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Apple Juice Clarification Using Microfiltration and Ultrafiltration Polymeric Membranes
Lebensm.-Wiss. u.-Technol., 32, 290}298 (1999)
Apple Juice Clari"cation Using Micro"ltration and Ultra"ltration Polymeric Membranes B. Girard* and L. R. Fukumoto
Agriculture and Agri-Food Canada, Paci"c Agri-Food Research Centre, 4200 Highway 97S, Summerland, B.C., V0H 1ZO (Canada) (Received July 7, 1998; accepted April 12, 1999)
The yux behavior of polyethersulfone (0.2 lm), polyvinylidene -uoride (0.2 lm), cellulose (0.2 lm, 100 kDa, 30 kDa, 10 kDa), and polysulfone (0.2 lm, 100 kDa, 30 kDa, 10 kDa) membranes was examined during dead-end ,ltration of apple juice. Membranes with molecular weight cut-o+ of 30 and 100 kDa had superior -ux performance to 0.2 lm or 10 kDa membranes. A cross-ow system equipped with various tubular polymeric membranes was also used to clarify apple juice at a temperature of 503C, a crossyow velocity of 3.3 m/s and a transmembrane pressure of 414 kPa. Steady state yuxes increased as the molecular weight cut-ow increased from 9 to 200 kDa. When challenged with P. diminuta, log reduction values between 6 and 7 were obtained with the cross-ow tubular polymeric system. Membranes between 20 and 200 kDa produced juices with similar initial characteristics that underwent comparable changes during storage at 4, 25 and 353C over 4}16 wk. The impact of xltration through the 9 kDa membrane was however noticeable on the physico-chemical properties since the apple juice had a green tint, lower soluble solids, lower yavanol content, and experienced minimal changes in browning and turbidity. Keywords: micro"ltration; ultra"ltration; polymeric membrane; apple juice; physicochemical properties; microbial challenge; sensory evaluation
Introduction The use of polymeric membranes is widespread for the clari"cation of apple juice by ultra"ltration. Padilla and McLellan (1) studied the "ltration of apple juice through 10, 50, 100 and 500 kDa polysulfone hollow "ber membranes. Initial #ux increased with molecular weight cuto! (MWCO) but the "ltered juice from all membranes had similar acidity, soluble solids and sensory quality. Although phenolic content was lower with the 500 kDa, it generally increased with MWCO as did total solids. The juice from the 100 and 500 kDa membranes had higher turbidity and brown color than the other membranes. All juices became turbid and browned during storage at 43 3C over 6 months but no signi"cant changes were observed at 18 3C. Sheu et al. (2) used 20 and 50 kDa polysulfone membranes in a plate and frame con"guration to "lter apple juice. The average #ux was 20 to 40 L/m h higher for the 50 than the 20 kDa membrane. Wu et al. (3) compared 0.1 km ceramic, 50 kDa polysulfone hollow "ber and 5 kDa polysulfone spiral-wound
PARC Contribution No. 1051 * To whom correspondence should be addressed.
0023-6438/99/050290#09 $30.00
membranes. The 0.1 km membrane permeate was darker and had higher turbidity, total solids and soluble solids. Rao et al. (4) found that apple juice permeate from a 30 kDa polyamide membrane contained more volatiles than permeate from a 50 kDa polysulfone membrane. Various membrane con"gurations of polymeric membranes have been reported for ultra"ltration of apple juice including hollow "ber (1, 3, 5), spiral-wound (3), and plate and frame (2). Although these con"gurations have better packing densities, they usually have narrow channels which make these designs more susceptible to membrane fouling than tubular con"gurations. Porter (6) lists various other advantages and disadvantages of the various con"gurations. Since freshly pressed apple juice contains a considerable amount of particulate matter, pre"ltration is often necessary when using designs with narrow channels. RoK sch (7) found apple juice could be concentrated approximately 70 times using a 25 mm diameter tubular system. Few studies have examined the e!ect of membrane material on ultra"ltration of apple juice. This paper focused on the application of polymeric membranes for apple juice "ltration and paralleled additional work on ceramic membrane published elsewhere (8). Initially, a stirred cell
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system was used for an examination of the e!ect of membrane material on apple juice "ltration. Apple juice was also "ltered through a pilot plant tubular system using ultra"ltration polymeric membranes. The e!ects of cross#ow velocity and transmembrane pressure on #ux were determined on the tubular system to optimize these operating parameters. A microbial challenge was carried out to establish the e!ectiveness of selected tubular polymeric ultra"ltration membranes in removing microorganisms. Batches of juice were also "ltered through di!erent membranes to determine the e!ect of MWCO on #ux behavior and on juice properties. Filtered juices were membrane sterilized, aseptically packaged and then stored at 4, 25 and 35 3C for up to 16 wk to examine their storage stability.
Materials and Methods Apple juice A blend of McIntosh, Spartan and Red Delicious apples (Malus domestica Borkh.) was obtained from the Summerland Research Centre orchards and from other orchards in the British Columbia Okanagan Valley. The apples were stored at 0 to 2 3C for several weeks until processed. Apples tested negative for starch using the method of Lau (9). Apples were brought to room temperature before processing. They were manually washed and then hammermilled through a 1.3 cm aperture screen. The mash was held for 60 min at room temperature to promote oxidation prior to juice extraction with a screw press (Vetter Model BA6006 Type 1/2, Postfach, Germany). Pectinex Ultra SP (0.012 mL/100 mL) and Pectinex 100L (0.006 mL/100 mL) enzymes (Novo Nordisk Biochem North America Inc., Franklinton, NC, U.S.A.) was added and the juice was kept overnight at 4 3C in a stainless steel tank after #ushing the headspace with N . Before "ltration, complete depectinization was ensured by increasing the temperature of the juice to 50 3C for 2 h using a tube-in-shell heat exchanger. The depectinization procedure was monitored using an alcohol test and a centrifugation test. The alcohol test consisted of mixing equal volumes of juice with ethanol (95 g/100 mL) in a test tube. The presence of a precipitate indicated that pectin was still present. The centrifugation test was modi"ed from the method of Ishii and Yokotsuka (10) and consisted of centrifuging 40 mL of juice at 2500;g for 5 min at 10 3C. The supernatant was "ltered through Whatman No. 41 "lter paper and the turbidity measured using a Hach Ratio/XR turbidimeter (Model 43900, Hach Co., Loveland, CO, U.S.A.). All batches of juices were depectinized to the same extent.
Stirred cell system Depectinized apple juice was "ltered through a Model 52 stirred cell of 43 mm diameter (Amicon Canada Ltd., Oakville, ON, Canada). Polyethersulfone (PES, Supor 200-60301) and polysulfone (PS, HT Tu!ryn 200-66199)
disc membranes (47 mm diam.) of 0.2 km pore size were obtained from Gelman Sciences Inc. (Montreal, PQ, Canada). Mixed cellulose esters (CE, MF type, GSWP04700) and hydrophilic polyvinylidene #uoride (PVDF, Durapore, GVWP-04700) disc membranes (47 mm diam.) of 0.2 km pore size were supplied by Millipore Corp. (Mississauga, ON, Canada). PS type membrane discs (43 mm diam.) of 10 kDa (PM10-13122) and 30 kDa (PM30-13222), and cellulosic type membrane discs (43 mm diam.) of 10 kDa (YM10, 13622), 30 kDa (YM30, 13722) and 100 kDa (YM100, 14422) were provided by Amicon Canada Ltd. (Oakville, ON, Canada). PS membrane discs of 100 kDa (GR40PP) were cut from DDS (De Danske Sukkerfabrikker) Lab 20 model membranes (Dow Danmark A/S, Separation Systems, Nakskov, Denmark). Membrane discs that were not designed for the Amicon system were carefully cut and "tted for use. Juice (50 mL) was "ltered through the stirred cell at room temperature with 276 kPa N pressure and a stirrer rotation of 750 rpm. The headspace in the stirred cell was #ushed with N before "ltration. The weight of permeate obtained from the cell was monitored over time to calculate #ux. The membrane and fouling layer resistances were determined using the following equation: *P J" k(R #R ) K D
Eqn [1]
where J is the average #ux (kg/ms) observed between concentration factors of 7.5 and 11, *P is the transmembrane pressure (Pa), k is the permeate viscosity (Pa ) s), R is the membrane resistance (m/kg), and R is K D the fouling layer resistance (m/kg). Water and juice #ux for the CE and PS micro"ltration membranes (0.2 km) were repeated six times for evaluating coe$cients of variation. All other measurements were done in duplicates.
Tubular polymeric membrane xltration system A B1 twin-entry tubular membrane module (PCI Membrane Systems Ltd., Eden Prairie, MN, U.S.A.) consisting of two parallel sets of nine tubes in series was connected to a membrane "ltration unit (APV Membrane Systems, Tonawanda, NY, U.S.A.). The tubes had a diameter of 12.5 mm and the total surface area for all 18 tubes was 0.9 m. Several PCI membranes of di!erent MWCOs and composition were tested including 200 kDa PVDF (FP200), 100 kDa PVDF (FP100), 25 kDa PES (ES625), 20 kDa PS (PU120) and 9 kDa PES (ES209). Due to the limited commercial availability, membranes made with di!erent material but the same MWCO could not be compared. The "ltration unit was setup to run in a batch mode for a feed and bleed circuit. The feed temperature was regulated by a heat exchanger connected to the feed tank. Both feed and recirculation pumps had variable drives. For #ux studies, depectinized juice (50 L) was recirculated through either 25 kDa PES or 200 kDa PVDF
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membranes. Only two tubes in parallel were used corresponding to a membrane area of 0.1 m. Both the permeate and retentate (bleed) were returned to the feed tank which was continuously #ushed with N . The feed pump was set to deliver 6 L/min and the recirculation pump was increased over 15 min to the desired cross#ow velocity (CFV). Cross#ow velocities of 3.3 and 6.7 m/s corresponded to recirculation rates of 49 and 98 L/min and pressure di!erentials of 83 and 276 kPa, respectively. The recycle #ux was continuously monitored using a graduated cylinder and stopwatch. The system was allowed to equilibrate for 30 to 50 min before increasing the transmembrane pressure (TMP) by adjusting the retentate outlet valve. Prior to batch concentration runs and microbial challenge tests, the membranes were sanitized by circulating a 50 mg/L chlorine solution at 50 3C for 30 min then rinsing with distilled water. The permeate tank and lines underwent a sterilization treatment at 121 3C and 104 kPa for 15 min. Sterile N was used to maintain a positive pressure in the system after cooling. Batches of apple juice (200 to 240 L) were "ltered through the above mentioned membranes. The operating conditions for these batch concentration runs were 3.3 m/s CFV, 414 kPa TMP, and 50 3C. The feed pump provided a #owrate of 6 L/min and the feed tank was continuously #ushed with N . The #ux was monitored using an elec tromagnetic #owmeter (Model M053724010R100A, ABB Kent-Taylor, Rochester, NY, U.S.A.) as the permeate was separately collected in a SS tank. The juice was processed to a concentration of three times for the 9 kDa membrane and 10 times for the other membranes. Prior to juice "ltration, the water #ux was measured under the same conditions. The membrane and fouling layer resistances were calculated using Eqn [1]. The "ltration unit was cleaned-in-place using a solution of 0.1 to 0.15 g/100 mL Ultrasil 10 (Klenzade, Mississauga, ON, Canada) with 100 to 200 mg/L free chlorine. Following a rinse with water, the cleaning cycle was repeated a second time.
Bottling and storage Immediately before bottling, the clari"ed juice was "ltered through a sterile 0.45 km Supor DCF capsule (Gelman Sciences Inc., Montreal, PQ, Canada). The capsule had a 0.8 km pre"lter and a 0.45 km hydrophillic PS "lter. This "ltration step ensured that microbial growth would not occur during storage. The juice was bottled into 250 mL glass jars with twist cap lids in a laminar #owhood. The jars and lids were steam sterilized prior to use. The headspace of the jars was #ushed with N "ltered through a 0.2 km PTFE membrane Hi Flo Sol-Vent capule (Gelman Sciences). Juices were stored at 35, 25 and 4 3C in the dark. At 35 3C, samples were removed after 1, 2, 3 and 4 wk storage. At 25 and 4 3C, samples were removed after 4, 8, 12 and 16 wk storage. Initial and stored samples were frozen at !30 3C until analysed.
Microbial challenge test The microbial retention of the 9 kDa and 25 kDa PES membranes was tested with Pseudomonas diminuta (Brevundimonas diminuta) ATCC 19146 (American Type Culture Collection, Rockville, MD, U.S.A.). The culture was propagated in nutrient broth at 30 3C for 24 h. The cells were separated by centrifugation at 5 000;g for 15 min at 4 3C and were resuspended in 50 L of 0.1 g/100 mL peptone to challenge a membrane with 1;10 cfu/cm. The bacterial test solution was circulated through the membrane at 25 3C, 2 m/s CFV and 207 kPa TMP with the feed pump delivering 6 L/min. The solution was recycled for 60 min before withdrawing permeate and retentate samples. CFV and TMP were then increased to 3.3 m/s and 414 kPa, respectively, and samples were taken again after 60 min. Aliquots of retentate were serially diluted with 0.1 g/100mL peptone and spread plated in duplicate on nutrient agar. Plates were incubated at 25 3C for 72 h. Two 1000 mL samples of permeate were membrane "ltered through sterile 0.45 km membranes. The membranes were incubated on absorbent pads soaked with nutrient broth at 25 3C for 72 h.
Analytical measurements Samples were analysed for color, turbidity, viscosity, soluble solids, titratable acidity, #avanols and protein. Brown color was measured at an absorbance of 420 nm using a Beckman DU 640 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA, U.S.A.). Color was also assessed spectrophotometrically with a CIE L*, a* and b* Color Determination and Matching program Version 1.0 (Beckman Instruments, Inc.). Values were calculated for standard illuminant C and the CIE 1964 Supplementary Standard Observer. Turbidity was determined with a Hach Ratio/XR turbidimeter (Model 43900, Hach Co., Loveland, CO, U.S.A.). A Brook"eld LVDV-II# viscometer with a UL adapter (Brook"eld Engineering Laboratories, Inc., Stoughton, MA, U.S.A.) was used to measure viscosity at a shear rate of 73 s\. Soluble solids were assessed with a Reichert Abbe Mark II digital refractometer (AO Scienti"c Instruments, Bu!alo, NY, U.S.A.). Juice samples (10 g) were titrated with 0.1 N NaOH to an endpoint of pH 8.1 using a Metrohm 686 Titroprocessor and 665 Dosimat (Metrohm Ltd. Switzerland) and titratable acidity was expressed as g malic acid per 100 mL juice. Flavanol content was determined by the acidi"ed vanillin method of Broadhurst and Jones (11) using (-)-epicatechin (Sigma Chemical Co., St. Louis, MO, U.S.A.) as standard. Initial protein content was measured using the Kjeldahl method. Juice samples (5 g) were dried at 70 3C in a vacuum oven (104 kPa) prior to digestion. Nitrogen was measured with a Technicon AutoAnalyzer II (Technicon Industrial Systems, Tarrytown, NY, U.S.A.) using a colorimetric assay based on the reaction of ammonia, sodium salicylate, sodium nitroprusside and sodium hypochlorite in a bu!ered alkaline medium which produced an ammonia-salicylate complex with an absorbance maximum at 660 nm. A factor of 6.25 was used to convert nitrogen concentration to
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protein concentration. For all analyses, measurements were made on two replicate samples.
Sensory Analysis Di!erences between juices "ltered through the various membranes were compared using triangle tests. Eighteen judges were selected from sta! at the Paci"c Agri-Food Research Centre. Juices were stored at !30 3C after initial processing. Prior to sensory evaluation, the juices were thawed at 4 3C. Juice samples (30 mL) were presented to each judge in covered black wine glasses with random three-digit codes. The juices were allowed to equilibrate at room temperature for 30 min before presentation. Judges were asked to smell and then taste a set of three glasses in the order given before selecting the odd sample. The triangle tests were completely randomized for the odd sample and the order of presentation. The level of signi"cance was determined according to Larmond (12).
Results and Discussion Stirred cell Micro"ltration (MF) membranes of 0.2 km pore size had similar steady state #ux for apple juice "ltered through the stirred cell. Based on water and juice #ux from cellulose (25267$2648 L/mh and 67.0$6.6 L/mh, respectively) and polysulfone (2588$243 L/mh and 64.3$ 7.2 L/mh, respectively) MF membranes, coe$cients of variation varied between 9.4 and 11.1%. Delineation between membranes were more apparent based on their relative #ux (permeate #ux/pure water #ux) (Fig. 1A). PVDF and PS comparatively gave higher values than PES. These di!erences were consistent with the results of Riedl et al. (13) and have been associated with membrane surface morphology rather than membrane surface hydrophobicity. Smooth membranes (e.g. PS and nylon) produced a dense surface layer whereas this same layer on rougher membranes (PES and PVDF) was more open (13). As with MF membranes, the initial relative #ux of 100 kDa membranes were high and the rates of permeate production decreased rapidly (Fig. 1B). Ultra"ltration (UF) membranes of 30 kDa had initial #ux declines intermediate to 100 and 10 kDa. Relative #ux of all UF membranes were also higher than that of MF membranes by at least "ve to ten fold (Fig. 1A and B). Once the membrane resistances were taken into account, less fouling was experienced by UF membranes of 30 and 100 kDa as evidenced by lower fouling layer resistances (Table 1). These results indicated that an optimum apple juice "ltration #ux exists between 0.2 km and 10 kDa membranes. Flux was expected to be lower for the 10 kDa due to high membrane resistance. Apple juice particulates of less than 0.2 km likely caused pore plugging and fouling in the MF membrane leading to increased #ux reduction compared to UF membranes.
Fig. 1 Relative #ux of depectinized apple juice in a stirred cell (TMP, 276 kPa; stirrer rotation, 750 rpm; temperature, 233C) through micro"ltration (A) membranes (}䉬}) 0.2 km cellulose; (}䉱}) 0.2 km polyvinylidene #uoride; (*;*) 0.2 km polysulfone; (}䊏}) 0.2 km polyethersulfone; and ultra"ltration, (B) membranes (}䉬}) 100 kDa cellulose; (}䉱}) 30 kDa cellulose; (*;*) 10 kDa cellulose; (}䊏}) 100 kDa polysulfone; (}䊉}) 30 kDa polysulfone; (}"}) 10 kDa polysulfone.
Flux studies with tubular membranes Typical behavior for cross#ow UF membranes is generally noted by an initial #ux increase followed by a plateau as TMP is ramped from low to high values. The pressure independent region of #ux is due to concentration polarization and the formation of a gel layer (14). Using the cross#ow tubular membrane system, the higher CFV (6.7 m/s) led to a large increase in recycle #ux with the 200 kDa PVDF membrane, but this #ux improvement was not observed with the 25 kDa PES membrane (results not shown). In cross#ow membrane "ltration processes, CFV is known to improve #ux due to higher shear rates promoting the removal of components deposited on the membrane surface (14). At higher MWCO (200 kDa), increasing CFV up to 414 kPa improved #ux because the fouling layer was a limiting factor. At lower MWCO (25 kDa), the pore size rather than the fouling layer was more restrictive and increased turbulence at the membrane surface was therefore not as e!ective.
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Table 1 Membrane resistance (R ), fouling layer resistance (R ) and total resistance (R ) for apple juice "ltration K D 2 with a stirred cell system. Membrane
Resistance? @ R K (;10 m/kg)
R D (;10 m/kg)
R 2 (;10 m/kg)
3.9 17.5 38.4 38.8 189.1 143.5 492.0 86.1 216.2 1118.3
1054.7 1233.7 1064.1 1088.6 624.4 760.9 1050.4 747.2 701.7 345.2
1058.6 1251.2 1102.4 1127.4 813.5 904.4 1542.4 833.3 917.9 1463.5
0.2 km cellulose (Millipore) 0.2 km polyethersulfone (Gelman) 0.2 km polysulfone (Gelman) 0.2 km hydrophilic polyvinylidene #uoride (Millipore) 100 kDa polysulfone (DDS) 30 kDa polysulfone (Amicon) 10 kDa polysulfone (Amicon) 100 kDa cellulose (Amicon) 30 kDa cellulose (Amicon) 10 kDa cellulose Amicon)
? Transmembrane pressure, 276 kPa; stirrer speed, 750 rpm; temperature, 23 3C @ Values for 0.2 km cellulose and polysulfone membranes are averages of six replicates while the remaining values are averages of two replicate samples
Table 2 Challenge tests of tubular polyethersulfone membranes with Pseudomonas diminuta Sampling time and conditions?
9 kDa membrane Initial After 1 h at 25 3C, 2 m/s CFV and 207 kPa TMP After 1 h at 25 3C, 3.3 m/s CFV and 414 kPa TMP 25 kDa membrane Initial After 1 h at 25 3C, 2 m/s CFV and 207 kPa TMP After 1 h at 25 3C, 3.3 m/s CFV and 414 kPa TMP
Microbial counts@ Retentate (cfu/L)
Permeate (cfu/L)
14.0;10
*
13.3;10
1.2;10
11.0;10
3.3;10
12.1;10
*
11.6;10
3.5;10
7.9;10
1.2;10
? CFV, cross#ow velocity; TMP, transmembrane pressure @ Results are the averages of two replicate samples
Microbial challenge test During the challenge tests of the 9 and 25 kDa PES membranes, the concentrations of P. diminuta in the retentate decreased slightly with time as cells adhered to the membrane and piping surfaces (Table 2). The low number of colony forming units (cfu) detected in the permeates indicated that the membranes "ltered out most microorganisms. As P. diminuta is one of the smallest known bacteria (around 0.2 km), the 9 and 25 kDa membranes were e!ective in removing microorganisms to log reduction values between 6 and 7. As the CFV and TMP were increased, more microorganisms passed through the membrane system. With a UF ceramic membrane module, a log reduction value greater than 9 can be obtained (8). The tubular polymeric membrane system used in this study can nevertheless meet the present FDA proposal of achieving a 100 000 fold reduc-
tion of microorganisms in "nished product compared to levels that may be present in untreated juice. A tangential #ow system using mineral membranes has been reported for the production of cold sterile apple juice (15). The use of a "nal dead-end "lter prior to bottling could however ensure microbial stability for both polymeric and ceramic membranes.
Batch concentration with tubular membranes A batch concentration study entails the monitoring of #ux as the retentate returns to the feed tank and the permeate is removed. The stabilized #ux increased with membrane MWCO notwithstanding the di!erences in membrane material and manufacture (Fig. 2). The highest steady state #ux (236 L/mh) was obtained with the 200 kDa PVDF membrane and the 9 kDa PES membrane had the lowest #ux (14 L/mh). Relative #ux for the UF membranes under cross#ow conditions were in the same range as those obtained with the stirred cell system. Although PVDF membranes were of larger pore size (100 kDa and 200 kDa), their #ux resistances due to the fouling layer (R ) were smaller than the 20 kDa PS and D 9 kDa PES (Table 3). Comparison of absolute #ux values with other studies is often di$cult because of di!erences in operating conditions, depectinization treatment and membrane type. The trends were consistent with that of Padilla and McLellan (1), although #ux results were lower for all membranes, except the 200 kDa membrane. Turbidity, viscosity, titratable acidity, and nitrogen (protein) content were similar in all juices (Table 4). The yellow/brown color of the juices decreased with membranes of smaller MWCOs. The visual appearance of the juice "ltered through the 9 kDa membrane was distinctly di!erent in that a slight green tint was evident. This membrane also retained more sugars and #avanols than the other membranes as noted by reduced soluble solids and #avanol content. Along with the #avanols, the 9 kDa
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Table 5 Triangle test comparisons of apple juice "ltered through various ultra"ltration membranes Comparison 200 200 200 100 100 20
vs. vs. vs. vs. vs. vs.
Correct responses?
Level of signi"cance
9 13 14 7 14 15
NS@ P(0.001 P(0.001 NS P(0.001 P(0.001
100 kDa 20 kDa 9 kDa 20 kDa 9 kDa 9 kDa
? a panel of 18 judges was used @ n.s."not signi"cant
Fig. 2 Permeate #ux and relative #ux of depectinized apple juice through ultra"ltration tubular membranes (TMP, 414 kPa; CFV, 3.3 m/s; temperature, 50 3C). (}䉬}) 200 kDa polyvinylidene #uoride; (;) 100 kDa polyvinylidene #uoride; (}䉱}) 20 kDa polysulfone; (}䊏}) 9 kDa polyethersulfone
membrane retained phenolic compounds responsible for the decrease in yellow color (A , b*). The green tinge possibly associated with the presence of chlorophyll likely became apparent once the masking e!ect of the yellow pigment was partially removed. Triangle tests were carried out to compare #avor characteristics of the juices "ltered through various membranes (Table 5). Although the juices from the 200 and 20 kDa membranes were signi"cantly di!erent from each other, their respective di!erences with the 100 kDa membrane were too small to be detected. All other tested membrane
Table 3 Membrane resistance (R ), fouling layer resistance (R ) and total resistance (R ) for apple juice "ltration K D 2 with a cross#ow tubular system Membrane
Resistance? @ R K (;10 m/kg)
R D (;10 m/kg)
R 2 (;10 m/kg)
128.5 164.3 442.3 699.7
322.6 772.3 1817.4 6670.4
451.1 936.6 2259.7 7370.1
200 kDa polyvinylidene #uoride 100 kDa polyvinylidene #uoride 20 kDa polysulfone 9 kDa polyethersulfone
? Transmembrane pressure, 414 kPa; cross#ow velocity, 3.3 m/s; temperature, 50 3C @ Results are averages of two replicate samples
Table 4 Properties of apple juice "ltered through di!erent tubular polymeric membranes Property?
A L* a* b* Turbidity (NTU) Viscosity at 253C (cps) Soluble solids (3Brix) Titratable acidity (g malic acid/100 mL juice) Flavanol content (mg/L) Protein content (mg/L)
Tubular polymeric membrane@ 200 kDa PVDF
100 kDa PVDF
20 kDa PS
9 kDa PES
0.44 95.9 !5.4 30.6 0.12 1.38 12.7 0.44
0.38 96.2 !4.9 27.7 0.09 1.37 12.3 0.37
0.34 96.6 !4.3 24.8 0.16 1.38 12.2 0.34
0.18 99.0 !4.8 14.3 0.15 1.36 11.4 0.39
66 29
80 29
? Results are the averages of two replicate samples @ PVDF, polyvinylidene #uoride; PS, polysulfone; PES, polyethersulfone
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Fig. 3 E!ect of storage temperature and time on turbidity of apple juice "ltered through (a) 200 kDa PVDF, (b) 100 kDa PVDF, (c) 20 kDa PS, and (d) 9 kDa PES membranes. (}䉬}) 35 3C; (}䊏}) 25 3C; (}䊉}) 4 3C
combinations were also found to be signi"cantly di!erent. Judges noticed the largest di!erences between juices "ltered through the 9 kDa membrane and the other membranes. They commented that the 9 kDa membrane juice was thin (low body/mouthfeel/viscosity), had less #avor and had an o!-#avor (medicinal/metallic). Besides retaining sugars and #avanols, the 9 kDa membrane may also have retained other #avor components.
Storage study The viscosity, soluble solids and titratable acidity of the juices did not change with either storage temperature or time. In addition, no microbial growth was observed during storage of the cold pasteurized juices. However, turbidity increased over time with higher storage temperatures and membranes of larger MWCOs (Fig. 3). Changes were more pronounced at 35 3C as the rates of chemical reactions generally increase with temperature. The 200 kDa membrane had the most turbidity development whereas the 9 kDa membrane had the least turbidity formation. Turbidity has previously been reported to increase with storage of juice (1). Haze formation in UF clari"ed apple juice is a potential problem. Polymerization of phenolics and interactions with other components (e.g. proteins) can lead to turbidity in fruit juice products (16, 17). Evidence that phenolics could be involved in turbidity development is supported by the #avanol content changes observed during storage (Fig. 4). Flavanol content in all membrane "ltered juices decreased in a similar fashion with storage temperature and time. On a comparative basis, #avanol content values among juices were an indicator of the degree of #avanol polymerization (18). Dur-
ing storage, changes in #avanols paralleled increases in turbidity and yellow/brown color. The 9 kDa membrane initially retained more #avanols than the other membranes and the juice consequently had the lowest changes in turbidity. The juices from all membranes had similar color changes as measured using the absorbance at 420 nm (Fig. 5). A pattern characterized by an initial decrease of about 0.1 absorbance unit followed by an increase of more than 0.1 unit was observed for most juices stored at 25 and 35 3C. The rates at which the changes occured were three times faster at 35 3C than at 25 3C. The oxidation and/or polymerization reactions involving the phenolic compounds may have created intermediate molecular species which absorbed less at 420 nm for a period of time. The length of this period was dependent on temperature. All juices stored at 4 3C consistently decreased in yellowness but did not increase again during the 15 wk period. The degradation products which progressively regained the partial loss of absorbance at 420 nm were not generated at 4 3C probably due to a slower reaction rate.
Conclusions Initial testing of di!erent membrane types during deadend "ltration of apple juice indicated that PVDF and PS membranes gave higher relative #ux than PES and CE membranes. In addition, 0.2 km or 10 kDa membranes had higher fouling layer resistances than 30 and 100 kDa membranes. With tubular polymeric UF membranes, the #ux for depectinized apple juice improved as the membrane MWCO increased from 9 to 200 kDa. Juices
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Fig. 4 E!ect of storage temperature and time on #avanol content of apple juice "ltered through (a) 200 kDa PVDF, (b) 100 kDa PVDF, (c) 20 kDa PS, and (d) 9 kDa PES membranes. (}䉬}) 35 3C; (}䊏}) 25 3C; (}䊉}) 4 3C
Fig. 5. E!ect of storage temperature and time on browning (A ) of apple juice "ltered through (a) 200 kDa PVDF, (b) 100 kDa PVDF, (c) 20 kDa PS, and (d) 9 kDa PES membranes. (}䉬}) 35 3C; (}䊏}) 25 3C; (}䊉}) 4 3C
"ltered through 200, 100 and 20 kDa membranes had similar properties, but juice "ltered through a 9 kDa membrane had lower soluble solids, #avanols and yellow/brown pigments. During storage at 4, 25 and 35 3C, the viscosity, soluble solids and titratable acidity did not
change for all juices. Under these conditions, the cold pasteurized juices were microbiologically stable. However, turbidity increased with storage especially for the higher MWCO membranes. A slight decrease in #avanol content and a slight increase in browning were also
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observed over time. Storage at refrigerated temperature (4 3C) minimized these changes. Although the juice from the 9 kDa membrane was physicochemically more stable during storage, #ux performance was comparatively low and the sensory characteristics were a!ected due to increased membrane retention of sugar, phenolic and other #avor components.
Acknowledgements This study was done in collaboration with Sun-Rype Products Limited of Kelowna, B.C. with the assistance of the Industrial Research Assistance Program. The authors would like to thank Dr M. Cli!, Dr P. Delaquis and Mr T. Kopp for their assistance. Written on behalf of the Department of Agriculture and Agri-Food, Government of Canada. Minister of Public Works and Government Services Canada 1999
References 1 PADILLA, O. I. AND MCLELLAN, M. R. Molecular weight cut-o! of ultra"ltration membranes and the quality and stability of apple juice. Journal of Food Science, 54, 1250}1254 (1989) 2 SHEU, M. J., WILEY, R. C. AND SCHLIMME, D. V. Solute and enzyme recoveries in apple juice clari"cation using ultra"ltration. Journal of Food Science, 52, 732}736 (1987) 3 WU, M. L., ZALL, R. R. AND TZENG, W. C. Micro"ltration and ultra"ltration comparison for apple juice clari"cation. Journal of Food Science, 55, 1162}1163 (1990) 4 RAO, M. A., ACREE, T. E., COOLEY, H. J. AND ENNIS, R. W. Clari"cation of apple juice by hollow "ber ultra"ltration: #uxes and retention of odor-active volatiles. Journal of Food Science, 52, 375}377 (1987) 5 HEATHERBELL, D. A., SHORT, J. L. AND STRUBI, P. Apple juice clari"cation by ultra"ltration. Confructa, 22, 157}169 (1977)
6 PORTER, M. C. Ultra"ltration. In: Porter, M. C. (Ed.), Handbook of Industrial Membrane ¹echnology. New Jersey: Noyes Publications, Park Ridge, Ch. 3., p. 136}259 (1990) 7 ROG SCH, E. Experience with ultra"ltration in the 1984 season. Confructa Studien, 29(1), 27}31 (1985) 8 FUKUMOTO, L. R., DELAQUIS, P. AND GIRARD, B. Micro"ltration and ultra"ltration ceramic membranes for apple juice clari"cation. Journal of Food Science, 63, 845}850 (1998) 9 LAU, O. L. Harvest guides for B.C. apples. B.C. Orchardist, 7(7), 1A}20A (1985) 10 ISHII, S. AND YOKOTSUKA, T. Susceptibility of fruit juice to enzymatic clari"cation by pectin lyase and its relation to pectin in fruit juice. Journal of Agricultural and Food Chemistry, 21(2), 269}272 (1973) 11 BROADHURST, R. B. AND JONES, W. T. Analysis of condensed tannins using acidi"ed vanillin. Journal of the Science of Food and Agriculture, 29, 788}794 (1978) 12 LARMOND, E. ¸aboratory Methods for Sensory Evaluation of Food. Ottawa: Publication 1637, Food Research Institute, Agriculture Canada, Ontario p. 63 (1977) 13 RIEDL, K., GIRARD, B. AND LENCKI, R. W. In#uence of membrane structure on fouling layer morphology during apple juice clari"cation. Journal of Membrane Science, 139, 155}166 (1998) 14 CHERYAN, M. Modelling of ultra"ltration processes. In: ;ltra,ltration Handbook. Technomic Publishing Co., Inc., Lancaster, PA. Ch. 4, p. 73}125 (1986) 15 GROHMANN, M. AND FEUERPEIL, H. P. Cold sterile apple juice-cold sterile "ltrates with tangential #ow "ltration. Confructa Studien, 31, 171}174 (1987) 16 NAGEL, C. W. AND SCHOBINGER, U. Investigation of the origin of turbidity in ultra"ltered apple and pear juice concentrate. Confructa Studien, 29(1), 16}22 (1985) 17 VAN BUREN, J. P. Causes and prevention of turbidity in apple juice. In: Downing, D. L. (Ed.), Processed Apple Products. New York: Van Nostrand Reinhold, Ch. 5, p. 97}120 (1989) 18 SINGLETON, V. L. Wine Phenols. In: Linskens, H. F. and Jackson, J. F. (Eds.), =ine Analysis. Berlin: Springer-Verlag, p. 198 (1988)
298
Apple Juice Clari"cation Using Micro"ltration and Ultra"ltration Polymeric Membranes B. Girard* and L. R. Fukumoto
Agriculture and Agri-Food Canada, Paci"c Agri-Food Research Centre, 4200 Highway 97S, Summerland, B.C., V0H 1ZO (Canada) (Received July 7, 1998; accepted April 12, 1999)
The yux behavior of polyethersulfone (0.2 lm), polyvinylidene -uoride (0.2 lm), cellulose (0.2 lm, 100 kDa, 30 kDa, 10 kDa), and polysulfone (0.2 lm, 100 kDa, 30 kDa, 10 kDa) membranes was examined during dead-end ,ltration of apple juice. Membranes with molecular weight cut-o+ of 30 and 100 kDa had superior -ux performance to 0.2 lm or 10 kDa membranes. A cross-ow system equipped with various tubular polymeric membranes was also used to clarify apple juice at a temperature of 503C, a crossyow velocity of 3.3 m/s and a transmembrane pressure of 414 kPa. Steady state yuxes increased as the molecular weight cut-ow increased from 9 to 200 kDa. When challenged with P. diminuta, log reduction values between 6 and 7 were obtained with the cross-ow tubular polymeric system. Membranes between 20 and 200 kDa produced juices with similar initial characteristics that underwent comparable changes during storage at 4, 25 and 353C over 4}16 wk. The impact of xltration through the 9 kDa membrane was however noticeable on the physico-chemical properties since the apple juice had a green tint, lower soluble solids, lower yavanol content, and experienced minimal changes in browning and turbidity. Keywords: micro"ltration; ultra"ltration; polymeric membrane; apple juice; physicochemical properties; microbial challenge; sensory evaluation
Introduction The use of polymeric membranes is widespread for the clari"cation of apple juice by ultra"ltration. Padilla and McLellan (1) studied the "ltration of apple juice through 10, 50, 100 and 500 kDa polysulfone hollow "ber membranes. Initial #ux increased with molecular weight cuto! (MWCO) but the "ltered juice from all membranes had similar acidity, soluble solids and sensory quality. Although phenolic content was lower with the 500 kDa, it generally increased with MWCO as did total solids. The juice from the 100 and 500 kDa membranes had higher turbidity and brown color than the other membranes. All juices became turbid and browned during storage at 43 3C over 6 months but no signi"cant changes were observed at 18 3C. Sheu et al. (2) used 20 and 50 kDa polysulfone membranes in a plate and frame con"guration to "lter apple juice. The average #ux was 20 to 40 L/m h higher for the 50 than the 20 kDa membrane. Wu et al. (3) compared 0.1 km ceramic, 50 kDa polysulfone hollow "ber and 5 kDa polysulfone spiral-wound
PARC Contribution No. 1051 * To whom correspondence should be addressed.
0023-6438/99/050290#09 $30.00
membranes. The 0.1 km membrane permeate was darker and had higher turbidity, total solids and soluble solids. Rao et al. (4) found that apple juice permeate from a 30 kDa polyamide membrane contained more volatiles than permeate from a 50 kDa polysulfone membrane. Various membrane con"gurations of polymeric membranes have been reported for ultra"ltration of apple juice including hollow "ber (1, 3, 5), spiral-wound (3), and plate and frame (2). Although these con"gurations have better packing densities, they usually have narrow channels which make these designs more susceptible to membrane fouling than tubular con"gurations. Porter (6) lists various other advantages and disadvantages of the various con"gurations. Since freshly pressed apple juice contains a considerable amount of particulate matter, pre"ltration is often necessary when using designs with narrow channels. RoK sch (7) found apple juice could be concentrated approximately 70 times using a 25 mm diameter tubular system. Few studies have examined the e!ect of membrane material on ultra"ltration of apple juice. This paper focused on the application of polymeric membranes for apple juice "ltration and paralleled additional work on ceramic membrane published elsewhere (8). Initially, a stirred cell
Article No. fstl.1999.0554 All articles available online at http://www.idealibrary.com on
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system was used for an examination of the e!ect of membrane material on apple juice "ltration. Apple juice was also "ltered through a pilot plant tubular system using ultra"ltration polymeric membranes. The e!ects of cross#ow velocity and transmembrane pressure on #ux were determined on the tubular system to optimize these operating parameters. A microbial challenge was carried out to establish the e!ectiveness of selected tubular polymeric ultra"ltration membranes in removing microorganisms. Batches of juice were also "ltered through di!erent membranes to determine the e!ect of MWCO on #ux behavior and on juice properties. Filtered juices were membrane sterilized, aseptically packaged and then stored at 4, 25 and 35 3C for up to 16 wk to examine their storage stability.
Materials and Methods Apple juice A blend of McIntosh, Spartan and Red Delicious apples (Malus domestica Borkh.) was obtained from the Summerland Research Centre orchards and from other orchards in the British Columbia Okanagan Valley. The apples were stored at 0 to 2 3C for several weeks until processed. Apples tested negative for starch using the method of Lau (9). Apples were brought to room temperature before processing. They were manually washed and then hammermilled through a 1.3 cm aperture screen. The mash was held for 60 min at room temperature to promote oxidation prior to juice extraction with a screw press (Vetter Model BA6006 Type 1/2, Postfach, Germany). Pectinex Ultra SP (0.012 mL/100 mL) and Pectinex 100L (0.006 mL/100 mL) enzymes (Novo Nordisk Biochem North America Inc., Franklinton, NC, U.S.A.) was added and the juice was kept overnight at 4 3C in a stainless steel tank after #ushing the headspace with N . Before "ltration, complete depectinization was ensured by increasing the temperature of the juice to 50 3C for 2 h using a tube-in-shell heat exchanger. The depectinization procedure was monitored using an alcohol test and a centrifugation test. The alcohol test consisted of mixing equal volumes of juice with ethanol (95 g/100 mL) in a test tube. The presence of a precipitate indicated that pectin was still present. The centrifugation test was modi"ed from the method of Ishii and Yokotsuka (10) and consisted of centrifuging 40 mL of juice at 2500;g for 5 min at 10 3C. The supernatant was "ltered through Whatman No. 41 "lter paper and the turbidity measured using a Hach Ratio/XR turbidimeter (Model 43900, Hach Co., Loveland, CO, U.S.A.). All batches of juices were depectinized to the same extent.
Stirred cell system Depectinized apple juice was "ltered through a Model 52 stirred cell of 43 mm diameter (Amicon Canada Ltd., Oakville, ON, Canada). Polyethersulfone (PES, Supor 200-60301) and polysulfone (PS, HT Tu!ryn 200-66199)
disc membranes (47 mm diam.) of 0.2 km pore size were obtained from Gelman Sciences Inc. (Montreal, PQ, Canada). Mixed cellulose esters (CE, MF type, GSWP04700) and hydrophilic polyvinylidene #uoride (PVDF, Durapore, GVWP-04700) disc membranes (47 mm diam.) of 0.2 km pore size were supplied by Millipore Corp. (Mississauga, ON, Canada). PS type membrane discs (43 mm diam.) of 10 kDa (PM10-13122) and 30 kDa (PM30-13222), and cellulosic type membrane discs (43 mm diam.) of 10 kDa (YM10, 13622), 30 kDa (YM30, 13722) and 100 kDa (YM100, 14422) were provided by Amicon Canada Ltd. (Oakville, ON, Canada). PS membrane discs of 100 kDa (GR40PP) were cut from DDS (De Danske Sukkerfabrikker) Lab 20 model membranes (Dow Danmark A/S, Separation Systems, Nakskov, Denmark). Membrane discs that were not designed for the Amicon system were carefully cut and "tted for use. Juice (50 mL) was "ltered through the stirred cell at room temperature with 276 kPa N pressure and a stirrer rotation of 750 rpm. The headspace in the stirred cell was #ushed with N before "ltration. The weight of permeate obtained from the cell was monitored over time to calculate #ux. The membrane and fouling layer resistances were determined using the following equation: *P J" k(R #R ) K D
Eqn [1]
where J is the average #ux (kg/ms) observed between concentration factors of 7.5 and 11, *P is the transmembrane pressure (Pa), k is the permeate viscosity (Pa ) s), R is the membrane resistance (m/kg), and R is K D the fouling layer resistance (m/kg). Water and juice #ux for the CE and PS micro"ltration membranes (0.2 km) were repeated six times for evaluating coe$cients of variation. All other measurements were done in duplicates.
Tubular polymeric membrane xltration system A B1 twin-entry tubular membrane module (PCI Membrane Systems Ltd., Eden Prairie, MN, U.S.A.) consisting of two parallel sets of nine tubes in series was connected to a membrane "ltration unit (APV Membrane Systems, Tonawanda, NY, U.S.A.). The tubes had a diameter of 12.5 mm and the total surface area for all 18 tubes was 0.9 m. Several PCI membranes of di!erent MWCOs and composition were tested including 200 kDa PVDF (FP200), 100 kDa PVDF (FP100), 25 kDa PES (ES625), 20 kDa PS (PU120) and 9 kDa PES (ES209). Due to the limited commercial availability, membranes made with di!erent material but the same MWCO could not be compared. The "ltration unit was setup to run in a batch mode for a feed and bleed circuit. The feed temperature was regulated by a heat exchanger connected to the feed tank. Both feed and recirculation pumps had variable drives. For #ux studies, depectinized juice (50 L) was recirculated through either 25 kDa PES or 200 kDa PVDF
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membranes. Only two tubes in parallel were used corresponding to a membrane area of 0.1 m. Both the permeate and retentate (bleed) were returned to the feed tank which was continuously #ushed with N . The feed pump was set to deliver 6 L/min and the recirculation pump was increased over 15 min to the desired cross#ow velocity (CFV). Cross#ow velocities of 3.3 and 6.7 m/s corresponded to recirculation rates of 49 and 98 L/min and pressure di!erentials of 83 and 276 kPa, respectively. The recycle #ux was continuously monitored using a graduated cylinder and stopwatch. The system was allowed to equilibrate for 30 to 50 min before increasing the transmembrane pressure (TMP) by adjusting the retentate outlet valve. Prior to batch concentration runs and microbial challenge tests, the membranes were sanitized by circulating a 50 mg/L chlorine solution at 50 3C for 30 min then rinsing with distilled water. The permeate tank and lines underwent a sterilization treatment at 121 3C and 104 kPa for 15 min. Sterile N was used to maintain a positive pressure in the system after cooling. Batches of apple juice (200 to 240 L) were "ltered through the above mentioned membranes. The operating conditions for these batch concentration runs were 3.3 m/s CFV, 414 kPa TMP, and 50 3C. The feed pump provided a #owrate of 6 L/min and the feed tank was continuously #ushed with N . The #ux was monitored using an elec tromagnetic #owmeter (Model M053724010R100A, ABB Kent-Taylor, Rochester, NY, U.S.A.) as the permeate was separately collected in a SS tank. The juice was processed to a concentration of three times for the 9 kDa membrane and 10 times for the other membranes. Prior to juice "ltration, the water #ux was measured under the same conditions. The membrane and fouling layer resistances were calculated using Eqn [1]. The "ltration unit was cleaned-in-place using a solution of 0.1 to 0.15 g/100 mL Ultrasil 10 (Klenzade, Mississauga, ON, Canada) with 100 to 200 mg/L free chlorine. Following a rinse with water, the cleaning cycle was repeated a second time.
Bottling and storage Immediately before bottling, the clari"ed juice was "ltered through a sterile 0.45 km Supor DCF capsule (Gelman Sciences Inc., Montreal, PQ, Canada). The capsule had a 0.8 km pre"lter and a 0.45 km hydrophillic PS "lter. This "ltration step ensured that microbial growth would not occur during storage. The juice was bottled into 250 mL glass jars with twist cap lids in a laminar #owhood. The jars and lids were steam sterilized prior to use. The headspace of the jars was #ushed with N "ltered through a 0.2 km PTFE membrane Hi Flo Sol-Vent capule (Gelman Sciences). Juices were stored at 35, 25 and 4 3C in the dark. At 35 3C, samples were removed after 1, 2, 3 and 4 wk storage. At 25 and 4 3C, samples were removed after 4, 8, 12 and 16 wk storage. Initial and stored samples were frozen at !30 3C until analysed.
Microbial challenge test The microbial retention of the 9 kDa and 25 kDa PES membranes was tested with Pseudomonas diminuta (Brevundimonas diminuta) ATCC 19146 (American Type Culture Collection, Rockville, MD, U.S.A.). The culture was propagated in nutrient broth at 30 3C for 24 h. The cells were separated by centrifugation at 5 000;g for 15 min at 4 3C and were resuspended in 50 L of 0.1 g/100 mL peptone to challenge a membrane with 1;10 cfu/cm. The bacterial test solution was circulated through the membrane at 25 3C, 2 m/s CFV and 207 kPa TMP with the feed pump delivering 6 L/min. The solution was recycled for 60 min before withdrawing permeate and retentate samples. CFV and TMP were then increased to 3.3 m/s and 414 kPa, respectively, and samples were taken again after 60 min. Aliquots of retentate were serially diluted with 0.1 g/100mL peptone and spread plated in duplicate on nutrient agar. Plates were incubated at 25 3C for 72 h. Two 1000 mL samples of permeate were membrane "ltered through sterile 0.45 km membranes. The membranes were incubated on absorbent pads soaked with nutrient broth at 25 3C for 72 h.
Analytical measurements Samples were analysed for color, turbidity, viscosity, soluble solids, titratable acidity, #avanols and protein. Brown color was measured at an absorbance of 420 nm using a Beckman DU 640 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA, U.S.A.). Color was also assessed spectrophotometrically with a CIE L*, a* and b* Color Determination and Matching program Version 1.0 (Beckman Instruments, Inc.). Values were calculated for standard illuminant C and the CIE 1964 Supplementary Standard Observer. Turbidity was determined with a Hach Ratio/XR turbidimeter (Model 43900, Hach Co., Loveland, CO, U.S.A.). A Brook"eld LVDV-II# viscometer with a UL adapter (Brook"eld Engineering Laboratories, Inc., Stoughton, MA, U.S.A.) was used to measure viscosity at a shear rate of 73 s\. Soluble solids were assessed with a Reichert Abbe Mark II digital refractometer (AO Scienti"c Instruments, Bu!alo, NY, U.S.A.). Juice samples (10 g) were titrated with 0.1 N NaOH to an endpoint of pH 8.1 using a Metrohm 686 Titroprocessor and 665 Dosimat (Metrohm Ltd. Switzerland) and titratable acidity was expressed as g malic acid per 100 mL juice. Flavanol content was determined by the acidi"ed vanillin method of Broadhurst and Jones (11) using (-)-epicatechin (Sigma Chemical Co., St. Louis, MO, U.S.A.) as standard. Initial protein content was measured using the Kjeldahl method. Juice samples (5 g) were dried at 70 3C in a vacuum oven (104 kPa) prior to digestion. Nitrogen was measured with a Technicon AutoAnalyzer II (Technicon Industrial Systems, Tarrytown, NY, U.S.A.) using a colorimetric assay based on the reaction of ammonia, sodium salicylate, sodium nitroprusside and sodium hypochlorite in a bu!ered alkaline medium which produced an ammonia-salicylate complex with an absorbance maximum at 660 nm. A factor of 6.25 was used to convert nitrogen concentration to
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protein concentration. For all analyses, measurements were made on two replicate samples.
Sensory Analysis Di!erences between juices "ltered through the various membranes were compared using triangle tests. Eighteen judges were selected from sta! at the Paci"c Agri-Food Research Centre. Juices were stored at !30 3C after initial processing. Prior to sensory evaluation, the juices were thawed at 4 3C. Juice samples (30 mL) were presented to each judge in covered black wine glasses with random three-digit codes. The juices were allowed to equilibrate at room temperature for 30 min before presentation. Judges were asked to smell and then taste a set of three glasses in the order given before selecting the odd sample. The triangle tests were completely randomized for the odd sample and the order of presentation. The level of signi"cance was determined according to Larmond (12).
Results and Discussion Stirred cell Micro"ltration (MF) membranes of 0.2 km pore size had similar steady state #ux for apple juice "ltered through the stirred cell. Based on water and juice #ux from cellulose (25267$2648 L/mh and 67.0$6.6 L/mh, respectively) and polysulfone (2588$243 L/mh and 64.3$ 7.2 L/mh, respectively) MF membranes, coe$cients of variation varied between 9.4 and 11.1%. Delineation between membranes were more apparent based on their relative #ux (permeate #ux/pure water #ux) (Fig. 1A). PVDF and PS comparatively gave higher values than PES. These di!erences were consistent with the results of Riedl et al. (13) and have been associated with membrane surface morphology rather than membrane surface hydrophobicity. Smooth membranes (e.g. PS and nylon) produced a dense surface layer whereas this same layer on rougher membranes (PES and PVDF) was more open (13). As with MF membranes, the initial relative #ux of 100 kDa membranes were high and the rates of permeate production decreased rapidly (Fig. 1B). Ultra"ltration (UF) membranes of 30 kDa had initial #ux declines intermediate to 100 and 10 kDa. Relative #ux of all UF membranes were also higher than that of MF membranes by at least "ve to ten fold (Fig. 1A and B). Once the membrane resistances were taken into account, less fouling was experienced by UF membranes of 30 and 100 kDa as evidenced by lower fouling layer resistances (Table 1). These results indicated that an optimum apple juice "ltration #ux exists between 0.2 km and 10 kDa membranes. Flux was expected to be lower for the 10 kDa due to high membrane resistance. Apple juice particulates of less than 0.2 km likely caused pore plugging and fouling in the MF membrane leading to increased #ux reduction compared to UF membranes.
Fig. 1 Relative #ux of depectinized apple juice in a stirred cell (TMP, 276 kPa; stirrer rotation, 750 rpm; temperature, 233C) through micro"ltration (A) membranes (}䉬}) 0.2 km cellulose; (}䉱}) 0.2 km polyvinylidene #uoride; (*;*) 0.2 km polysulfone; (}䊏}) 0.2 km polyethersulfone; and ultra"ltration, (B) membranes (}䉬}) 100 kDa cellulose; (}䉱}) 30 kDa cellulose; (*;*) 10 kDa cellulose; (}䊏}) 100 kDa polysulfone; (}䊉}) 30 kDa polysulfone; (}"}) 10 kDa polysulfone.
Flux studies with tubular membranes Typical behavior for cross#ow UF membranes is generally noted by an initial #ux increase followed by a plateau as TMP is ramped from low to high values. The pressure independent region of #ux is due to concentration polarization and the formation of a gel layer (14). Using the cross#ow tubular membrane system, the higher CFV (6.7 m/s) led to a large increase in recycle #ux with the 200 kDa PVDF membrane, but this #ux improvement was not observed with the 25 kDa PES membrane (results not shown). In cross#ow membrane "ltration processes, CFV is known to improve #ux due to higher shear rates promoting the removal of components deposited on the membrane surface (14). At higher MWCO (200 kDa), increasing CFV up to 414 kPa improved #ux because the fouling layer was a limiting factor. At lower MWCO (25 kDa), the pore size rather than the fouling layer was more restrictive and increased turbulence at the membrane surface was therefore not as e!ective.
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Table 1 Membrane resistance (R ), fouling layer resistance (R ) and total resistance (R ) for apple juice "ltration K D 2 with a stirred cell system. Membrane
Resistance? @ R K (;10 m/kg)
R D (;10 m/kg)
R 2 (;10 m/kg)
3.9 17.5 38.4 38.8 189.1 143.5 492.0 86.1 216.2 1118.3
1054.7 1233.7 1064.1 1088.6 624.4 760.9 1050.4 747.2 701.7 345.2
1058.6 1251.2 1102.4 1127.4 813.5 904.4 1542.4 833.3 917.9 1463.5
0.2 km cellulose (Millipore) 0.2 km polyethersulfone (Gelman) 0.2 km polysulfone (Gelman) 0.2 km hydrophilic polyvinylidene #uoride (Millipore) 100 kDa polysulfone (DDS) 30 kDa polysulfone (Amicon) 10 kDa polysulfone (Amicon) 100 kDa cellulose (Amicon) 30 kDa cellulose (Amicon) 10 kDa cellulose Amicon)
? Transmembrane pressure, 276 kPa; stirrer speed, 750 rpm; temperature, 23 3C @ Values for 0.2 km cellulose and polysulfone membranes are averages of six replicates while the remaining values are averages of two replicate samples
Table 2 Challenge tests of tubular polyethersulfone membranes with Pseudomonas diminuta Sampling time and conditions?
9 kDa membrane Initial After 1 h at 25 3C, 2 m/s CFV and 207 kPa TMP After 1 h at 25 3C, 3.3 m/s CFV and 414 kPa TMP 25 kDa membrane Initial After 1 h at 25 3C, 2 m/s CFV and 207 kPa TMP After 1 h at 25 3C, 3.3 m/s CFV and 414 kPa TMP
Microbial counts@ Retentate (cfu/L)
Permeate (cfu/L)
14.0;10
*
13.3;10
1.2;10
11.0;10
3.3;10
12.1;10
*
11.6;10
3.5;10
7.9;10
1.2;10
? CFV, cross#ow velocity; TMP, transmembrane pressure @ Results are the averages of two replicate samples
Microbial challenge test During the challenge tests of the 9 and 25 kDa PES membranes, the concentrations of P. diminuta in the retentate decreased slightly with time as cells adhered to the membrane and piping surfaces (Table 2). The low number of colony forming units (cfu) detected in the permeates indicated that the membranes "ltered out most microorganisms. As P. diminuta is one of the smallest known bacteria (around 0.2 km), the 9 and 25 kDa membranes were e!ective in removing microorganisms to log reduction values between 6 and 7. As the CFV and TMP were increased, more microorganisms passed through the membrane system. With a UF ceramic membrane module, a log reduction value greater than 9 can be obtained (8). The tubular polymeric membrane system used in this study can nevertheless meet the present FDA proposal of achieving a 100 000 fold reduc-
tion of microorganisms in "nished product compared to levels that may be present in untreated juice. A tangential #ow system using mineral membranes has been reported for the production of cold sterile apple juice (15). The use of a "nal dead-end "lter prior to bottling could however ensure microbial stability for both polymeric and ceramic membranes.
Batch concentration with tubular membranes A batch concentration study entails the monitoring of #ux as the retentate returns to the feed tank and the permeate is removed. The stabilized #ux increased with membrane MWCO notwithstanding the di!erences in membrane material and manufacture (Fig. 2). The highest steady state #ux (236 L/mh) was obtained with the 200 kDa PVDF membrane and the 9 kDa PES membrane had the lowest #ux (14 L/mh). Relative #ux for the UF membranes under cross#ow conditions were in the same range as those obtained with the stirred cell system. Although PVDF membranes were of larger pore size (100 kDa and 200 kDa), their #ux resistances due to the fouling layer (R ) were smaller than the 20 kDa PS and D 9 kDa PES (Table 3). Comparison of absolute #ux values with other studies is often di$cult because of di!erences in operating conditions, depectinization treatment and membrane type. The trends were consistent with that of Padilla and McLellan (1), although #ux results were lower for all membranes, except the 200 kDa membrane. Turbidity, viscosity, titratable acidity, and nitrogen (protein) content were similar in all juices (Table 4). The yellow/brown color of the juices decreased with membranes of smaller MWCOs. The visual appearance of the juice "ltered through the 9 kDa membrane was distinctly di!erent in that a slight green tint was evident. This membrane also retained more sugars and #avanols than the other membranes as noted by reduced soluble solids and #avanol content. Along with the #avanols, the 9 kDa
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Table 5 Triangle test comparisons of apple juice "ltered through various ultra"ltration membranes Comparison 200 200 200 100 100 20
vs. vs. vs. vs. vs. vs.
Correct responses?
Level of signi"cance
9 13 14 7 14 15
NS@ P(0.001 P(0.001 NS P(0.001 P(0.001
100 kDa 20 kDa 9 kDa 20 kDa 9 kDa 9 kDa
? a panel of 18 judges was used @ n.s."not signi"cant
Fig. 2 Permeate #ux and relative #ux of depectinized apple juice through ultra"ltration tubular membranes (TMP, 414 kPa; CFV, 3.3 m/s; temperature, 50 3C). (}䉬}) 200 kDa polyvinylidene #uoride; (;) 100 kDa polyvinylidene #uoride; (}䉱}) 20 kDa polysulfone; (}䊏}) 9 kDa polyethersulfone
membrane retained phenolic compounds responsible for the decrease in yellow color (A , b*). The green tinge possibly associated with the presence of chlorophyll likely became apparent once the masking e!ect of the yellow pigment was partially removed. Triangle tests were carried out to compare #avor characteristics of the juices "ltered through various membranes (Table 5). Although the juices from the 200 and 20 kDa membranes were signi"cantly di!erent from each other, their respective di!erences with the 100 kDa membrane were too small to be detected. All other tested membrane
Table 3 Membrane resistance (R ), fouling layer resistance (R ) and total resistance (R ) for apple juice "ltration K D 2 with a cross#ow tubular system Membrane
Resistance? @ R K (;10 m/kg)
R D (;10 m/kg)
R 2 (;10 m/kg)
128.5 164.3 442.3 699.7
322.6 772.3 1817.4 6670.4
451.1 936.6 2259.7 7370.1
200 kDa polyvinylidene #uoride 100 kDa polyvinylidene #uoride 20 kDa polysulfone 9 kDa polyethersulfone
? Transmembrane pressure, 414 kPa; cross#ow velocity, 3.3 m/s; temperature, 50 3C @ Results are averages of two replicate samples
Table 4 Properties of apple juice "ltered through di!erent tubular polymeric membranes Property?
A L* a* b* Turbidity (NTU) Viscosity at 253C (cps) Soluble solids (3Brix) Titratable acidity (g malic acid/100 mL juice) Flavanol content (mg/L) Protein content (mg/L)
Tubular polymeric membrane@ 200 kDa PVDF
100 kDa PVDF
20 kDa PS
9 kDa PES
0.44 95.9 !5.4 30.6 0.12 1.38 12.7 0.44
0.38 96.2 !4.9 27.7 0.09 1.37 12.3 0.37
0.34 96.6 !4.3 24.8 0.16 1.38 12.2 0.34
0.18 99.0 !4.8 14.3 0.15 1.36 11.4 0.39
66 29
80 29
? Results are the averages of two replicate samples @ PVDF, polyvinylidene #uoride; PS, polysulfone; PES, polyethersulfone
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108 23
17 34
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Fig. 3 E!ect of storage temperature and time on turbidity of apple juice "ltered through (a) 200 kDa PVDF, (b) 100 kDa PVDF, (c) 20 kDa PS, and (d) 9 kDa PES membranes. (}䉬}) 35 3C; (}䊏}) 25 3C; (}䊉}) 4 3C
combinations were also found to be signi"cantly di!erent. Judges noticed the largest di!erences between juices "ltered through the 9 kDa membrane and the other membranes. They commented that the 9 kDa membrane juice was thin (low body/mouthfeel/viscosity), had less #avor and had an o!-#avor (medicinal/metallic). Besides retaining sugars and #avanols, the 9 kDa membrane may also have retained other #avor components.
Storage study The viscosity, soluble solids and titratable acidity of the juices did not change with either storage temperature or time. In addition, no microbial growth was observed during storage of the cold pasteurized juices. However, turbidity increased over time with higher storage temperatures and membranes of larger MWCOs (Fig. 3). Changes were more pronounced at 35 3C as the rates of chemical reactions generally increase with temperature. The 200 kDa membrane had the most turbidity development whereas the 9 kDa membrane had the least turbidity formation. Turbidity has previously been reported to increase with storage of juice (1). Haze formation in UF clari"ed apple juice is a potential problem. Polymerization of phenolics and interactions with other components (e.g. proteins) can lead to turbidity in fruit juice products (16, 17). Evidence that phenolics could be involved in turbidity development is supported by the #avanol content changes observed during storage (Fig. 4). Flavanol content in all membrane "ltered juices decreased in a similar fashion with storage temperature and time. On a comparative basis, #avanol content values among juices were an indicator of the degree of #avanol polymerization (18). Dur-
ing storage, changes in #avanols paralleled increases in turbidity and yellow/brown color. The 9 kDa membrane initially retained more #avanols than the other membranes and the juice consequently had the lowest changes in turbidity. The juices from all membranes had similar color changes as measured using the absorbance at 420 nm (Fig. 5). A pattern characterized by an initial decrease of about 0.1 absorbance unit followed by an increase of more than 0.1 unit was observed for most juices stored at 25 and 35 3C. The rates at which the changes occured were three times faster at 35 3C than at 25 3C. The oxidation and/or polymerization reactions involving the phenolic compounds may have created intermediate molecular species which absorbed less at 420 nm for a period of time. The length of this period was dependent on temperature. All juices stored at 4 3C consistently decreased in yellowness but did not increase again during the 15 wk period. The degradation products which progressively regained the partial loss of absorbance at 420 nm were not generated at 4 3C probably due to a slower reaction rate.
Conclusions Initial testing of di!erent membrane types during deadend "ltration of apple juice indicated that PVDF and PS membranes gave higher relative #ux than PES and CE membranes. In addition, 0.2 km or 10 kDa membranes had higher fouling layer resistances than 30 and 100 kDa membranes. With tubular polymeric UF membranes, the #ux for depectinized apple juice improved as the membrane MWCO increased from 9 to 200 kDa. Juices
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Fig. 4 E!ect of storage temperature and time on #avanol content of apple juice "ltered through (a) 200 kDa PVDF, (b) 100 kDa PVDF, (c) 20 kDa PS, and (d) 9 kDa PES membranes. (}䉬}) 35 3C; (}䊏}) 25 3C; (}䊉}) 4 3C
Fig. 5. E!ect of storage temperature and time on browning (A ) of apple juice "ltered through (a) 200 kDa PVDF, (b) 100 kDa PVDF, (c) 20 kDa PS, and (d) 9 kDa PES membranes. (}䉬}) 35 3C; (}䊏}) 25 3C; (}䊉}) 4 3C
"ltered through 200, 100 and 20 kDa membranes had similar properties, but juice "ltered through a 9 kDa membrane had lower soluble solids, #avanols and yellow/brown pigments. During storage at 4, 25 and 35 3C, the viscosity, soluble solids and titratable acidity did not
change for all juices. Under these conditions, the cold pasteurized juices were microbiologically stable. However, turbidity increased with storage especially for the higher MWCO membranes. A slight decrease in #avanol content and a slight increase in browning were also
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observed over time. Storage at refrigerated temperature (4 3C) minimized these changes. Although the juice from the 9 kDa membrane was physicochemically more stable during storage, #ux performance was comparatively low and the sensory characteristics were a!ected due to increased membrane retention of sugar, phenolic and other #avor components.
Acknowledgements This study was done in collaboration with Sun-Rype Products Limited of Kelowna, B.C. with the assistance of the Industrial Research Assistance Program. The authors would like to thank Dr M. Cli!, Dr P. Delaquis and Mr T. Kopp for their assistance. Written on behalf of the Department of Agriculture and Agri-Food, Government of Canada. Minister of Public Works and Government Services Canada 1999
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