Journal of Food Engineering 47 (2001) 195±202
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Concentration of passion fruit juice on an industrial pilot scale using osmotic evaporation F. Vaillant a,c, E. Jeanton a,b, M. Dornier a,b,*, G.M. OÕBrien c, M. Reynes a, M. Decloux b a b
Centre International de Recherche Agronomique pour le D eveloppement/FLHOR Dept., B.P. 5085, 34032 Montpellier Cedex 1, France Ecole Nationale Sup erieure des Industries Alimentaires/Tropical Food Engineering Dept., B.P. 5098, 34033 Montpellier Cedex 1, France c Universidad del Valle, Depto. de Ciencia y Tecnologõa de Alimentos, Sede Mel endez, Cali, Colombia Received 21 February 2000; accepted 1 July 2000
Abstract Osmotic evaporation to concentrate clari®ed passion fruit juice was tried out on an industrial scale. A pilot plant that was equipped with a module containing 10.2 m2 of polypropylene hollow ®bres was used to concentrate passion fruit juice up to a total soluble solids (TSS) content higher than 60 g/100 g at 30°C. Tangential velocity, temperature and concentration of solutions signi®cantly in¯uenced evaporation ¯ux. An average evaporation ¯ux of almost 0.75 kg hÿ1 mÿ2 was obtained with water, 0.65 kg hÿ1 mÿ2 when juice was concentrated to 40 g TSS/100 g and 0.50 kg hÿ1 mÿ2 when it reached 60 g TSS/100 g. A long-term trial, lasting 28 h, was successfully carried out without membrane fouling. Osmotic evaporation can be also conducted as a multistage procedure, giving a constant evaporation ¯ux of around 0.62 kg hÿ1 mÿ2 when juice was concentrated from 14 to 60 g TSS/100 g. Sensory quality and vitamin C content were well preserved in the concentrated juice. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Concentration; Fruit juice; Membrane contactor; Osmotic evaporation
1. Introduction For economic reasons (reduced transport and storage costs), fruit juices are routinely concentrated. This is especially true in the case of tropical fruit juices for which centres of production and consumption are normally far apart geographically. Classical thermal concentration techniques lead to subsequent losses of aromatic compounds and vitamins. Especially for tropical fruits, which are usually valued for their distinctive aromas, these losses are a serious marketing problem. For passion fruit, Casimir, Keord and Whit®eld (1981) have reported important losses of the initial aromatic compounds when classical concentration was applied, even when an aroma recuperation unit was used. Additionally, technological improvements to thermal concentration methods, while lessening the damage they cause, have more or less reached their peak. Despite improvements, thermal processing continues to lead to an inevitable loss of ¯avour and nutrients, and the re*
Corresponding author. Tel.: +33-467-61-4432; fax: +33-467-614433. E-mail address:
[email protected] (M. Dornier).
sulting concentrates tend towards the low-quality end of the market. Meanwhile, a demand for fruit juices with better conserved nutritional and sensory qualities is increasing in industrialised countries (Ganlmann, 1993). During the last three decades, eorts have been made to develop new technologies such as cryoconcentration and reverse osmosis that would more satisfactorily conserve the original qualities of thermosensitive aromatic fruit juices. Nevertheless, these methods have been relatively less used in industry because of the diculties in reaching juice concentration levels beyond 40 g TSS/100 g (Gostoli, 1998; Jariel, Reynes, Courel, Durand, Dornier, & Deblay, 1996). Osmotic evaporation (OE) is a relatively new technology based on the use of a hydrophobic microporous membrane to separate two liquid phases that dier greatly in terms of solute concentration (Deblay, 1991; Hogan, Canning, Peterson, Johnson, & Michaels, 1998; Lefebvre, 1988). The membraneÕs hydrophobic nature prevents penetration of the pores by aqueous solutions, creating air gaps within the membrane. The dierence in water activity (Aw ) between the two sides of the membrane induces a partial pressure gradient in the vapour phase. Vapour is transferred across the pores from the high-vapour pressure phase to the low one. This transfer
0260-8774/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 0 - 8 7 7 4 ( 0 0 ) 0 0 1 1 5 - 1
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Notation F Jw L M P T
¯ow (kg hÿ1 ) evaporation ¯ux (kg hÿ1 mÿ2 ) liquid level (m) mass (kg) pressure (bar) temperature (°C)
is isothermal. OE can be carried out at low temperatures without the need for a pressure dierential, thus improving preservation of volatile compounds (Barbe, Bartley, Jacobs, & Johnson, 1998). Osmotic evaporation has been studied mainly in the laboratory, using sucrose solutions, orange, grape or tomato juices (Courel, Dornier,Herry, Rios, & Reynes, 2000; Durham & Nguyen, 1994; Sheng, Johnson, & Lefebvre, 1991). Some work on an industrial level has been carried out on the concentration of wine production in Australia (Johnson, Valks, & Lefebvre, 1989; Thompson, 1991) but little is known implement OE on a process line. The aim of this study is to evaluate the potential of OE for concentrating clari®ed passion fruit juice on an industrial scale, taking into account the relevant impact on the overall product quality.
2. Materials and methods 2.1. Passion fruit juice and quality evaluation Processed in the PASSICOL S.A. plant (Chinchina, Colombia), raw passion fruit juice was clari®ed, using a cross¯ow micro®ltration plant, ®tted with a 0.2 lm ceramic membrane (Vaillant, Millan, O'brien, Dornier, & Decloux, 1999). To reduce viscosity and facilitate micro®ltration, the juice was ®rst lique®ed with enzymes. The clari®ed juice was then stored at )20°C until needed. Juice was analysed for total soluble solids (TSS) content, titratable acidity and density, using standard AOAC methods (AOAC, 1990). Vitamin C was analysed by the PelletierÕs iodine method (Pelletier, 1985). The viscosity was determined with a glass, capillary, routine viscometer (Cannon-Fenske) in a thermostatically controlled water bath. The water activity was determined with an Aw -meter (Novasina). A panel of 24 tasters, who were speci®cally trained to taste passion fruit juice, made a sensory evaluation of dierently processed juices. Owing to the passion fruitÕs high acidity, tests were done with a soft drink prepared with 15% of juice standardised at 14 g TSS/100 g with 8.5% sucrose and water added. The tasters were invited to distinguish between a drink containing fresh clari®ed
TSS
total soluble solids (g/100 g)
Greek Symbols l dynamic viscosity (Pa s) Subscripts b brine c concentrate f feed
juice and a drink made from juice concentrate according to the triangle dierence method. According to statistical analysis, we assumed that no dierences were detected between the samples (5% con®dence level) when 15 or more tasters failed to distinguish the right sample. Another test was done by comparing soft drinks prepared from (1) fresh juice, (2) juice pasteurised at 85°C for 55 s in a plate heat exchanger, (3) juice thermally concentrated in a Centritherm CT9 evaporator (AlphaLaval), ®tted with an aroma recovery system, and (4) OE-concentrated juice. Additionally, the juice from the OE concentrate was reconstituted with insoluble solids (pulp) from pasteurised juice that had previously been centrifuged at 3000 g. The supernatant was removed and replaced by an equal volume of OE concentrate reconstituted at 14 g TSS/100 g. The new supernatant blend with pulp was homogenised with a vortex. The tasters were asked to qualify the dierent drinks, on a scale of 0±10 according to their aroma, colour, taste and overall impression, against a check sample of fresh juice to which the score 10 was attributed for all the characteristics. 2.2. Concentration procedure The OE unit was furnished by the COGIA Company (Palaiseau, France) and features a module that contains polypropylene hollow ®bres. The internal diameter of the ®bres was 1.8 mm, the external diameter was 2.6 mm, and the average pore diameter was 0.2 lm. The total eective area was 10.2 m2 (Fig. 1). The juice to be concentrated was continuously circulated in a closed loop, which included the membrane cartridge. The juice circulated inside the hollow ®bres (hold-up volume of 12.9 L) with an average velocity of 0.24 m sÿ1 . The closed loop was continuously fed with fresh juice and the concentrate was extracted when the TSS set-up was reached. Compared with the batch con®guration already tested in previous studies (Courel, 1999), this continuous-feed process minimises microbiological and oxidation risks. Passing along the membraneÕs other face, brine was circulated between the cartridge and the thermal evaporation tank for regeneration. Circulation of both brine and juice was co-current. Calcium chloride was chosen
F. Vaillant et al. / Journal of Food Engineering 47 (2001) 195±202
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Fig. 1. Schematic of the industrial pilot plant of osmotic evaporation.
as the extracting solution because it is not toxic and because of its low Aw at saturation (0.33 at 25°C), its ready availability and low cost. The brine was maintained close to 5.3 M (45% w/w) by keeping it at an almost constant volume in the rig with level sensors controlling the heat in the evaporation tank. Before entering the membrane cartridge, the concentrated boiling brine from the thermal evaporation tank is cooled to the set-up temperature in a heat exchanger fed with chilled water. Before going back the evaporator, the diluted brine emerging from the cartridge is preheated by another exchanger fed with hot water vapours. Only the brine temperature could be controlled by modulating the ¯ow of chilled water into the ®rst heat exchanger. Tangential velocity of the brine was maintained constant and estimated at 1.8 ´ 10ÿ3 m sÿ1 . Pressures at the entrances to both the concentrate loop and the brine rig were registered with two pressure gauges, mainly to control the pressure dierentials between the two sides of the membrane. Dierential pressure can aect membrane integrity if the value is above the intrusion pressure given by the Laplace equation. The tolerance limit of the dierential pressure was estimated as being 1.5 bar with water for the used membrane. The feed ¯ow rate was registered with an electromagnetic ¯owmeter (Krohne, Duisburg, Germany), connected to a computer via a programmable logic controller (FPO, Matsushita Electric Works, Osaka, Japan). The ¯ow of extracted concentrate is measured with a balance (Mettler 0.01 kg) placed under the collecting tank and after a density adjustment. These ¯ow rates were used to calculate an average evaporation ¯ux (Jw ) every minute and reported against time. After each trial, the pilot plant was cleaned by ®rst thoroughly rinsing the concentrate loop with pre-®ltered tap water at 40°C until the emerging water produced a
TSS near zero. Then, an alkaline solution (NaOH ± 0.38 N) was circulated for 30 min at 50°C. Finally, the circuit was rinsed with de-ionised and distilled water. 3. Results and discussion 3.1. Preliminary tests with water The ®rst set of experiments was carried out with tap water at around 30°C to evaluate the evaporation performance of the pilot plant. The evaporation ¯ux ¯uctuated between 0.72 and 0.81 kg hÿ1 mÿ2 , whereas the water temperature ¯uctuated between 28°C and 31°C, and the brine concentration ¯uctuated between 5.1 and 5.6 M. The relative pressures inside both the concentrate loop and the brine rig remained constant, at around 0.1 bar, which corresponded to the pressure drop on both sides of the membrane. The water temperature inside the closed loop closely followed that of the brine, including ¯uctuations, keeping a stable dierence of around 2°C. The water temperature is therefore indirectly controlled by changes in the brineÕs temperature. A temperature decline in water submitted to OE negatively aects the evaporation rate. Under similar conditions of brine concentration, evaporation ¯ux drops 9% from when water was 31°C to when it was 28°C. Under the same operating conditions, water evaporation ¯ux diered by only 3%. 3.2. Concentrating juice by osmotic evaporation 3.2.1. Concentration without removing concentrate Experimental results are shown in Fig. 2. With a constant feeding of the clari®ed juice, raising the
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Fig. 2. Concentration of clari®ed passion fruit juice using osmotic evaporation without removing the concentrate (28°C < Tc < 31°C, Tb 28°C).
concentration within the closed loop from 14 to 60 g TSS/100 g took almost 12 h. This long period resulted from the high ratio between the total hold-up volume of the concentrate loop (12.9 L) and the average ¯ow of evaporation (about 6 kg hÿ1 ). At ®rst, the temperature of both the juice and brine was 28°C, and the brine concentration was 5.3 M, giving rise to an evaporation ¯ux of 0.62 kg hÿ1 mÿ2 . The evaporation ¯ux started to increase steadily towards a peak of 0.73 kg hÿ1 mÿ2 as a result of the brine concentration peaking at 5.5 M. The ¯ux then tended to decrease, following the steady decrease in brine concentration. The important in¯uence of the brineÕs concentration on evaporation ¯ux was highlighted during these ®rst hours of operation. When concentration inside the loop reached 45 g TSS/100 g, even as the brine concentration was increasing, ¯ux continued to decrease, corresponding to the juiceÕs increased viscosity (Fig. 2). Evaporation ¯ux reached a minimum of 0.50 kg hÿ1 mÿ2 when juice concentration peaked at 63 g TSS/100 g. These results indicate the strong in¯uence that TSS content of the
concentrate has on the evaporation ¯ux as, during the last part of this trial, concentration of the brine remained almost a constant at 5:4 0:1 M. Throughout the trial, brine temperature was maintained almost constant at 28°C 1. Even so, juice temperature increased steadily to 31°C, because of generation of heat from the viscous ¯uid circulation in the pump. Pressures inside the brine rig remained constant because the solutionÕs concentration in the range of variation found in the trial did not aect the solutionÕs viscosity. In contrast, in the concentrate loop, when concentration reached a value of about 42 g TSS/100 g, pressure began to increase exponentially. When concentration was 63 g TSS/100 g, pressure increased to almost 1.0 bar, which, when compared with the pressure drop in the brine rig, had still remained safe for the membrane. Consequently, this trial proved that OE can concentrate clari®ed juice to at least 63 g TSS/100 g at about 30°C. Complementary trials showed it was possible to reach a concentration of 69 g TSS/100 g. At the same brine concentration (5.45 M) and temperature (30°C), evaporation ¯ux decreased from 0.73 kg hÿ1 mÿ2 at 30 g TSS/100 g to 0.55 kg hÿ1 mÿ2 at 60 g TSS/100 g. This 25% decrease showed that evaporation ¯ux is strongly aected by concentration, particularly when it is more than 40 g TSS/100 g. Indeed, the break-even point of the viscosity curve with respect to concentration corresponds to this TSS value. It is the point where the juice viscosity increases exponentially and the evaporation ¯ux decreases steadily, even though brine concentration is either increasing or is a constant. As viscosity is also heavily dependent on temperature, its in¯uence on the evaporation rate also increases at high concentration values. We can therefore deduce that, at low TSS, as in the case of water, evaporation ¯ux seems to depend mainly on brine concentration. At concentration values higher than 40 g TSS/100 g, evaporation rate depends predominantly on juice viscosity and consequently on juice concentration and temperature. These observations corroborate results obtained for sucrose solutions at the laboratory scale (Courel et al., 2000). 3.2.2. Concentration with continuous extraction of concentrate The following experiments were carried out over 28 h while continuously extracting the concentrate at dierent TSS levels, to show that OE can be continuously conducted, and to ®nd the average values of the evaporation rate when concentration inside the loop was at 40 and 60 g TSS/100 g. These concentration values were chosen in function of, respectively, hygiene during the process and commercial usage. Indeed, 40 g TSS/100 g is the lowest concentration at which growth of microor-
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ganisms is limited, at least during the time needed for the process, as it corresponds to a water activity of below 0.92. However, a concentration level of 60 g TSS/100 g is needed for commercial operation, even though the evaporation ¯ux would be signi®cantly lower, as found before. Experimental results (Fig. 3) show that, thanks to an important inertia, the TSS of the extracted concentrate could be easily controlled to within 1.5 g TSS/100 g of the desired value during various hours of operation. As observed before, the evaporation ¯ux follows a general decreasing trend when juice concentration increases. Nonetheless, we can observe three distinct phases where evaporation rate ¯uctuates around an average value. The ®rst phase corresponds to the extraction of concentrate at 40 g TSS/100 g, the second when concentrate was extracted at 60 g TSS/100 g and the third when the tangential velocity on the concentrate side was abruptly decreased. During these phases, when concentration and circulation ¯ows were constant during extraction, ¯ux depended mainly on the ¯uctuations of brine concentration. Under almost identical conditions of operation (brine concentration
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at 5:3 0:2 M and juice temperature at 30°C 0:7), the average evaporation rate was almost 0.66 kg hÿ1 mÿ2 at 40 g TSS/100 g and only 0.49 kg hÿ1 mÿ2 at 60 g TSS/100 g. The average decrease in evaporation rate is about 26%, comparing concentrations at 40 and 60 g TSS/100 g, under similar operational conditions. At the end of an almost steady state operation, when 60 g TSS/100 g concentration was achieved and concentrates were collected in over various hours, we deliberately decreased the circulation ¯ow abruptly, reducing the tangential velocity from 0.24 to 0.09 m sÿ1 . The evaporation ¯ux also dropped by almost 20% from an average 0.49 to 0.40 kg hÿ1 mÿ2 . At high concentration levels, tangential velocity strongly in¯uences the evaporation rate. In fact, we can assume that, as mass transfer occurs only at the membraneÕs surface, the liquid near the surface becomes increasingly concentrated. Once it reaches a critical concentration level, its viscosity begins to rise rapidly with an increased polarisation eect that is highly sensitive to hydrodynamic parameters and, consequently, to further water removal. The water evaporation ¯ux ¯uctuations in this experiment can be explained by operating parameters (tangential velocity, temperature and concentration of both brine and juice). Almost the same evaporation ¯uxes were achieved under similar conditions, but independently of time. This fact tends to show that, at least over the trial period of almost 28 h, no dynamic fouling layer was formed at the membraneÕs surface. 3.3. Conducting multistage concentration
Fig. 3. Concentration of clari®ed passion fruit juice using osmotic evaporation with the continuous extraction of concentrate (26°C < Tc < 29°C, Tb 30°C).
During OE, as applied to clari®ed juice, the concentration ¯ux is aected negatively by the concentration level achieved. Even so, compared with other Ôlow temperatureÕ concentration techniques such as reverse osmosis or cryoconcentration, the decrease is not as dramatic. Under similar conditions, the average evaporation ¯ux decreased by only about 12% between 0 and 40 g TSS/100 g and by 26% between 40 and 60 g TSS/100 g. To obtain a better overall performance during concentration, OE can be conducted within a multistage con®guration. An experiment was conducted with the concentrated juice at 40 g 1 TSS/100 g extracted from previous OE trials, to raise the concentration to 60 g TSS/100 g. Fig. 4 shows that when a concentrate of 40 g TSS/100 g circulates inside the loop, the average water ¯ux is about 0.65 kg hÿ1 mÿ2 . Under similar conditions, when the concentrate is increased to 60 g TSS/100 g, the water ¯ux decreases to an average 0.50 kg hÿ1 mÿ2 . About the same average values were found in the previous experiment, proving that, under similar conditions, experimental evaporation rate is repeatable.
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3.4. Cleaning the membranes
Fig. 4. Concentration of clari®ed passion fruit juice using osmotic evaporation with the continuous feed of concentrate at 40 g TSS/100 g.
We compared the production of concentrate at 60 g TSS/100 g between the one-stage and a two-stage process (Fig. 5). In the two-stage process, in the ®rst stage, the concentration level in the closed loop can be maintained at 40 g TSS/100 g by constantly feeding with freshly clari®ed juice and removing the concentrate at 40 g TSS/ 100 g. The removed concentrate is, in its turn, used to feed a second stage in which concentration in the loop is kept at 60 g TSS/100 g. The two-stage procedure saves almost 20% of the membraneÕs surface area. The juiceÕs average residence time is also considerably reduced.
After each experiment, the entire equipment was thoroughly rinsed with warm tap water (40°C). For some trials, after rinsing, an evaporation test, in which the loop was ®lled with water, was carried out. Evaporation rates after a simple rinsing did not result in signi®cant dierences between the usual water evaporation rate expected under the same conditions when the membrane was completely clean. The same observation was found after cleaning with soda solution, although the cleaning solution turned slightly yellow, suggesting that some molecules attached to the membraneÕs surface were removed. They may have corresponded to some hydrophobic pigments from the passion fruit juice that had stuck to the membraneÕs surface, but without noticeably interfering with the evaporation process. Van Gassel and Schneider (1986) had similar ®ndings in experiments on membrane distillation. After 25 continuous processing and cleaning cycles and almost 250 h of accumulated operation, we did not detect salt leakage into the feed juice. This proved that the hydrophobic integrity of the membrane and the module was maintained, even under industrial conditions. Thus, OE can apparently be conducted for various hours without needing to clean the membrane, at least not for operational reasons. Reasons of hygiene, however, may oblige the insertion of a cleaning cycle at regular intervals. To avoid this problem, an aseptic connection between the previous step of micro®ltration and OE could also be envisaged.
Fig. 5. Scheme of one- or two-stage continuous-feed osmotic evaporation process and membrane area required.
F. Vaillant et al. / Journal of Food Engineering 47 (2001) 195±202
3.5. Concentrate quality All samples of the juice concentrated at 60 g TSS/100 g for sensory quality tests were obtained after at least 3 h of continuous extraction of concentrate, at a time when we could consider that the hold-up volume was completely renewed. Once the hold-up volume was renewed, the average residence time of the juice to be concentrated from 14 to 60 g TSS/100 g was calculated as being 1.3 h. The quality of the concentrates produced was ®rst assessed by comparing fresh, clari®ed juice with clari®ed juice reconstituted from OE concentrate. A simple triangular dierence test, carried out with highly trained tasters, showed that no signi®cant dierences (5% con®dence level) could be noticed. The same tasters could
201
easily recognise fresh juice from juice reconstituted from concentrate obtained by thermal processing. A second test was done, comparing the juice reconstituted from OE concentrate with pulp obtained from pasteurised juice. This juice was compared with fresh juice, pasteurised juice and juice reconstituted from thermal concentrate. The tasters were asked to qualify aroma, taste and colour, and to give an overall score (Fig. 6). Reconstituted juice from OE concentrate is very similar to pasteurised juice as far as aroma, taste and colour are concerned. For these factors, scores obtained by the OE sample concentrate are higher than for the thermally concentrated juice and con®rms results already obtained under similar conditions (Shaw, Lebrun, Dornier, Ducamp, Courel, & Reynes, 2000). As far as biochemical properties are concerned, OE concentrate at 60 g TSS/100 g was comparable with juice thermally concentrated at 49 g TSS/100 g, as is sold in the market (Table 1). Directly comparing the results expressed in kg of TSS, we show that the OE-concentrate had the added advantage of retaining almost all the vitamin C content of the original juice because of the low processing temperature.
4. Conclusions
Fig. 6. Sensorial pro®le of dierently processed juices made from passion fruit.
Osmotic evaporation readily concentrates a clari®ed juice up to 60 g TSS/100 g, a value that is higher than is obtained with other Ôlow temperatureÕ concentration techniques. When juice concentration reaches 40 g TSS/ 100 g, evaporation ¯ux is only 12% less than the ¯ow rate initially registered with water. Continuous OE with constant extraction of concentrate and feeding with extemporarily processed, fresh, clari®ed juice is more hygienic and performs well. The same procedure can be done at 60 g TSS/100 g, although the evaporation rate is 33% less than the evaporation rate obtained with water. Nonetheless, by implementing a two-stage process, the average overall eciency of OE can be maintained at around 0.62 kg hÿ1 mÿ2 when concentrating clari®ed
Table 1 Comparison of the main physico-chemical characteristics of the initial clari®ed passion fruit juice, osmotic evaporation and thermal concentrates Characteristic
Unit
Initial juice
OE concentrate
Thermal concentrate
Total soluble solids (TSS) pH (20°C) Vitamin C
g/100 g
14 3.1 11.4 781 59 4.0 0.99 1.3 1043
60 2.8 60 769 350 4.5 0.81 1500 1300
49 2.7 5.3 86 260 4.2 0.90 32 1255
Titratable acidity Water activity (25°C) Viscosity (25°C) Density
mg/100 mL mg/kg TSS meq/100 mL eq/kg TSS mPa s kg mÿ3
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juice from 14 to 60 g TSS/100 g. Quality of the OE concentrate was signi®cantly better preserved than for the thermal concentrate. Evaporation ¯ux obtained at 40 and 60 g TSS/100 g are, respectively, 0.65 and 0.50 kg hÿ1 mÿ2 , which is nonetheless low when compared with reverse osmosis where ¯uxes are almost 10 times higher. These low values can be probably explained by the high membrane thickness (800 lm). The hydrophobic membrane used in this study was not optimised for this application because in the laboratory, evaporation ¯uxes that were from 10 to 20 times higher, have been attained with other membrane (Courel et al., 2000). Even so, we could see how OE can be applied on an industrial level and thus be a genuine alternative for concentrating thermosensitive products to the high concentration values required by markets with less changes of the original nutritional and sensorial qualities. Further developments of OE applications now need the input of membrane manufacturers to design industrial modules with hydrophobic membranes that are much more suited to the process.
Acknowledgements The authors wish to thank Victor Amu R. for his valuable technical help, Passicol S.A. (Chinchina, Colombia), Colciencias (Colombia) and the French Embassy in Santa Fe de Bogota for providing this project with ®nancial assistance.
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