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Clarification of high-added value products from olive mill wastewater
Journal of Food Engineering 99 (2010) 190–197
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Clarification of high-added value products from olive mill wastewater Charis M. Galanakis a,*, Eva Tornberg b,1, Vassilis Gekas c,2 a
Department of Environmental Engineering, Technical University of Crete, Politechnioupolis, GR-73100 Chania, Greece Department of Food Technology, Engineering and Nutrition, Faculty of Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden c Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, P.O. Box 50329, Lemesos, CY 3603, Cyprus b
a r t i c l e
i n f o
Article history: Received 4 December 2009 Received in revised form 7 February 2010 Accepted 13 February 2010 Available online 25 February 2010 Keywords: Membrane Ultrafiltration Nanofiltration Pectin Phenols Antioxidant properties
a b s t r a c t The objective of the current study is to investigate the clarification of two high-added value products (pectin containing solution and phenol containing beverage) recovered from olive mill wastewater. For this purpose, both liquids were processed with four types of ultrafiltration (100, 25, 10 and 2 kDa) and one nanofiltration membranes under optimum transmembrane pressure. Retention coefficients and performance parameters were monitored for each experiment. The membranes of 25 and 100 kDa showed very satisfying results with regard to the concentration of pectin solutions as they were able to separate it from cations and phenols. The membrane of 25 kDa was also able to partially remove the heavier fragments of hydroxycinnamic acid derivatives and flavonols, and simultaneously to sustain the antioxidant properties of the phenol containing beverage in the permeate stream. Finally, nanofiltration clarified the beverage from cations that passed in the permeate stream, but this process resulted in loss of antioxidant compounds, too. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Membrane processes like ultrafiltration and nanofiltration have been successfully used for over three decades in food and beverage industries, by treating several substrates, fluids and agricultural wastewaters (Cuperus and Nijhuis, 1993; Gekas et al., 1998; Susanto et al., 2009). The most popular applications with regard to the fruit related sources target the separation, concentration or recovery of high-added value food ingredients like pectin and phenols. Clarification of fruit juices is a typical example as the purpose is to retain high molecular weight macromolecules (pectin or protein) in the concentrate stream and allow low molecular weight solutes (sucrose, acids, salts, aroma or flavor compounds) to permeate through the membrane and enhance product yield (Carvalho et al., 1998, 2008; Yazdanshenas et al., 2005; Sarkar et al., 2009). Ultrafiltration has also been used to concentrate and purify pectin crude aqueous extracts prior its recovery with alcohol precipitation (Yapo et al., 2007). On the other hand, fruit juices have been processed using ultrafiltration to obtain fractions enriched in phenols (i.e. flavonoids and hydroxycinnamic acid derivatives) with satisfying antioxidant activity and health promoting effects (Mangas et al., 1997; Gökmen et al., 2003; Cassano et al., 2007,
* Corresponding author. Tel.: +30 28210 37827; fax: +30 28210 88981. E-mail addresses: [email protected] (C.M. Galanakis), eva.tornberg@ food.lth.se (E. Tornberg), [email protected] (V. Gekas). 1 Tel.: +46 046 2224821. 2 Tel.: +357 25002301. 0260-8774/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2010.02.018
2008). Besides, aqueous extracts from pressed grape pomace have been treated using nanofiltration membranes for the same purpose (Díaz-Reinoso et al., 2009). Among the several fruits, olive is known to contain an appreciable amount of phenols with good antioxidant properties (Boskou et al., 2006) and dietary fibers with promising water holding capacity (Jiménez et al., 2000). Nevertheless, the majority of these compounds are lost in olive mill wastewater (OMW) during olive oil production. For example, OMW typically contains 98% of the total phenols in the olive fruit (Obied et al., 2005). OMW has also been referred to possess soluble dietary fibers and especially pectin material with satisfying gelling ability (Vierhuis et al., 2003; Cardoso et al., 2003). In our earlier studies, we recovered dietary fibers from OMW, based on the thermal extraction, prior the precipitation of macromolecules with 85 mL ethanol/100 mL (Galanakis et al., in press-b, in press-c). The developed method proposed the simultaneous recovery of dietary fibers and phenols in two different streams: (i) an alcohol insoluble residue rich in dietary fibers and (ii) an ethanolic liquid rich in phenols. The ethanolic liquid has been proposed for a direct application in the beverage industry, i.e. as an additive to produce soft drinks (Tornberg and Galanakis, 2008). Moreover, the water soluble fraction of the alcohol insoluble residue (WSAIR) has been proposed as fat replacer in meatballs achieved by dissolving the alcohol insoluble residue in water and sequentially separating it from the corresponding water insoluble fraction (Galanakis et al., in press-a). Despite the promising applications, these rather crude materials should be further clarified or
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separated before utilization in order to optimize their functional properties and improve their taste. For example, WSAIR was shown to contain only 5.3 g pectin/100 g, while the corresponding ash and potassium contents were nine- (46.2 g/100 g) and twofold (10.4 g/ 100 g) higher, respectively. The very high concentration of ions affected negatively both the gelling properties and the taste of this material. Thereby, membrane technologies could be utilized in order to purify WSAIR by separating pectin and ions. Similarly, the same technique could be applied in order to concentrate the phenol content of the proposed soft drinks, to modify their antioxidant profile or to clarify them from other low molecular weight (MW) compounds like salts and free sugars. The objective of the current study is to investigate the clarification as well as the recovery of high-added value ingredients like pectin and phenols from the aqueous solutions derived by the aforementioned WSAIR material and the ethanolic extract, respectively. For this purpose, both materials were processed with different ultrafiltration and nanofiltration membranes under constant conditions, i.e. temperature and circulation flux. Parameters like permeate flux and retention coefficients of several components were monitored for each feed-membrane combination. 2. Experimental 2.1. Materials Samples of OMW were collected from a local three phase olive mill production plant (Chania, Greece). The used extraction process requires 10–20 L fresh water/100 kg of olives processed to separate the olive oil from the crushed olive cake and the wastewater. Crushing and kneading temperature was kept below 30 °C, while the residence period of the OMW in the sequential extractor was 40 min. Fresh samples were collected from the output of the decanter and kept in plastic containers in the freezer ( 20 °C) until usage. Thereafter, OMW was processed according to the method described by Galanakis et al. (in press-a) for the recovery of two different materials: (i) a pectin containing water soluble, alcohol insoluble residue (WSAIR) and (ii) an ethanolic extract (85 mL/ 100 mL) rich in olive phenols. WSAIR was preserved in the freezer ( 20 °C) and ethanolic extracts were stored in the dark at 4 °C until usage. 2.2. Preparation of the feed liquids The above materials were further processed for the preparation of two different feed liquids. The first feed liquid (A) was prepared from the solubilization of WSAIR in water (6 g in 3 L) and it was named as ‘‘pectin containing solution”. The second feed liquid (B) was prepared from the ethanolic extract (85 mL/100 mL). In particular, the extract (B) was concentrated fivefold in a rotary evaporator (model Laborota 4011, Heidolph, Germany) at 60 °C and then it was diluted with water in order to obtain an ethanol concentration up to 2.0 mL/100 mL (90 mL of the condensed extract were diluted up to 3 L). This process was performed in order to increase the ratio of total phenols per ethanol concentration inside the beverage. The resulted solution was vacuum filtered sequentially with three filters (10 mm, 0.45 lm and 0.2 lm) before its utilization as feed liquid in membrane experiments in order to remove any residual fats and oils from OMW. This feed liquid was named as ‘‘phenol containing beverage”. 2.3. Membranes Five commercial membranes (Alfa Laval, Nakskov, Denmark) were investigated at the current study as presented in Table 1:
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four ultrafiltration membranes (GR40PP, GR60PP, GR81PP and GR95PP) and one nanofiltration membrane (NF99). The GR40PP and GR60PP membranes were made of polysulphone, while the GR81PP and GR95PP membranes were made of polyethersulphone. The NF99 membrane was a polymeric three-layer thin film membrane with an active layer of semiaromatic/aliphatic polyamide (polypiperazine). 2.4. Experimental set-up and operation control The membrane experiments were carried out in a plate frame module (DSS Labstak M20, Alfa Laval Nakskov, Denmark) as shown in Fig. 1. This unit is a cross-flow membrane filtration system, where membrane filter sheets as well as support- and spacer plates are stacked and compressed in a vertical frame. Plates were compressed with a hydraulic hand pump at 320 bar (model P392, Enerpac, USA). The membrane area was 0.036 m2 (two membrane sheets of 0.018 m2) and the processed feed volume was 3 L. Feed was circulated through compressed membrane plate with an auxiliary pump (hydra cell industrial pump, model G13XDSGHHEMA, Wanner Engineering Inc., USA) equipped with a motor (Varmeca10, model 013XDSGHHEMA, Leroy Somer, USA). The circulation flux was constant at 38 mL s 1. The feed inlet and the outlet temperature were kept constant at 25 ± 0.5 °C, by placing feed tank into a water bath, and controlled with temperature sensors (Pt100 class A, Pentronic AB, Sweden). The transmembrane pressure (TMP) of the feed liquid was adjusted to the appropriate level with a retentate adjusting valve. The inlet and the outlet TMPs were measured with module self-contained. The permeate flux was measured gravimetrically as the change of permeate weight with time by using a laboratory scale balance (XT120A, Precisa Instruments Ltd., Switzerland) which was monitored via a computer. Permeate flux was expressed in L/h m2. 2.5. Experimental procedure 2.5.1. Membrane pretreatment Each membrane was packed into Labstak M20 module and pretreated with de-ionised water as feed liquid (5 L) in order to minimize membrane compaction during the experiments. The GR40PP and GR60PP membranes were both pressurized at 2, 3, 4 and 5 bar in two sequential rounds (15 min duration) using fresh de-ionised water in each one. Permeate was discharged continuously. Fresh de-ionised water was added continuously in order to retain a volume of 4–5 L feed liquid. The GR81PP and GR95PP membranes were pressurized with the same process at 3, 4, 5 and 6 as well as 5, 6, 7 and 8 bar, respectively. The NF99 membrane was similarly pressurized at 4, 8, 12 and 16 bar. It is assumed that glycerine, which is used as membrane preservative is washed out with this process (Nilsson et al., 2006). 2.5.2. Process optimization Pretreated membranes were pressurized in triplicates using separately the pectin containing solution and the phenol containing beverage with the same protocol and tested pressures as above. A new pretreated membrane was utilized for all the combinations of membranes with each type of feed liquid. Permeate flux was monitored for both feed liquids and a permeate sample (10 mL) was kept for analysis at each tested pressure (Table 1). Thereafter, pectin and total phenols (determined with Folin–Ciocalteau reagent) retention coefficients were determined for the pectin containing solution and the phenol containing beverage, respectively. The optimum TMPs were selected according to the higher permeate flux as well as the higher pectin and phenol retention coefficients in each feed liquid, respectively. Permeate flux was enhanced with increasing TMP for all the tested membranes and
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Table 1 Characteristics of the utilized membranes as recommended from the supplier and corresponding operation conditions during the experiments. Membrane name
Type
Support material and active layer
Typical MWCOa
GR40PP GR60PP GR81PP GR95PP NF99
UFe UFe UFe UFe NFf
Polysulphone Polysulphone Polyethersulphone Polyethersulphone Polypiperazine
100,000 25,000 10,000 2000 120g
pH rangeb
(Da)
a b c d e f g
1–13 1–13 1–13 1–13 2–10
Temperature rangeb
Pressure rangeb
Tested pressurec
Applied pressured
(°C)
(bar)
(bar)
(bar)
0–75 0–75 0–75 0–75 0–50
1–10 1–10 1–10 1–10 1–55
2, 2, 3, 5, 4,
3, 3, 4, 6, 8,
4, 5 4, 5 5, 6 7, 8 12, 16
2 3 5 8 12
‘‘MWCO” for ‘‘molecular weight cut off”. Recommended specifications during continuous operation. During membrane pretreatment and process optimization. During clarification experiments of both feed solutions. ‘‘UF” for ‘‘ultrafiltration”. ‘‘NF” for ‘‘nanofiltration”. The manufacturer measured retention of 2000 mg/L MgSO4 solution is P99% using 9 bar and 25 °C.
Temperature sensor
Manometer
Membrane Permeate
Cooling water inlet
Digital balance, permeate flux control Control valve Outlet Manometer Temperature sensor
Pump Feed solution
Retentate
Temperature sensor Feed tank Water bath Fig. 1. Schematic diagram of the experimental setup.
both feed liquids. Finally, the same optimum TMP (Table 1) was applied for both feed liquids as it was found that pectin and phenol retention coefficients, respectively, were lower with increasing TMP above a certain value for all the tested membranes (2, 3, 5, 8 and 12 bar for GR40PP, GR60PP, GR81PP, GR95PP and NF99, respectively). 2.5.3. Clarification experiments Triplicate pretreated membranes were again utilized for each feed-membrane experiment. Three liters of feed liquid (phenol beverage or pectin solution) were processed in membrane apparatus with the optimum TMP until the recovery of 1.5 L permeate and 1.5 L retentate. Permeate flux was determined during the process and the relative flux (RF) of the feed liquid was calculated in percentage according to the following equation: RF = (Jv/Jw0)100 (%),
where Jv is the permeate flux at steady state and Jw0 is the pure water flux. Samples of feed and permeate streams were kept in the freezer ( 20 °C) until analysis. After the completion of each clarification experiment, feed liquid was replaced with de-ionised water and processed for 60 min at the same TMP in order to clean the membrane. Permeate flux was again determined with fresh deionised water and the flux recovery (FR) was calculated in percentage according to the following equation: FR = (Jwf/Jw0)100 (%), where Jwf and Jw0 are the pure water flux after and prior the clarification experiment, respectively (Gekas et al., 1993). 2.6. Analyses Samples from the treatment of the pectin containing solution were assayed for the determination of the pH and conductivity
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value, as well as the potassium, sodium, total phenols and pectin content. Total sugars were not determined for this feed liquid as it was expected to contain only traces of them. Besides, the initial material (WSAIR) for the production of this feed liquid was isolated from the OMW after a precipitation step with ethanol that is known to remove low MW compounds like free sugars into the ethanolic extract (Qi et al., 2000). Other parameters like different phenolic classes and antioxidant capacity were also not determined for this feed liquid as the main purpose was the purification of the contained pectin from the high concentrations of ions that are known to contribute negatively to its gelling properties (Galanakis et al., in press-a). On the other hand, samples from the treatment of the phenol containing beverage were assayed for the determination of the pH and conductivity value, potassium, sodium, ethanol and total sugars concentration. Moreover, the phenolic content of these samples was measured with several parameters (total phenols with Folin–Ciocalteau reagent, o-diphenols at 370 nm, total phenols at 280 nm, hydroxycinnamic acid derivatives at 320 nm and flavonols at 360 nm), while their antioxidant capacity was also determined by assaying two different activities: 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging efficacy and ferric ion reduction ability. Pectin content of the beverage was not determined as polysaccharides were expected to be removed after the precipitation of the alcohol insoluble residue (Qi et al., 2000). The determination and classification of the phenols in the extracts were measured colorimetrically using several methods. Total phenols content was determined colorimetrically using the Folin– Ciocalteau reagent. A diluted sample was mixed with 0.25 mL Folin–Ciocalteau reagent. After 3 min stirring, 1 mL saturated sodium carbonate solution was added and the final solution was left in the dark for 1 h. The absorbance of the solution was measured at 725 nm using a Shimadzu UV-mini-1240 spectrophotometer. A standard curve was prepared using 0–50 mg/L solutions of caffeic acid in methanol/water. The standard solutions were prepared with several dilutions of a mother solution in water. Mother solution was prepared as follows: 1 g of caffeic acid was solubilized in 100 mL of methanol and then 1 mL of the resulted solution was diluted to 100 mL of water. Total phenol values were expressed as caffeic acid equivalents (mg/L). Extracts were used after dilution (1:20) with de-ionised water. The determination of o-diphenols was performed following a modification of the method described by Mateos et al. (2001). In particular, 1 mL of diluted extract (1:5 in 50 mL ethanol/100 mL solution) was vigorously mixed with 1 mL of sodium molybdate dihydrate solution (5 g/100 mL in 50 mL ethanol/100 mL solution) into a 10 mL volumetric flask and the volume was made up to 10 mL with 50 mL ethanol/100 mL solution. After 15 min, the absorbance was measured at 370 nm. A blank was prepared by mixing the sodium molybdate dihydrate solution with 50 mL ethanol/100 mL solution. A standard curve was prepared using solutions of caffeic acid (0–200 mg/L) in 50 mL ethanol/100 mL solution. Results were expressed as caffeic acid equivalents (mg/L). The determination of different polyphenol classes was performed according to Obied et al. (2005). One mL of diluted ethanolic extract (1:10 in water) was mixed with 1 mL of HCl–ethanol solution (0.1 mL HCl/100 mL in 95 mL ethanol/100 mL) into a 10 mL volumetric flask and the volume was made up to 10 mL with 2 mL HCl/100 mL. After mixing, the absorbance was measured at 280, 320, and 360 nm to determine total phenols, hydroxycinnamic acid derivatives and flavonols, respectively. A blank was prepared by mixing the HCl–ethanol solution with 2 mL HCl/100 mL. The corresponding standard curves to the above determinations were prepared using solutions (10 mL ethanol/100 mL water) of gallic acid (0–200 mg/L), caffeic acid (0–100 mg/L) and quercetin (0–150 mg/L), respectively.
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Antioxidant ability of ferric ion reduction was performed following a modification of the method described by SzydlowskaCzerniak et al. (2008). In particular, 1 mL of FeCl3 solution (0.2 g/ 100 mL of absolute ethanol) and 0.5 mL of 1,10-phenanthroline solution (0.5 g/100 mL ethanol) were vigorously mixed into a 10 mL volumetric flask. After mixing, 0.6 mL of diluted extract (1:10 in absolute ethanol) was added and the volume was made up to 10 mL with absolute ethanol. The obtained solution was mixed and left in the dark. After 20 min, the absorbance was measured at 510 nm. A blank was prepared by mixing FeCl3 solution with phenanthroline reagent and the volume made up to 10 mL with absolute ethanol. Standard curve was prepared using solutions of FeSO47H2O (0–0.8 mmol/L) and the results were expressed as FeSO4 equivalents/L. DPPH radical scavenging activity of the extracts was performed following a modification of the method described by Kulisic et al. (2004). Several dilutions (100 lL) of each extract (1:5, 1:10, 1:20, 1:30 and 1:40) were vigorously mixed with 1.5 mL methanolic solution of DPPH radical (32 mg/L) in 2-mL plastic tubes. After 1 h, the absorbance at 517 nm of the resulting mixtures was measured against methanol, which was used to zero the absorbance. A blank solution of the DPPH radical without antioxidant was utilized as control sample. The percentage inhibition of the DPPH radical by the samples was calculated according to the equation: % inhibition = ((AC(0) AA(t))/AC(0)) 100, where AC(0) is the absorbance of the control at t = 0 min and AA(t) (mg DPPH/g antioxidant) is the absorbance of the antioxidant at t = 1 h. Results were expressed in Antioxidant Efficacy (AE) values: AE = 1/EC50, where EC50 is the effective concentration of the antioxidant extract that resulted in 50% scavenging of DPPH radical. Galacturonic acid (GalA), potassium, sodium and pH determinations of the samples were performed as previously described (Galanakis et al., in press-a). Conductivity values (lS/cm) of the samples were measured with a digital conductometer (K720, Consort, Belgium). Ethanol was determined in the distillate of the samples using control Gay-Lussac alcohol meters (Dujardin-Salleron Instruments, France) and expressed as alcoholic degrees (mL ethanol/100 mL of sample). Samples (100 mL) were distilled with an Alcodest module (JP Selecta, Spain). Total sugars were determined following a modification of the method described by Dubois et al. (1956). In particular, 1 mL of diluted sample was mixed vigorously with 1 mL of phenol solution (5 g/100 mL) into a tube. After mixing, 5 mL of concentrated H2SO4 were added and tube was mixed rapidly with vortex. After 10 min, the tube was shaken again and chilled in a water bath (20–25 °C). After 5 min, the absorbance was measured at 490 nm against a blank solution prepared according to the same protocol using de-ionised water instead of the sample. Standard curve was prepared using glucose solutions (5– 40 mg/L) instead of the sample. All the above determinations were performed in triplicates. Retention coefficients (R%) were calculated by the following equation: R% = 100 (Afeed Aperm)/Afeed, where R% is the retention coefficient of a parameter, while Afeed and Aperm are the values of any parameter in the feed and permeate, respectively. This value corresponded to mg/L for total sugars, total phenol (determined at 725 as well as 280 nm), o-diphenols, hydroxycinnamic acid derivatives, flavonols, potassium and sodium concentrations. 2.7. Statistical analysis Clarification experiments were performed in triplicates and the mean ± standard deviation of the three corresponding values for each variable was calculated. Data were analyzed using Students t-test (pair wise comparisons, Office Excel 2007). Significant differences between samples were detected when the acceptable level of probability was 5% (P 6 0.05) for all the comparisons.
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3. Results
3.2. Performance parameters of the clarification experiments
3.1. Characterization of the feed liquids
Performance parameters with regard to the flux of the feed liquids obtained as a function of the different tested membranes are shown in Table 3. Pure water flux before the clarification experiments was not significantly different for the GR40PP, GR60PP and NF99 membranes (95, 95 and 99 L/h m2, respectively) and more than sevenfold lower for the GR81PP and GR95PP membranes (11 and 14 L/h m2, respectively). Permeate flux of the pectin containing solution was rather low (4–15 L/h m2) for all the tested ultrafiltration membranes and higher (56 L/h m2) for the NF99 membrane. Likewise, pure water flux was decreased after the clarification experiments of the pectin containing solution for all the tested membranes (44, 46, 7, 8 and 82 L/h m2 for GR40PP, GR60PP, GR81PP, GR95PP and NF99, respectively). Moreover, FR value was ranged from 47% to 58% for all the ultrafiltration membranes (without significant differences) and was equal to 83% for the NF99. On the other hand, permeate flux of the phenol containing beverage was rather high for the GR40PP, GR60PP and NF99 membranes (76, 74 and 47 L/h m2, respectively) and very low for the GR81PP and GR95PP (3 and 2 L/h m2, respectively). After the clarification experiments of the phenol containing beverage, pure water flux was reduced up to 63, 61, 6, 9 and 81 L/h m2 for the GR40PP, GR60PP, GR81PP, GR95PP and NF99 membrane, respectively. These fluxes resulted in FR values ranging from 48% to 66% for all the ultrafiltration membranes and equal to 82% for the NF99.
The composition of both feed liquids that were utilized in the clarification experiments can be seen in Table 2. Pectin containing solution was a weakly acidic medium (pH 6.3) with rather high value of conductivity (784 lS/cm). This value was originated from the high amounts of contained cations like potassium and sodium (262 and 42 mg/L, respectively). Pectin and total phenol concentrations were ranged between the aforementioned cation contents (87 and 68 mg/L, respectively). On the other hand, the phenol containing beverage was an acidic (pH 4.9) soft drink (ethanol concentration equal to 1.6 mL/100 mL) with lower values of potassium and sodium contents (96 and 26 mg/L, respectively) compared to the pectin containing solution. These cations contents contributed to a lower value of conductivity (507 lS/cm). Moreover, this medium was found to be rich in total sugars (384 mg/L) and total phenols (280 and 112 mg/L as determined at 725 and 280 nm, respectively). Different phenolic classes like o-diphenols, hydroxycinnamic acid derivatives and flavonols were determined and found to be equal to 57, 19 and 18 mg/L, respectively. The contained phenols contributed to a beverage that was possessing antioxidant capacity, i.e. radical scavenging efficacy was equal to 1.7 mg DPPH/g and ferric ion reduction ability corresponded to 1.6 mmol FeSO4/L.
Table 2 Characteristics of the feed liquids. Values represent mean ± standard deviation (n = 3). Parameter
Unit
pH Conductivity Potassium Sodium Ethanol Total sugars Pectin Total phenols (determined at 725 nm) Total phenols (determined at 280 nm) o-Diphenols Hydroxycinnamic acid derivatives Flavonols
–
a
c
Pectin containing solution
Phenol containing beverage
mg/L mg/L mL/100 mL mg/L mg GalAa/L mg/L mg/L mg/L mg/L mg/L
6.3 ± 0.1 784 ± 17 262 ± 6 42 ± 1 n.d.b n.d.b 87 ± 7 68 ± 2 n.d.b n.d.b n.d.b n.d.b
4.9 ± 0.1 507 ± 26 96 ± 5 26 ± 2 1.6 ± 0.1 384 ± 25 n.d.b 280 ± 18 112 ± 11 57 ± 5 19 ± 2 18 ± 2
mg DPPHc/g mmol FeSO4/L
n.d.b n.d.b
1.7 ± 0.1 1.6 ± 0.2
lS/cm
Antioxidant capacity Antiradical efficacy Ferrous ions equivalents b
Feed liquid
‘‘GalA” for ‘‘galacturonic acid”. ‘‘n.d.” for ‘‘not determined”. ‘‘DPPH” for ‘‘2,2-diphenyl-1-picrylhydrazyl”.
Table 3 Performance parameters of the clarification experiments of both feed liquids obtained with different membranes. Values represent mean ± standard deviation (n = 3).
a
Membrane
Water
Pectin containing solution
Type
Jw0a (L/h m2)
Jv b (L/h m2)
Jwfc (L/h m2)
RFd (%)
FRe (%)
Jv b (L/h m2)
Phenol containing beverage Jwfc (L/h m2)
RFd (%)
FRe (%)
GR40PP GR60PP GR81PP GR95PP NF99
95 ± 5f 95 ± 7f 11 ± 1g 14 ± 2g 99 ± 3f
15 ± 2f 12 ± 3f 5 ± 1g 4 ± 1g 56 ± 1
44 ± 2f 46 ± 4f 7 ± 1g 8 ± 1g 82 ± 2
16 ± 1f 13 ± 3f 44 ± 10 g 31 ± 11g 57 ± 2
47 ± 4f 48 ± 7f 58 ± 4f 56 ± 9f 83 ± 1
76 ± 7f 74 ± 1f 3 ± 1g 2 ± 1g 47 ± 2
63 ± 4f 61 ± 3f 6 ± 1g 9 ± 1g 81 ± 4
80 ± 4f 78 ± 6f 27 ± 3 17 ± 1 48 ± 1
66 ± 2f 64 ± 5f 48 ± 9 63 ± 5f 82 ± 2
‘‘Jw0” for the ‘‘pure water flux” before clarification experiments of the feed solutions. ‘‘Jv” for the ‘‘permeate flux” at steady state. c ‘‘Jwf” for the ‘‘pure water flux” after clarification experiments of the feed solutions. d ‘‘RF” for the ‘‘relative flux” of the feed solutions. e ‘‘FR” for the ‘‘flux recovery”. f,g Values possessing the same superscripted letter within a column are not significantly different (P 6 0.05). b
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3.3. Clarification of the pectin containing solution The retention coefficients obtained for several parameters of pectin containing solution as a function of the different tested membranes are shown in Table 4. In particular, pectin retentions were quantitative (98–99%) for all the tested membranes possessing MWCO below 25 kDa (GR60PP, GR81PP, GR95PP and NF99) and the values were not significant different. Besides, pectin retention was also very high (79%) for the membrane (GR40PP) with the higher MWCO (100 kDa). On the other hand, retentions of other parameters like total phenols, potassium, sodium and conductivity were practically quantitative (99%, 96%, 91% and 93%, respectively) only for the nanofiltration membrane (NF99) and rather negligible (13%, 2%, 2% and 5%, respectively) for the GR40PP membrane. The corresponding retentions obtained for the three membranes possessing MWCO between 2 and 25 kDa (GR60PP, GR81PP and GR95PP) showed different behavior among the aforementioned characteristics. For example, ion recovery as expressed with potassium, sodium and conductivity coefficients was minor (14%, 10% and 12%, respectively) for the GR60PP membrane (MWCO equal to 25 kDa), while total phenols recovery was rather important (40%). Moreover, total phenols coefficients were very high (71% and 81%) for the membranes possessing MWCO of 10 kDa (GR81PP) and 2 kDa (GR95PP), respectively. The corresponding retentions of potassium, sodium and conductivity were a bit lower (49% and 55%, 56% and 60%, as well as 58% and 61%, respectively), while the values obtained for each parameter were not significantly different. 3.4. Clarification of the phenol containing beverage The retention coefficients obtained for several parameters of phenol containing beverage as a function of the different tested membranes are shown in Table 5. Definitely, all the retention coefficients were negligible (<1) for the GR40PP membrane (MWCO of 100 kDa) except for the flavonols recovery that was a bit higher (10%). For the rest of the tested membranes, retention coefficients showed different values among the several parameters. For example, potassium and sodium retentions were ranged from 23% to 28% and from 17% to 20%, respectively, for the tested membranes possessing MWCO between 2 and 25 kDa (GR60PP, GR81PP and GR95PP), while the values obtained for each parameter were not significantly different. Nevertheless, the corresponding retentions for the NF99 membrane were significantly higher (55% and 45%, respectively). Total sugars retention was rather low (18%, 32% and 38%) for the membranes possessing MWCO between 2 and 25 kDa (GR60PP, GR81PP and GR95PP, respectively), but quantitative (98%) for the nanofiltration membrane (NF99). Total phenols (determined at 725 and 280 nm) retention showed more or less the same tendency than total sugars: low values (10% and 19%, 21% and 33% as well as 25% and 42%, Table 4 Retention coefficients obtained for several parameters of pectin containing solution as a function of different utilized membranes. Membrane
Retention coefficienta (%)b
Type
Pectin
Total phenolsc
Potassium
Sodium
Conductivity
GR40PP GR60PP GR81PP GR95PP NF99
79 ± 3 98 ± 2* 98 ± 2* 99 ± 1* 99 ± 1*
13 ± 3 40 ± 4 71 ± 3 81 ± 4 99 ± 1
2±1 10 ± 2 49 ± 2* 55 ± 4* 96 ± 2
2±1 14 ± 2 56 ± 3* 60 ± 4* 91 ± 3
5±2 12 ± 3 58 ± 4* 61 ± 2* 93 ± 3
a Retention coefficient = 100 (Afeed Aperm)/Afeed, where Afeed and Aperm are the values of any parameter in the feed and permeate, respectively. b Values represent mean ± standard deviation (n = 3). Values possessing a superscripted star within a column are not significantly different (P 6 0.05). c Total phenols as determined at 725 nm with Folin–Ciocalteau reagent.
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respectively) for the GR60PP, GR81PP and GR95PP membranes, respectively, and very high values (70% and 91%, respectively) for the NF99 membrane. Retention coefficients of the different phenolic classes followed the same tendency as the corresponding coefficients of total phenols, but the values were generally a bit higher. In particular, o-diphenols, hydroxycinnamic derivatives and flavonols retentions were equal to 6%, 32% and 37%, respectively, for the GR60PP membrane (MWCO of 25 kDa), significantly higher (32%, 44% and 56% as well as 48%, 53% and 62%, respectively) for the membranes possessing MWCO between 2 and 10 kDa (GR81PP and GR95PP) and rather quantitative (85%, 99% and 99%, respectively) for the nanofiltration membrane (NF99). On the other hand, antiradical efficacy and ferrous ions equivalents retentions were rather low (8% and 4%, 24% and 15% as well as 36% and 24%, respectively) for the membranes possessing MWCO between 2 and 25 kDa (GR95PP, GR81PP and GR60PP) and importantly higher but not quantitative (63% and 75%, respectively) for the NF99.
4. Discussion When the pectin containing solution was supplied in the feed stream, the two membranes (GR40PP and GR60PP) possessing the highest MWCO (100 and 25 kDa, respectively) showed very good results with regard to the separation of the pectin from the ions and their recovery in the concentrate and the permeate streams, respectively. The GR40PP membrane was also able to remove quantitatively the contained phenols (retention equal to 13%) in contrast to the GR60PP that retained 40% of them. Thus, a membrane possessing MWCO between 100 and 25 kDa could hypothetically be utilized in order to optimize the clarification of the contained pectin from ions and phenols simultaneously. A drawback for the application of both membranes was the low permeate flux and RF (12–15 L/h m2 and 13–16%, respectively) as well as the limited FR (47–48%). A higher TMP could be utilized for both membranes in order to increase the efficacy of the ultrafiltration. Nevertheless, this parameter could negatively affect the recovery of the pectin, especially for the GR40PP membrane that did not possess so high retention value (79%). With regard to the application of the other ultrafiltration membranes (GR81PP and GR95PP), the separation of pectin from ions was not so distinct, as the retention of the latest was relatively high (49–60%). The corresponding retention values of the phenols were even higher (71–81%). These results were not so expected, as the pores of the membranes (MWCO of 10 and 2 kDa, respectively) were still very wide for the size of simple phenolic compounds and ions. A possible explanation is that some of the phenols in OMW could be bound to dietary fibers like pectin (Bravo et al., 1994) and thereby they could retain in association with them inside the concentrate stream. Besides, the retention of macromolecular gelling substances is generating the formation of a second or dynamic membrane that increases the retention of lower MW solutes like ions (Mulder, 1996). This hypothesis was confirmed by the very low permeate flux (4–5 L/h m2) and the restricted FR (56–58%) for both membranes, despite the rather high applied TMPs (5 and 8 bar for GR81PP and GR95PP, respectively). Nanofiltration process showed much higher permeate flux (56 L/h m2) as well as FR (83%) due to the higher applied TMP (12 bar), but the separation was rather impossible as the retention of the ingredients was quantitative (91–99%) for all the tested parameters. The high retention values (even for the monovalent ions) could be due to the concentration polarization layer of retained species (macromolecular and micromolecular) on the NF99 membrane pores. Therefore, all the membranes possessing MWCO of 10 kDa or lower could not be preferred for the clarification of the pectin.
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Table 5 Retention percentages of several parameters for phenol containing beverage obtained with different membranes.
a b c d e
Membrane
Retention coefficienta (%)b
Type
Potassium
Sodium
Conductivity
Total sugars
Total phenolsc
Total phenolsd
oDiphenols
Hydr-cinn. acidse
Flavonols
Antiradical efficacy
Ferrous ions equivalents
GR40PP GR60PP GR81PP GR95PP NF99
<1 26 ± 4* 23 ± 4* 28 ± 5*
<1 19 ± 3* 17 ± 4* 20 ± 4*
<1 21 ± 3 36 ± 3* 40 ± 4*
<1 18 ± 3 32 ± 4* 38 ± 5*
<1 10 ± 3 21 ± 3* 25 ± 5*
45 ± 5
73 ± 2
98 ± 1
70 ± 3
<1 6±3 32 ± 4* 48 ± 4* 85 ± 5
<1 32 ± 3 44 ± 3 53 ± 3 99 ± 1
10 ± 2 37 ± 4 56 ± 4* 62 ± 5*
55 ± 4
<1 19 ± 3 33 ± 3 42 ± 2 91 ± 4
<1 8±2 24 ± 3 36 ± 4 63 ± 5
<1 4±2 15 ± 3* 24 ± 5* 75 ± 5
99 ± 1
‘‘Retention coefficient” = 100 (Afeed Aperm)/Afeed, where Afeed and Aperm are the values of any parameter in the feed and permeate, respectively. Values represent mean ± standard deviation (n = 3). Values possessing a superscripted star (*) within a column are not significantly different (P 6 0.05). Determined at 725 nm. Determined at 280 nm. ‘‘Hydr-cinn. acids” for ‘‘hydroxycinnamic acid derivatives”.
The clarification of the ingredients in the phenol containing beverage was, however, proved to be more difficult. The existence of smaller compounds (i.e. sugars) and the absence of pectin macromolecules complicated their separation, as the solutes possessed MW within the same order of magnitude. For example, the membrane with the highest MWCO (GR40PP) was unable to recover any substances in the concentrate, as all of them (except for a 10% of flavonols) were quantitatively passed in the permeate stream. The separation of solutes with membranes possessing MWCO of 10 and 2 kDa (GR81PP and GR95PP) was inappreciable as the retention values were ranging from 20% to 60% for all the tested parameters. Moreover, the separation of sugars and phenols was rather impossible as the retention values of total sugars and total phenols (determined with both methods) were similar for all the assayed membranes (Table 5). This result indicates that the contained phenols possessed similar MW with sugars and subsequently they were not as much polymerized. Nevertheless, the assayed beverage is basically an aqueous medium (1.6 mL ethanol/100 mL) that cannot secure the preservation of the phenols like the aforementioned ethanolic extract. Besides, autoxidation and polymerization of low MW phenols are known to produce darkly colored polymers with characteristic off-flavor present in OMW (Niaounakis and Halvadakis, 2004). This is important to state as autoxidation should be avoided in order to obtain a beverage with satisfying odor and taste. For this purpose, the clarification of the ‘‘heavier” phenolic compounds could be conducted in order to improve the organoleptic characteristics of the beverage and to extend its self-life as a product. Hydroxycinnamic acid derivatives and flavonols seem to be ‘‘heavier” compared to o-diphenols, as the corresponding retention values showed the same tendency (flavonols > hydroxycinnamic acid derivatives > o-diphenols) for all the levels of MWCO. The separation of these phenols seem to be possible with the application of GR60PP membrane, as the o-diphenols were quantitatively passed into the permeate stream (6% retention), while the flavonols and hydroxycinnamic acid derivatives were partially retained in the concentrate stream (37% and 32% retention, respectively). Likewise, the permeate stream sustained the antioxidant properties of the beverage, as the retentions of antiradical efficacy and ferrous ions equivalents were rather negligible (8% and 4%, respectively). The last observation could be explained by the fact that o-diphenols were contained in higher amounts compared to flavonols and hydroxycinnamic acid derivatives (Table 2) inside the phenol containing beverage and generally they are known to possess higher antioxidant capacities due to their structure (Kiokias et al., 2008). Although, the performance parameters of this membrane processing were very satisfying, since permeate flux, RF and FR were relatively high (74 L/h m2, 78% and 64%, respectively). These retention data denote that the beverage could hypothetically
be concentrated more than twice, as there were no severe problems of concentration polarization or fouling. Eventually, nanofiltration of the phenol containing beverage could be utilized in order to concentrate specific phenol classes and sugars with a simultaneous clarification of them from small cations like potassium and sodium. In particular, the recovery of sugars, hydroxycinnamic acid derivatives and flavonols was quantitative (99%) and at the same time the retention of potassium and sodium was much lower (55% and 45%, respectively). The permeate flux was rather high (47 L/h m2) due to the applied TMP (12 bar) and the FR was satisfying (82%), too. Nevertheless, the main disadvantage of this application is the induced loss of the antioxidant properties (retention values of antiradical efficacy and ferrous ions equivalents was equal to 63% and 75%, respectively) due to the lower retention of o-diphenols (85%).
5. Conclusion The current study suggests that the clarification as well as the recovery of valuable compounds from pectin containing solution and phenol containing beverage is possible with the utilization of membrane technologies. The separation of these ingredients was mainly governed from the characteristics of each feed liquid as well as the MWCO of the applied membranes. With regard to the pectin containing solution, the membranes possessing MWCO between 25 and 100 kDa (GR60PP and GR40PP, respectively) were able to quantitatively recover pectin in the concentrate stream and separate it from smaller solutes like potassium, sodium and phenols that pass into the permeate stream. The clarified pectin material could be utilized as gelling agent after concentration in low fat meatballs as it was proposed in a previous study (Galanakis et al., in press-a). On the other hand, the GR60PP membrane could also be utilized for the clarification of the phenol containing beverage. This membrane was able to partially remove the heavier fragments of hydroxycinnamic acid derivatives and flavonols in the concentrate stream and at the same time to sustain the antioxidant properties of the beverage in the permeate stream. This process would improve the organoleptic characteristics and self-life of the beverage. Finally, nanofiltration could be used in order to clarify the beverage from the potassium and sodium, but this process would reduce the antioxidant ability of the product due to a loss of o-diphenols in the permeate stream.
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Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Clarification of high-added value products from olive mill wastewater Charis M. Galanakis a,*, Eva Tornberg b,1, Vassilis Gekas c,2 a
Department of Environmental Engineering, Technical University of Crete, Politechnioupolis, GR-73100 Chania, Greece Department of Food Technology, Engineering and Nutrition, Faculty of Engineering, Lund University, P.O. Box 124, S-221 00 Lund, Sweden c Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology, P.O. Box 50329, Lemesos, CY 3603, Cyprus b
a r t i c l e
i n f o
Article history: Received 4 December 2009 Received in revised form 7 February 2010 Accepted 13 February 2010 Available online 25 February 2010 Keywords: Membrane Ultrafiltration Nanofiltration Pectin Phenols Antioxidant properties
a b s t r a c t The objective of the current study is to investigate the clarification of two high-added value products (pectin containing solution and phenol containing beverage) recovered from olive mill wastewater. For this purpose, both liquids were processed with four types of ultrafiltration (100, 25, 10 and 2 kDa) and one nanofiltration membranes under optimum transmembrane pressure. Retention coefficients and performance parameters were monitored for each experiment. The membranes of 25 and 100 kDa showed very satisfying results with regard to the concentration of pectin solutions as they were able to separate it from cations and phenols. The membrane of 25 kDa was also able to partially remove the heavier fragments of hydroxycinnamic acid derivatives and flavonols, and simultaneously to sustain the antioxidant properties of the phenol containing beverage in the permeate stream. Finally, nanofiltration clarified the beverage from cations that passed in the permeate stream, but this process resulted in loss of antioxidant compounds, too. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Membrane processes like ultrafiltration and nanofiltration have been successfully used for over three decades in food and beverage industries, by treating several substrates, fluids and agricultural wastewaters (Cuperus and Nijhuis, 1993; Gekas et al., 1998; Susanto et al., 2009). The most popular applications with regard to the fruit related sources target the separation, concentration or recovery of high-added value food ingredients like pectin and phenols. Clarification of fruit juices is a typical example as the purpose is to retain high molecular weight macromolecules (pectin or protein) in the concentrate stream and allow low molecular weight solutes (sucrose, acids, salts, aroma or flavor compounds) to permeate through the membrane and enhance product yield (Carvalho et al., 1998, 2008; Yazdanshenas et al., 2005; Sarkar et al., 2009). Ultrafiltration has also been used to concentrate and purify pectin crude aqueous extracts prior its recovery with alcohol precipitation (Yapo et al., 2007). On the other hand, fruit juices have been processed using ultrafiltration to obtain fractions enriched in phenols (i.e. flavonoids and hydroxycinnamic acid derivatives) with satisfying antioxidant activity and health promoting effects (Mangas et al., 1997; Gökmen et al., 2003; Cassano et al., 2007,
* Corresponding author. Tel.: +30 28210 37827; fax: +30 28210 88981. E-mail addresses: [email protected] (C.M. Galanakis), eva.tornberg@ food.lth.se (E. Tornberg), [email protected] (V. Gekas). 1 Tel.: +46 046 2224821. 2 Tel.: +357 25002301. 0260-8774/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2010.02.018
2008). Besides, aqueous extracts from pressed grape pomace have been treated using nanofiltration membranes for the same purpose (Díaz-Reinoso et al., 2009). Among the several fruits, olive is known to contain an appreciable amount of phenols with good antioxidant properties (Boskou et al., 2006) and dietary fibers with promising water holding capacity (Jiménez et al., 2000). Nevertheless, the majority of these compounds are lost in olive mill wastewater (OMW) during olive oil production. For example, OMW typically contains 98% of the total phenols in the olive fruit (Obied et al., 2005). OMW has also been referred to possess soluble dietary fibers and especially pectin material with satisfying gelling ability (Vierhuis et al., 2003; Cardoso et al., 2003). In our earlier studies, we recovered dietary fibers from OMW, based on the thermal extraction, prior the precipitation of macromolecules with 85 mL ethanol/100 mL (Galanakis et al., in press-b, in press-c). The developed method proposed the simultaneous recovery of dietary fibers and phenols in two different streams: (i) an alcohol insoluble residue rich in dietary fibers and (ii) an ethanolic liquid rich in phenols. The ethanolic liquid has been proposed for a direct application in the beverage industry, i.e. as an additive to produce soft drinks (Tornberg and Galanakis, 2008). Moreover, the water soluble fraction of the alcohol insoluble residue (WSAIR) has been proposed as fat replacer in meatballs achieved by dissolving the alcohol insoluble residue in water and sequentially separating it from the corresponding water insoluble fraction (Galanakis et al., in press-a). Despite the promising applications, these rather crude materials should be further clarified or
C.M. Galanakis et al. / Journal of Food Engineering 99 (2010) 190–197
separated before utilization in order to optimize their functional properties and improve their taste. For example, WSAIR was shown to contain only 5.3 g pectin/100 g, while the corresponding ash and potassium contents were nine- (46.2 g/100 g) and twofold (10.4 g/ 100 g) higher, respectively. The very high concentration of ions affected negatively both the gelling properties and the taste of this material. Thereby, membrane technologies could be utilized in order to purify WSAIR by separating pectin and ions. Similarly, the same technique could be applied in order to concentrate the phenol content of the proposed soft drinks, to modify their antioxidant profile or to clarify them from other low molecular weight (MW) compounds like salts and free sugars. The objective of the current study is to investigate the clarification as well as the recovery of high-added value ingredients like pectin and phenols from the aqueous solutions derived by the aforementioned WSAIR material and the ethanolic extract, respectively. For this purpose, both materials were processed with different ultrafiltration and nanofiltration membranes under constant conditions, i.e. temperature and circulation flux. Parameters like permeate flux and retention coefficients of several components were monitored for each feed-membrane combination. 2. Experimental 2.1. Materials Samples of OMW were collected from a local three phase olive mill production plant (Chania, Greece). The used extraction process requires 10–20 L fresh water/100 kg of olives processed to separate the olive oil from the crushed olive cake and the wastewater. Crushing and kneading temperature was kept below 30 °C, while the residence period of the OMW in the sequential extractor was 40 min. Fresh samples were collected from the output of the decanter and kept in plastic containers in the freezer ( 20 °C) until usage. Thereafter, OMW was processed according to the method described by Galanakis et al. (in press-a) for the recovery of two different materials: (i) a pectin containing water soluble, alcohol insoluble residue (WSAIR) and (ii) an ethanolic extract (85 mL/ 100 mL) rich in olive phenols. WSAIR was preserved in the freezer ( 20 °C) and ethanolic extracts were stored in the dark at 4 °C until usage. 2.2. Preparation of the feed liquids The above materials were further processed for the preparation of two different feed liquids. The first feed liquid (A) was prepared from the solubilization of WSAIR in water (6 g in 3 L) and it was named as ‘‘pectin containing solution”. The second feed liquid (B) was prepared from the ethanolic extract (85 mL/100 mL). In particular, the extract (B) was concentrated fivefold in a rotary evaporator (model Laborota 4011, Heidolph, Germany) at 60 °C and then it was diluted with water in order to obtain an ethanol concentration up to 2.0 mL/100 mL (90 mL of the condensed extract were diluted up to 3 L). This process was performed in order to increase the ratio of total phenols per ethanol concentration inside the beverage. The resulted solution was vacuum filtered sequentially with three filters (10 mm, 0.45 lm and 0.2 lm) before its utilization as feed liquid in membrane experiments in order to remove any residual fats and oils from OMW. This feed liquid was named as ‘‘phenol containing beverage”. 2.3. Membranes Five commercial membranes (Alfa Laval, Nakskov, Denmark) were investigated at the current study as presented in Table 1:
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four ultrafiltration membranes (GR40PP, GR60PP, GR81PP and GR95PP) and one nanofiltration membrane (NF99). The GR40PP and GR60PP membranes were made of polysulphone, while the GR81PP and GR95PP membranes were made of polyethersulphone. The NF99 membrane was a polymeric three-layer thin film membrane with an active layer of semiaromatic/aliphatic polyamide (polypiperazine). 2.4. Experimental set-up and operation control The membrane experiments were carried out in a plate frame module (DSS Labstak M20, Alfa Laval Nakskov, Denmark) as shown in Fig. 1. This unit is a cross-flow membrane filtration system, where membrane filter sheets as well as support- and spacer plates are stacked and compressed in a vertical frame. Plates were compressed with a hydraulic hand pump at 320 bar (model P392, Enerpac, USA). The membrane area was 0.036 m2 (two membrane sheets of 0.018 m2) and the processed feed volume was 3 L. Feed was circulated through compressed membrane plate with an auxiliary pump (hydra cell industrial pump, model G13XDSGHHEMA, Wanner Engineering Inc., USA) equipped with a motor (Varmeca10, model 013XDSGHHEMA, Leroy Somer, USA). The circulation flux was constant at 38 mL s 1. The feed inlet and the outlet temperature were kept constant at 25 ± 0.5 °C, by placing feed tank into a water bath, and controlled with temperature sensors (Pt100 class A, Pentronic AB, Sweden). The transmembrane pressure (TMP) of the feed liquid was adjusted to the appropriate level with a retentate adjusting valve. The inlet and the outlet TMPs were measured with module self-contained. The permeate flux was measured gravimetrically as the change of permeate weight with time by using a laboratory scale balance (XT120A, Precisa Instruments Ltd., Switzerland) which was monitored via a computer. Permeate flux was expressed in L/h m2. 2.5. Experimental procedure 2.5.1. Membrane pretreatment Each membrane was packed into Labstak M20 module and pretreated with de-ionised water as feed liquid (5 L) in order to minimize membrane compaction during the experiments. The GR40PP and GR60PP membranes were both pressurized at 2, 3, 4 and 5 bar in two sequential rounds (15 min duration) using fresh de-ionised water in each one. Permeate was discharged continuously. Fresh de-ionised water was added continuously in order to retain a volume of 4–5 L feed liquid. The GR81PP and GR95PP membranes were pressurized with the same process at 3, 4, 5 and 6 as well as 5, 6, 7 and 8 bar, respectively. The NF99 membrane was similarly pressurized at 4, 8, 12 and 16 bar. It is assumed that glycerine, which is used as membrane preservative is washed out with this process (Nilsson et al., 2006). 2.5.2. Process optimization Pretreated membranes were pressurized in triplicates using separately the pectin containing solution and the phenol containing beverage with the same protocol and tested pressures as above. A new pretreated membrane was utilized for all the combinations of membranes with each type of feed liquid. Permeate flux was monitored for both feed liquids and a permeate sample (10 mL) was kept for analysis at each tested pressure (Table 1). Thereafter, pectin and total phenols (determined with Folin–Ciocalteau reagent) retention coefficients were determined for the pectin containing solution and the phenol containing beverage, respectively. The optimum TMPs were selected according to the higher permeate flux as well as the higher pectin and phenol retention coefficients in each feed liquid, respectively. Permeate flux was enhanced with increasing TMP for all the tested membranes and
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Table 1 Characteristics of the utilized membranes as recommended from the supplier and corresponding operation conditions during the experiments. Membrane name
Type
Support material and active layer
Typical MWCOa
GR40PP GR60PP GR81PP GR95PP NF99
UFe UFe UFe UFe NFf
Polysulphone Polysulphone Polyethersulphone Polyethersulphone Polypiperazine
100,000 25,000 10,000 2000 120g
pH rangeb
(Da)
a b c d e f g
1–13 1–13 1–13 1–13 2–10
Temperature rangeb
Pressure rangeb
Tested pressurec
Applied pressured
(°C)
(bar)
(bar)
(bar)
0–75 0–75 0–75 0–75 0–50
1–10 1–10 1–10 1–10 1–55
2, 2, 3, 5, 4,
3, 3, 4, 6, 8,
4, 5 4, 5 5, 6 7, 8 12, 16
2 3 5 8 12
‘‘MWCO” for ‘‘molecular weight cut off”. Recommended specifications during continuous operation. During membrane pretreatment and process optimization. During clarification experiments of both feed solutions. ‘‘UF” for ‘‘ultrafiltration”. ‘‘NF” for ‘‘nanofiltration”. The manufacturer measured retention of 2000 mg/L MgSO4 solution is P99% using 9 bar and 25 °C.
Temperature sensor
Manometer
Membrane Permeate
Cooling water inlet
Digital balance, permeate flux control Control valve Outlet Manometer Temperature sensor
Pump Feed solution
Retentate
Temperature sensor Feed tank Water bath Fig. 1. Schematic diagram of the experimental setup.
both feed liquids. Finally, the same optimum TMP (Table 1) was applied for both feed liquids as it was found that pectin and phenol retention coefficients, respectively, were lower with increasing TMP above a certain value for all the tested membranes (2, 3, 5, 8 and 12 bar for GR40PP, GR60PP, GR81PP, GR95PP and NF99, respectively). 2.5.3. Clarification experiments Triplicate pretreated membranes were again utilized for each feed-membrane experiment. Three liters of feed liquid (phenol beverage or pectin solution) were processed in membrane apparatus with the optimum TMP until the recovery of 1.5 L permeate and 1.5 L retentate. Permeate flux was determined during the process and the relative flux (RF) of the feed liquid was calculated in percentage according to the following equation: RF = (Jv/Jw0)100 (%),
where Jv is the permeate flux at steady state and Jw0 is the pure water flux. Samples of feed and permeate streams were kept in the freezer ( 20 °C) until analysis. After the completion of each clarification experiment, feed liquid was replaced with de-ionised water and processed for 60 min at the same TMP in order to clean the membrane. Permeate flux was again determined with fresh deionised water and the flux recovery (FR) was calculated in percentage according to the following equation: FR = (Jwf/Jw0)100 (%), where Jwf and Jw0 are the pure water flux after and prior the clarification experiment, respectively (Gekas et al., 1993). 2.6. Analyses Samples from the treatment of the pectin containing solution were assayed for the determination of the pH and conductivity
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value, as well as the potassium, sodium, total phenols and pectin content. Total sugars were not determined for this feed liquid as it was expected to contain only traces of them. Besides, the initial material (WSAIR) for the production of this feed liquid was isolated from the OMW after a precipitation step with ethanol that is known to remove low MW compounds like free sugars into the ethanolic extract (Qi et al., 2000). Other parameters like different phenolic classes and antioxidant capacity were also not determined for this feed liquid as the main purpose was the purification of the contained pectin from the high concentrations of ions that are known to contribute negatively to its gelling properties (Galanakis et al., in press-a). On the other hand, samples from the treatment of the phenol containing beverage were assayed for the determination of the pH and conductivity value, potassium, sodium, ethanol and total sugars concentration. Moreover, the phenolic content of these samples was measured with several parameters (total phenols with Folin–Ciocalteau reagent, o-diphenols at 370 nm, total phenols at 280 nm, hydroxycinnamic acid derivatives at 320 nm and flavonols at 360 nm), while their antioxidant capacity was also determined by assaying two different activities: 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging efficacy and ferric ion reduction ability. Pectin content of the beverage was not determined as polysaccharides were expected to be removed after the precipitation of the alcohol insoluble residue (Qi et al., 2000). The determination and classification of the phenols in the extracts were measured colorimetrically using several methods. Total phenols content was determined colorimetrically using the Folin– Ciocalteau reagent. A diluted sample was mixed with 0.25 mL Folin–Ciocalteau reagent. After 3 min stirring, 1 mL saturated sodium carbonate solution was added and the final solution was left in the dark for 1 h. The absorbance of the solution was measured at 725 nm using a Shimadzu UV-mini-1240 spectrophotometer. A standard curve was prepared using 0–50 mg/L solutions of caffeic acid in methanol/water. The standard solutions were prepared with several dilutions of a mother solution in water. Mother solution was prepared as follows: 1 g of caffeic acid was solubilized in 100 mL of methanol and then 1 mL of the resulted solution was diluted to 100 mL of water. Total phenol values were expressed as caffeic acid equivalents (mg/L). Extracts were used after dilution (1:20) with de-ionised water. The determination of o-diphenols was performed following a modification of the method described by Mateos et al. (2001). In particular, 1 mL of diluted extract (1:5 in 50 mL ethanol/100 mL solution) was vigorously mixed with 1 mL of sodium molybdate dihydrate solution (5 g/100 mL in 50 mL ethanol/100 mL solution) into a 10 mL volumetric flask and the volume was made up to 10 mL with 50 mL ethanol/100 mL solution. After 15 min, the absorbance was measured at 370 nm. A blank was prepared by mixing the sodium molybdate dihydrate solution with 50 mL ethanol/100 mL solution. A standard curve was prepared using solutions of caffeic acid (0–200 mg/L) in 50 mL ethanol/100 mL solution. Results were expressed as caffeic acid equivalents (mg/L). The determination of different polyphenol classes was performed according to Obied et al. (2005). One mL of diluted ethanolic extract (1:10 in water) was mixed with 1 mL of HCl–ethanol solution (0.1 mL HCl/100 mL in 95 mL ethanol/100 mL) into a 10 mL volumetric flask and the volume was made up to 10 mL with 2 mL HCl/100 mL. After mixing, the absorbance was measured at 280, 320, and 360 nm to determine total phenols, hydroxycinnamic acid derivatives and flavonols, respectively. A blank was prepared by mixing the HCl–ethanol solution with 2 mL HCl/100 mL. The corresponding standard curves to the above determinations were prepared using solutions (10 mL ethanol/100 mL water) of gallic acid (0–200 mg/L), caffeic acid (0–100 mg/L) and quercetin (0–150 mg/L), respectively.
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Antioxidant ability of ferric ion reduction was performed following a modification of the method described by SzydlowskaCzerniak et al. (2008). In particular, 1 mL of FeCl3 solution (0.2 g/ 100 mL of absolute ethanol) and 0.5 mL of 1,10-phenanthroline solution (0.5 g/100 mL ethanol) were vigorously mixed into a 10 mL volumetric flask. After mixing, 0.6 mL of diluted extract (1:10 in absolute ethanol) was added and the volume was made up to 10 mL with absolute ethanol. The obtained solution was mixed and left in the dark. After 20 min, the absorbance was measured at 510 nm. A blank was prepared by mixing FeCl3 solution with phenanthroline reagent and the volume made up to 10 mL with absolute ethanol. Standard curve was prepared using solutions of FeSO47H2O (0–0.8 mmol/L) and the results were expressed as FeSO4 equivalents/L. DPPH radical scavenging activity of the extracts was performed following a modification of the method described by Kulisic et al. (2004). Several dilutions (100 lL) of each extract (1:5, 1:10, 1:20, 1:30 and 1:40) were vigorously mixed with 1.5 mL methanolic solution of DPPH radical (32 mg/L) in 2-mL plastic tubes. After 1 h, the absorbance at 517 nm of the resulting mixtures was measured against methanol, which was used to zero the absorbance. A blank solution of the DPPH radical without antioxidant was utilized as control sample. The percentage inhibition of the DPPH radical by the samples was calculated according to the equation: % inhibition = ((AC(0) AA(t))/AC(0)) 100, where AC(0) is the absorbance of the control at t = 0 min and AA(t) (mg DPPH/g antioxidant) is the absorbance of the antioxidant at t = 1 h. Results were expressed in Antioxidant Efficacy (AE) values: AE = 1/EC50, where EC50 is the effective concentration of the antioxidant extract that resulted in 50% scavenging of DPPH radical. Galacturonic acid (GalA), potassium, sodium and pH determinations of the samples were performed as previously described (Galanakis et al., in press-a). Conductivity values (lS/cm) of the samples were measured with a digital conductometer (K720, Consort, Belgium). Ethanol was determined in the distillate of the samples using control Gay-Lussac alcohol meters (Dujardin-Salleron Instruments, France) and expressed as alcoholic degrees (mL ethanol/100 mL of sample). Samples (100 mL) were distilled with an Alcodest module (JP Selecta, Spain). Total sugars were determined following a modification of the method described by Dubois et al. (1956). In particular, 1 mL of diluted sample was mixed vigorously with 1 mL of phenol solution (5 g/100 mL) into a tube. After mixing, 5 mL of concentrated H2SO4 were added and tube was mixed rapidly with vortex. After 10 min, the tube was shaken again and chilled in a water bath (20–25 °C). After 5 min, the absorbance was measured at 490 nm against a blank solution prepared according to the same protocol using de-ionised water instead of the sample. Standard curve was prepared using glucose solutions (5– 40 mg/L) instead of the sample. All the above determinations were performed in triplicates. Retention coefficients (R%) were calculated by the following equation: R% = 100 (Afeed Aperm)/Afeed, where R% is the retention coefficient of a parameter, while Afeed and Aperm are the values of any parameter in the feed and permeate, respectively. This value corresponded to mg/L for total sugars, total phenol (determined at 725 as well as 280 nm), o-diphenols, hydroxycinnamic acid derivatives, flavonols, potassium and sodium concentrations. 2.7. Statistical analysis Clarification experiments were performed in triplicates and the mean ± standard deviation of the three corresponding values for each variable was calculated. Data were analyzed using Students t-test (pair wise comparisons, Office Excel 2007). Significant differences between samples were detected when the acceptable level of probability was 5% (P 6 0.05) for all the comparisons.
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3. Results
3.2. Performance parameters of the clarification experiments
3.1. Characterization of the feed liquids
Performance parameters with regard to the flux of the feed liquids obtained as a function of the different tested membranes are shown in Table 3. Pure water flux before the clarification experiments was not significantly different for the GR40PP, GR60PP and NF99 membranes (95, 95 and 99 L/h m2, respectively) and more than sevenfold lower for the GR81PP and GR95PP membranes (11 and 14 L/h m2, respectively). Permeate flux of the pectin containing solution was rather low (4–15 L/h m2) for all the tested ultrafiltration membranes and higher (56 L/h m2) for the NF99 membrane. Likewise, pure water flux was decreased after the clarification experiments of the pectin containing solution for all the tested membranes (44, 46, 7, 8 and 82 L/h m2 for GR40PP, GR60PP, GR81PP, GR95PP and NF99, respectively). Moreover, FR value was ranged from 47% to 58% for all the ultrafiltration membranes (without significant differences) and was equal to 83% for the NF99. On the other hand, permeate flux of the phenol containing beverage was rather high for the GR40PP, GR60PP and NF99 membranes (76, 74 and 47 L/h m2, respectively) and very low for the GR81PP and GR95PP (3 and 2 L/h m2, respectively). After the clarification experiments of the phenol containing beverage, pure water flux was reduced up to 63, 61, 6, 9 and 81 L/h m2 for the GR40PP, GR60PP, GR81PP, GR95PP and NF99 membrane, respectively. These fluxes resulted in FR values ranging from 48% to 66% for all the ultrafiltration membranes and equal to 82% for the NF99.
The composition of both feed liquids that were utilized in the clarification experiments can be seen in Table 2. Pectin containing solution was a weakly acidic medium (pH 6.3) with rather high value of conductivity (784 lS/cm). This value was originated from the high amounts of contained cations like potassium and sodium (262 and 42 mg/L, respectively). Pectin and total phenol concentrations were ranged between the aforementioned cation contents (87 and 68 mg/L, respectively). On the other hand, the phenol containing beverage was an acidic (pH 4.9) soft drink (ethanol concentration equal to 1.6 mL/100 mL) with lower values of potassium and sodium contents (96 and 26 mg/L, respectively) compared to the pectin containing solution. These cations contents contributed to a lower value of conductivity (507 lS/cm). Moreover, this medium was found to be rich in total sugars (384 mg/L) and total phenols (280 and 112 mg/L as determined at 725 and 280 nm, respectively). Different phenolic classes like o-diphenols, hydroxycinnamic acid derivatives and flavonols were determined and found to be equal to 57, 19 and 18 mg/L, respectively. The contained phenols contributed to a beverage that was possessing antioxidant capacity, i.e. radical scavenging efficacy was equal to 1.7 mg DPPH/g and ferric ion reduction ability corresponded to 1.6 mmol FeSO4/L.
Table 2 Characteristics of the feed liquids. Values represent mean ± standard deviation (n = 3). Parameter
Unit
pH Conductivity Potassium Sodium Ethanol Total sugars Pectin Total phenols (determined at 725 nm) Total phenols (determined at 280 nm) o-Diphenols Hydroxycinnamic acid derivatives Flavonols
–
a
c
Pectin containing solution
Phenol containing beverage
mg/L mg/L mL/100 mL mg/L mg GalAa/L mg/L mg/L mg/L mg/L mg/L
6.3 ± 0.1 784 ± 17 262 ± 6 42 ± 1 n.d.b n.d.b 87 ± 7 68 ± 2 n.d.b n.d.b n.d.b n.d.b
4.9 ± 0.1 507 ± 26 96 ± 5 26 ± 2 1.6 ± 0.1 384 ± 25 n.d.b 280 ± 18 112 ± 11 57 ± 5 19 ± 2 18 ± 2
mg DPPHc/g mmol FeSO4/L
n.d.b n.d.b
1.7 ± 0.1 1.6 ± 0.2
lS/cm
Antioxidant capacity Antiradical efficacy Ferrous ions equivalents b
Feed liquid
‘‘GalA” for ‘‘galacturonic acid”. ‘‘n.d.” for ‘‘not determined”. ‘‘DPPH” for ‘‘2,2-diphenyl-1-picrylhydrazyl”.
Table 3 Performance parameters of the clarification experiments of both feed liquids obtained with different membranes. Values represent mean ± standard deviation (n = 3).
a
Membrane
Water
Pectin containing solution
Type
Jw0a (L/h m2)
Jv b (L/h m2)
Jwfc (L/h m2)
RFd (%)
FRe (%)
Jv b (L/h m2)
Phenol containing beverage Jwfc (L/h m2)
RFd (%)
FRe (%)
GR40PP GR60PP GR81PP GR95PP NF99
95 ± 5f 95 ± 7f 11 ± 1g 14 ± 2g 99 ± 3f
15 ± 2f 12 ± 3f 5 ± 1g 4 ± 1g 56 ± 1
44 ± 2f 46 ± 4f 7 ± 1g 8 ± 1g 82 ± 2
16 ± 1f 13 ± 3f 44 ± 10 g 31 ± 11g 57 ± 2
47 ± 4f 48 ± 7f 58 ± 4f 56 ± 9f 83 ± 1
76 ± 7f 74 ± 1f 3 ± 1g 2 ± 1g 47 ± 2
63 ± 4f 61 ± 3f 6 ± 1g 9 ± 1g 81 ± 4
80 ± 4f 78 ± 6f 27 ± 3 17 ± 1 48 ± 1
66 ± 2f 64 ± 5f 48 ± 9 63 ± 5f 82 ± 2
‘‘Jw0” for the ‘‘pure water flux” before clarification experiments of the feed solutions. ‘‘Jv” for the ‘‘permeate flux” at steady state. c ‘‘Jwf” for the ‘‘pure water flux” after clarification experiments of the feed solutions. d ‘‘RF” for the ‘‘relative flux” of the feed solutions. e ‘‘FR” for the ‘‘flux recovery”. f,g Values possessing the same superscripted letter within a column are not significantly different (P 6 0.05). b
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3.3. Clarification of the pectin containing solution The retention coefficients obtained for several parameters of pectin containing solution as a function of the different tested membranes are shown in Table 4. In particular, pectin retentions were quantitative (98–99%) for all the tested membranes possessing MWCO below 25 kDa (GR60PP, GR81PP, GR95PP and NF99) and the values were not significant different. Besides, pectin retention was also very high (79%) for the membrane (GR40PP) with the higher MWCO (100 kDa). On the other hand, retentions of other parameters like total phenols, potassium, sodium and conductivity were practically quantitative (99%, 96%, 91% and 93%, respectively) only for the nanofiltration membrane (NF99) and rather negligible (13%, 2%, 2% and 5%, respectively) for the GR40PP membrane. The corresponding retentions obtained for the three membranes possessing MWCO between 2 and 25 kDa (GR60PP, GR81PP and GR95PP) showed different behavior among the aforementioned characteristics. For example, ion recovery as expressed with potassium, sodium and conductivity coefficients was minor (14%, 10% and 12%, respectively) for the GR60PP membrane (MWCO equal to 25 kDa), while total phenols recovery was rather important (40%). Moreover, total phenols coefficients were very high (71% and 81%) for the membranes possessing MWCO of 10 kDa (GR81PP) and 2 kDa (GR95PP), respectively. The corresponding retentions of potassium, sodium and conductivity were a bit lower (49% and 55%, 56% and 60%, as well as 58% and 61%, respectively), while the values obtained for each parameter were not significantly different. 3.4. Clarification of the phenol containing beverage The retention coefficients obtained for several parameters of phenol containing beverage as a function of the different tested membranes are shown in Table 5. Definitely, all the retention coefficients were negligible (<1) for the GR40PP membrane (MWCO of 100 kDa) except for the flavonols recovery that was a bit higher (10%). For the rest of the tested membranes, retention coefficients showed different values among the several parameters. For example, potassium and sodium retentions were ranged from 23% to 28% and from 17% to 20%, respectively, for the tested membranes possessing MWCO between 2 and 25 kDa (GR60PP, GR81PP and GR95PP), while the values obtained for each parameter were not significantly different. Nevertheless, the corresponding retentions for the NF99 membrane were significantly higher (55% and 45%, respectively). Total sugars retention was rather low (18%, 32% and 38%) for the membranes possessing MWCO between 2 and 25 kDa (GR60PP, GR81PP and GR95PP, respectively), but quantitative (98%) for the nanofiltration membrane (NF99). Total phenols (determined at 725 and 280 nm) retention showed more or less the same tendency than total sugars: low values (10% and 19%, 21% and 33% as well as 25% and 42%, Table 4 Retention coefficients obtained for several parameters of pectin containing solution as a function of different utilized membranes. Membrane
Retention coefficienta (%)b
Type
Pectin
Total phenolsc
Potassium
Sodium
Conductivity
GR40PP GR60PP GR81PP GR95PP NF99
79 ± 3 98 ± 2* 98 ± 2* 99 ± 1* 99 ± 1*
13 ± 3 40 ± 4 71 ± 3 81 ± 4 99 ± 1
2±1 10 ± 2 49 ± 2* 55 ± 4* 96 ± 2
2±1 14 ± 2 56 ± 3* 60 ± 4* 91 ± 3
5±2 12 ± 3 58 ± 4* 61 ± 2* 93 ± 3
a Retention coefficient = 100 (Afeed Aperm)/Afeed, where Afeed and Aperm are the values of any parameter in the feed and permeate, respectively. b Values represent mean ± standard deviation (n = 3). Values possessing a superscripted star within a column are not significantly different (P 6 0.05). c Total phenols as determined at 725 nm with Folin–Ciocalteau reagent.
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respectively) for the GR60PP, GR81PP and GR95PP membranes, respectively, and very high values (70% and 91%, respectively) for the NF99 membrane. Retention coefficients of the different phenolic classes followed the same tendency as the corresponding coefficients of total phenols, but the values were generally a bit higher. In particular, o-diphenols, hydroxycinnamic derivatives and flavonols retentions were equal to 6%, 32% and 37%, respectively, for the GR60PP membrane (MWCO of 25 kDa), significantly higher (32%, 44% and 56% as well as 48%, 53% and 62%, respectively) for the membranes possessing MWCO between 2 and 10 kDa (GR81PP and GR95PP) and rather quantitative (85%, 99% and 99%, respectively) for the nanofiltration membrane (NF99). On the other hand, antiradical efficacy and ferrous ions equivalents retentions were rather low (8% and 4%, 24% and 15% as well as 36% and 24%, respectively) for the membranes possessing MWCO between 2 and 25 kDa (GR95PP, GR81PP and GR60PP) and importantly higher but not quantitative (63% and 75%, respectively) for the NF99.
4. Discussion When the pectin containing solution was supplied in the feed stream, the two membranes (GR40PP and GR60PP) possessing the highest MWCO (100 and 25 kDa, respectively) showed very good results with regard to the separation of the pectin from the ions and their recovery in the concentrate and the permeate streams, respectively. The GR40PP membrane was also able to remove quantitatively the contained phenols (retention equal to 13%) in contrast to the GR60PP that retained 40% of them. Thus, a membrane possessing MWCO between 100 and 25 kDa could hypothetically be utilized in order to optimize the clarification of the contained pectin from ions and phenols simultaneously. A drawback for the application of both membranes was the low permeate flux and RF (12–15 L/h m2 and 13–16%, respectively) as well as the limited FR (47–48%). A higher TMP could be utilized for both membranes in order to increase the efficacy of the ultrafiltration. Nevertheless, this parameter could negatively affect the recovery of the pectin, especially for the GR40PP membrane that did not possess so high retention value (79%). With regard to the application of the other ultrafiltration membranes (GR81PP and GR95PP), the separation of pectin from ions was not so distinct, as the retention of the latest was relatively high (49–60%). The corresponding retention values of the phenols were even higher (71–81%). These results were not so expected, as the pores of the membranes (MWCO of 10 and 2 kDa, respectively) were still very wide for the size of simple phenolic compounds and ions. A possible explanation is that some of the phenols in OMW could be bound to dietary fibers like pectin (Bravo et al., 1994) and thereby they could retain in association with them inside the concentrate stream. Besides, the retention of macromolecular gelling substances is generating the formation of a second or dynamic membrane that increases the retention of lower MW solutes like ions (Mulder, 1996). This hypothesis was confirmed by the very low permeate flux (4–5 L/h m2) and the restricted FR (56–58%) for both membranes, despite the rather high applied TMPs (5 and 8 bar for GR81PP and GR95PP, respectively). Nanofiltration process showed much higher permeate flux (56 L/h m2) as well as FR (83%) due to the higher applied TMP (12 bar), but the separation was rather impossible as the retention of the ingredients was quantitative (91–99%) for all the tested parameters. The high retention values (even for the monovalent ions) could be due to the concentration polarization layer of retained species (macromolecular and micromolecular) on the NF99 membrane pores. Therefore, all the membranes possessing MWCO of 10 kDa or lower could not be preferred for the clarification of the pectin.
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Table 5 Retention percentages of several parameters for phenol containing beverage obtained with different membranes.
a b c d e
Membrane
Retention coefficienta (%)b
Type
Potassium
Sodium
Conductivity
Total sugars
Total phenolsc
Total phenolsd
oDiphenols
Hydr-cinn. acidse
Flavonols
Antiradical efficacy
Ferrous ions equivalents
GR40PP GR60PP GR81PP GR95PP NF99
<1 26 ± 4* 23 ± 4* 28 ± 5*
<1 19 ± 3* 17 ± 4* 20 ± 4*
<1 21 ± 3 36 ± 3* 40 ± 4*
<1 18 ± 3 32 ± 4* 38 ± 5*
<1 10 ± 3 21 ± 3* 25 ± 5*
45 ± 5
73 ± 2
98 ± 1
70 ± 3
<1 6±3 32 ± 4* 48 ± 4* 85 ± 5
<1 32 ± 3 44 ± 3 53 ± 3 99 ± 1
10 ± 2 37 ± 4 56 ± 4* 62 ± 5*
55 ± 4
<1 19 ± 3 33 ± 3 42 ± 2 91 ± 4
<1 8±2 24 ± 3 36 ± 4 63 ± 5
<1 4±2 15 ± 3* 24 ± 5* 75 ± 5
99 ± 1
‘‘Retention coefficient” = 100 (Afeed Aperm)/Afeed, where Afeed and Aperm are the values of any parameter in the feed and permeate, respectively. Values represent mean ± standard deviation (n = 3). Values possessing a superscripted star (*) within a column are not significantly different (P 6 0.05). Determined at 725 nm. Determined at 280 nm. ‘‘Hydr-cinn. acids” for ‘‘hydroxycinnamic acid derivatives”.
The clarification of the ingredients in the phenol containing beverage was, however, proved to be more difficult. The existence of smaller compounds (i.e. sugars) and the absence of pectin macromolecules complicated their separation, as the solutes possessed MW within the same order of magnitude. For example, the membrane with the highest MWCO (GR40PP) was unable to recover any substances in the concentrate, as all of them (except for a 10% of flavonols) were quantitatively passed in the permeate stream. The separation of solutes with membranes possessing MWCO of 10 and 2 kDa (GR81PP and GR95PP) was inappreciable as the retention values were ranging from 20% to 60% for all the tested parameters. Moreover, the separation of sugars and phenols was rather impossible as the retention values of total sugars and total phenols (determined with both methods) were similar for all the assayed membranes (Table 5). This result indicates that the contained phenols possessed similar MW with sugars and subsequently they were not as much polymerized. Nevertheless, the assayed beverage is basically an aqueous medium (1.6 mL ethanol/100 mL) that cannot secure the preservation of the phenols like the aforementioned ethanolic extract. Besides, autoxidation and polymerization of low MW phenols are known to produce darkly colored polymers with characteristic off-flavor present in OMW (Niaounakis and Halvadakis, 2004). This is important to state as autoxidation should be avoided in order to obtain a beverage with satisfying odor and taste. For this purpose, the clarification of the ‘‘heavier” phenolic compounds could be conducted in order to improve the organoleptic characteristics of the beverage and to extend its self-life as a product. Hydroxycinnamic acid derivatives and flavonols seem to be ‘‘heavier” compared to o-diphenols, as the corresponding retention values showed the same tendency (flavonols > hydroxycinnamic acid derivatives > o-diphenols) for all the levels of MWCO. The separation of these phenols seem to be possible with the application of GR60PP membrane, as the o-diphenols were quantitatively passed into the permeate stream (6% retention), while the flavonols and hydroxycinnamic acid derivatives were partially retained in the concentrate stream (37% and 32% retention, respectively). Likewise, the permeate stream sustained the antioxidant properties of the beverage, as the retentions of antiradical efficacy and ferrous ions equivalents were rather negligible (8% and 4%, respectively). The last observation could be explained by the fact that o-diphenols were contained in higher amounts compared to flavonols and hydroxycinnamic acid derivatives (Table 2) inside the phenol containing beverage and generally they are known to possess higher antioxidant capacities due to their structure (Kiokias et al., 2008). Although, the performance parameters of this membrane processing were very satisfying, since permeate flux, RF and FR were relatively high (74 L/h m2, 78% and 64%, respectively). These retention data denote that the beverage could hypothetically
be concentrated more than twice, as there were no severe problems of concentration polarization or fouling. Eventually, nanofiltration of the phenol containing beverage could be utilized in order to concentrate specific phenol classes and sugars with a simultaneous clarification of them from small cations like potassium and sodium. In particular, the recovery of sugars, hydroxycinnamic acid derivatives and flavonols was quantitative (99%) and at the same time the retention of potassium and sodium was much lower (55% and 45%, respectively). The permeate flux was rather high (47 L/h m2) due to the applied TMP (12 bar) and the FR was satisfying (82%), too. Nevertheless, the main disadvantage of this application is the induced loss of the antioxidant properties (retention values of antiradical efficacy and ferrous ions equivalents was equal to 63% and 75%, respectively) due to the lower retention of o-diphenols (85%).
5. Conclusion The current study suggests that the clarification as well as the recovery of valuable compounds from pectin containing solution and phenol containing beverage is possible with the utilization of membrane technologies. The separation of these ingredients was mainly governed from the characteristics of each feed liquid as well as the MWCO of the applied membranes. With regard to the pectin containing solution, the membranes possessing MWCO between 25 and 100 kDa (GR60PP and GR40PP, respectively) were able to quantitatively recover pectin in the concentrate stream and separate it from smaller solutes like potassium, sodium and phenols that pass into the permeate stream. The clarified pectin material could be utilized as gelling agent after concentration in low fat meatballs as it was proposed in a previous study (Galanakis et al., in press-a). On the other hand, the GR60PP membrane could also be utilized for the clarification of the phenol containing beverage. This membrane was able to partially remove the heavier fragments of hydroxycinnamic acid derivatives and flavonols in the concentrate stream and at the same time to sustain the antioxidant properties of the beverage in the permeate stream. This process would improve the organoleptic characteristics and self-life of the beverage. Finally, nanofiltration could be used in order to clarify the beverage from the potassium and sodium, but this process would reduce the antioxidant ability of the product due to a loss of o-diphenols in the permeate stream.
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