Novel solvent stable micro-porous membrane made of whey protein isolate gel

Journal of Membrane Science 192 (2001) 71–82

Novel solvent stable micro-porous membrane made of whey protein isolate gel Jiunn Yeong Teo, Robert Beitle∗ a

Department of Chemical Engineering, 3202 Bell Engineering Center, University of Arkansas, Fayetteville, AR 72701, USA

Received 3 October 2000; received in revised form 23 April 2001; accepted 26 April 2001

Abstract Novel micro-porous membranes made of whey protein isolate (WPI) were developed. Aggregates of WPI comprised the bulk of the membrane, the size and packing density of which were varied by changing CaCl2 concentration (0.3–0.05 M) and WPI concentration (30–40 wt.%), respectively. Aggregate sizes of the membranes made with 0.3, 0.1, 0.05 M CaCl2 were roughly 1.5, 1, and 0.8 ␮m, respectively. Skin layer of thickness about 0.5 ␮m was found on either side of the membrane, but the thickness could reach 5 ␮m at 0.3 M CaC12 . Additionally, the porosity of the skin layer was shown to be modifiable with the addition of surfactant. Membranes were stable in hexane with flux values on the order of 1–1000 gal/ft2 d depending on the morphology of the membrane. A discussion concerning the formation and use of protein gel as a membrane is included. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Micro-porous and porous membrane; Hydrogels; Membrane preparation and structure; Protein-based membrane

1. Introduction This study introduces a novel micro-porous membrane made of heat-induced whey protein isolate (WPI) gel. In contrast to the use of polymers such as polyimide [1,2], WPI gels are reliant upon their hydrophilic nature and inherent cross-linking potential to enhance solvent stability. WPI gels also may be formed that are less fragile than cross-linked hydrogels, and be rendered porous without the use of porogens. With membrane characteristics primarily dependent upon temperature, pH, and ionic strength of casting solution, preparation of WPI membranes ∗ Corresponding author. Tel.: +1-501-575-4951; fax: +1-501-575-7926. E-mail addresses: [email protected] (J.Y. Teo), [email protected] (R. Beitle).

are robust in nature. Moreover, organic solvents are not required for membrane formation, allowing WPI membranes to be classified as environmentally benign. This represents a novel approach to the development of non-polymeric, solvent resistant membranes, which is of particular interest to the chemical process industry. Protein molecules like those comprising WPI are complex and active molecules that undergo conformational and net charge changes in response to environmental factors [3]. The changes in structure and net charge will generally induce covalent and/or non-covalent reaction(s) due to exposure of different groups (local unfolding of the protein) or activation of some other functional moieties (protonation). Balancing their extent influences the formation of protein gels with different morphologies [4–11]. There are many books and chapters outlining protein chemistry in detail [12–17], wherein gel formation is described

0376-7388/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 6 - 7 3 8 8 ( 0 1 ) 0 0 4 7 3 - 2

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within the context of food sensory qualities (i.e. mouth feel) and food coatings, but not quantitative metrics of value to assess their use in chemical separation applications. Adaptation of protein gelation chemistry in order to develop a membrane with controllable morphology represents a new avenue of membrane technology sought after in this study. After a brief review of protein gel chemistry/theory, the effects of (i) CaCl2 concentration, (ii) WPI concentration, and (iii) surfactant addition on the morphology of membranes cross-linked with formaldehyde are described. Membrane morphology was characterized by scanning electron microscopy (SEM), and flux and stability were determined using hexane as a challenge fluid. Result indicated that membrane morphology can be controlled, and the material prepared had high hexane flux.

2. Theory 2.1. Formation of protein gel Protein molecules contain hydrophobic and hydrophilic amino acid residues in their structures and exhibit amphiphilic characteristics. When protein molecules are in a hydrophilic environment, hydrophobic residues typically are folded inside a three dimensional structure. Germane to WPI is the tendency to adopt a barrel-like conformation with hydrophobic interior. An abundance of cysteine residue (Cys) are commonly found in globular food proteins. Cys has a thiol group (–SH), which is capable of forming covalent disulfide bonds with another thiol group by an oxidation reaction [18]. Free thiol groups may also participate in thiol-disulfide interchange reactions with disulfide bonds [19]. The formation of the covalent disulfide bonds within the protein molecule is one primary factor in reducing the extent of protein unfolding. Other solvent exposed Cys are responsible for the formation of intermolecular disulfide bonds with another protein. Stable covalent disulfide linkages between protein molecules are responsible for thermoset gel formation (e.g. custard) in contrast to a (less stable) thermoplastic gel formation (e.g. gelatin). Understandably, formation of thermoset gels requires unfolding and denaturation of protein molecules, achievable by

the addition of heat, denaturant, and other materials [12]. Salt bridging, hydrophobic/hydrophilic interaction, electrostatic interaction, van der Waals interaction, ionic bonding, and hydrogen bonding are some other reactions involved in the formation of protein gel [20]. 2.2. Morphology of protein gel The net charge of a protein greatly affects electrostatic attractive and repulsive forces, and thus, can be used to attenuate the level of interactions among protein, neighbor, and solvent during gel formation [12]. If protein molecules repel each other under the condition of high repulsive force, bonding reactions among the molecules will not happen even if the protein molecules are denatured and the Cys residues are revealed. On the other hand, at high attractive force, protein molecules aggregate and turn the protein solution turbid. Precipitation of protein molecules may even occur. Langton and Hermansson [14] found that heating turbid protein solutions led to the formation of opaque aggregated micro-porous gel while the heating of transparent solutions resulted in transparent fine stranded non-porous gels. It is thus, reasonable to view the protein gelation process as a series of events involving aggregation, unfolding, and association (cross-linking) of protein molecules to form a network. In the case where aggregation of protein molecules precedes denaturation, cross-linking of this protein solution results in a porous aggregate structure due to the fact that it begins as a dispersed, but non-homogeneous system. The formation of non-porous fine stranded gel, conversely, begins with homogeneously distributed protein molecules. In order to produce a protein gel with desired morphology, it is therefore, important to manipulate the electrostatic attractive and repulsive forces. Changing the ionic strength and pH of the solution are two common and effective ways to induce such morphological change. 2.2.1. Effect of pH The electrostatic attractive and repulsive forces are influenced by the net charge carried by each protein molecule. Net charge, instead of charge, is considered since protein molecule contains many different amino acid residues and each of these residues can be either

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protonated or deprotonated depending on the pH of the solution [18]. At a certain pH corresponding to the isoelectric point, the net positive and negative charges of these amino acid residues balance out each other and diminish the electrostatic repulsive forces. However, individual amino acid residues on the protein molecule are not necessarily neutral. The numerous charged groups present on protein molecule could then provide an overall stability by the electrostatic attraction on opposite charges, hydrophobic interactions or other forces. Without the repulsive force, protein molecules are ‘free’ to associate with each other leading to the formation of protein aggregates at the isoelectric point [5]. When pH increases or decreases from the isoelectric point, the net charge on protein molecules will turn negative or positive, respectively. The charges on the surface of a protein molecule give rise to a diffuse layer of ions, the electrical double layer [21]. The overlapping of electrical double layer that occurs as two proteins approach each other give rise to electrostatic repulsion. 2.2.2. Effect of salts The presence of salts affects gelation and gel properties via charge neutralization of protein molecules and, thus, diminishes the electrical double layer and generally enhances the interaction between macromolecules outside of their isoelectric point. Mulvihill and Kinsella [6] studied the effects of NaCl and CaCl2 on the structural properties of ␤-lactoglobulin gel at pH 8, reporting that self-supporting gels were not formed unless salts were added. In the absence of NaCl or CaCl2 , excessive repulsive force prevented the denatured protein molecules from associating to form a network strong enough be self-supporting. Increasing ionic strength of the solution by addition of NaCl or CaCl2 would diminish the repulsive force and self-supporting gels were then formed. Gel strength was at a maximum when NaCl and CaCl2 concentrations were at 100 and 10 mM, respectively. At a CaCl2 concentration below that required for maximum gel strength, protein aggregates were finely dispersed in the matrix and linked together by fine strands. At the CaCl2 concentration corresponding to maximum gel strength, the protein was still evenly dispersed in the matrix, but with a greater level of aggregation with aggregates more strongly linked. At a CaCl2 concentration greater than that required for

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maximum gel strength, proteins were no longer evenly dispersed, but formed large aggregates that was separated from other aggregate families by the aqueous phase. The size of aggregate-family, and the area of free aqueous space increased with increasing CaCl2 concentration. Barbut [7] studied the effect of sodium level on the micro-structure and texture of whey protein isolate gels and arrived at a same conclusion that gel strength was maximum when NaCl concentration was 100 mM. Specifically, Barbut [7] observed that the free aqueous space, or size of macropores, of said gel was about 0.1–0.2 ␮m at 50 mM NaCl and 0.5 ␮m at 100 mM NaCl. 2.3. Strength of protein gel The strength of protein gel is primarily enhanced by the formation of covalent disulfide bonds with adjacent protein molecules. Intermolecular covalent bonds in protein gel may increase the chain length of the polypeptide and increase the molecular entanglements within the gel structure, thereby restricting relative thermal motions [12]. Logically, increasing the density of covalent bonds by increasing the protein concentration or the fraction of protein that has the most Cys residue would improve the strength of the gel. It had been demonstrated that the strength or hardness of ␤-lactoglobulin gel [12] and whey protein gel [5] increased proportionally with the protein concentration. For aggregate gel, the increase in protein concentration also implies an increase in packing density of protein aggregate, therefore, an increase in gel strength and hardness. Changing heating conditions will also alter the strength of protein gel as well. ␤-lactoglobulin formed self-supporting and firm gels in the temperature range 70–80◦ C with the gel hardness increase as the temperature increased up to 90◦ C [8]. Maximum hardness of whey protein isolate gels was obtained at temperatures of 80 and 90◦ C and pH 6.0–6.4, respectively [9]. Preheating a protein solution in an excess repulsive force condition could ensure that proteins unfold and reveal their thiol groups before the protein molecules collapse into network [6]. Upon neutralizing the repulsive force by adding the appropriate amount of salt, this preheated protein solution formed a gel, credited to the cross-linking of the revealed thiol groups. Such gel had lower gel penetration force

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values than normal heat-induced gel, but higher shear stress at fracture [10]. Bridging between two negatively charge groups on adjacent protein molecules by metal ions is also very effective in strengthening the gel matrix. CaCl2 was among the best of 25 different salts that had been tested for their ability to strengthen whey protein gel [11]. Other metal salts not as effective included sodium chloride, potassium chloride and others. 2.4. Formation and control of selective skin layer As protein molecules possess amphiphilic characteristics, it is natural for the protein molecules to get adsorbed at the air water interface. Dickinson and McClements [18] explained that protein molecules diffuse from the bulk solution and collide with the interface and may eventually led to adsorption when the hydrophilic segments of the protein molecules are oriented towards the interface. Once a segment or more segments of the protein molecule have successfully adsorbed at an interface, there is usually a process of unfolding and rearrangement of the protein structure such that the contact area between non-polar groups and water at the interface is reduced. Doing so, the protein reduces the free energy of the system and hence the interfacial tension. The net protein charge and charge distribution along the polypeptide chain play an important role in the rate and extent of protein adsorption. When protein molecules are carrying charges, the electrostatic repulsion between the adsorbed proteins and the approaching proteins will discourage any further adsorption at the interface. On the other hand, electrostatic attractive forces at isoelectric point will encourage the protein adsorption. Addition of salt will neutralize the charges carried by the protein molecules and enhance protein adsorption as well. It is widely known that surfactant will destabilize protein foam due to the competitive adsorption of surfactant at the air–water interface [22]. Recent studies on mixed surfactant/protein adsorption layers have shown that addition of surfactant in protein solution can disturb the formation of skin layer on the protein solution and air interface [23–25]. No study has shown the influence of surfactant on protein gel, particularly that related to the interfacial skin layer.

3. Experimental 3.1. Materials and equipment WPI was provided by NZMP (North America), Inc. (32W895). The dry WPI contains 97.4% protein, 1.6% ash, 0.4% fat, and 0.6% lactose. Tween-20 was purchased from Sigma (P9416). MilliQ® water was used throughout the study for protein solution preparation. Hexane was purchased from Fisher Scientific (AC26836-0025). The filtration setup used in the characterization of membrane flux consisted of a 5 l pressure vessel (Millipore, XX6700P05), a 47 mm diameter stainless steel membrane holder (Gelman Sciences, 2220), compressed air supply, and graduated cylinder. A schematic diagram of the setup is shown in Fig. 1. 3.2. Membrane preparation Different composition of protein casting solutions were prepared by dissolving the desired weight percent of WPI power into CaCl2 /Tween-20 (surfactant) buffer of desired composition. A very small amount of 5 M HCl solution was used to adjust the pH of the protein solution to 6.15 (pI of WPI = 5.2) such that no appreciable dilution was introduced. Each protein solution was then centrifuged at about 160g to remove bubbles. It is important to maintain a low speed such that settlement of protein aggregates could be avoided. Each protein solution was then transferred carefully

Fig. 1. Apparatus for flux measurement.

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Table 1 Composition of membranes studied Label

WPI concentration (wt.%)

CaCl2 concentration (M)

Tween-20 concentration (wt.%)

30WPI–0.1Ca–0T20 35WPI–0.1Ca–0T20 40WPI–0.1Ca–0T20 35WPI–0.015Ca–0T20 35WPI–0.05Ca–0T20 35WPI–0.3Ca–0T20 35WPI–0.05Ca–0.05T20

30 35 40 35 35 35 35

0.1 0.1 0.1 0.015 0.05 0.3 0.05

0 0 0 0 0 0 0.05

onto a flat non-stick baking sheet (Teflon® coated) and drawn to an even thickness of about 1 mm. The baking sheet was then covered and heated in an autoclave at 121◦ C to initiate and complete the gelation process. The autoclave was exhausted at the end of 1 h: WPI membranes were removed and immediately immersed in water to prevent the hot membranes from drying. The membranes, thus, made were immersed in 20% formaldehyde solution overnight to further cross-link and preserve the membrane. Table 1 summarizes the composition of the membranes made for this study. SEM was used to observe the morphologies of the membranes. 3.3. Flux measurement The membrane was cut into a 47 mm diameter circle and loaded into the membrane holder. The process tank was filled with hexane and the pressure in the process tank was regulated to a desired level using compressed air. Hexane was filtered through the membrane for five minutes before the measurement of flux, where the volume of hexane collected for a period of 2 min was obtained. Four different membranes of the same type were tested in the study. Flux was calculated by dividing the permeate flow rate by the effective membrane area.

4. Results 4.1. WPI concentration effect Figs. 2–4 show the effect of WPI concentration on the morphology of the membrane. Three membranes made with the same CaCl2 concentration of 0.1 M, but

Fig. 2. SEM of 30WPI–0.1Ca–0T20: (a) front; (b) back.

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Fig. 3. SEM of 35WPI–0.1Ca–0T20: (a) front; (b) back.

Fig. 4. SEM of 40WPI–0.1Ca–0T20: (a) front; (b) back.

different WPI concentrations of 30, 35, and 40 wt.% were compared. These membranes were shown to be asymmetric with surface skin layer formed on either side of the membrane, covering a mono-dispersed spherical aggregate structure. At 40 wt.% WPI, the aggregates were so densely packed that the skin layer could not be distinguished as clearly as those made of lower protein concentrations. Much of the aggregates appear to be connected and formed rather irregular shape of aggregate families. Nonetheless, all these membranes have roughly the same aggregate size of

about 1 ␮m. The packing density of the aggregates also increased correspondingly to the increasing WPI concentration, and therefore, provided a stronger feel to the membranes made with a higher WPI concentration. These observations are in agreement with the literatures that described about the enhancement of protein gel strength by increasing protein concentration [5,12]. The differences of the WPI membranes made of different WPI concentrations can also be revealed by their hexane fluxes as shown in Fig. 5. In general, hexane

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Fig. 5. WPI concentration effect on hexane flux: 30WPI–0.1Ca–0T20 (䊐); 35WPI–0.1Ca–0T20 (); 40WPI–0.1Ca–0T20 (䊊).

fluxes of these membranes were inversely related to WPI concentration, with a higher flux for those made of lower WPI concentration. While it is obvious that higher packing density of WPI aggregate would generate more resistance to the hexane flux, the skin layers (about 0.5 ␮m) on both sides of the membrane should be the flux limiting layers. For that reason, the fluxes of these membranes are not significantly different despite their variations in aggregate packing density. 4.2. CaCl2 concentration effect Figs. 3 and 6–8 show the CaC12 concentration effect on the morphology of the membrane. Membranes made of same WPI concentration of 35 wt.%, but different CaC12 concentrations of 0.015, 0.05, 0.1, and 0.3 M were compared. The morphology of the membranes changed significantly under the influence of salt concentration as that discussed in the literature [6,7]. At pH = 6.5 (pI = 5.2), the charges carried by the protein molecules could not be completely neutralized by the 0.015 M CaC12 solution. The protein molecules become more finely dispersed, and the structure of the membrane is, therefore, finer and

denser as that shown in Fig. 6. At a higher salt concentration of 0.05 M CaC12 and above, neutralization and the subsequent aggregation of protein molecules occurred, resulting in the formation of porous membrane shown in Figs. 3 and 7–8. The aggregate sizes of the membranes made with 0.3, 0.1, 0.05 M CaCl2 were roughly 1.5, 1, and 0.8 ␮m, respectively. Mulvihill and Kinsella [6] discussed about the formation of large aggregate-family at a high salt concentration due to excessive salt bridging effect. This phenomenon may be related to the formation of thick skin layer of about 5 ␮m on the membrane made with 0.3 M CaC12 (Fig. 8), where excessive salt bridging effect may have also resulted in enhanced adsorption at the interface. In fact, variables like pH and salt concentration are known to influence the protein adsorption at interface [18]. The much thicker skin formed on the top of the membrane compared to the skin layer at the bottom also suggested a preferential adsorption at the top air/liquid interface versus the bottom liquid/solid (Teflon® coated surface) interface at 0.3 M salt concentration. Differences in surface tension of the two interfaces are believed to be the reason.

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determined. It should be noted that membrane made with 0.3 M CaCl2 appears stronger, presumably due to its thick skin layer. However, the thick skin cracked relatively easily under stress, suggesting an inflexible skin. Therefore, unnoticeable cracks may have contributed to the higher than expected flux obtained. 4.3. Surfactant concentration effect Figs. 7 and 10–11 show the effect of Tween-20 concentration on the morphology of skin layer. Three

Fig. 6. SEM of 35WPI–0.015Ca–0T20: (a) front; (b) back.

With the exception of the membrane made with 0.015 M CaCl2 that did not show appreciable hexane flux within the tested pressure range, all membranes show similar hexane flux up to about 100 gal/ft2 d as shown in Fig. 9. Despite its skin thickness that is about 10 times greater than membranes made with 0.1 and 0.05 M CaCl2 , membrane made with 0.3 M CaCl2 produced a surprisingly comparable flux. It is suspected that the thick skin layer may have macro voids caused by gapping between the adsorbed aggregate families, but the true micro-structure of the skin is yet to be

Fig. 7. SEM of 35WPI–0.05Ca–0T20: (a) front; (b) back.

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Fig. 8. SEM of 35WPI–0.3Ca–0T20: (a) front; (b) back.

membranes made with same CaCl2 concentration of 0.05 M and WPI concentration of 35 wt.%, but different Tween-20 concentrations of 0, 0.05, and 0.1% were compared. The bulk gel phase of these membranes have the same morphology, but their

skin layers changed significantly with the addition of Tween-20. As expected, Tween-20 was found to have disrupted the formation of skin layer by competitive adsorption at the interface due to their higher interfacial property. At a Tween-20 level of 0.05 wt.%,

Fig. 9. CaCl2 concentration effect on hexane flux: 35WPI–0.05Ca–0T20 (䊊); 35WPI–0.1Ca–0T20 (䊐); 35WPI–0.3Ca–0T20 ().

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Fig. 10. SEM of 35WPI–0.05Ca–0.05T20: (a) front; (b) back.

as in Fig. 10, the skin layer was still present, but its surface was much rougher and showed possible pores compared to membrane made without Tween-20, as in Fig. 6. The skin layer was completely disrupted at a Tween-20 concentration of 0.1 wt.%, as in Fig. 11. The disruption of adsorbed protein film on protein solution by surfactant have been studied recently [23–25], and the SEM images of skin layers obtained in this study on protein gel have provided a visible demonstration on this competitive adsorption phenomenon.

Fig. 11. SEM of 35WPI–0.05Ca–0.1T20: (a) front; (b) back.

Without the skin layer, the hexane flux of membrane made with 0.1 wt.% Tween-20 showed markedly increased flux at around 1000 gal/ft2 d as shown in Fig. 12. Membranes made with 0.05 and 0 wt.% Tween-20 have fluxes about an order of magnitude and two orders of magnitude lower than that made with 0.1 wt.% Tween-20, respectively. The results have shown that the surfactant can indeed be used to modify the skin layer of WPI membrane. However, the disruption of skin would come at the expense of the rejection capability of the membrane.

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Fig. 12. Surfactant concentration effect on hexane flux: 35WPI–0.05Ca–0.1T20 (䊐); 35WPI–0.05Ca–0.05T20 (); 35WPI–0.05Ca–0T20 (䊊).

5. Conclusions WPI membranes are hexane stable with flux value greater than its polymeric counterparts that would generally swell under similar challenge [26]. The morphology of the membrane was shown to be asymmetric and controllable for a specific application by adjusting the concentration of one or many of the following components: CaCl2 , WPI, Tween-20. In particular, increasing WPI concentration was shown to increase the aggregate packing density of the membrane, and decreasing CaC12 concentration was shown to decrease the size of the aggregate/aggregate-family. Although not shown in this study, other factors such as salt type and pH should also induce similar morphological changes as suggested in the literature [5,18]. The porosity of the selective skin layer was also improved by increasing the surfactant concentration in the membrane. Regardless of its excellent hexane flux, many other characterizations are needed to assess the potential of WPI membranes for separation. Experiments to determine molecular weight cut-off will immediately

address the rejection characteristics and guide the work towards future applications and will be reported in a later submission. Such applications are likely to be found in edible oil and petroleum refineries that require hydrocarbon stable micro-porous membranes. The fouling resistance of the membranes in aqueous-based applications is also an interesting characteristic yet to be further explored. Since protein coated membranes are known to resist non-specific adsorption and fouling, theoretically, these desired characteristics are extensible to membranes made entirely of protein. Besides membrane, a porous protein-based separation matrix could also be appreciated in other separation technologies, namely chromatographic separation. References [1] A. Iwama, Y. Kasuse, New polyimide ultrafiltration membrane for organic use, J. Membr. Sci. 11 (1982) 297–309. [2] D.T. Friesen, S.B. McCray, D.D. Newbold, Solvent resistant hollow fiber vapor permeation membrane and modules, US Patent no. 5,753,008, 19 May 1998.

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