Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes

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Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes Mariana T.C. Machado ∗ , Beatriz C.B.S. Mello, Miriam D. Hubinger Department of Food Engineering, School of Food Engineering, University of Campinas, 80, Monteiro Lobato Street, P.O. Box 6121, 13083-862, Campinas, SP, Brazil

a b s t r a c t Pequi (Caryocar Brasiliense Camb.) is a typical fruit from Brazilian Cerrado, rich in antioxidant compounds, such as carotenoids and phenolic substances. Membrane processes allow concentrating extracts at low temperatures, preserving its functional properties. This work reports the results on the carotenoids and polyphenols concentration from pequi aqueous extract by ultrafiltration and nanofiltration processes, in order to develop a natural product rich in biologically active compounds suitable for functional applications. Experimental assays were performed in a dead-end stirred cell and membrane performance was evaluated in terms of permeate flux and permeate and retentate composition in relation to carotenoids and, phenolic substances content as well as antioxidant capacity. The ultrafiltration and the two different nanofiltration membranes presented rejection coefficients around 98%, 100% and 100% of the carotenoids and 65%, 94% and 97% of phenolic compounds, respectively. Thus, the nanofiltration showed a better performance to recovery the antioxidants compounds of pequi aqueous extract. © 2014 Published by Elsevier B.V. on behalf of The Institution of Chemical Engineers.

Keywords: Pequi; Polyphenols; Carotenoids; Ultrafiltration; Pore blocking; Nanofiltration

1.

Introduction

Pequi (Caryocar brasiliense Camb.) is a typical fruit from Brazilian Cerrado, with great occurrence and economical importance in this region. The fruit has high level of phenolic compounds, ␤-carotene, zeaxanthin, violaxanthin and lutein, that benefit human health due to their antioxidant capacity (Azevedo-Meleiro and Rodriguez-Amaya, 2004; Roesler et al., 2007). However, pequi is not widely used in Brazil, as it is highly perishable and its inside contains a lot of thorns that becomes its manipulation difficult, being its use limited to regional cuisine. Therefore, there is a need to developing new ways of pequi utilization, in order to better explore its potential and add value to this Brazilian product. Solid-liquid extraction process by organic solvents is a widely used technique to recover the antioxidants compounds from vegetal material (Spigno and De Faveri, 2007). The water has been studied as extraction solvent, since it is environmentally friendly, safe for its use and efficient to recover bioactive

compounds (Cissé et al., 2011; Roesler et al., 2007). Phenolic compounds are water-soluble, whereas carotenoids are oilsoluble, although some carotenoids can be extracted, in a lower amount, by water such as ␣ and ␤-carotene, lycopene, lutein and zeaxanthin (Sánchez et al., 2008). Therefore, water is an interesting solvent to this extraction study. Pequi extract may be widely used by food companies in functional food products with positive health benefits. The global market for functional foods is estimated to be worth about US$ 167 billion and is expanding with a yearly growth around 10% due to consumers’ demand, for a healthy life style (Granato et al., 2010; Menrad, 2003). However, for extract application, a concentration step is needed, as the solid–liquid extraction of natural products (vegetables, herbs, etc.) usually results in a very dilute extract (Sohrabi et al., 2010; Tylkowski et al., 2010). Membrane separation processes is a good alternative to concentrate these kinds of solutions due to the low temperatures used, absence of phase transition and low energy demand. The technique,

∗

Corresponding author. Tel.: +55 19 3521 4036; fax: +55 19 3521 4027. E-mail address: [email protected] (M.T.C. Machado). Received 4 December 2013; Received in revised form 12 September 2014; Accepted 20 October 2014 http://dx.doi.org/10.1016/j.fbp.2014.10.013 0960-3085/© 2014 Published by Elsevier B.V. on behalf of The Institution of Chemical Engineers.

Please cite this article in press as: Machado, M.T.C., et al., Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.10.013

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Table 1 – Relevant composition of the pequi pulp, seed and endocarp. Analyses

Pulp −1

Moisture (g 100 g ) Protein (g 100 g−1 ) Lipids (g 100 g−1 ) Vitamin C (mg 100 g−1 )

51.17 2.83 26.30 6.63

± ± ± ±

Seed 32.13 ± 2.23 17.52 ± 1.64 30.02 ± 1.21

3.78 0.47 1.47 1.02

*

Endocarp 46.47 ± 0.56 2.93 ± 0.13 19.13 ± 0.40 *

Data presented correspond to an average of five determinations, with standard deviations. ∗

Amount is not significant.

mainly ultrafiltration and nanofiltration, has been widely used to concentrate bioactive compounds (Cissé et al., 2011; Conidi et al., 2012; Mello et al., 2010; Prudêncio et al., 2012; Tsibranska and Tylkowski, 2013; Tylkowski et al., 2010). However, few works studied carotenoids retention (Chiu et al., 2009; Darnoko and Cheryan, 2006; Krupa et al., 2010; Ongaratto and Viotto, 2009; Tsui and Cheryan, 2007) by membrane process and no reports are readily available on the performance of membrane concentration of carotenoids and polyphenols together. The objective of the present work was to investigate the effect of ultrafiltration and nanofiltration processes on the carotenoids and phenolic acids concentration of pequi aqueous extract, in order to develop a natural product which could be used as functional ingredient. Besides the antioxidants compounds retention, the performance of the selected membranes was also evaluated in terms of permeate flux, flux resistances and membrane surface before and after filtration.

volume of the stirred cell is 0.25 L, the effective membrane area is 0.00159 m2 and the maximum operational pressure for this cell was 8 bar. Before the experiments, the membranes were rinsed with deionized water. 100 mL of feed were added into the deadend stirred cell, which was pressurized using compressed nitrogen. Pressure in the permeate side was approximately atmospheric under all conditions. The stirring rate was kept constant at 750 rpm. The filtrations were performed at 25 ◦ C and 7 bar for ultrafiltration and 8 bar for nanofiltration. During the process, permeate from the bottom of the cell was continuously collected and its volume was measured. Permeate was removed until obtaining a concentration factor of around 1.50. The concentration factor (VRF) is calculated according to the following equation:

Fc =

2.

Experimental

2.1.

Pequi extract

Frozen pequi (pulp, seed and endocarp) was purchased from Grande Sertão cooperative (Montes Claros, Minas Gerais, Brazil). Table 1 shows its proximal composition. The fruit was stored in a freezing chamber at −18 ◦ C until the trials. The pequi aqueous extract was prepared according to the protocol described by (Machado et al., 2013). Pequi was thawed and homogenized with distilled water in the weight proportion of 1:3 fruit:water. Extraction was performed for 1 h at 25 ◦ C with constant magnetic agitation (750 rpm); after that, extract was filtered using a strainer to remove rough particles, then centrifuged at 25 ◦ C, 10,000 rpm for 10 min and the supernatant was filtered with paper filter at room temperature for oil removal, and the residue was re-extracted in the same conditions. The mixture of the two extracts was stored at 5 ◦ C until use for feeding the membrane filtration experiments. Table 2 shows the physical–chemical characteristics of the raw extract.

2.2.

UF and NF membranes

Pequi extract was concentrated in three flat sheet membranes: one of ultrafiltration (UF, US100, Microdyn-NADIR, Piracicaba, São Paulo, Brazil) and two of nanofiltration (NF90 and NF270, Filmtec, Dow Chemical Company, São Paulo, SP, Brazil). Table 3 provides the specification of the membranes as given by the manufacturers.

2.3.

Procedure dead-end stirred cell

The selection of membranes was performed in a dead-end stirred cell (ARTINOX, São José, Santa Catarina, Brazil). The

Vf

(1)

Vc

where Vf is the total volume used in the feed and Vc is the volume collected in the concentrate fraction. The concentration factor of 1.50 was chosen because, with this factor, we can evaluate the membrane behavior in relation to bioactive compounds retention, permeate flux and flux resistance. In addition, this factor is sufficient to conduct studies in bench scale, and it can provide the necessary information for selecting the suitable membrane.

Table 2 – Physico-chemical characteristics of aqueous pequi extract (fresh weight). Analyses Moisture (g 100 g−1 ) ◦ Brix pH Zeta Potential (mV) Acidity (g 100 g−1 ) Ash (g 100 g−1 ) Protein (g 100 g−1 ) Lipids (g 100 g−1 ) Carbohydrates (g 100 g−1 ) Vitamin C (mg 100 g−1 ) Carotenoids ␤-carotene Zeaxanthin Polyphenols Gallic acid P-coumaric Ferulic acid Ellagic acid

98.55 2.3 5.94 −40.9 0.02 0.02 0.45 0.23 0.75 3.57

± ± ± ± ± ± ± ± ± ±

0.03 0.1 0.02 2.9 0.01 0.01 0.06 0.04 0.04 0.13

103.41 ± 4.92 153.54 ± 3.86 277.25 ± 59.48 16.91 ± 3.03 7.85 ± 0.65 130.13 ± 21.21

Data presented correspond to an average of five determinations, with standard deviations.

Please cite this article in press as: Machado, M.T.C., et al., Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.10.013

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Table 3 – Specification of the used membranes. Membrane

UF

NF90

NF270

Material

Polysulfone

Polyamide and Polysulfone

Maximum operating pressure Maximum operating temperature pH Molecular weight cut off (MWCO) Rejection (%)a Pure water permeability

*

41 bar 45 ◦ C 3–10 200–300 Da >97a 6.4 L h−1 bar−1 m−2 c

Polypiperazine polyamide and polysulfone 41 bar 45 ◦ C 3–10 200–300 Da >97a 13.5 L h−1 bar−1 m−2 c

∗ a b c

95 ◦ C 1–14 100 kDa *

>300 L h−1 m−2 b

Information not given by the manufacturer. MgSO4 2000 mg L−1 at 7.8 bar. Testing conditions: 3 bar, 20 ◦ C. Nghiem and Hawkes (2007).

A curve J versus t was obtained for each run; the permeate flux to build the curve was calculated in time intervals according to the following equation:

J=

Vp t×A

(2)

where J is the permeate flux (L h−1 m−2 ), A is the effective membrane area (m2 ) and Vp is the permeate volume collected (L) during the filtration time t (h). The feed, retentate and permeates were analyzed for color characteristics and content of total phenolic substances, total carotenoids and antioxidant capacity. The retention coefficient (R) of these compounds was calculated using the following equation:

 R=

Cp 1− Cf

 × 100%

The permeate flux decay has been the main drawback to using membrane separation process. This decline is a critical reason for acceptance of membrane technology in the industry. Therefore, it is important to identify the most prominent fouling mechanisms during the filtration process for improving the control and minimizing the permeate flux decay. To investigate about membrane fouling mechanisms, a mathematic model of permeate flux decline phenomenon during constant-pressure filtration process can be suggested (Hermia, 1982):



ln (J) = ln (J0 ) − k1 × t

2.4.2.

(5)

Standard pore blocking (n = 1.5)

Standard blocking or internal pore blocking mechanism is produced due to the adsorption or deposit of small size solute onto the membrane pore, reducing its volume. The correlation between the flow and time is given according to the following equation: J−1 = J0−1 + k2 × t

Blocking filtration law or Hermia’s model

d2 t dt =k dV dV 2

Complete pore blocking (n = 2)

Complete pore blocking model considers that this type of fouling occurs when solid particles or macromolecules are greater than the membranes pores and might seal an open pore. Blocked membrane pores increase as the permeate flow rate will decrease exponentially with time (Ng et al., 2014). The filtration flow can be related to the time using the following equation:

(3)

where Cf and Cp were the feed and permeate concentrations of the biologically active compound (␮g mL−1 ).

2.4.

2.4.1.

n (4)

2.4.3.

Intermediate pore blocking (n = 1)

Intermediate pore blocking is used when the diameter of the solutes are very similar to the pore size. The material rejected may seal the entrance of some pores or settle on the other particles, being less restrictive than complete pore blocking. The filtration flow can be related to the time using the following equation: −1/2

J−1/2 = J0

2.4.4.

+ k3 × t

(7)

Cake formation (n = 0)

Cake formation occurs when the solutes diameter is larger than membrane pores size. Particles will settle on other predeposited material on membrane surface, causing a high concentration of solute and forming a cake layer. The correlation between the flow and time is given according to the following equation: J−2 = J0−2 + k4 × t

where t is the time of process, V is the cumulative volume of filtrate, k is a constant and n is the type of filtration mechanism. Four empirical models have been proposed by Hermia (1982), as cited in the following sessions.

(6)

(8)

When the experimental data is fitted to the equations in filtration models, ln(J), J−1 , J−1/2 , J−2 , against time (t) and a linear correlation is assumed, the slope for each curve gives rates of pore blocking by each mechanism (k1 , k2 , k3 and k4 ).

Please cite this article in press as: Machado, M.T.C., et al., Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.10.013

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Moreover, the regression coefficients (R2 ) are determinate from these model equations.

spectrophotometer at a wavelength of 450 nm, and the total carotenoids content was calculated by the following equation:

2.5.

Analytical determinations

Total carotenoids =

2.5.1.

Physical–chemical characterization

Pequi fruit, raw extract (feed of filtration processes), and permeate and concentrate (from the most appropriate membrane separation process) were analyzed in relation to their physical–chemical characteristics. Moisture, acidity, ash, protein, vitamin C content were determined by AOAC (1998). ◦ Brix, pH and zeta potential was assessed using refractometer, pH meter and Zetasizer NanoZS, respectively. Lipids were measured by Soxhlet method for pulp, and Bligh and Dyer (Checchi, 2003) for seed, endocarp and filtration products. Carbohydrates were estimated by difference (meaning 100—the sum of moisture, protein, fat and ash). Besides that, the carotenoids and polyphenols majority were identified and quantified using high performance liquid chromatography (HPLC) with photodiode array (DAD) (Dionex, UltiMate 3000 Standard LC, California, EUA), controlled by Software Chromeleon 6.8. Carotenoids were extracted according to Rodriguez-Amaya (1999) and analyzed by methodology described by AzevedoMeleiro and Rodriguez-Amaya (2004) with modifications. An Acclaim 120 C18 column (250 mm × 4.6 mm e 5 ␮m) was used at 25 ◦ C. The mobile phase consisted of acetonitrile, methanol and ethyl acetate. The mobile phase was maintained in 95:5:0 until 20 min, after a concave gradient (curve 9) was employed from 95:5:0 to 60:20:20 until 25 min, maintaining the last proportion during 45 min. Stabilization was done during 15 min and the flow rate was 1 mL min−1 . And for polyphenols, extract was injected directly and analyzed according to Chisté et al. (2012) with modifications. A Poroshell 120 EC-C18 column was used (100 mm × 4.6 mm e 2.7 ␮m) at 29 ◦ C. The mobile phase was composed by water and acetonitrile, both with formic acid (0.5%) and a linear gradient was used from 99:1 to 50:50 in 50 min, following 1:99 in 5 min and maintaining the last proportion during 5 min. The mobile phase flux was 0.9 mL min−1 . Both identifications were done by comparison of retention times and absorption spectrums, using the standards.

Abs × V × 106 A1% 1cm × v × 100

(9)

where Total carotenoids were expressed in ␮g of ␤carotene mL−1 of extract, Abs was absorbance maximum measure, V was the dilution volume (mL), A1% 1cm was the absorption coefficient of the carotenoid in petroleum ether (2592) and v was sample volume (mL). The analyses were done in triplicate.

2.5.4.

Total phenolic substances

Total phenolics in pequi solutions were determined by the Folin–Ciocalteau colorimetric method (Swain and Hillis, 1969). 0.5 mL of pequi solution previously diluted in the proportion of 1:10 was mixed with 2.5 mL of 10% Folin–Ciocalteau reagent. After 3 min, 2 mL of 7.5% sodium carbonate solution was added. The absorbance was read at 760 nm after 1 h of incubation at room temperature. Gallic acid was used as the standard to build the calibration curve. The mean of three readings was used and the total phenolic content expressed in ␮g of gallic acid equivalents (␮g GAE mL−1 of extract). The analyses were done in triplicate.

2.5.5.

Antioxidant capacity determination

The antioxidant capacities were determined by two methods, DPPH (Nagai et al., 2003) and FRAP (Benzie and Strain, 1996). For DPPH assay, the pequi solution previously diluted in the proportion of 1:10 (0.3 mL) was mixed with 0.3 mL of ethanol solution containing 0.5 mM DPPH and 2.4 mL of ethanol (99.5%). For the control, 0.3 mL of distilled water was used in place of diluted extract. The absorbance was read at 517 nm after 40 min of incubation at room temperature. The ability of scavenging free radicals was expressed as inhibition percentage (IP) of radical oxidation and calculated according to the following equation:

IP (%inhibition) =

A



(0) − A (t) × 100 A (0)

(10)

The surface morphology of the membranes before and after filtration was evaluated by scanning electron microscopy (SEM). Membranes were attached to a double-sided adhesive tape mounted on SEM stubs, coated with 3–5 mA gold/palladium under vacuum and examined with a LEO440i scanning electron microscope (LEICA Electron Microscopy Ltd., Cambridge, England). SEM was operated at 15 kV with magnifications of 3000×.

where A(0) was control absorbance and A(t) was pequi solution absorbance. For FRAP method, the extracted solution previously diluted in the proportion of 1:10 (100 ␮L) was mixed with 3 mL of freshly prepared FRAP reagent (TPTZ, FeCl3 , acetate buffer). The absorbance was read at 593 nm after 30 min of incubation at 37 ◦ C. Fe(II) water solutions were used as the standard to produce the calibration curve. The mean of three readings was used and the antioxidant capacity expressed in ␮M of FeSO4 (␮g mL−1 of extract). The analyses were conducted in triplicate.

2.5.3.

2.5.6.

2.5.2.

Surface morphology of the membranes

Total carotenoids

Total carotenoids were quantified spectrophotometrically (Rodriguez-Amaya, 1999). Initially, carotenoids were extracted with acetone. The mixture (acetone and extract) was vacuum filtered. Then, the filtrate was placed in separating funnel with petroleum ether. The solution was washed with distilled water and separated into two phases. The petroleum ether phase was collected in 25 mL flask and petroleum ether was added until the volume is full. Absorbance was measured in a UV–vis

Color

The color of feed, permeate and retentate from filtration processes was measured using a Hunter Lab colorimeter (model Color Quest II, Reston, USA), with reflectance mode (RSIN), CIELab scale (L*, a*, b*), D65 as illuminant and a 10◦ observer angle as a reference system, at 25 ◦ C of temperature. The color measurements were expressed in terms of lightness L*, and the chromaticity parameters a* and b*. From these parameters, the cylindrical coordinates C* (chroma) and H* (hue angle)

Please cite this article in press as: Machado, M.T.C., et al., Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.10.013

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Fig. 1 – Permeate flux versus filtration time of pequi aqueous extract using three different membranes. () UF; (*) NF90; () NF270. were calculated according to Eqs. (11) and (12), respectively. The analyses were done in triplicate. C∗ =



(a∗2 + b∗2 )

H∗ = arctan

2.6.

 b∗  a∗

(11) (12)

Statistical analysis

The responses were analyzed using the software Statistica® 8.0 (Statsoft Inc. Tulsa, OK, USA). The coded variable effects for all responses were analyzed at a significance level of 5% (p ≤ 0.050).

3.

Results and discussion

3.1.

Dead-end stirred cell filtration

The experimental assays were performed to identify the most appropriate membrane for the concentration of antioxidant compounds from pequi aqueous extract. The permeate flux and the compounds recovery in the concentrate were adopted as selection criteria. Figs. 1 and 2 show the profiles of the permeate flux in two different forms, flux versus time (Fig. 1) and the corresponding normalized flux versus cumulative permeate volume (Fig. 2), obtained for the three membranes (UF, NF90 and NF270) processing the pequi extract in the selected operational conditions. Regardless the membrane, permeate flux

Fig. 2 – Normalized flux versus accumulated permeate volume of pequi aqueous extract using three different membranes. () UF; (*) NF90; () NF270.

5

has declined quickly in the initial period (around 10 min); after that, the permeate flux smoothly decreased. Particularly for NF90 membrane, the permeate flux declined immediately after the starting of the process, and after 5 min, the initial permeate flux of 18.5 L h−1 m−2 fell to about 10 L h−1 m−2 . Although the initial sharp decline in permeate flux, this membrane has achieved the concentration factor in a smaller time and its normalized flux curve was almost linear, suggesting that it permeate flux is more stable than the others. Permeate flux values at the end of experiments (concentration factor of around 1.50) were for UF membrane: 7.32 L h−1 m−2 ; NF90 membrane: 8.55 L−1 h m−2 ; NF270 membrane: 8.32 L h−1 m−2 . For the membrane with the highest MWCO (UF) a gradual flux decline and the lowest flux in the end of process were obtained. This peculiar behavior probably can be explained by two facts: the membrane material and the fouling mechanism. UF membrane material is composed by polysulfone, which has a hydrophobic character and in this way, it caused more resistance to water flux than to other solvents (Chen et al., 1996; Michelon et al., 2014; Susanto et al., 2009) and some solutes, such as protein and lipids, are more susceptible to depose in the membrane surface and pores (Blank et al., 1998; Metsämuuronen and Nyström, 2009). The second fact that reduces permeate flux is the pore blocking. For investigating the fouling mechanisms involved in filtration, Hermia’s models were used. The R2 values and fitted parameters (k) for each mechanism are displayed in Table 4. A high R2 value indicates a good fit of the experimental data in relation to Hermia’s model. Moreover, a high value of fit parameter suggests a more severe flux decline for the membrane. The NF270 membrane led to the highest R2 values for all Hermia’s model (>0.97). Furthermore, for all membranes, cake layer formation mechanism gives the best goodness of fit based on correlation coefficient (R2 ), indicating that this model was predominant during filtration. In addition, regarding the fit parameters, the highest values were obtained for UF membrane, suggesting that cake formation was more severe. As cited previously, this membrane has higher MWCO and hydrophobic character, being more susceptible to fouling (Chhaya et al. (2012); Nghiem and Hawkes, 2007). This observation is in good agreement with the flux decline. This membrane, although with the larger pore size, showed the same final flux of NF membranes. The occurrence of particles deposition can be seen by the SEM images. SEM analyses were carried out on the used membranes in order to evaluate the changes after filtration of pequi extract on each membrane surface. In Figs. 3–5, images of the membranes surface before and after filtration can be compared. Regardless the membrane, the micrographs showed smoothes membranes surfaces before filtration (Figs. 3A, 4A and 5A) and heterogeneous surfaces with particles deposition after the process (Figs 3B, 4B and 5B). On UF surface (Fig. 3B), it can be seen organic foulants, that could be attributed to lipids, proteins and carbohydrate extracted from the pequi. As previously mentioned, this membrane presents the more severe cake layer formation and its material is more susceptible to deposition of some solutes, such as these macromolecules. On NF270 surface (Fig. 4B), it can be observed organic and inorganic foulants, which can be related to lipids, proteins and mineral presented in pequi extract. And NF90 surface presented only inorganic foulants (Fig. 5B), that can be attributed to minerals. This fact is according to fit parameters obtained

Please cite this article in press as: Machado, M.T.C., et al., Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.10.013

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Table 4 – Evaluation of fouling parameters by applying Hermia’s models at UF and NF membranes. Model

Membrane Complete blocking

UF NF90 NF270

Intermediate blocking

R2

Kc × 10−5 (s−1 )

R2

0.844 0.971 0.909

9.00 3.00 6.00

0.924 0.978 0.951

Ki × 10−6 (m−1 ) 8.00 3.00 6.00

Standard blocking R2 0.888 0.975 0.932

Ks × 10−6 (s−0.5 m−0.5 ) 10.00 4.00 9.00

Cake layer R2

Kgl × 10−6 (sm−2 )

0.972 0.982 0.977

2.00 0.70 1.00

Fig. 3 – Microphotographs of UF membrane surface before (A) and after (B) filtration. Scale = 20 ␮m.

Fig. 4 – Microphotographs of NF90 membrane surface before (A) and after (B) filtration. Scale = 20 ␮m. from Hermia’s model. This membrane showed the lowest fit parameters, indicating lower cake layer formation. Beyond MWCO and membrane material, another parameter that could have influenced on particles deposit in membrane surface is the charge density. Solution pH can modify the membrane charge and pore size and hence, affect the performance of NF and UF membranes (Metsämuuronen

and Nyström, 2009; Teixeira et al., 2005). Nghiem and Hawkes (2007) and Metsämuuronen and Nyström (2009) reported the zeta potential of NF and UF virgin membranes, respectively. Both NF membrane (NF90 and NF270) have negative charge (−17.8 mV and −19.4 mV, respectively) at pH 6 (around extract pH, 5.94 ± 0.02) and UF membrane showed zeta potentials varying in the range of 0 to −3 mV in the pH range 3.5–6.8.

Fig. 5 – Microphotographs of NF270 membrane surface before (A) and after (B) filtration. Scale = 20 ␮m. Please cite this article in press as: Machado, M.T.C., et al., Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.10.013

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Table 5 – Physico-chemical characteristics of pequi extract subjected to three membranes treatment. Parameter

UF

Feed Retentate

Carotenoids (␮g mL−1 ) Total phenolics (␮g mL−1 ) DPPH (% inhibition) FRAP (mM mL−1 ) Color L* C* H*

2.31 216.38 23.03 2.62 30.80 13.58 101.28

± ± ± ± ± ± ±

0.06b 1.42d 0.36c 0.01d 0.18d 0.23d 0.41c

3.41 292.49 24.53 3.16 37.43 19.31 96.62

± ± ± ± ± ± ±

0.02a 2.90c 0.30b 0.07c 0.49b 0.46c 0.37a

NF90 Permeate

c

0.05 74.76 7.44 1.42 91.86 3.41 116.56

± ± ± ± ± ± ±

Retentate

0.01c 1.13e 0.83d 0.01e 2.45a 0.67b 2.35d

3.47 338.40 29.48 3.71 35.51 18.79 97.46

± ± ± ± ± ± ±

0.04a 6.93a 0.66a 0.05a 0.41c 0.93c 0.37b

Permeate

NF270 Retentate

0.00c 3.46 ± 0.02a 6.29 ± 0.10f 319.09 ± 2.34b 3.07 ± 0.49e 26.25 ± 0.87b 0.54 ± 0.0g 3.47 ± 0.06b 90.80 ± 0.77a 35.35 ± 0.52c 1.41 ± 0.11a 17.93 ± 0.16c 246.65 ± 4.43e 97.73 ± 0.37b

Permeate 0.00c 14.53 ± 0.77d 4.02 ± 0.20e 0.73 ± 0.01f 89.40 ± 0.51a 1.15 ± 0.13a 238.51 ± 5.57e

f

Data presented correspond to an average of five determinations, with standard deviations. Same letters in the same line mean that samples did not differ statistically and different ones mean that they differ at 95% of confidence level.

The pequi extract showed −40.9 ± 2.9 mV. Hence, electrostatic repulsion could have happened between the charged NF membranes and a charged organic compound, indicating that fewer components could be deposited on membrane surfaces. However, between UF membrane and extract compounds, it can be considered that no charge interactions occurred, since this membrane surface is nearly not charged. Therefore, more components could have deposited on membrane surface. These observations are in good agreement with SEM images and Hermia’s model. On nanofiltration membranes surface there are few foulants and by Hermia’s model, these membranes showed less cake formation. Whereas on UF membrane surface, there is a higher amount of foulant and the cake formation was more severe. Therefore, the particles charge can have influenced and affected the fouling.

3.2.

Compounds retention

As the membrane selection also depends on the antioxidants compounds concentration, Table 5 shows carotenoids and phenolic substances contents, the antioxidant capacity, as well as the color measures of the feed, retentate and permeates from ultrafiltration and nanofiltration process. It is observed from this Table that the three membranes showed a very good rejection toward carotenoids. For ultrafiltration, permeate contained small amounts of carotenoids (2% of initial solution), while for nanofiltration process (NF90 and NF270 membranes), there was no loss of compounds to the permeate solution. The obtained results are in agreement with experimental data reported by other authors. Ongaratto and Viotto (2009) clarified pitanga (Eugenia uniflora L.) juice by micro and ultrafiltration membranes (with nominal MWCOs of 200, 150 and 30 KDa), and in the trials they have obtained rejection toward carotenoids of 100%. Moreover, Tsui and Cheryan (2007) concentrated xanthophylls (lutein and zaexanthin) of corn alcoholic extract by nanofiltration process and reported a recovery of these compounds in the retentate around 98% for the two used membranes (with nominal MWCOs of 200 and 300 Da). Carotenoids with molecular weights (from 536 to 600 Da) smaller than nominal MWCO of UF membrane (100 KDa) were retained on the retentate side of the membrane. This behavior can be attributed to cake layer formation phenomenon, which may form a secondary barrier. This barrier acts as a membrane and prevents the passage of other molecules, increasing retention of permeable solutes. Besides that, as carotenoids have a hydrophobic character, they can be attached to cell debris and

fats and can be rejected by the membrane (Razi et al., 2012; Rodriguez-Amaya, 1999). In relation to polyphenols, it is observed from Table 5 that the NF90 and NF270 membranes also showed a rejection higher than 90%, with a better performance for concentrating pequi aqueous extract than UF membrane, which retained 65% of total phenols on the retentate side of the membrane. The higher rejection toward phenolic substances for the nanofiltration membranes can be explained by nominal MWCOs of these membranes, which were specified as 200–300 Da. It could be anticipated that organic compounds with molecular weight between 164 and 302 Da would be rejected only by the nanofiltration membranes used. Similar results were obtained by other authors, as Mello et al. (2010), which worked with concentration of propolis aqueous extract by nanofiltration process, with the same membrane of the present study, NF90, and achieved a rejection toward phenolic compounds of 84%. Conidi et al. (2012) concentrated flavonoids with nanofiltration membranes from the same manufactory (N70 and NF200). They obtained between 88 and 95% of rejection toward these compounds. Conidi et al. (2011) concentrated bergamot juice (Citrus Bergamia Risso) and obtained a rejection toward

Table 6 – Physical–chemical characteristics of permeate and concentrate products from NF90 process (fresh weight). Analyses Moisture (g 100 g−1 ) ◦ Brix pH Zeta Potential (mV) Acidity (g 100 g−1 ) Ash (g 100 g−1 ) Protein (g 100 g−1 ) Lipids (g 100 g−1 ) Carbohydrates (g 100 g−1 ) Vitamin C (mg 100 g−1 ) Carotenoids ␤-carotene Zeaxanthin Polyphenols Gallic acid P-coumaric Ferulic acid Ellagic acid

Concentrate 97.92 ± 0.01 3.0 ± 0.1 5.53 ± 0.02 −35.7 ± 3.0 0.03 ± 0.01 0.03 ± 0.01 0.59 ± 0.07 0.30 ± 0.02 1.16 ± 0.03 3.71 ± 0.13 264.78 ± 8.19 181.35 ± 6.92 360.43 22.83 10.20 162.67

± ± ± ±

77.33 4.09 0.84 26.51

Permeate 99.98 ± 0.01 0.4 ± 0.1 6.86 ± 0.03 −32.8 ± 1.5 * * * * *

2.35 ± 0.29 * *

* * * *

Data presented correspond to an average of five determinations, with standard deviations. ∗

Amount is not significant.

Please cite this article in press as: Machado, M.T.C., et al., Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.10.013

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polyphenols of 2 and 84% by ultra and nanofiltration (membrane with MWCO of 100 KDa e 450 Da), respectively. Bernardo et al. (2011) worked with partition of cork industry wastewater by ultra and nanofiltration process. For the ultrafiltration process (membrane with MWCO of 25 KDa), polyphenolic substances recovery in retentate was around 68% and for nanofiltration (membrane with cut-off of 125 Da) was 91.7%. Onsekizoglu et al. (2010) used an ultrafiltration membrane (MWCO of 10 and 100 KDa) for apple juice filtration and did not observe any difference on polyphenols concentration between the permeate and retentate side. By comparing the present study with the works previously cited, the ultrafiltration process using a membrane with MWCO 100 KDa showed a higher retention toward phenolic compounds (with molecular weight of 164–302 Da). The same explanation for rejection toward carotenoids may be applied to polyphenols, e.g., cake layer formation phenomenon increases the retention. Besides that, the aromatic ring of polyphenols may form aggregates with proteins, becoming bigger particles that stay retained in the membrane filter layer (Charlton et al., 2002). In addition, hydrogen bonding between hydroxyl groups from polyphenol and oxygen atom from SO2 group in polyethersulfone (membrane surface) may also be possible (Susanto et al., 2009), reducing the polyphenols passage through this membrane. Bioactive compounds content presented by solutions is straightly associated to its color as it can be seen in Table 5. Regardless the membrane, permeates were clear and had higher L* values than retentates, that were opaque and yellowcolored darker than feed solution. All retentates were yellower than feed solution, in good agreement with the increase on bioactive compounds concentration observed in the retentates. For UF process, permeate had Hue angle of 116.6◦ , being slightly yellow-colored, due to a lower compounds concentration. The permeates from nanofiltration process (NF90 and NF270) were colorless and had Hue angle of 246.7◦ and 238.5◦ , respectively, showing slightly blue color, due to the absence of carotenoids and low polyphenols content. All chroma retentates were more intense, having a darker yellow color than the feed solution. And permeate coming from ultrafiltration had chroma of 3.41, being clearer than the feed solution, while the NF90 and NF270 permeates presented chroma around 1.0. The observed rejection toward polyphenols and carotenoids for the three membranes is also confirmed by the antioxidant capacity analyses, showed in Table 5. All retentates showed higher antioxidant capacity than the feed solution, as a consequence of the higher carotenoids and polyphenols concentration obtained. In particular, the lower antioxidant capacity value was measured in the retentate coming from ultrafiltration process where phenolic compounds and carotenoids concentration were lower than in the retentate obtained by nanofiltration. The lowest antioxidant capacity was detected in permeates originated from nanofiltration treatment, due to the smaller content of polyphenols and absence of carotenoids in this stream. It can be noted that the rejection toward carotenoids and polyphenols, and, consequently, the antioxidant capacity value, has increased by decreasing the nominal MWCO. The obtained results are in agreement with the experimental data reported by Conidi et al. (2011), which worked with filtration of bergamot juice by ultra and nanofiltration. The obtained results suggest that the biologically active compounds from pequi aqueous extract were better

recovered by NF90 process. The main physical–chemical properties of permeate and concentrate products from NF90 process are shown in Table 6. Products pH and zeta potential have increased in relation to the feed. The acidity of feed and concentrate was similar, however, for permeate, this amount was not significant, indicating that at NF90 process a concentration of hydrogen ions has occurred, without altering the amount of non-dissociated acids. In addition, the compounds that could interact between themselves and with the membrane (proteins, lipids, carbohydrates), as previously cited, were rejected by membrane and slightly increased in concentrate in relation to feed, whereas in permeate was also not significant. Carotenoids and polyphenols could not be identified in permeate, whereas in concentrate their amount increased about 50 and 30%, respectively. The vitamin C retention through NF90 membrane was only 34%. The molecular weight of vitamin C is 180 Da, which is lower than membrane MWCO. Therefore, both concentrate and permeate can be used as food ingredients riches in antioxidants compounds.

4.

Conclusions

Results showed that the additional resistance in filtration mass transfer caused by associated cake layer formation in UF membrane allowed the increase in carotenoids and polyphenols retention by this membrane. However, nanofiltration process is better for concentrating pequi extracts; due to its good permeate flux and good polyphenols and carotenoids concentration, since almost 100% of the compounds were retained. Furthermore, this process allows removal of the solvent from the extract, reducing the disadvantages associated to the solvents extractions. It should be noted that compared with other concentration methods, in the membrane process the product is not submitted to high temperatures (saving energy) and there is no change in the physical state of the solvent. The concentrated pequi extract could increase the use of pequi in many industrial applications and in development of new products with functional properties.

Acknowledgments The authors thank FAPESP (Process no. 09/50593-2), CNPq (304475/2013-0 and 138039/2009-7) and CAPES for the financial support.

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Please cite this article in press as: Machado, M.T.C., et al., Evaluation of pequi (Caryocar Brasiliense Camb.) aqueous extract quality processed by membranes. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.10.013