Potential of ultrafiltration for separation and ...

Separation and Purification Technology 125 (2014) 120–125

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Potential of ultrafiltration for separation and purification of ellagitannins in blackberry (Rubus adenotrichus Schltdl.) juice Oscar Acosta a, Fabrice Vaillant b, Ana M. Pérez a, Manuel Dornier c,⇑ a

Centro Nacional de Ciencia y Tecnología de Alimentos (CITA), Universidad de Costa Rica (UCR), Ciudad Universitaria Rodrigo Facio, código postal 11501-2060, San José, Costa Rica Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), UMR95 QualiSud, 73 rue Jean-François Breton, TA B-95/16, F-34398 Montpellier cedex 5, France c Montpellier SupAgro, UMR95 QualiSud, 1101 avenue Agropolis, B.P. 5098, F-34093 Montpellier cedex 5, France b

a r t i c l e

i n f o

Article history: Received 23 September 2013 Received in revised form 21 January 2014 Accepted 23 January 2014 Available online 4 February 2014 Keywords: Ultrafiltration Fractionation Blackberry Ellagitannins Anthocyanins

a b s t r a c t Tropical highland blackberry is characterized by its high content of ellagitannins, which are considered to be potentially beneficial for human health, but relatively rare in food sources. Due to its relatively high molecular weight, the feasibility of selective separation of ellagitannins by ultrafiltration was assessed on clarified tropical highland blackberry juice. Six organic tight ultrafiltration membranes with nominal molecular weight cut-off (MWCO) ranging from 1 to 150 kDa were tested. Filtration temperature was set constant to 30 °C, cross-flow velocity to 0.3 m s1 and transmembrane pressures varied at 0.5, 1, 1.5, 2 and 3 MPa, for each membrane tested. Experiments were performed at a constant volumetric reduction ratio (1 < VRR < 1.1) and quantitative analyses of total ellagitannins and anthocyanins were determined by HPLC/DAD. Retention of ellagitannins reached 100% for all membranes except for that showing a nominal MWCO of 150 kDa, when transmembrane pressure was over 1 MPa. Retention of total anthocyanins increased with transmembrane pressure and reached values over 90% for all membranes tested at 3 MPa. A membrane with a nominal MWCO of 2 kDa presented the highest anthocyanin flux at 2 MPa and thus appeared to be the most promising for fractionation of the polyphenolic compounds. A model used to predict total ellagitannin purification with constant volume diafiltration confirmed that this membrane presented one of the lowest diavolumes needed to achieve a purity of 90% at higher transmembrane pressures. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Phytochemicals are bioactive non-nutrient plant compounds found in fruits, vegetables, grains, and other plant materials that potentially have the ability to reduce the risk of major chronic diseases [1]. Polyphenols, a group of phytochemicals, are secondary metabolites of plants and are generally involved in defense against ultraviolet radiation or aggression by pathogens. In addition to having antioxidant properties, polyphenols have several other specific biological actions that have not been completely understood [2]. Studies have confirmed that blackberries are a good source of dietary antioxidants, and are known to contain appreciable levels of phenolic compounds [3–5]. Two recent reviews have extensively covered the chemical composition and potential health benefits of blackberries and other Rubus fruits [6,7]. It has been described that ellagitannins and anthocyanins are the major polyphenolic compounds found in Rubus adenotrichus, ⇑ Corresponding author. Tel.: +33 467616597; fax: +33 467614434. E-mail address: [email protected] (M. Dornier). http://dx.doi.org/10.1016/j.seppur.2014.01.037 1383-5866/Ó 2014 Elsevier B.V. All rights reserved.

being the ellagitannins lambertianin C and sanguiin H-6, and the anthocyanins cyanidin-3-glucoside and cyanidin-3-malonyl glucoside, the predominant ones [5]. Anthocyanins are glycosides with an anthocyanidin (flavonoid) C6–C3–C6 skeleton. In plant tissues they produce blue, purple, red and intermediate hues [8]. Apart from their physiological roles in plants, anthocyanins are regarded as important components in human nutrition, which is supported by numerous studies that report a high positive correlation of fruit or vegetable pigment content and antioxidant capacities [9]. Ellagitannins, which belong to the hydrolyzable tannin class of polyphenols, are complex derivatives of ellagic acid. They contain one or more hexahydroxydiphenic acid moieties esterified to a polyol (most often b-D-glucose) [10]. The occurrence of these compounds has been extensively reviewed [11,12] and recent studies have focused on the potential health effects of ellagitannins, ellagic acid and their derived metabolites [13]. The optimization of methods for fractionation, purification and concentration of polyphenolic compounds, particularly ellagitannins, are an opportunity for the food and drug industry. The removal of anthocyanins (and therefore the color) from the extract

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might increase its range of applicability. Functional food products are one of the fastest growing food sectors, recently valued at US$168 billion [14], and new ingredients to fortify and enrich current or new products are an important part of this market [15]. These innovations can potentially improve wellness of the general population, besides promoting economic growth throughout the value chain [16]. While the application of membrane technologies for production of functional foods and nutraceuticals is widespread [17], not many studies have addressed the use of membrane filtration to fractionate polyphenolic compounds [18–21]. No information was found in the literature related to the performance of membrane process filtration for the separation of anthocyanins and ellagitannins. The objective of this study was to determine the feasibility of separation and purification of ellagitannins found in a clarified blackberry juice by use of ultrafiltration membranes. 2. Materials and methods 2.1. Raw material Frozen, organically grown, ripe tropical highland blackberries (R. adenotrichus Schltdl.) were purchased from a local growers association in Cartago, Costa Rica. Fruits were thawed in portions at 5 °C for 24 h and then pressed using an OTC 25-Ton hydraulic press (SPX Corporation, Owatonna, MN, USA) to extract the juice. After pressing the fruits, the juice was heated to 35 ± 2 °C, and the commercial enzymatic solution KlerzymeÒ 150 (DSM Food Specialties, México) was added to the juice in concentrations up to 0.19 g kg1. The juice was macerated during 1 h at 35 ± 1 °C. To obtain a clarified juice, the cross-flow microfiltration unit and membrane described by Vaillant et al. [22] were used, with transmembrane pressures that ranged from 90 to 290 kPa. Temperature was fixed at 35 ± 2 °C, cross-flow velocity at 6 m s1, and trials were carried out with continuous juice feed. Clarified juice was packaged immediately and stored at 20 °C. Before concentration, all juice fractions were mixed together. To obtain a concentrated juice, the homogenized microfiltered juice was concentrated by osmotic evaporation by using the pilot unit, membrane and conditions described by Vaillant et al. [22]. The temperature of the clarified juice fed to the system was always under 5 °C. Concentrated clarified juice was stored at 20 °C until diluted with deionised water (conductivity < 5 lS cm1) immediately before the ultrafiltration experiments. Portions of 2.5 L of the diluted juice were used for each filtration trial. 2.2. Filtration system and membranes Cross-flow ultrafiltration experiments were performed using a pilot unit, which incorporated a SepaÒ CF II Membrane Cell System (GE Osmonics, Minnetonka, MN, USA) with an effective membrane area of 0.0155 m2. A schematic of the pilot unit has been previously described [23]. Temperature was kept constant by using a water bath (±0.5 °C), and permeate flux was determined by measuring

the weight of permeate with a balance. Weight and pressure values were acquired and recorded every 2 s by an electronic system. The characteristics of the six different flat-sheet tight ultrafiltration membranes tested (supplied by the manufacturers), as well as the measured water permeability values are listed in Table 1. Only new membranes were used in the experiments. 2.3. Evaluation of membrane performance Pre-conditioning of the membranes was performed with deionised water (conductivity < 5 lS cm1) for 60 min, at 30 °C, 2 MPa of transmembrane pressure and 0.3 m s1 of cross-flow velocity. Hydraulic permeability was determined during the last 10 min of pre-conditioning. Permeability for the UP150 membrane was measured at 0.14 MPa of transmembrane pressure, and this membrane was not pre-conditioned. Experiments were performed immediately after pre-conditioning, with permeate and retentate being recycled back to the feed tank in order to maintain a constant feed concentration (all experiments were performed at a volumetric reduction ratio (VRR) that varied between 1 and 1.1). The concentrated raw material was diluted to 100 g kg1 of total soluble solids (TSS) (100.8 ± 0.5 g kg1 for eight experiments). Filtration temperature was set constant to 30 °C and cross-flow velocity to 0.3 m s1. The transmembrane pressures tested for each membrane were 0.5, 1, 1.5, 2 and 3 MPa, and pressure was held constant during 55 min. All permeate flux values were acquired during the last 10 min for each pressure. After that time, the pressure was increased to the next value. Samples of permeate were collected for each pressure at the last possible moment (after the stabilization period) and the amounts never exceeded 50 mL. Samples of raw material and permeate collected were immediately frozen and kept at 20 °C until analyzed. In order to measure membrane selectivity towards a compound i (ACY for anthocyanins, ET for ellagitannins, and TSS for total soluble solids), the observed retention ri (%) was calculated according to Eq. (1).

ri ¼ 1

C pi C fi

100

ð1Þ

where Cpi and Cfi are the concentration of the compound i in permeate and in feed, respectively. 2.4. Modeling total ellagitannin purification with constant volume diafiltration A model was used to simulate the impact of purification of total ellagitannins by constant volume diafiltration [24] of a juice that had been previously fractionated by ultrafiltration. The concentration of the compound i in the retentate (Ci) can be expressed using Eq. (2). This approach is based on an ideal mass balance (no creation or degradation) and assumes that (i) compound retention ri is constant and independent of VRR and (ii) the system behaves like an ideal stirred reactor.

Table 1 Characteristics of the tested ultrafiltration membranes.

a

Designation, manufacturer

Membrane type

Nominal MWCO (kDa)

Water permeability (kg h1 m2 MPa1)

GH, GE Osmonics, USA GK, GE Osmonics, USA UP005, Microdyn-Nadir, France UP020, Microdyn-Nadir, France UH050, Microdyn-Nadir, France UP150, Microdyn-Nadir, France

Thin film Thin film Polyethersulphone Polyethersulphone Permanently hydrophilic polyethersulphone Polyethersulphone

1 2 5 20 50 150

38 57 137 257 255 4170 ± 40a

Without pre-conditioning, measured at 0.14 MPa transmembrane pressure, value is the mean ± standard deviation (n = 3).

122


C i ¼ C i0 VRRri eDVð1ri Þ

ð2Þ

where Ci0 is the initial concentration of the compound i, and ri corresponds to the observed retention. VRR and the reduced diavolume of water (DV) are given by Eqs. (3) and (4).

Vf Vr

ð3Þ

Vw Vr

ð4Þ

VRR ¼

DV ¼

where Vf, Vr and Vw correspond to volumes of the feed, retentate and water added throughout diafiltration, respectively. In order to compare membrane performance, the DV that allowed obtaining a purity p (defined according to Eq. (5)) was selected as the criterion (Eq. (6)). We used the value of DV needed at a fixed VRR of 10 to reach a purity target of 0.9 kg ET kg TSS1, at the different transmembrane pressures tested.

p¼

C ET C TSS

ln DV ¼

ð5Þ p p0

þ ðrTSS rET Þ lnðVRRÞ

rET rTSS

ð6Þ

3. Results and discussion 3.1. Raw material composition The main components (as well as the antioxidant capacity) of the microfiltered blackberry juice used as raw material for the ultrafiltration experiments are presented in Table 2. The clarified juice presents relatively low contents of sugars, and high titratable acidity. When comparing concentrations (in a dry matter basis) of the main polyphenolic compounds present in the standardized juice used in this study with reported concentrations in whole blackberry fruits [3], values are considerably lower (recovery of 42% of ellagitannins and 49% of anthocyanins), probably due to retention and degradation during extraction, clarification, and concentration. 3.2. Permeate flux Using the microfiltered juice, the measured permeate fluxes plotted against applied transmembrane pressure for the six membranes tested are presented in Fig. 1. GE Osmonics membranes in general presented higher permeate fluxes when compared to Microdyn-Nadir membranes with similar nominal MWCO. Moreover, it can be noted that a thin film membrane from GE Osmonics with 2 kDa MWCO (GK) presented the highest permeation flux at 3 MPa (around 40 kg h1 m2) while a polyethersulphone Microdyn-Nadir membrane with 5 kDa MWCO (UP005) presented the lowest permeation flux in the same conditions (around 24 kg h1 m2). 50

40

-1

TSS were measured using a digital PAL-3 refractometer (Atago Co., Ltd., Tokyo, Japan) with automatic temperature compensation. The methods for determination of total titratable acidity, as well as sucrose, fructose, and glucose have been previously described [3]. Quantitative analyses of ellagitannins and anthocyanins were performed by HPLC/DAD with the system, column, conditions, standards and solvents described by Mertz et al. [5], with the following changes: solvent flow rate was set at 0.8 mL min1, gradient conditions of solvents A (aqueous formic acid) and B (acetonitrile/water/formic acid, 80:18:2, v/v/v) were from 5% to 10% B in 4 min, from 10% to 16% B in 4 min, from 16% to 25% B in 32 min and from 25% to 100% B in 3 min, then the column was washed at 100% B during 5 min and equilibrated at the initial gradient conditions for 20 min. Antioxidant capacity was measured in terms of HORAC, following the method described in [25], using a Synergy™ HT microplate reader (Biotek Instruments, Inc., Winooski, VT, USA).

-2

)

2.5. Analyses

30

20 GH GK UP005 UP020 UH050 UP150

10

0 0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Transmembrane pressure (MPa) Fig. 1. Evolution of permeate flux for the tested ultrafiltration membranes (error bars of UP150 membrane represent standard deviation for n = 3).

Table 2 Relevant composition and antioxidant capacity of the raw material used in the filtration experiments.

a b c d

Component

Value

Molecular mass (Da)

Cyanidin-3-glucoside (mg kg1) Cyanidin-3-malonyl glucoside (mg cyanidin-3-glucoside equivalents kg1) Lambertianin C (mg ellagic acid equivalents kg1) Sanguiin H-6 (mg ellagic acid equivalents kg1) Total titratable acidity (g malic acid equivalents kg1) Sucrose (g kg1) Fructose (g kg1) Glucose (g kg1) Total soluble solids (g kg1) Antioxidant capacity as H-ORAC value (mmol Trolox equivalents kg1)

410 ± 10a 30 ± 1a 380 ± 50a 230 ± 20a 31.5 ± 0.2b NDc 20 ± 2b 20 ± 1b 100.8 ± 0.5a 43 ± 2b

449 535 2806 1871 134d 342 180 180 – –

Values are the mean ± standard deviation (n = 8). Values are the mean ± standard deviation (n = 3). ND: not detected (60.2 g 100 mL1). Malic acid.

123


3.3. Solutes retention

90

80

70

50

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Transmembrane pressure (MPa) Fig. 3. Retention of total ellagitannins for the tested ultrafiltration membranes (error bars of UP150 membrane represent standard deviation for n = 3).

pressures over 1 MPa presented 100% of ellagitannin retention. For the UP150 membrane, an increase in the retention of total ellagitannins is observed with higher transmembrane pressures (Fig. 3), as it has been already discussed for total anthocyanin retention. Consequently, the use of any of the tested membranes (with exception of UP150) can theoretically make possible the separation of the major polyphenolic compounds in a clarified blackberry juice, since ellagitannins can be completely retained while anthocyanins are only partially rejected. It has been noticed that membranes retain phenolic compounds that present lower molecular weight than the nominal MWCO. This result could be explained by steric effects, physicochemical interactions with the membrane or association with other bigger compounds such as colloidal fractions. Retention values of TSS calculated from Eq. (1), for all tested membranes and transmembrane pressures are presented in Fig. 4. Results are similar to the reported behavior of retention of total anthocyanins: an increase in transmembrane pressure causes the retention to increase. As expected (due to the lower molecular weight of sugars and organic acids) retentions are lower than those observed for total anthocyanins, ranging from 12% for UP150 membrane at 0.5 MPa transmembrane pressure to 80% for UP005 at 3 MPa transmembrane pressure.

100

100

90

80

80

60

70

GH GK UP005 UP020 UH050 UP150

60

TSS

ACY

The retention values of total anthocyanins and total ellagitannins calculated from Eq. (1), for different membranes and applied transmembrane pressures are presented in Figs. 2 and 3, respectively. Retention of total anthocyanins ranged between 60% for UP150 membrane (150 kDa MWCO) at 0.5 MPa transmembrane pressure and 99% for UP005 (5 kDa MWCO) at 3 MPa. As pressure increases, so does the retention of total anthocyanins, presenting values of over 90% for every membrane, when 3 MPa of transmembrane pressure was reached. Retention of total anthocyanins linearly increased with decreasing nominal MWCO of MicrodynNadir ultrafiltration membranes tested (data not shown), but differences between membranes become less perceptible at higher transmembrane pressure. Membranes with higher MWCO were found to be more sensitive to transmembrane pressure changes, in terms of total anthocyanin retention. This effect can also be explained by membrane compaction, which induces pore size reduction [26]. Kalbasi and Cisneros-Zevallos [20] found a similar behavior when evaluating anthocyanin retention from concord grape juice, and similar results obtained using the same membranes and experimental setup, but different source of anthocyanins have been published [23]. Concerning total ellagitannin retention, with the exception of the UP150 membrane, every membrane at transmembrane

100

ET

As a function of pressure, membranes with nominal MWCO of 20 kDa or lower, present a similar tendency with a regular permeate flux increase when transmembrane pressure augmented. The UH050 membrane with 50 kDa MWCO presented a slightly different behavior: flux seemed to stabilize as pressure increased. On the contrary, flux surprisingly decreased with pressure for the higher MWCO membrane (UP150). Impact of pressure on permeate flux could be explained by an increase in the driving force for the mass transfer trough the porous medium, but pressure could also change the total hydraulic resistance of the membrane, as well as affect fouling. Therefore, the effect of pressure on resistance appears to be complex and distinctive, depending on the membrane’s material and particularly its nominal MWCO. These results could be related to the compaction of the porous system, i.e. membrane and fouling [26,27]. This compaction leads simultaneously to reduction of the membrane’s thickness (decreasing total hydraulic resistance) and restriction of its porosity (increasing resistance). So pressure could affect resistance in different ways according to the prominent phenomenon.

40 GH GK UP005 UP020 UH050 UP150

60


20

50

0 0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Transmembrane pressure (MPa) Fig. 2. Retention of total anthocyanins for the tested ultrafiltration membranes (error bars of UP150 membrane represent standard deviation for n = 3).

0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Transmembrane pressure (MPa) Fig. 4. Retention of total soluble solids for the tested ultrafiltration membranes (error bars of UP150 membrane represent standard deviation for n = 3).


2500

16


-1

-2

)

124

2000


14 12

DV

1500

1000

8

500

0

10

6 4 0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0

Fig. 5. Flux of total anthocyanins for the tested ultrafiltration membranes (results of UP150 membrane are intentionally cut; error bars of UP150 membrane represent standard deviation for n = 3).

Fig. 5 shows the flux of total anthocyanins (mg cyanidin-3-glucoside equivalents h1 m2) through the tested membranes, at various transmembrane pressures. It can be observed that GE Osmonics membranes behave differently, as total anthocyanin flux increases with transmembrane pressure, contrary to what is observed with Microdyn-Nadir membranes. The UP005 membrane presents the lowest anthocyanin flux, remaining steady for all transmembrane pressures, as an increase in permeate flux is offset by an increase in anthocyanin rejection. For all the other MicrodynNadir membranes the increase in permeate flux is not totally counterbalanced by the increase of solute rejection, and anthocyanin fluxes tend to decrease with transmembrane pressure. For GE Osmonics membranes, the contrary occurs and it appears that the increase in permeate flux is more important, allowing to overcome the decrease of anthocyanin concentration in the permeate. Consequently, these membranes are favorable for fractionation of blackberry polyphenolic compounds. For example, the GK membrane presents the highest anthocyanin flux at 2 MPa of transmembrane pressure (membrane UP150 not taken into account because of its incomplete ellagitannin retention), and thus appears to be the most promising membrane for the separation of ellagitannins from anthocyanins. 3.4. Modeling total ellagitannin purification with constant volume diafiltration The results of modeling total ellagitannin purification with constant volume diafiltration are shown in Fig. 6, using purity calculated as a ratio of total ellagitannin to TSS concentrations. It can be observed that UP150 membrane needs the lowest diavolume to achieve the desired purity at almost every transmembrane pressure tested. Although GE Osmonics and UH050 membranes present a different behavior when compared to membrane UP150, the resulting diavolumes are not that dissimilar. Membranes with the lowest nominal MWCO (UP005 and UP020) would require the highest diavolumes at almost every pressure in order to reach the chosen purity. It must be stressed that while this model is useful to predict membrane performance based on purity of the final product, losses of the compounds of interest can occur, as is the case with membrane UP150, which presents an incomplete retention of ellagitannins (Fig. 3) and therefore would present the lowest final concentration of the compounds in the retentate. Consequently, membranes with complete retention of total ellagitannins that also present low diavolumes needed should be

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Transmembrane pressure (MPa)

Transmembrane pressure (MPa)

Fig. 6. Evolution of diavolume needed to achieve total ellagitannin purity of 0.9 (with respect to TSS), when a VRR of 10 has been achieved previously by ultrafiltration for the tested membranes (results of UP005 membrane are intentionally cut; error bars of UP150 membrane represent standard deviation for n = 3).

preferred. Theoretically, these membranes would allow a high purification of blackberry ellagitannins at feasible conditions (VRR of 10 and DV lower than 10). Predictions should however be validated using further experiments with increasing VRR, because concentration levels could affect retention rates. 4. Conclusions The organic ultrafiltration membrane GK from GE Osmonics (2 kDa nominal MWCO) presents the highest potential for selective separation and concentration of ellagitannins from blackberry juice. At 2 MPa of transmembrane pressure, the highest value of anthocyanin flux is reached, while 100% of ellagitannin retention is achieved. Modeling total ellagitannin purification with constant volume diafiltration also confirmed that GE Osmonics membranes are the most promising, since they presented some of the lowest diavolumes needed to achieve the desired purity (90%), at higher transmembrane pressures. Acknowledgments Authors thank Christian Mertz (CIRAD, Montpellier) for his valuable technical assistance with HPLC analyses, Marie-Pierre Belleville (Institut Européen des Membranes, Montpellier) for her expertise in diafiltration and Alternative Marketing SAS/MICRODYN-NADIR for providing membranes. This research project was funded by PAVUC-FP6-INCO project DEV-2, Contract 015279. Financial support was also provided by MICIT/CONICIT (Costa Rican Government). References [1] R.H. Liu, Potential synergy of phytochemicals in cancer prevention: mechanism of action, J. Nutr. 134 (2004) 3479S–3485S. [2] C. Manach, A. Scalbert, C. Morand, C. Remesy, L. Jimenez, Polyphenols: food sources and bioavailability, Am. J. Clin. Nutr. 79 (2004) 727–747. [3] O. Acosta-Montoya, F. Vaillant, S. Cozzano, C. Mertz, A.M. Perez, M.V. Castro, Phenolic content and antioxidant capacity of tropical highland blackberry (Rubus adenotrichus Schltdl.) during three edible maturity stages, Food Chem. 119 (2010) 1497–1501. [4] K.L. Wolfe, X.M. Kang, X.J. He, M. Dong, Q.Y. Zhang, R.H. Liu, Cellular antioxidant activity of common fruits, J. Agric. Food Chem. 56 (2008) 8418– 8426. [5] C. Mertz, V. Cheynier, Z. Gunata, P. Brat, Analysis of phenolic compounds in two blackberry species (Rubus glaucus and Rubus adenotrichus) by highperformance liquid chromatography with diode array detection and


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