Desalination 278 (2011) 381386

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Desalination
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d e s a l

Concentration and purication of whey proteins by ultraltration

C. Baldasso , T.C. Barros, I.C. Tessaro
Laboratory of Membrane Separation Processes Departamento de Engenharia Qumica, Universidade Federal do Rio Grande do Sul R. Eng. Luiz Englert, s/n. CEP: 90040-040, Porto Alegre, RS, Brazil

a r t i c l e

i n f o

a b s t r a c t
Whey is a liquid by-product of the dairy industry produced during the manufacture of cheeses and casein. As a raw material, it has many applications in food technology due to the functional and nutritional properties of its proteins. Membrane technology, especially ultraltration (UF), has been used in the dairy industry to produce whey-protein concentrates, because this technology allows the selective concentration of the proteins in relation to the other components. In this context, the objective of this work was to concentrate and to purify the whey proteins using UF in association with discontinuous dialtration (DF). The two strategies were tested by changing the volumetric-concentration factor (VCF), the DF water volume and the number of DF steps. The results showed that the UF process is adequate for the production of protein concentrates; in the best experimental strategy, the protein concentrate obtained was greater than 70% by weight (dry basis). 2011 Elsevier B.V. All rights reserved.

Article history: Received 26 November 2010 Received in revised form 5 May 2011 Accepted 23 May 2011 Available online 23 June 2011 Keywords: Whey Protein Concentration Purication Ultraltration

1. Introduction Whey, the by-product of cheese or casein production, is of relative importance in the dairy industry due to the large volumes produced and the nutritional composition; the production of 12 kg of cheese yields 89 kg of whey. Whey contains more than half of the solids present in the original whole milk, including whey proteins (20% of the total protein) and most of the lactose, minerals, water-soluble vitamins and minerals [1,2]. Worldwide whey production is estimated at around 180 to 190 10 6 ton/year; of this amount only 50% is processed. The whey can be considered a valuable by-product with several applications in the food and pharmaceutical industries; however, it is often treated as a dairy wastewater. The treatment of whey represents a serious problem due to its high organic load, which can reach a chemical oxygen demand (COD) of 100,000 mg O2 L 1 [3,4]. Many techniques have been developed to selectively concentrate whey proteins, because the whey is not a balanced source of nutrients, containing a high lactose content compared to the protein, and thus does not have the nutritional benets of more typical protein sources. Whey proteins have a high nutritional value, due to the high content of essential amino acids, especially sulfur-containing ones [5]. Besides the nutritional properties, the whey proteins have functional properties which impart benecial physical properties when used as ingredients in food, mainly due to its high solubility, water absorption, gelatinization and emulsifying capacities [6]. Due to the higher specicity of the product, and the excellent functional and nutritional

Corresponding author. Fax: +55 51 3308 3983. E-mail address: cbaldasso@gmail.com (C. Baldasso). 0011-9164/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2011.05.055

value, the commercial value of whey protein concentrate is from 3 to 40 times greater than that of whey powder [3]. The conventional method of whey concentration is by thermal evaporation. The main disadvantages of this method are the high energy consumption and the high content of ashs and lactose that remains in the concentrate; additionally, the heat treatment can change the characteristics of whey components, mainly the proteins, which are thermolabile and can lose their nutritional and functional properties during heating. Ultraltration (UF) is a very attractive alternative method, as it does not use heat and as a consequence does not involve a phase change, which makes the concentration process more economical. UF is a membrane separation process (MSP) typically used to retain macromolecules, and has been used in the dairy industry in the recovery and fractionation of milk components. UF allows a variation in the ratio of concentration between the whey components, due to the retention of protein and selective permeation of lactose, minerals, water and compounds of low molar mass [7]. Dialtration (DF) is used for the production of whey-protein concentrate (WPC) with a high protein content. DF is used for protein purication to eliminate problems association with high concentrations in the retained product, generating high purication, while retaining good performance process [8]. Also, it should be pointed out that the addition of small DF volumes several times is more effective than a big volume at one time only. The operability studies provide important information about the capabilities and limitations of the whey ultraltration process. The whey ultraltration process is well designed to deliver the desired total solids and protein concentrations for the production of whey protein concentrates. However, the process becomes less capable of delivering the desired product specications after long hours of operation when long-term fouling is more signicant [9,10].

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Further studies involving ultraltration operated dialtration mode are needed to reveal dialtration volumes that are effective. More than that, which may take into account what level and how many cycles dialtration are needed, one can optimize the work. The desired total solids and protein concentrations cannot be achieved if the volume concentration ratio alone is used to control the owrate and composition of the product stream. The limitations of the whey ultraltration process highlight the necessity for direct monitoring of the total solids and protein concentration in the product stream during the production of whey protein concentrates. Monitoring of total solids and protein concentration also helps determine how the amounts of dialtration water and recycled permeate need to be adjusted to achieve the desired product owrate and composition from the ultraltration plant. Such adjustments are necessary either after deviations from the desired specications are observed or after changes in the desired specications are made. Furthermore, the development of technologies capable of solving the problem of whey utilization can bring economic and environmental benets as the whey is a good source of protein for human consumption, thus justifying and studying the possibility of using it commercially. In this context, the aims of the present work were the concentration and purication of whey proteins by UF associated with DF. In this work, different strategies were tested, including varying the volume of water added to the concentrate and the number of DF steps and modifying the volumetric concentration factor (VCF; the ratio of the initial volume of the whey solution and volume retained). Thus, the main objective is to obtain a large protein purication, with the minimum DF solution (in this case water).

with a total solids content of approximately 6%. Liquid whey was reconstituted by manually dissolving the whey powder in distilled water at neutral pH and a temperature of 50 C. The initial volume of whey for ultraltration was approximately 30 L (29.6 L water and 1.86 kg whey powder). The average initial contents of lactose, protein and ashs were 42 kg m 3 (72.4% - w/w), 9 kg m 3 (15.6%) and 7 kg m 3 (12%), respectively; the amount of fat was considered negligible, since it is removed before the whey was dried in a spray dryer. 2.2. Membrane The UF membrane was UF-6001, made of polyethersulfone, in a spiral module manufactured by Koch Membrane Systems. The molar weight cut-off was 10 kDa, the feed spacer was 80 mils (thousandths of inch) and permeation area was 0.28 m 2. 2.3. UF equipment Experiments were performed in a pilot plant, WGM-KOCH PROTOSEP IV, shown schematically in Fig. 1. The pilot plant comprises the following equipments: feed tank (1), stainless steel, with a volume of 75 L, manufactured by SULINOX. The tank has an agitator and a temperature-control system that operates in the range of 25 to 150 C; pneumatic pump (2), diaphragm type, model Versamatic VM50, operated with compressed air through a system comprising an FLR kit (lter, air regulator and lubricator); pre-lter (3), manufactured by CUNO, consisting of a PVC housing and a polypropylene lter element with a nominal pore size of 1 m; housing for module spiral membrane (5), 30 cm in length and 5.8 cm in diameter, of 316 stainless steel; manometers (4) and (6), 316 stainless steel, scale from 0 to 10.5 bar.

2. Materials and methods 2.1. Whey The sweet whey powder used in this study was supplied by Eleva Alimentos (Teutonia, RS), from the manufacture of mozzarella cheese,

Fig. 1. Schematic of the membrane unit used for the experiments. (1) tank, (2) pump, (3) pre-lter, (4) and (6) manometers, (5) membrane module.

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2.4. Analytical methods The concentration and purication characteristics of concentrate and permeate samples were determined by analysis of the following parameters: concentrations of protein and lactose, total solids content, electrical conductivity and pH. Determination of total solids content was carried by a gravimetric technique in accordance with the procedures given in LANARA [11]. The concentration of lactose was determined by the dinitrosalicylic acid (DNS) method by Miller [12]. Protein concentration was determined by the Lowry method [13]. These methods are spectrophotometric and the absorbance readings were performed in a UVvisible spectrophotometer (Varian Cary I). pH analysis was with a Digimed DM20 pH meter. Electrical conductivity was determined with a Digimed - DM31 equipped with electrode model DMC-010M. 2.5. Experimental The rst experiments were performed by measuring the permeate ux at different transmembrane pressures (P) with water and with whey. The UF permeation tests were carried out in total recirculation mode (for pressure selection) and in concentration mode. In the total recirculation mode, both concentrate and permeate, are recirculated to the feed tank, to achieve homogenization of the solution and thermal equilibrium in the system. In the concentration mode only the concentrate is recirculated to the feed tank. The concentrationmode experiments were performed at a transmembrane pressure of 2 bar and a feed owrate of 840 L h 1; these operating conditions were determined in previous studies [14]. The whey temperature was kept constant at 50 C, based on the temperature output of the whey in the cheese manufacturing process and the maximum allowable temperature for the membrane (55 C). In all experiments, measurements of water ux were carried out before and after ultraltration with whey to quantify the formation of fouling on the membrane. Two experiments were conducted with different strategies to verify which condition would allow for greater purication of proteins. The experiments consisted of one step of UF in batch mode, where the initial volume was reduced to a certain VCF. And another stage, it operated ultraltration in discontinuous dialtration mode, which was to add some incremental volumes of water distilled to concentrate and remove this added volume, in the permeate, to remove most of the lactose and other low molecular weight compounds, increasing the degree protein purication. The experiments were made as are described below. Experiment 1 the inicial whey solution (30 L) was concentrated to a volume of 6 L in the feed tank (VCF = 5); subsequently, ve DF steps were done: two DFs (DF1 and DF2) with 6 L of distilled water each and three DFs (DF3, DF4 and DF5) with 3 L of distilled water each, ve cycles of DF, totaled 21 L of water, were added in the concentrate, this order: 6 L + 6 L + 3 L + 3 L + 3 L.* Experiment 2 the inicial whey solution (30 L) was concentrated to a volume of 5 L (VCF = 6); subsequently, four DF steps were done: DF1 and the DF2 with 5 L of distilled water each, DF4 and the DF5 with 2.5 L of distilled water each. Four dialtration cycles of distilled water were added in the concentrate, so: 5 L + 5 L + 2.5 L + 2.5 L, in total were added during the dialtration 15 L of water.* *Each DF cycle is equivalent to add the volume of distilled water indicated in each experiment in the concentrate, and to keep constant the volume of concentrate. The membrane was not cleaned between the concentration and purication stages, i.e., the process was continuous.

A chemical cleaning procedure was performed at the end of each experiment to restore the ux and retention characteristics of the membrane and prevent the growth of microorganisms in the system. The cleaning consisted of the following steps: a rinse with distilled water, an alkaline cleaning, an alkaline chlorine cleaning and an acid cleaning. These steps were always within the limits of pH and temperature tolerance of the membrane. 3. Results and discussion Fig. 2 shows the water-permeate and whey-permeate uxes as functions of transmembrane pressure; observe that the ux of water increased linearly with transmembrane pressure (r 2 = 0.9982). Moreover, for same operating conditions there was a signicant difference in the whey and water-permeate uxes. This same behavior was found by Rektor and Vatai [1], Atra et al. [2] and Butylina et al. [15] in their works, i.e., the whey permeate ux was lower than the water ux at all pressures. Possible causes for these different uxes include lower interactions between the membrane and solution, effects of mass diffusivity and mainly the higher viscosity () of the solution. The whey-permeate ux being smaller than the water ux shows that the concentration-polarization effect is very signicant for whey in the initial concentration and this effect tends to increase as the whey is being concentrated. In this case, we can say that the increase of ux is limited by an increased polarization layer, i.e., the increase of transmembrane pressure is counterbalanced by the increase in total resistance. In the plots the start of UF is identied by UFi, and the end by UFf. Dialtrations are identied by DF and the corresponding stage (1, 2, 3, 4 or 5). In Experiment 1, the UF lasted 225 min; DF1 and DF2 about 75 min each, and DF3, DF4 and DF5 about 45 min each. In Experiment 2, the UF lasted 265 min, DF1 and DF2 about 65 min each, and DF3 and DF4, 35 min each. The UF step of Exp. 1 was shorter, because the nal concentrate volume was bigger (6 L) than for Exp 2 (5 L). In the other hand, the DFs of Exp. 1 took more time than the DFs of Exp. 2. Fig. 3 presents a comparison between the two concentration experiments with respect to the percentages of protein on a dry basis (protein content/total solids content) versus the stage of the process. The percentage of initial protein mass in both experiments was about 15%. The experiment with the higher VCF resulted in a slightly higher protein percentage at the end of the UF and this difference was accentuated with the DF steps, and signicant for both DFs, the larger volume and the smaller volume. In experiment 2 the DFS reached the nal protein concentration of 71% versus 62% in Experiment 1.

Fig. 2. Water and whey ux vs transmembrane pressure. Membrane UF-6001, T = 50 C, feed owrate= 840 L h1. Legend: () water, () whey. *coefcient of variation: 0.5%.

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Fig. 5. Permeate ux vs volumetric concentration factor (VCF) for Experiment 2. UF membrane UF-6001, P = 2 bar, T = 50 C, feed owrate = 840 L h1.

Fig. 3. Percentage of protein (w/w, dry basis) versus the stage of the process for Experiments 1 and 2. Membrane UF-6001, T = 50 C, P = 2 bar, feed owrate = 840 L h1. Legend: () Experiment 1; () Experiment 2. *coefcient of variation: 0.05%.

Fig. 4 shows the percentage mass of lactose on a dry basis (lactose content/total solids content) in the concentrate versus the stage of the process. In the early experiments the percentage by mass (dry basis) of lactose was 72%; at the end of the UF, the percentages dropped to 64 and 57% for Experiments 1 and 2, respectively. During the dialtrations these percentages were further reduced to 37% for Experiment 1 and 29% for Experiment 2. In relation to ash content, the initial samples initially had 12% ash on a dry basis. In both experiments the process was efcient for the removal of ashs, with percentages close to zero at the end of the UF, showing that nearly all of the ash was removed from the protein concentrate. It was found that the DFs of smaller volumes were very effective for the removal of ashs and lactose; in Experiment 2, for example, there was a large reduction in the concentration of other elements relative to the amount of protein in the last DFs of 2.5 L. The protein-concentration results in Experiment 2 were better than those obtained them in Experiment 1. It is noteworthy that in

Fig. 4. Percentage of lactose (w/w, dry basis ) versus the stage of the process, for Experiments 1 and 2. Membrane UF-6001, T = 50 C, P = 2 bar, feed owrate = 840 L h1. Legend: () Experiment 1; () Experiment 2. *coefcient of variation: 0.06%.

Experiment 2, the UF concentrate volume before DF was 5 L, and this volume decrease resulted in a higher nal protein concentration and a lower volume of water to the DF stage. For Experiment 1 was used 21 L of water to the stage of DF, while for Experiment 2 was used 15 L of water in DFS and protein percentage was higher than achieved. Therefore, in Experiment 2 was obtained a higher content of protein, with fewer steps of DF, with a smaller total volume of water used to purify these, this result shows that this method is promising and can be used to obtain protein concentrates. The present results are in agreement with those found in the literature; Rektor and Vatai [1], Butylina et al. [15], and Zydney [16], performed studies showing that components of low molecular weight (lactose and ashs) preferentially permeate the membranes of UF, which retain the protein molecules. Fig. 5 shows the ux of whey permeate as a function of the volumetric concentration factor for the UF stage of Experiment 2, which resulted in better protein purication. It was observed that the permeate ux decreased as the volumetric concentration factor was increased. The VCF reached a maximum of 6, because the concentrate was to be used in subsequent dialtrations and these volumes and concentrations were appropriate for that purpose. The data in Fig. 5 conrm what several authors have already observed. According to Smith [17], the permeate ux generally decreases with the increase in VCF. According Rektor and Vatai [1], Bacchin et al. [18] and Cheryan [19], the more concentrated the solution of protein is, the lower the permeate ux is, due to the higher osmotic pressure and the greater accumulation of solute molecules in the polarized layer, increasing its thickness and, consequently, its resistance to permeation. According Atra et al. [2], at higher VCF there was a deposit of the largest and most dense layer that reduced the permeate ux until it reached a static condition. The permeate ux in the DF steps was below the initial ux in UF due to the fouling formed in the protein-concentration stage, and the dilution factor was not very high. Fig. 6 shows the total solids (TS) concentrations of the concentrate and permeate samples over time for Experiment 2 in the concentration and dialtration stages. There was an increase of TS in the concentrate during UF due to the increased concentration of protein. In the permeate the concentration of TS was approximately constant until the end of the UF, increasing slightly in the last hour of the experiment. The concentrate had higher concentrations of TS in both the UF and DF stages. During the DF a reduction of TS in the concentrate occurred due to the removal of lactose and ashs and in the permeate due to the dilution.

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Fig. 6. Total solids concentrations of the concentrate and permeate samples (w/v) vs time for Experiment 2. Membrane UF-6001, T = 50 C, feed owrate = 840 L h1, P = 2 bar. Legend: () permeate UF, () concentrate UF, () permeate DF, () concentrate DF. * coefcient of variation: 6%.

Fig. 8. Lactose concentrations of the concentrate and permeate samples (w/v) vs time for Experiment 2. Membrane UF-6001, T = 50 C, feed owrate = 840 L h1, P = 2 bar. Legend: () permeate UF, () concentrate UF, () permeate DF, () concentrate DF. * coefcient of variation: 6%.

Fig. 7 shows the concentration of protein versus time for the concentrate and permeate in the concentration and purication stages. The concentration of protein increased in the retentate throughout the UF stage of the experiment. The initial concentration of protein was about 9 kg.m 3 and was about 36 kgm 3 at the end of the UF. Note that the concentration of protein in the DF did not show much variation however the level of contaminants, ashs and lactose decreased signicantly in DF, thus resulting in a purication of the proteins. Similar behavior was reported by Leite et al. [20]. The lactose concentrations of the concentrate and permeate samples versus time are shown in Fig. 8. The curve shows a behavior similar to that found for total solids, because the lactose, the most abundant component in whey, with a molecular weight lower than the molar mass cut-off of the membrane, showed a low retention and had a decisive impact on the behavior of total solids over time in both permeate and concentrate. In the UF stage the permeate concentration of lactose remained around 40 kg.

Fig. 7. Protein concentrations of the concentrate and permeate samples (w/v) vs time for Experiment 2. Membrane UF-6001, T = 50 C, feed owrate = 840 L h1, P = 2 bar. Legend: () permeate UF, () concentrate UF, () permeate DF, () concentrate DF. * coefcient of variation: 5%.

m 3; the retentate started with 42 kg.m 3 and reached 50 kg.m 3 at the nal stage of UF. At the start of DF the concentration of lactose decreased signicantly in both concentrate and permeate. At the end of the four DFs the concentration of lactose in the retentate reached 15 kg.m 3 , while the concentration of lactose in the permeate was around 10 kg.m 3 , almost the same as the concentration of total solids, indicating that virtually all ash was removed, and the solids remaining in the permeate were almost all lactose. Some authors revealed results similar to this job. Roman et al. [21,22] performed experiments of NF associated with DF for demineralization of cottage cheese whey considering two important factors: the duration of the process and water consumption. The concentration of feeding solution was performed with different values of the ratio between the volume of water and dialtrante permeate volume. The degree of demineralization of monovalent ions reached 70% and 90% at 2.5 volume concentration ratio, and at dialtration and permeate volume ratios () of 0.5 and 0.75, respectively. For = 0.75, the experiment ended after 18.9 h, and hit a VCF = 2. For = 0.5, the experiment lasted 10 h, and the VCF was 2.65. The degree of purication of the detained, was two times higher for = 0.75, but this process has used three times more water than the = 0.5. The comparison of the two processes revealed that greater dilution retained from beginning to end of the experiment, contributed to further purify but increased the process time and water consumption. These results are consistent with the ndings of Jaffrin and Charrier [23]. These authors suggested that in an ideal process a pre-concentration would be interesting, where a certain amount of macrosolute was reached before the onset of DF, the DF following the completion of continuing with the decrease in volume would increase the degree of purication, the which was also suggested by Foley [24]. The latter researcher performed DF continued after an initial concentration step, and achieved a signicant reduction in water consumption without increasing the process time too. The pH did not signicantly change throughout the process; samples of concentrate and permeate pH remained between 6.2 and 6.4. This behavior is a good indication that the solution was not degraded during the time of the experiment. The measurement of electrical conductivity remained almost the same for the permeate and concentrate throughout the UF stage, because the membrane is not selective for ashs, which are the compounds that contribute most to the electrical conductivity. During the DF stage the electrical conductivity of the samples was decreased after the addition of distilled water (dilution effect).

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The results show that UF associated with DF is a technology that can be successfully used for demineralizing the whey, and still concentrate components such as proteins. And still has the advantage of not degrading its main nutrients. 4. Conclusions Based on the results of this work, we obtained the ndings listed below: For the same operating conditions, there was a signicant difference in the permeate ux of the whey and of the water; the ux of water is greater than the ux of whey permeate. The increase in ux was limited by an increased polarization layer, i.e., the increase of transmembrane pressure was counterbalanced by the increase in total resistance. The higher VCF generated the best results for protein purication. For Experiment 2, with a VCF equal to 6, after the DFs a protein concentration of 71% by weight (dry basis) was reached. The process proved to be efcient for the removal of ashs; the ash concentration approached zero end of the UF stage, i.e., practically all the ash was removed from the concentrate. The DF was very effective when performed with small volumes and a greater number of times. Decreasing the volume of concentrate from 6 to 5 L before the DF steps resulted in a higher nal protein concentration and a lower volume of water to the DF stage. The measurements of pH and electrical conductivity showed that the solution was not degraded during the experiments. The ux of whey permeate decreased with the increase in VCF due to several effects: the increase of viscosity, the fouling, the higher osmotic pressure and the greater accumulation of solute molecules in the polarization layer, which increased the resistance to permeation. The permeate generated can be treated by nanoltration for the recovery of lactose for use in the food and pharmaceutical industries. It is also possible to promote the demineralization of the process water by reverse osmosis and electrodialysis for the recovery of ashs and water in the process, generating nancial and environmental benets. Acknowledgments The authors thank the CNPq (National Counsel of Technological and Scientic Development) and CAPES (Coordination for the Improvement of Higher Education Personnel) for nancial support.

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