polyethersulfone Polypropylene
hydrophilic hydrophilic hydrophobic
0.22 μm 5 kDa 500–600 Da
10449 7.59 1.90
solution (5%, v:v) at a concentration of 10 g L−1 (Domingues et al., 2012). After coagulation with chitosan, the aqueous extract was centrifuged at 8000 rpm for 10 min. The supernatant was then filtered through a paper filter (Prolab, 10 μm) for oil and solids removal.
(2015) elucidated the main mechanisms responsible for the retention of bioactive molecules by ultrafiltration membranes. Conidi et al. (2017) evaluated the performance of ultra and nanofiltration membranes with different nominal molecular weight cut-off (MWCO) for the recovery of phenolic compounds from pomegranate juice. Despite the recognized efficiency of membrane filtration processes for the treatment of fruit extracts, including the recovery of bioactive compounds, few papers are reported in the literature focusing on integrated membrane processes that are properly designed for specific food matrices (Conidi et al., 2018). The present study evaluated sequential and direct membrane filtration processes for the clarification and concentration of the phenolic compounds from pequi fruit extract. We evaluated the effect of micro, ultra and nanofiltration of pequi fruit extract on permeate flux, membrane fouling and concentration of bioactive compounds. Ultra and nanofiltration processes were sequentially and directly performed in order to investigate different operation modes to clarify and concentrate pequi fruit extract.
2.2. Membrane separation processes Three different flat membranes were evaluated for the pequi fruit pulp extract treatment in a sequential or direct filtration, as presented in Fig. 1(a). The characteristics of each used polymeric membrane are presented in Table 1. Microfiltration experiments were performed at 0.6 bar of transmembrane pressure until a concentration factor of 1.7. Ultra and nanofiltrations were performed at 6 and 8 bar of transmembrane pressure, respectively, both until a concentration factor of 2.0. For all experiments, the dead-end filtration module was pressurized with nitrogen. Microfiltration membrane area was of 5.40 × 10−3 m2, while ultra and nanofiltration membrane areas were both of 1.93 × 10−3 m2. Microfiltration process required a larger filtration area than ultra and nanofiltrations due to its higher permeate flux. Permeate flux was recorded according to filtration time and total permeate samples were collected for further analyses. Fig. 1(b) presents a scheme of the used filtration module. Equation (1) was used to model the flux decay during the dead-end membrane filtrations, as proposed by Hermia (1982). The original Hermia equation was properly written in terms of flux, following the alternative form presented by Field et al. (1995) and (Field and Wu, 2011).
2. Material and methods 2.1. Extraction and pre-treatment process Pequi aqueous extract was prepared from pequi fruit pulp (Minas Gerais state, Brazil) at the conditions proposed by de Santana Magalhães et al. (2018). Water was used as solvent since it is recognized as eco-friendly technology for extraction of bioactive compounds (Azmir et al., 2013). Briefly, pequi fruit pulp was added to distilled water at a concentration of 25 g 100 mL−1. Then, extraction process was carried out for 1 h at 80 °C at constant magnetic agitation (750 rpm). After that, the aqueous extract was filtered through a stainless steel strainer to remove rough particles. The extract was pre-treated with chitosan (from shrimp shells, ≥75% (deacetylated, Sigma-Aldrich)) at a concentration of 0.1 g L−1 for 20 min in a jar test equipment (Milan, Model JT 203) for a previous clarification and lipid removal, as proposed by de Santana Magalhães et al. (2018). Chitosan hydrolysis was carried out with an acetic acid
−
dJ = K A2 − n J 3 − n dt
(1)
where J is the volumetric flux (m s-l), t is the filtration time (s), K is a constant in a dead-end fouling equation (units depend on mechanism), A is the membrane surface area (m2) and n is the number indicating fouling mechanism (dimensionless). According to the “n” index of Equation (1), four different fouling mechanisms can be assigned (complete pore blocking for n = 2, 27
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Direct processes
Sequential processes
internal pore blocking for n = 1.5, partial pore blocking for n = 1.0 and cake formation for n = 0). Experimental flux data were adjusted to the differential equations proposed by Hermia by applying the Levenberg–Marquardt numerical method, as proposed by Domingues et al. (2014) and Sousa et al. (2016). The residual sum of squares between numerical calculations and experimental data were considered to determine the main fouling mechanisms (Nor et al., 2017). The resistance-in-series model was also used to describe fouling occurrences. The membrane hydraulic resistance (RM) and the resistances due to cake formation and pore blockage (RC and RP, respectively) were considered to determine the total resistance (RT) (Sun et al., 2018). Water filtrations through a clean membrane were carried out to determine the membrane hydraulic resistance, as presented in Equation (2).
RM =
ΔP μ JW 0
(2) −1
where RM is the membrane hydraulic resistance (m ), ΔP is the transmembrane pressure (Pa), μ is the water viscosity at 25 °C (Pa s), and JW0 is pure water volumetric flux through a clean membrane (m sl ). After pequi extract filtration, water flux through the used membrane was once again recorded in order to determine the sum of the resistances due to pore blockage and cake formation, as presented in Equation (3).
RP + RC =
ΔP μ JW1
(3)
where RP and RC are resistances due to pore blockage and cake formation (m−1), ΔP is the transmembrane pressure (Pa), μ is the water viscosity at 25 °C (Pa s), and JW1 is water volumetric flux after filtration procedure (m s-l). After physically removing the cake layer by gently brushing the membrane surface, the individual resistance due to pore blockage was determined by a third water flux measurement, as presented in Equation (4).
Mean values denoted by different letters at the same column are significantly different at p ≤ 0.05.
0.31a ± 0.03 0.28a ± 0.06 0.28a ± 0.04 0.18b ± 0.01 0.01c ± 0.00 0.01c ± 0.00 0.02c ± 0.01 7.14a ± 0.07 4.71b ± 0.85 3.95b ± 0.53 0.61c ± 0.12 0.34c ± 0.18 0.88c ± 0.80 0.48c ± 0.40 18.97a ± 1.93 12.58b ± 1.50 9.19c ± 0.07 3.48d ± 0.45 1.09f + 0.07 2.55d.e ± 0.84 1.56e.f ± 0.98 108.56a ± 0.44 77.52b ± 0.57 54.53c ± 0.50 31.44d ± 0.89 21.43e ± 0.30 29.01d ± 0.29 22.10e ± 0.87 1.07a ± 0.20 0.46b ± 0.35 0.16c ± 0.03 0.08d ± 0.06 0.00e ± 0.00 0.01e ± 0.00 0.00e ± 0.00 998.33a ± 2.89 985a ± 15.00 649b ± 9.00 58.33c ± 0.58 7.40d ± 0.20 63.67c ± 2.52 1.47d ± 0.33 Crude extract Pre-treated extract Microfiltered (MS) Ultrafiltered (US) Nanofiltered (NS) Ultrafiltered (UD) Nanofiltered (ND)
20.6 a ± 0.39 16.73b ± 0.18 8.30c ± 0.84 6.33c ± 0.81 2.93e ± 0.17 4.30d ± 0.25 2.97e ± 0.21
p-Coumaric acid (mg/100 g FW) Ellagic acid (mg/100 g FW) Gallic acid (mg/100 g FW) TPC (mgGAE/100 g FW) TSC (g L−1) LC (%) Turbidity (NTU) Extract
Table 2 Characteristics of feed and permeate samples of pequi fruit extract.
F. de Santana Magalhães, et al.
RP =
ΔP μ JW 2
(4) −1
where RP is the resistance due to pore blockage (m ), ΔP is the transmembrane pressure (Pa), μ is the water viscosity at 25 °C (Pa s), and JW2 is water volumetric flux after cake removal (m s-l). Individual values of the resistance due to cake formation were determined by subtracting the values found in Equations (3) and (4). Additionally, morphological characteristics of the cake that was formed on the membrane outer surface were verified by scanning electron microscopy (SEM, Carl Zeiss, Model EVO MA 10). 2.3. Physical chemical analyses Analyses of turbidity, total solid content (TSC), total polyphenol content (TPC), lipid content and concentrations of gallic, p-coumaric and ellagic acids were carried out to characterize feed and permeate samples. Turbidity was verified in a calibrated Nova Organica HD 114 turbidimeter. The weight of the samples (a volume of 2 mL) before and after drying at 105 °C for 24 h was used to determine the total solid content. The Folin–Ciocalteu method (Singleton and Rossi, 1965) was applied to determine total polyphenol content, as described in de Santana Magalhães et al. (2018). Total lipid content was determined by the Bligh and Dyer method (Bligh and Dyer, 1959). The chromatographic methodology proposed by Chisté and Mercadante (2012) was adapted to determine concentrations of gallic, p-coumaric and ellagic acids (Sigma-Aldrich) in a Shimadzu HPLC (model LC20AT Prominence) and using a VP-ODS C18 (4.6 × 150 mm) column. All physicalchemical analyses were done in triplicate and were statically analyzed according to the Tukey test at a significance level of 5%. 28
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Fig. 2. Retention factors of total solids and polyphenols and gallic, ellagic and p-coumaric acids.
identified (gallic acid, quinic acid, quercetin, and quercetin 3-O-arabinose). Rocha et al. (2015) reported that pequi fruit peel presents considerable concentrations of gallic acid. The crude pequi fruit extract presented similar concentrations of gallic, ellagic and p-coumaric acids than mango fruit (Palafox-Carlos et al., 2012), walnut kernel (Colaric et al., 2005) and piquiá (Caryocar villosum) (Chisté and Mercadante, 2012). Before membrane filtrations, crude pequi extract was pre-treated with chitosan, as proposed by de Santana Magalhães et al. (2018). The pre-treatment with chitosan was particularly efficient in reducing the lipid content of crude extract (reduction of 57%). Jarto et al. (2015) also verified a great decrease in lipid concentration after the treatment of whey protein concentrate with chitosan. According to Hwang and Damodaran (1995), chitosan is able to form of a chitosan-fat globule membrane complex due to electrostatic interactions between chitosan and the negatively charged fat globules. The chitosan pre-treatment also decreased the total solid content, which is favorable for further membrane filtrations. However, the chitosan pre-treatment caused a decrease of 28% in total polyphenol content. Reductions on gallic and ellagic acids after the chitosan pre-treatment were also of approximately 34%. Concentration of p-coumaric acid after the chitosan pretreatment was reduced on less than 10%. The smaller polar characteristic of p-coumaric acid if compared to gallic and ellagic acids probably resulted in a lower attraction of p-coumaric acid by the positively charged chitosan. In conclusion, there is a trade-off between applying a pre-treatment that will probably contribute to increase permeate fluxes through the membrane, but that retains bioactive compounds. de
Retention factors of defined components (gallic, p-coumaric and ellagic acids, solids and polyphenols) were calculated according to Equation (5).
C Retention factor = ⎛1 − P ⎞ CF ⎠ ⎝ ⎜
⎟
(5)
where CP and CF are the concentrations of a specific component in permeate and feed streams, respectively. 3. Results and discussion 3.1. Characterizations of pequi fruit extracts Table 2 presents the physico-chemical characteristics of the pequi fruit extract before and after the proposed filtration processes. The applied conditions of extraction resulted in a crude pequi extract with considerable amounts of polyphenols, lipids and solids. Pequi is a locally important fruit, but under-exploited or totally unknown elsewhere, although this fruit could make major contributions to world nutrition. Crude pequi extract presented greater polyphenol content than pineapple aqueous extract (34.7 mgGAE 100 g−1 pulp), but lower than aqueous guava extract (153 mgGAE 100 g−1 pulp) (Alothman et al., 2009). Additionally, crude pequi extract presented significant concentrations of gallic, ellagic and p-coumaric acids. Roesler et al. (2008) investigated the concentration of polar components of pequi ethanolic extract by direct infusion electrospray ionization mass spectrometry (ESI-MS) and showed that important bioactive compounds were 29
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Fig. 3. Mass balance for the proposed processes for pequi extract membrane filtration.
extract with lower solid content than the sequential process. Total solid contents in direct and sequential nanofiltrations were statistically equivalent. Retentions of individual acids were also similar in direct and sequential processes, excepted to p-coumaric retention by sequential and direct ultrafiltration. Thus, direct and sequential processes produced permeate streams with similar characteristics. Arend et al. (2017) also reported that microfiltration previous to nanofiltration did not change the characteristics of permeate and retentate samples of strawberry juice. Reductions in turbidity, total solids and lipids are desirable to obtain a clarified extract, but reductions in polyphenols compromise the nutrition quality of the extract. Fig. 2 presents retention factors for total solids and polyphenols and individual acids. The microfiltration process reduced in 50% the total solid content of the pre-treated pequi extract and reductions in total polyphenol content was lower than 30%. Retention of polyphenol compounds by microfiltration membranes is probably associated to the retention of larger solutes and, thus, membranes with large pore sizes may be able to retain micromolecules (Arend et al., 2017). During the microfiltration process, the retention factor towards ellagic and gallic acids were higher if compared to p-coumaric acid. Compared to gallic and ellagic acid, p-coumaric acid presents the lowest molecular and, thus, completely passed through the microfiltration membrane. However, the permeate sample from the microfiltration process still presented a cloudy appearance with a large turbidity value (649 NTU). In fact, the turbidity value of microfiltered extract was much greater than the
Santana Magalhães et al. (2018) showed that chitosan pre-treatment in pequi fruit extract is recommended to improve further membrane filtration processes. In fact, high lipid concentrations represent a great concern due to substantial decreases in membrane permeability. The pre-treated pequi extract still presented high turbidity value (985 ± 15 NTU). Chandini et al. (2013) recommended turbidity values lower than 50 NTU to ready-to-drink beverages. Thus, clarification steps in addition to the chitosan pre-treatment are required. Results presented in Table 2 shows that all the applied processes were able to significantly reduce turbidity, total polyphenol, total solid and lipid contents from the feed extract, as well as concentrations of individual acids. Microfiltration process significantly reduced the turbidity of pretreated pequi fruit extract (reduction of 34%), but the turbidity value of the microfiltered extract was still high (649 ± 9 NTU). Ultrafiltration processes reduced the turbidity of pequi extract to values close to 50 NTU. Turbidity values of the nanofiltered pequi extract was lower than 10 NTU. Lipid content was almost totally reduced by ultra and nanofiltration processes. Sequential ultrafiltration process did not significantly reduce the solid content of the microfiltered extract, but the sequential nanofiltration was quite efficient in total solid reduction. Direct nano and ultrafiltration processes significantly reduced the total solid content of the pre-treated extract. All processes significantly reduced total polyphenol content and concentrations of individual acids. Comparing sequential and direct processes, the direct processes reduced turbidity, lipids and total polyphenols at the same extend than the sequential processes. The direct ultrafiltration process resulted in an 30
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Fig. 4. Permeate fluxes of pequi extract through (a) micro, (b) ultra and (c) nanofiltration membranes.
cut-off of the ultrafiltration membrane (5 kDa) can not directly justify the high retention value towards ellagic acid, but micromolecules may be clustered by other compounds. Galanakis (2015) indicated that the expected theoretically behavior according to the sieving mechanism may be not observed during the permeation of micromolecules due to molecule associations and the asymmetric characteristics of the membranes. The sequential nanofiltration process was more efficient in retaining the smallest component (p-coumaric acid) with retention factor for this component around 94%. During the sequential nanofiltration and direct ultra and nanofiltrations, the retention factor towards the lowest molecular weight polyphenol (p-coumaric acid) was higher if compared with gallic and ellagic acid. The low concentration of pcoumaric acid in the pequi extract may also have contributed to its association with macromolecules and consequent cluster formations, which were easily retained by ultra and nanofiltration membranes. The direct ultrafiltration process resulted in greater retention factors than the sequential processes. More than 60% of the polyphenolic compounds were retained by the direct ultrafiltration process and this process retained more than 74% of the total solids. Retention factors by the direct nanofiltration towards individual phenolic acids process were close to 0.9. Thus, the direct nanofiltration process is the most efficient for the concentration of phenolic compounds. A mass balance on the membrane processes was carried out in order to express the amount of bioactive compounds recovered in the different permeate fractions for a basis set of 1000 L of pre-treated feed
Table 3 Steady state fluxes and hydraulic resistances (Membrane resistance (RM), Cake resistance (RC), Pore blockage resistance (RP) and Total resistance (RT)). Process
Microfiltration Sequential ultrafiltration Sequential nanofiltration Direct ultrafiltration Direct nanofiltration
Final flux (L h−1 m−2)
8.48 5.67 2.99 4.84 1.17
Resistances (1013 m2 m−3) RM
RC
RP
RT
0.004 1.88 22.20 1.88 22.20
4.79 3.96 34.50 8.19 72.10
0.045 3.69 8.00 3.26 16.90
4.84 9.53 64.70 13.33 111.20
recommended value for clarified beverages (approximately 50 NTU) ((Chandini et al., 2013)). Thus, a sequential process is recommended to produce a clarified pequi extract and/or to concentrate the bioactive compounds. The sequential ultrafiltration process produced a permeate stream with low value of turbidity, solid and lipid contents, but also retained the phenolic compounds at a large extend. As presented in Fig. 2, retention factor of total polyphenol content by the sequential ultrafiltration process was of 42%, with the lowest retention for the pcoumaric acid, which presents the lowest molecular weight if compared to gallic and ellagic acids. Then, the ultrafiltration membrane of 5 kDa was especially efficient in retaining the ellagic acid, with retention factor for this substance greater than 80%. In fact, the molecular weight
31
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Fig. 5. Cross-section SEM images of microfiltration membranes.
The final fluxes achieved in the sequential processes are greater than the final fluxes in the direct processes. Additionally, the reported fluxes are in agreement with those reported in the literature for other fruit juices (Arend et al., 2017; Arriola et al., 2014). The nanofiltration membrane presented the greatest total resistance probably due to its hydrophobic nature in addition to its greater rejection coefficient. For all processes, the major resistance contribution was due to cake formation. The cake resistance accounts for 99% of the total resistance during microfiltration, but this contribution decreased to lower than 54% in the sequential ultra and nanofiltration processes. Thus, the previous microfiltration process resulted in the removal of larger particles that caused the formation of a significant cake layer on the membrane surface. Additionally, resistance values are greater in direct than in sequential processes. Thus, sequential processes are suggested to decrease total hydraulic resistances. Fig. 5 presents cross-section SEM images of clean and used microfiltration membranes. A dense cake layer was formed on the top surface of the microfiltration membrane. This cake layer presented a thickness of approximately 57 μm, as presented in Fig. 5. None visual difference was observed in the ultra and nanofiltration membranes, which confirms that the previous microfiltration was efficient in retaining most of the macromolecules.
stream, as presented in Fig. 3. Although direct and sequential processes have produced permeate with similar characteristics (Table 2), direct processes enabled to obtain larger volumes and quantities than the sequential processes, as presented in Fig. 3. The yields of polyphenols in the retentate stream of the nanofiltrations were of 5.5% and 85.7% in the sequential and direct processes, respectively. Conidi et al. (2017) reported similar yields for the retention of polyphenol compounds from pomegranate juice by nanofiltration membranes. The visual aspects of pequi extracts are also presented in Fig. 3. The pre-treated pequi extract presented a yellowish, cloudy and oily appearance, while the permeate samples are clearer. Nanofiltration processes produced a transparent permeate, while its retentate was with similar visual aspect than the pre-treated pequi extract. Due to its high antioxidant activity, the retentate of the direct nanofiltration process is suggested to be used in the formulation of nutraceutical products, as well as natural colorant due to its intense yellowish color. Permeate samples are clear and they are suggested to be used as food additives or as bases for soft drinks.
3.2. Flux declines during direct and sequential filtration processes Fig. 4 presents the flux declines during dead-end filtrations of pretreated pequi extract by micro, ultra and nanofiltration membranes at both sequential and direct mode. A pronounced flux decline was observed in the first 20 min of filtration for all processes, which is a typical behavior of dead-end filtrations. According to Pereira et al. (2005), a continuous decrease in the permeate flux is frequently observed, indicating other phenomena that could be happening besides the polarization of the concentration, such as adsorption of solute molecules on the membrane surface and blockage of membrane pores. The adjustment of flux data according to Hermia model (Hermia, 1982) showed that cake formation was the main fouling mechanism for all the evaluated filtrations. As presented in Fig. 4, direct and sequential filtrations presented similar flux behavior. However, fluxes in sequential ultrafiltration were up to 14% greater than in direct ultrafiltration. Fluxes in sequential nanofiltration were up to 51% greater than in direct nanofiltration. Thus, the sequential process was especially efficient in increasing the permeate flux through ultra and nanofiltration membranes. Additionally, the direct processes required greater times to achieve the same concentration factor, especially in the nanofiltration process. Thus, although the direct process had been more efficient in concentrating the phenolic compounds, permeate fluxes were mitigated. Table 3 presents the final fluxes for all the proposed membrane filtrations and the calculated hydraulic resistances.
4. Conclusions We compared the efficiency of sequential and direct membrane filtration processes for the treatment of pequi fruit extract. This fruit extract presented a considerable amount of phenolic compounds, in addition to solid and lipid contents. Thus, proper clarification processes should be suggested. The microfiltration process was efficient in clarifying the pre-treated pequi extract (reduction of 50% in total solids and of 65% and total lipids). Additionally, the microfiltration process did not retain the phenolic compounds at a large extend. However, the turbidity value of the permeate from the microfiltration process was still right, and sequential clarification steps were required. The sequential ultrafiltration process produced a clear permeate and retained 42% of the phenolic compounds from the feed solution. Thus, the sequential ultrafiltration process is suitable to produce a clear permeate and a concentrate which is rich in phenolic compounds. Additionally, the sequential ultrafiltration process was particularly efficient in retain the phenolic acid with the greatest molecular weight (ellagic acid). The direct ultrafiltration process resulted in a greater retention factor of phenolic compounds than the sequential ultrafiltration, since the retention of phenolic compounds is probably associated to the retention 32
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of larger solutes. However, the permeate flux in the sequential ultrafiltration was 14% greater than in the direct process, since a previous microfiltration was applied to retain larger molecules. Thus, the direct ultrafiltration process is recommended to concentrate the bioactive compounds from pequi extract, since this process presents greater productivity and retention of polyphenol compounds than the sequential process, even that the permeate flux is a bit lower. The direct nanofiltration process resulted in almost 100% retention of phenolic compounds, but the permeate flux is quite low. In the sequential nanofiltration process (with micro and ultrafiltration processes as pretreatments), the permeate flux is twice greater than the permeate flux in the direct process. However, the yield of the sequential process is lower than in the direct process. Thus, the sequential process is recommended to fractionate the phenolic compounds and to obtain a clarified permeate, while the direct ultra and nanofiltration processes are recommend as proper concentration steps.
Domingues, R.C.C., Faria Junior, S.B., Silva, R.B., Cardoso, V.L., Reis, M.H.M., 2012. Clarification of passion fruit juice with chitosan: effects of coagulation process variables and comparison with centrifugation and enzymatic treatments. Process Biochem. 47 (3), 467–471. Domingues, R.C.C., Ramos, A.A., Cardoso, V.L., Reis, M.H.M., 2014. Microfiltration of passion fruit juice using hollow fibre membranes and evaluation of fouling mechanisms. J. Food Eng. 121 (1), 73–79. Field, R.W., Wu, D., Howell, J.A., Gupta, B.B., 1995. Critical flux concept for microfiltration fouling. J. Membr. Sci. 100 (3), 259–272. Field, R.W., Wu, J.J., 2011. Modelling of permeability loss in membrane filtration: Reexamination of fundamental fouling equations and their link to critical flux. Desalination 283, 68–74. Galanakis, C.M., 2015. Separation of functional macromolecules and micromolecules: from ultrafiltration to the border of nanofiltration. Trends Food Sci. Technol. 42 (1), 44–63. Ghosh, P., Pradhan, R.C., Mishra, S., 2018. Clarification of jamun juice by centrifugation and microfiltration: analysis of quality parameters, operating conditions, and resistance. J. Food Process. Eng. 41 (1), e12603. Hermia, J., 1982. Constant pressure blocking filtration laws - application to power-law non-Newtonian fluids. Trans. Inst. Chem. Eng. 60 (3), 183–187. Hwang, D.-C., Damodaran, S., 1995. Selective precipitation and removal of lipids from cheese whey using chitosan. J. Agric. Food Chem. 43 (1), 33–37. Jarto, I., Lucey, J.A., Molitor, M.S., Smith, K.E., 2015. Utilisation of chitosan flocculation of residual lipids and microfiltration for the production of low fat, clear WPC80. Int. J. Dairy Technol. 68 (4), 471–477. Khouri, J., Resck, I.S., Poças-Fonseca, M., Sousa, T.M.M., Pereira, L.O., Oliveira, A.B.B., Grisolia, C.K., 2007. Anticlastogenic potential and antioxidant effects of an aqueous extract of pulp from the pequi tree (Caryocar brasiliense Camb). Genet. Mol. Biol. 30 (2), 442–448. Machado, M.T.C., Mello, B.C.B.S., Hubinger, M.D., 2015. Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes. Food Bioprod. Process. 95, 304–312. Machado, M.T.C., Trevisan, S., Pimentel-Souza, J.D.R., Pastore, G.M., Hubinger, M.D., 2016. Clarification and concentration of oligosaccharides from artichoke extract by a sequential process with microfiltration and nanofiltration membranes. J. Food Eng. 180, 120–128. Nath, K., Dave, H.K., Patel, T.M., 2018. Revisiting the recent applications of nanofiltration in food processing industries: progress and prognosis. Trends Food Sci. Technol. 73, 12–24. Ng, C.Y., Mohammad, A.W., Ng, L.Y., Jahim, J.M., 2015. Sequential fractionation of value-added coconut products using membrane processes. J. Ind. Eng. Chem. 25, 162–167. Nor, M.Z.M., Ramchandran, L., Duke, M., Vasiljevic, T., 2017. Integrated ultrafiltration process for the recovery of bromelain from pineapple waste mixture. J. Food Process. Eng. 40 (3), e12492. Palafox-Carlos, H., Yahia, E.M., González-Aguilar, G.A., 2012. Identification and quantification of major phenolic compounds from mango (Mangifera indica, cv. Ataulfo) fruit by HPLC–DAD–MS/MS-ESI and their individual contribution to the antioxidant activity during ripening. Food Chem. 135 (1), 105–111. Pereira, C.C., Rufino, J.R.M., Habert, A.C., Nobrega, R., Cabral, L.M.C., Borges, C.P., 2005. Aroma compounds recovery of tropical fruit juice by pervaporation: membrane material selection and process evaluation. J. Food Eng. 66 (1), 77–87. Rocha, L.B., Melo, A.M., Paula, S.L.A., Nobre, S.A.M., Abreu, I.N., 2015. Gallic acid as the major antioxidant in pequi (Caryocar brasiliense camb.) fruit peel. Rev. Bras. Plantas Med. 17 (4), 592–598. Roesler, R., Catharino, R.R., Malta, L.G., Eberlin, M.N., Pastore, G., 2008. Antioxidant activity of Caryocar brasiliense (pequi) and characterization of components by electrospray ionization mass spectrometry. Food Chem. 110 (3), 711–717. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am. J. Enol. Vitic. 16 (3), 144–158. Sousa, L.D.S., Cabral, B.V., Madrona, G.S., Cardoso, V.L., Reis, M.H.M., 2016. Purification of polyphenols from green tea leaves by ultrasound assisted ultrafiltration process. Separ. Purif. Technol. 168, 188–198. Sun, Y., Qin, Z., Zhao, L., Chen, Q., Hou, Q., Lin, H., Jiang, L., Liu, J., Du, Z., 2018. Membrane fouling mechanisms and permeate flux decline model in soy sauce microfiltration. J. Food Process. Eng. 41 (1), e12599. Urošević, T., Povrenović, D., Vukosavljević, P., Urošević, I., Stevanović, S., 2017. Recent developments in microfiltration and ultrafiltration of fruit juices. Food Bioprod. Process. 106, 147–161.
Acknowledgements This research was supported by FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). References Alothman, M., Bhat, R., Karim, A.A., 2009. Antioxidant capacity and phenolic content of selected tropical fruits from Malaysia, extracted with different solvents. Food Chem. 115 (3), 785–788. Arend, G.D., Adorno, W.T., Rezzadori, K., Di Luccio, M., Chaves, V.C., Reginatto, F.H., Petrus, J.C.C., 2017. Concentration of phenolic compounds from strawberry (Fragaria X ananassa Duch) juice by nanofiltration membrane. J. Food Eng. 201, 36–41. Arriola, N.A., Santos, G.D., Prudêncio, E.S., Vitali, L., Petrus, J.C.C., Castanho Amboni, R.D.M., 2014. Potential of nanofiltration for the concentration of bioactive compounds from watermelon juice. Int. J. Food Sci. Technol. 49 (9), 2052–2060. Azmir, J., Zaidul, I.S.M., Rahman, M.M., Sharif, K.M., Mohamed, A., Sahena, F., Jahurul, M.H.A., Ghafoor, K., Norulaini, N.A.N., Omar, A.K.M., 2013. Techniques for extraction of bioactive compounds from plant materials: a review. J. Food Eng. 117 (4), 426–436. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37 (8), 911–917. Castro-Muñoz, R., 2018. Separation, Fractionation and Concentration of High-AddedValue Compounds from Agro-Food By-Products through Membrane-Based Technologies. Reference Module in Food Science. Elsevier. Chandini, S.K., Rao, L.J., Subramanian, R., 2013. Membrane clarification of black tea extracts. Food Bioprocess Technol. 6 (8), 1926–1943. Chisté, R.C., Mercadante, A.Z., 2012. Identification and quantification, by HPLC-DADMS/MS, of carotenoids and phenolic compounds from the amazonian fruit caryocar villosum. J. Agric. Food Chem. 60 (23), 5884–5892. Colaric, M., Veberic, R., Solar, A., Hudina, M., Stampar, F., 2005. Phenolic acids, syringaldehyde, and juglone in fruits of different cultivars of juglans regia L. J. Agric. Food Chem. 53 (16), 6390–6396. Conidi, C., Cassano, A., Caiazzo, F., Drioli, E., 2017. Separation and purification of phenolic compounds from pomegranate juice by ultrafiltration and nanofiltration membranes. J. Food Eng. 195, 1–13. Conidi, C., Drioli, E., Cassano, A., 2018. Membrane-based agro-food production processes for polyphenol separation, purification and concentration. Curr. Opin. Food Sci. 23, 149–164. de Santana Magalhães, F., Cardoso, V.L., Reis, M.H.M., 2018. Sequential process with bioadsorbents and microfiltration for clarification of pequi (Caryocar brasiliense Camb.) fruit extract. Food Bioprod. Process. 108, 105–116.
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Recovery of phenolic compounds from pequi (Caryocar brasiliense Camb.) fruit extract by membrane filtrations: Comparison of direct and sequential processes
T
Flávia de Santana Magalhães, Marcelo de Souza Martins Sá, Vicelma Luiz Cardoso, Miria Hespanhol Miranda Reis∗ Faculdade de Engenharia Química, Universidade Federal de Uberlândia, Av. João Naves de Ávila, 2121, 38400-902, Uberlândia, Minas Gerais, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Fruit Pequi Polyphenols Membrane Microfiltration Ultrafiltration Nanofiltration
Pequi is a local important Brazilian fruit, which presents substantial phenolic compounds that justify the processing of this fruit. Here we investigated the clarification and concentration of pequi fruit extract by membrane processes. Microfiltration through a membrane of 0.22 μm was applied to clarify the pequi extract and total solids and lipids were reduced in 50 and 65%, respectively. However, the microfiltered extract still presented a cloudy appearance (turbidity of 649 NTU) and sequential processes (ultra and nanofiltration) were applied for a further clarification and concentration. Sequential ultra and nanofiltrations presented retention factor lower than 45% for total polyphenol compounds. Ultrafiltration was especially efficient in retaining larger molecules (ellagic acid), while the smallest molecule (p-coumaric acid) was retained by the sequential nanofiltration process. Direct ultra and nanofiltration processes presented greater retention factors and yields than the sequential processes. However, the permeate flux in the direct process were lower than in the sequential membrane filtration processes. Thus, sequential membrane filtration processes are recommended to clarify and fractionate pequi fruit extract, while direct ultra and nanofiltrations presented the greatest retention factors and yields for bioactive compounds from pequi extract.
1. Introduction Pequi (Caryocar brasiliense Camb.) is a drupe fruit with a seed in the mesocarp and covered with thin and tough thorns. This fruit is found in the Brazilian Cerrado (a tropical savanna ecoregion) and it is usually consumed as local dishes. However, pequi fruit presents a great potential to be explored as a nutrient source. Khouri et al. (2007) suggested the effectiveness of aqueous pequi fruit extract against clastogenicity due to its high concentration of antioxidant compounds. Also due to its health-promoting benefits, some studies have been reported in the literature about the processing of pequi fruit (de Santana Magalhães et al., 2018; Machado et al., 2015; Machado et al., 2016). According to Machado et al. (2015), membrane filtration processes can be successfully applied to concentrate carotenoids and phenolic acids from pequi aqueous extract. de Santana Magalhães et al. (2018) evaluated the clarification of pequi fruit extract with different bioadsorbents (chitosan and moringa seeds). However, the number of studies about clarification of pequi extract and concentration of its bioactive compounds is still scarce. Moreover, the efficiency of the membrane
∗
processes for the processing of fruit extracts should be deeply investigated focused on the juice clarification and on the recovery of bioactive compounds. Recent reviews are published in the literature about application of membrane processes for the treatment of fruit juices (Castro-Muñoz, 2018; Nath et al., 2018; Urošević et al., 2017). Some strategies are suggested to improve the membrane performance, such as the integration of membrane operations (Urošević et al., 2017). Ng et al. (2015) proposed the sequential fractioning of bioactive compounds from coconut products by membrane processes. Galanakis (2015) highlighted the importance of macroscopic pre-treatments prior to ultrafiltration to improve the performance of the UF membrane. Ghosh et al. (2018) compared the efficiency of centrifugation and microfiltration on the clarification of jamun juice. In addition to the concentration and clarification of fruit juices, membrane processes have been also studied for the purification of phenolic compounds from food matrices. Conidi et al. (2018) published a review about membrane-based processes applied for the recovery of polyphenols from different natural sources. More specifically, Galanakis
Corresponding author. E-mail address: [email protected] (M. Hespanhol Miranda Reis).
https://doi.org/10.1016/j.jfoodeng.2019.03.025 Received 30 October 2018; Received in revised form 15 March 2019; Accepted 30 March 2019 Available online 04 April 2019 0260-8774/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. (a) Sequential and direct membrane processes applied for pequi fruit extract filtration, (b) Membrane filtration apparatus. Table 1 Characteristics of the selected flat membranes for pequi fruit extract treatment. Membrane type
Manufacturer
Material
Characteristic
Pore size/MWCO
Water permeability (L h−1 m−2 bar−1)
Microfiltration Ultrafiltration Nanofiltration
Millipore™ Microdyn-Nadir® Microdyn-Nadir®
Mixed cellulose esters Polyethersulfone Polypropylene
hydrophilic hydrophilic hydrophobic
0.22 μm 5 kDa 500–600 Da
10449 7.59 1.90
solution (5%, v:v) at a concentration of 10 g L−1 (Domingues et al., 2012). After coagulation with chitosan, the aqueous extract was centrifuged at 8000 rpm for 10 min. The supernatant was then filtered through a paper filter (Prolab, 10 μm) for oil and solids removal.
(2015) elucidated the main mechanisms responsible for the retention of bioactive molecules by ultrafiltration membranes. Conidi et al. (2017) evaluated the performance of ultra and nanofiltration membranes with different nominal molecular weight cut-off (MWCO) for the recovery of phenolic compounds from pomegranate juice. Despite the recognized efficiency of membrane filtration processes for the treatment of fruit extracts, including the recovery of bioactive compounds, few papers are reported in the literature focusing on integrated membrane processes that are properly designed for specific food matrices (Conidi et al., 2018). The present study evaluated sequential and direct membrane filtration processes for the clarification and concentration of the phenolic compounds from pequi fruit extract. We evaluated the effect of micro, ultra and nanofiltration of pequi fruit extract on permeate flux, membrane fouling and concentration of bioactive compounds. Ultra and nanofiltration processes were sequentially and directly performed in order to investigate different operation modes to clarify and concentrate pequi fruit extract.
2.2. Membrane separation processes Three different flat membranes were evaluated for the pequi fruit pulp extract treatment in a sequential or direct filtration, as presented in Fig. 1(a). The characteristics of each used polymeric membrane are presented in Table 1. Microfiltration experiments were performed at 0.6 bar of transmembrane pressure until a concentration factor of 1.7. Ultra and nanofiltrations were performed at 6 and 8 bar of transmembrane pressure, respectively, both until a concentration factor of 2.0. For all experiments, the dead-end filtration module was pressurized with nitrogen. Microfiltration membrane area was of 5.40 × 10−3 m2, while ultra and nanofiltration membrane areas were both of 1.93 × 10−3 m2. Microfiltration process required a larger filtration area than ultra and nanofiltrations due to its higher permeate flux. Permeate flux was recorded according to filtration time and total permeate samples were collected for further analyses. Fig. 1(b) presents a scheme of the used filtration module. Equation (1) was used to model the flux decay during the dead-end membrane filtrations, as proposed by Hermia (1982). The original Hermia equation was properly written in terms of flux, following the alternative form presented by Field et al. (1995) and (Field and Wu, 2011).
2. Material and methods 2.1. Extraction and pre-treatment process Pequi aqueous extract was prepared from pequi fruit pulp (Minas Gerais state, Brazil) at the conditions proposed by de Santana Magalhães et al. (2018). Water was used as solvent since it is recognized as eco-friendly technology for extraction of bioactive compounds (Azmir et al., 2013). Briefly, pequi fruit pulp was added to distilled water at a concentration of 25 g 100 mL−1. Then, extraction process was carried out for 1 h at 80 °C at constant magnetic agitation (750 rpm). After that, the aqueous extract was filtered through a stainless steel strainer to remove rough particles. The extract was pre-treated with chitosan (from shrimp shells, ≥75% (deacetylated, Sigma-Aldrich)) at a concentration of 0.1 g L−1 for 20 min in a jar test equipment (Milan, Model JT 203) for a previous clarification and lipid removal, as proposed by de Santana Magalhães et al. (2018). Chitosan hydrolysis was carried out with an acetic acid
−
dJ = K A2 − n J 3 − n dt
(1)
where J is the volumetric flux (m s-l), t is the filtration time (s), K is a constant in a dead-end fouling equation (units depend on mechanism), A is the membrane surface area (m2) and n is the number indicating fouling mechanism (dimensionless). According to the “n” index of Equation (1), four different fouling mechanisms can be assigned (complete pore blocking for n = 2, 27
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Direct processes
Sequential processes
internal pore blocking for n = 1.5, partial pore blocking for n = 1.0 and cake formation for n = 0). Experimental flux data were adjusted to the differential equations proposed by Hermia by applying the Levenberg–Marquardt numerical method, as proposed by Domingues et al. (2014) and Sousa et al. (2016). The residual sum of squares between numerical calculations and experimental data were considered to determine the main fouling mechanisms (Nor et al., 2017). The resistance-in-series model was also used to describe fouling occurrences. The membrane hydraulic resistance (RM) and the resistances due to cake formation and pore blockage (RC and RP, respectively) were considered to determine the total resistance (RT) (Sun et al., 2018). Water filtrations through a clean membrane were carried out to determine the membrane hydraulic resistance, as presented in Equation (2).
RM =
ΔP μ JW 0
(2) −1
where RM is the membrane hydraulic resistance (m ), ΔP is the transmembrane pressure (Pa), μ is the water viscosity at 25 °C (Pa s), and JW0 is pure water volumetric flux through a clean membrane (m sl ). After pequi extract filtration, water flux through the used membrane was once again recorded in order to determine the sum of the resistances due to pore blockage and cake formation, as presented in Equation (3).
RP + RC =
ΔP μ JW1
(3)
where RP and RC are resistances due to pore blockage and cake formation (m−1), ΔP is the transmembrane pressure (Pa), μ is the water viscosity at 25 °C (Pa s), and JW1 is water volumetric flux after filtration procedure (m s-l). After physically removing the cake layer by gently brushing the membrane surface, the individual resistance due to pore blockage was determined by a third water flux measurement, as presented in Equation (4).
Mean values denoted by different letters at the same column are significantly different at p ≤ 0.05.
0.31a ± 0.03 0.28a ± 0.06 0.28a ± 0.04 0.18b ± 0.01 0.01c ± 0.00 0.01c ± 0.00 0.02c ± 0.01 7.14a ± 0.07 4.71b ± 0.85 3.95b ± 0.53 0.61c ± 0.12 0.34c ± 0.18 0.88c ± 0.80 0.48c ± 0.40 18.97a ± 1.93 12.58b ± 1.50 9.19c ± 0.07 3.48d ± 0.45 1.09f + 0.07 2.55d.e ± 0.84 1.56e.f ± 0.98 108.56a ± 0.44 77.52b ± 0.57 54.53c ± 0.50 31.44d ± 0.89 21.43e ± 0.30 29.01d ± 0.29 22.10e ± 0.87 1.07a ± 0.20 0.46b ± 0.35 0.16c ± 0.03 0.08d ± 0.06 0.00e ± 0.00 0.01e ± 0.00 0.00e ± 0.00 998.33a ± 2.89 985a ± 15.00 649b ± 9.00 58.33c ± 0.58 7.40d ± 0.20 63.67c ± 2.52 1.47d ± 0.33 Crude extract Pre-treated extract Microfiltered (MS) Ultrafiltered (US) Nanofiltered (NS) Ultrafiltered (UD) Nanofiltered (ND)
20.6 a ± 0.39 16.73b ± 0.18 8.30c ± 0.84 6.33c ± 0.81 2.93e ± 0.17 4.30d ± 0.25 2.97e ± 0.21
p-Coumaric acid (mg/100 g FW) Ellagic acid (mg/100 g FW) Gallic acid (mg/100 g FW) TPC (mgGAE/100 g FW) TSC (g L−1) LC (%) Turbidity (NTU) Extract
Table 2 Characteristics of feed and permeate samples of pequi fruit extract.
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RP =
ΔP μ JW 2
(4) −1
where RP is the resistance due to pore blockage (m ), ΔP is the transmembrane pressure (Pa), μ is the water viscosity at 25 °C (Pa s), and JW2 is water volumetric flux after cake removal (m s-l). Individual values of the resistance due to cake formation were determined by subtracting the values found in Equations (3) and (4). Additionally, morphological characteristics of the cake that was formed on the membrane outer surface were verified by scanning electron microscopy (SEM, Carl Zeiss, Model EVO MA 10). 2.3. Physical chemical analyses Analyses of turbidity, total solid content (TSC), total polyphenol content (TPC), lipid content and concentrations of gallic, p-coumaric and ellagic acids were carried out to characterize feed and permeate samples. Turbidity was verified in a calibrated Nova Organica HD 114 turbidimeter. The weight of the samples (a volume of 2 mL) before and after drying at 105 °C for 24 h was used to determine the total solid content. The Folin–Ciocalteu method (Singleton and Rossi, 1965) was applied to determine total polyphenol content, as described in de Santana Magalhães et al. (2018). Total lipid content was determined by the Bligh and Dyer method (Bligh and Dyer, 1959). The chromatographic methodology proposed by Chisté and Mercadante (2012) was adapted to determine concentrations of gallic, p-coumaric and ellagic acids (Sigma-Aldrich) in a Shimadzu HPLC (model LC20AT Prominence) and using a VP-ODS C18 (4.6 × 150 mm) column. All physicalchemical analyses were done in triplicate and were statically analyzed according to the Tukey test at a significance level of 5%. 28
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Fig. 2. Retention factors of total solids and polyphenols and gallic, ellagic and p-coumaric acids.
identified (gallic acid, quinic acid, quercetin, and quercetin 3-O-arabinose). Rocha et al. (2015) reported that pequi fruit peel presents considerable concentrations of gallic acid. The crude pequi fruit extract presented similar concentrations of gallic, ellagic and p-coumaric acids than mango fruit (Palafox-Carlos et al., 2012), walnut kernel (Colaric et al., 2005) and piquiá (Caryocar villosum) (Chisté and Mercadante, 2012). Before membrane filtrations, crude pequi extract was pre-treated with chitosan, as proposed by de Santana Magalhães et al. (2018). The pre-treatment with chitosan was particularly efficient in reducing the lipid content of crude extract (reduction of 57%). Jarto et al. (2015) also verified a great decrease in lipid concentration after the treatment of whey protein concentrate with chitosan. According to Hwang and Damodaran (1995), chitosan is able to form of a chitosan-fat globule membrane complex due to electrostatic interactions between chitosan and the negatively charged fat globules. The chitosan pre-treatment also decreased the total solid content, which is favorable for further membrane filtrations. However, the chitosan pre-treatment caused a decrease of 28% in total polyphenol content. Reductions on gallic and ellagic acids after the chitosan pre-treatment were also of approximately 34%. Concentration of p-coumaric acid after the chitosan pretreatment was reduced on less than 10%. The smaller polar characteristic of p-coumaric acid if compared to gallic and ellagic acids probably resulted in a lower attraction of p-coumaric acid by the positively charged chitosan. In conclusion, there is a trade-off between applying a pre-treatment that will probably contribute to increase permeate fluxes through the membrane, but that retains bioactive compounds. de
Retention factors of defined components (gallic, p-coumaric and ellagic acids, solids and polyphenols) were calculated according to Equation (5).
C Retention factor = ⎛1 − P ⎞ CF ⎠ ⎝ ⎜
⎟
(5)
where CP and CF are the concentrations of a specific component in permeate and feed streams, respectively. 3. Results and discussion 3.1. Characterizations of pequi fruit extracts Table 2 presents the physico-chemical characteristics of the pequi fruit extract before and after the proposed filtration processes. The applied conditions of extraction resulted in a crude pequi extract with considerable amounts of polyphenols, lipids and solids. Pequi is a locally important fruit, but under-exploited or totally unknown elsewhere, although this fruit could make major contributions to world nutrition. Crude pequi extract presented greater polyphenol content than pineapple aqueous extract (34.7 mgGAE 100 g−1 pulp), but lower than aqueous guava extract (153 mgGAE 100 g−1 pulp) (Alothman et al., 2009). Additionally, crude pequi extract presented significant concentrations of gallic, ellagic and p-coumaric acids. Roesler et al. (2008) investigated the concentration of polar components of pequi ethanolic extract by direct infusion electrospray ionization mass spectrometry (ESI-MS) and showed that important bioactive compounds were 29
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Fig. 3. Mass balance for the proposed processes for pequi extract membrane filtration.
extract with lower solid content than the sequential process. Total solid contents in direct and sequential nanofiltrations were statistically equivalent. Retentions of individual acids were also similar in direct and sequential processes, excepted to p-coumaric retention by sequential and direct ultrafiltration. Thus, direct and sequential processes produced permeate streams with similar characteristics. Arend et al. (2017) also reported that microfiltration previous to nanofiltration did not change the characteristics of permeate and retentate samples of strawberry juice. Reductions in turbidity, total solids and lipids are desirable to obtain a clarified extract, but reductions in polyphenols compromise the nutrition quality of the extract. Fig. 2 presents retention factors for total solids and polyphenols and individual acids. The microfiltration process reduced in 50% the total solid content of the pre-treated pequi extract and reductions in total polyphenol content was lower than 30%. Retention of polyphenol compounds by microfiltration membranes is probably associated to the retention of larger solutes and, thus, membranes with large pore sizes may be able to retain micromolecules (Arend et al., 2017). During the microfiltration process, the retention factor towards ellagic and gallic acids were higher if compared to p-coumaric acid. Compared to gallic and ellagic acid, p-coumaric acid presents the lowest molecular and, thus, completely passed through the microfiltration membrane. However, the permeate sample from the microfiltration process still presented a cloudy appearance with a large turbidity value (649 NTU). In fact, the turbidity value of microfiltered extract was much greater than the
Santana Magalhães et al. (2018) showed that chitosan pre-treatment in pequi fruit extract is recommended to improve further membrane filtration processes. In fact, high lipid concentrations represent a great concern due to substantial decreases in membrane permeability. The pre-treated pequi extract still presented high turbidity value (985 ± 15 NTU). Chandini et al. (2013) recommended turbidity values lower than 50 NTU to ready-to-drink beverages. Thus, clarification steps in addition to the chitosan pre-treatment are required. Results presented in Table 2 shows that all the applied processes were able to significantly reduce turbidity, total polyphenol, total solid and lipid contents from the feed extract, as well as concentrations of individual acids. Microfiltration process significantly reduced the turbidity of pretreated pequi fruit extract (reduction of 34%), but the turbidity value of the microfiltered extract was still high (649 ± 9 NTU). Ultrafiltration processes reduced the turbidity of pequi extract to values close to 50 NTU. Turbidity values of the nanofiltered pequi extract was lower than 10 NTU. Lipid content was almost totally reduced by ultra and nanofiltration processes. Sequential ultrafiltration process did not significantly reduce the solid content of the microfiltered extract, but the sequential nanofiltration was quite efficient in total solid reduction. Direct nano and ultrafiltration processes significantly reduced the total solid content of the pre-treated extract. All processes significantly reduced total polyphenol content and concentrations of individual acids. Comparing sequential and direct processes, the direct processes reduced turbidity, lipids and total polyphenols at the same extend than the sequential processes. The direct ultrafiltration process resulted in an 30
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Fig. 4. Permeate fluxes of pequi extract through (a) micro, (b) ultra and (c) nanofiltration membranes.
cut-off of the ultrafiltration membrane (5 kDa) can not directly justify the high retention value towards ellagic acid, but micromolecules may be clustered by other compounds. Galanakis (2015) indicated that the expected theoretically behavior according to the sieving mechanism may be not observed during the permeation of micromolecules due to molecule associations and the asymmetric characteristics of the membranes. The sequential nanofiltration process was more efficient in retaining the smallest component (p-coumaric acid) with retention factor for this component around 94%. During the sequential nanofiltration and direct ultra and nanofiltrations, the retention factor towards the lowest molecular weight polyphenol (p-coumaric acid) was higher if compared with gallic and ellagic acid. The low concentration of pcoumaric acid in the pequi extract may also have contributed to its association with macromolecules and consequent cluster formations, which were easily retained by ultra and nanofiltration membranes. The direct ultrafiltration process resulted in greater retention factors than the sequential processes. More than 60% of the polyphenolic compounds were retained by the direct ultrafiltration process and this process retained more than 74% of the total solids. Retention factors by the direct nanofiltration towards individual phenolic acids process were close to 0.9. Thus, the direct nanofiltration process is the most efficient for the concentration of phenolic compounds. A mass balance on the membrane processes was carried out in order to express the amount of bioactive compounds recovered in the different permeate fractions for a basis set of 1000 L of pre-treated feed
Table 3 Steady state fluxes and hydraulic resistances (Membrane resistance (RM), Cake resistance (RC), Pore blockage resistance (RP) and Total resistance (RT)). Process
Microfiltration Sequential ultrafiltration Sequential nanofiltration Direct ultrafiltration Direct nanofiltration
Final flux (L h−1 m−2)
8.48 5.67 2.99 4.84 1.17
Resistances (1013 m2 m−3) RM
RC
RP
RT
0.004 1.88 22.20 1.88 22.20
4.79 3.96 34.50 8.19 72.10
0.045 3.69 8.00 3.26 16.90
4.84 9.53 64.70 13.33 111.20
recommended value for clarified beverages (approximately 50 NTU) ((Chandini et al., 2013)). Thus, a sequential process is recommended to produce a clarified pequi extract and/or to concentrate the bioactive compounds. The sequential ultrafiltration process produced a permeate stream with low value of turbidity, solid and lipid contents, but also retained the phenolic compounds at a large extend. As presented in Fig. 2, retention factor of total polyphenol content by the sequential ultrafiltration process was of 42%, with the lowest retention for the pcoumaric acid, which presents the lowest molecular weight if compared to gallic and ellagic acids. Then, the ultrafiltration membrane of 5 kDa was especially efficient in retaining the ellagic acid, with retention factor for this substance greater than 80%. In fact, the molecular weight
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Fig. 5. Cross-section SEM images of microfiltration membranes.
The final fluxes achieved in the sequential processes are greater than the final fluxes in the direct processes. Additionally, the reported fluxes are in agreement with those reported in the literature for other fruit juices (Arend et al., 2017; Arriola et al., 2014). The nanofiltration membrane presented the greatest total resistance probably due to its hydrophobic nature in addition to its greater rejection coefficient. For all processes, the major resistance contribution was due to cake formation. The cake resistance accounts for 99% of the total resistance during microfiltration, but this contribution decreased to lower than 54% in the sequential ultra and nanofiltration processes. Thus, the previous microfiltration process resulted in the removal of larger particles that caused the formation of a significant cake layer on the membrane surface. Additionally, resistance values are greater in direct than in sequential processes. Thus, sequential processes are suggested to decrease total hydraulic resistances. Fig. 5 presents cross-section SEM images of clean and used microfiltration membranes. A dense cake layer was formed on the top surface of the microfiltration membrane. This cake layer presented a thickness of approximately 57 μm, as presented in Fig. 5. None visual difference was observed in the ultra and nanofiltration membranes, which confirms that the previous microfiltration was efficient in retaining most of the macromolecules.
stream, as presented in Fig. 3. Although direct and sequential processes have produced permeate with similar characteristics (Table 2), direct processes enabled to obtain larger volumes and quantities than the sequential processes, as presented in Fig. 3. The yields of polyphenols in the retentate stream of the nanofiltrations were of 5.5% and 85.7% in the sequential and direct processes, respectively. Conidi et al. (2017) reported similar yields for the retention of polyphenol compounds from pomegranate juice by nanofiltration membranes. The visual aspects of pequi extracts are also presented in Fig. 3. The pre-treated pequi extract presented a yellowish, cloudy and oily appearance, while the permeate samples are clearer. Nanofiltration processes produced a transparent permeate, while its retentate was with similar visual aspect than the pre-treated pequi extract. Due to its high antioxidant activity, the retentate of the direct nanofiltration process is suggested to be used in the formulation of nutraceutical products, as well as natural colorant due to its intense yellowish color. Permeate samples are clear and they are suggested to be used as food additives or as bases for soft drinks.
3.2. Flux declines during direct and sequential filtration processes Fig. 4 presents the flux declines during dead-end filtrations of pretreated pequi extract by micro, ultra and nanofiltration membranes at both sequential and direct mode. A pronounced flux decline was observed in the first 20 min of filtration for all processes, which is a typical behavior of dead-end filtrations. According to Pereira et al. (2005), a continuous decrease in the permeate flux is frequently observed, indicating other phenomena that could be happening besides the polarization of the concentration, such as adsorption of solute molecules on the membrane surface and blockage of membrane pores. The adjustment of flux data according to Hermia model (Hermia, 1982) showed that cake formation was the main fouling mechanism for all the evaluated filtrations. As presented in Fig. 4, direct and sequential filtrations presented similar flux behavior. However, fluxes in sequential ultrafiltration were up to 14% greater than in direct ultrafiltration. Fluxes in sequential nanofiltration were up to 51% greater than in direct nanofiltration. Thus, the sequential process was especially efficient in increasing the permeate flux through ultra and nanofiltration membranes. Additionally, the direct processes required greater times to achieve the same concentration factor, especially in the nanofiltration process. Thus, although the direct process had been more efficient in concentrating the phenolic compounds, permeate fluxes were mitigated. Table 3 presents the final fluxes for all the proposed membrane filtrations and the calculated hydraulic resistances.
4. Conclusions We compared the efficiency of sequential and direct membrane filtration processes for the treatment of pequi fruit extract. This fruit extract presented a considerable amount of phenolic compounds, in addition to solid and lipid contents. Thus, proper clarification processes should be suggested. The microfiltration process was efficient in clarifying the pre-treated pequi extract (reduction of 50% in total solids and of 65% and total lipids). Additionally, the microfiltration process did not retain the phenolic compounds at a large extend. However, the turbidity value of the permeate from the microfiltration process was still right, and sequential clarification steps were required. The sequential ultrafiltration process produced a clear permeate and retained 42% of the phenolic compounds from the feed solution. Thus, the sequential ultrafiltration process is suitable to produce a clear permeate and a concentrate which is rich in phenolic compounds. Additionally, the sequential ultrafiltration process was particularly efficient in retain the phenolic acid with the greatest molecular weight (ellagic acid). The direct ultrafiltration process resulted in a greater retention factor of phenolic compounds than the sequential ultrafiltration, since the retention of phenolic compounds is probably associated to the retention 32
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of larger solutes. However, the permeate flux in the sequential ultrafiltration was 14% greater than in the direct process, since a previous microfiltration was applied to retain larger molecules. Thus, the direct ultrafiltration process is recommended to concentrate the bioactive compounds from pequi extract, since this process presents greater productivity and retention of polyphenol compounds than the sequential process, even that the permeate flux is a bit lower. The direct nanofiltration process resulted in almost 100% retention of phenolic compounds, but the permeate flux is quite low. In the sequential nanofiltration process (with micro and ultrafiltration processes as pretreatments), the permeate flux is twice greater than the permeate flux in the direct process. However, the yield of the sequential process is lower than in the direct process. Thus, the sequential process is recommended to fractionate the phenolic compounds and to obtain a clarified permeate, while the direct ultra and nanofiltration processes are recommend as proper concentration steps.
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