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Highly Stable Foams from Block Oligomers Synthesized by Enzymatic Reactions Leonard M. C. Sagis,*,† Carmen G. Boeriu,§ Guus E. Frissen,§ Henk A. Schols,‡ and Peter A. Wierenga† Food Physics Group and Laboratory of Food Chemistry, Wageningen UniVersity and Research Center, Bomenweg 2, 6703 HD Wageningen, The Netherlands, and AFSG, Bornsesteeg 59, 6708 PD Wageningen, The Netherlands ReceiVed October 2, 2007. In Final Form: December 6, 2007 We have synthesized a new amphiphilic block oligomer by the enzymatic linking of a fatty acid (lauric acid) to a fructan oligomer (inulin) and tested the functionality of this carbohydrate derivative in foam stabilization. The structure of the modified oligosaccharide was found to be (Fruc)n(Glc)1CO-C11H23, which implies that on average one lauric acid molecule was linked to one inulin molecule. The new component produces foams with exceptional stability. Our results show that enzymatic acylation can produce an entirely new class of amphiphilic materials, with functionality comparable to that of synthetic block copolymers.
Introduction Block copolymers and block oligomers consisting of hydrophilic and hydrophobic blocks are excellent stabilizers for foam and emulsions. They can self-assemble into highly stable vesicles,1-4 which can be used for encapsulation and controlled release purposes in drug delivery1-4 and food products.5 They can also form stable microbubbles for application in ultrasonic diagnostics.6,7 In addition, they can form hydrogels, which can be used in controlled release applications, and for the stabilization of aerated products and solid dispersions.8,9 In view of their functional properties, block amphiphiles could potentially be used to design a new class of low-calorie, low-fat food products. The incorporation of these products into a healthy diet could contribute to solving the growing problem of obesity in the modern western industrial world. A major drawback of currently available synthetic block amphiphiles is that they are not food grade and therefore cannot be incorporated into food products. For pharmaceutical applications, it is in general acceptable that the block amphiphiles are merely biocompatible or biotolerable (i.e., they do not evoke an immune response10), but for application in food products, this is not sufficient. A possible method to create food-grade block amphiphiles is the linking of food-grade components. For example, hydrophobic fatty acids could be linked to hydrophilic polysaccharides,10 * Corresponding author. E-mail:
[email protected]. † Food Physics Group, Wageningen University. ‡ Laboratory of Food Chemistry, Wageningen University. § AFSG. (1) Discher, B. M.; Won, Y.-Y.; Ege, D. S.; Lee, J. C.-M.; Bates, F. S.; Discher, D. E.; Hammer, D. A. Science 1999, 284, 1143-1146. (2) Discher, D.; Eisenberg, A. Science 2002, 297, 967-973. (3) Antonietti, M.; Fo¨rster, S. AdV. Mater. 2003, 15, 1323-1333. (4) Holowka, E. P.; Pochan, D. J.; Deming, T. J. J. Am. Chem. Soc. 2005, 127, 12423-12428. (5) Gibbs, B. F.; Kermasha, S.; Alli, I.; Mulligan, C. N. Int. J. Food Sci. Nutr. 1999, 50, 213-224. (6) Forsberg, F.; Lathia, J. D.; Merton, D. A.; Liu, J.-B.; Le, N. T.; Goldberg, B. B.; Weatley, M. A. Ultrasound Med. Biol. 2004, 30, 1281-1287. (7) Rapoport, N.; Gao, Z.; Kennedy, A. J. Natl. Cancer Inst. 2007, 99, 10951106. (8) Shim, W. S; Yoo, J. S.; Bae, Y. H.; Lee, D. S. Biomacromolecules 2005, 6, 2930-2934. (9) Nagapudi, K.; Brinkman, W. T.; Leisen, J.; Thomas, B. S.; Wright, E. R.; Haller, C.; Wu, X.; Apkarian, R. P.; Conticello, V. P.; Chaikof, E. L. Macromolecules 2005, 38, 345-354. (10) Besheer, A.; Hause, G.; Kressler, J.; Ma¨der, K. Biomacromolecules 2007, 8, 359-367.
hydrophobic polysaccharides to hydrophilic proteins (or protein segments) or vise versa, or hydrophilic polysaccharides to hydrophobic proteins. An example of such a conjugate was recently developed by Besheer et al.10 They produced biocompatible block copolymers by coupling several types of fatty acids to hydroxyethyl starch and showed that these amphiphilic polymers can form micelles and vesicles. Although in view of their synthesis route these vesicles are not food grade, their work shows that this type of conjugate has functionality comparable to that of synthetic block copolymers. Magdassi et al.11 present a review of several hydrophobically modified biomolecules, including polysaccharides such as carboxymethylcellulose (CMC) and pullulan and proteins such as glucose oxidase and immunoglobulins, and discuss the effect of this modification on the interfacial behavior of these biomolecules. Another example of the hydrophobic modification of biopolymers is the chemical esterification of inulin described by Verraest et al.,12 Rogge and Stevens,13 and Grandtner et al.,14 although these authors do not discuss the effect of the modification on interfacial behavior. The heat-induced linking of polysaccharides, proteins, and fatty acids leads to nonspecific substitutions and renders a product with a wide compositional range and generally only a marginal increase in functionality.15 Enzymatic synthesis can give a higher specificity in substitutions and can produce products with substantially increased functionality. For these reasons, we have opted here for the enzymatic route to produce functional oligomers. In this work, we have focused on the development of new polysaccharide-based conjugates with surfactant properties, consisting of a hydrophilic polysaccharide backbone and a hydrophobic fatty acid side chain. For the hydrophobic block, lauric acid was used. For the hydrophilic block, we used inulin (Raftiline LS), a linear-chain fructo-oligosaccharide formed by β-(2,1) linkages of fructose and terminated by a glucose unit at one end.16 The molecular structure of Raftiline can be represented (11) Magdassi, S.; Kamyshny, A.; Baszkin, A. J. Dispersion Sci. Technol. 2001, 22, 331-322. (12) Verraest, D. L.; Zitha-Bovens, E.; Peters, J. A.; Bekkum, H. van. Carbohydr. Res. 1998, 310, 109-115. (13) Rogge, T. M.; Stevens, C. V. Biomacromolecules 2004, 5, 1799-1803. (14) Grandtner, G.; Joly, N.; Cavrot, J.-P., Granet, R.; Bandur, G.; Rusnac, L.; Martin, P.; Krausz, P. Polym. Bull. 2005, 55, 235-241. (15) Oliver, C. M.; Melton, L. D.; Stanley, R. A. Crit. ReV. Food Sci. Nutr. 2006, 46, 337-350. (16) Coussement, P. Lebensmittelind. Milchwirtsch. 1997, 118, 526-527.
10.1021/la7030494 CCC: $40.75 © 2008 American Chemical Society Published on Web 12/15/2007
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by the formula (Fruc)nGlc. For the sample used here, the average value of n is 10, as determined by MALDI-TOF and HPSEC (data not shown). Inulin, a water-soluble dietary fiber, is commercially extracted from chicory root but is also found in artichokes, asparagus, onions, garlic, and wheat. It has a low caloric value (1 kcal/g) and also acts as a prebiotic.17 Because the modifications were performed using an enzymatic route and all solvents used (such as DMSO) are accepted for use in the production of food ingredients, the modified samples can be considered food grade. We determined the effect of the enzymatic modification on the foam-stabilizing properties of the material. We first measured the dilatational elastic modulus of interfaces stabilized with the modified sample. This modulus is often an important parameter for the stability of foams. Low values for this modulus imply that the interfaces are very mobile and exhibit viscous behavior, whereas high values mean that the interfaces behave more like an elastic solid. The latter interfaces tend to produce more stable foams. We also tested the foam stability itself, using foam tubes, and found that the modification significantly enhances the foamstabilizing properties of inulin. Experimental Section Raftiline LS was obtained from Orafti (Tienen, Belgium) and was used without any further purification. Sucrose monolaurate was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Lipase from Candida antarctica, immobilized (Novozym-435), with an activity of 2 U/mg was a kind gift from Novozymes Netherlands (Bunnik, The Netherlands). For the synthesis of lauryl esters of Raftiline, 500 mg of Raftiline LS and 846 mg (3.8 mmoles) of vinyl laurate were dissolved in 75 mL of a mixture of tert-butanol/DMSO (80/20 v/v) at 80 °C. Then, 5 g of molecular sieves (4 Å) and 500 mg of Novozym-435 were added, and the reaction mixture was maintained for 24 h at 80 °C with mixing. The reaction was terminated by removing the biocatalyst by filtration, after cooling to approximately 35-40 °C. The solvent was evaporated under vacuum, and the solid residue was thoroughly washed with acetone to remove all traces of residual vinyl laurate and dried at 50 °C in vacuum. The reaction yielded 395 mg (product yield of 76%) of a light-yellow semicrystalline material. Control experiments were conducted without enzyme. For the characterization of lauryl esters of Raftiline, FTIR spectra were recorded with a Bruker Vector-22 FTIR spectrophotometer equipped with a transmittance accessory, at 4 cm-1 resolution, using a KBr pellet as reference. Interferograms (64) were co-added to obtain a high signal-to-noise ratio. The concentration of samples in the KBr pellet was 1%. NMR spectra were recorded on a Bruker Avance DPX300 spectrometer operating at 300 MHz (1H) and 75 MHz (13C). DMSO-d6 (99.8 atom % D, Merck, Uvasol) was used as received. The lauryl content in the product was determined according to the GC-FAME method.18 The samples were saponified with methanolic sodium hydroxide during 30 min at 100 °C and subsequently methylated with 6 N HCl in methanol at 80 °C. The FAMEs were extracted with hexan/methyl-tert-butyl ether and analyzed by GC. Lauric acid was determined from GC data on the basis of a calibration constructed with pure lauric acid and treated in a similar way as for the samples. The degree of substitution was determined using DS (mol/mol) ) mLA(162 + ∆M)/MLAmRL, where mLA is the amount of lauric acid (in milligrams) determined by the GC-FAME method, MLA is the molecular weight of lauric acid (200.32 g/mol), mRL is the amount of sample (in milligrams) present as a dry substance, 162 is the molecular weight of the hexose residue, and ∆M is the molecular weight of the lauryl residue (183 g/mol). The dilatational elastic modulus, Ed, of the samples was determined using an automated drop tensiometer (ADT, ITCONCEPT, Longessaigne, France). All samples were dissolved overnight in a 10 mM phosphate buffer (pH 7) at a concentration of 0.1% w/w. The (17) Coussement, P. Food Technol. 1996, 2, 102-104. (18) Sasser, M. MIDI Technical Note No. 101, 1990.
Figure 1. (A) FTIR spectra of Raftiline LS (line 1) and Raftiline LS laurate (line 2). The inset shows the difference spectrum (line 2 - line 1) for the 1660-1780 cm-1 spectral region, which is characteristic of ester absorption. (B) Possible structure of a lauryl ester of Raftiline LS. solutions were centrifuged to remove any insoluble materials. Surface pressure Π was measured as a function of time, and cycles of sinusoidal deformations were applied to determine the dilatational elastic modulus. From the data of the surface pressure and dilatational elastic modulus as a function of time, a Π-Ed curve was constructed. The drop volume was 7 µL, and the deformation amplitude ∆A/A was equal to 0.04. For the foam tests, a 0.3% w/w solution of modified Raftiline LS and a 0.66% w/w solution of sucrose monolaurate were prepared in 10 mM phosphate buffer (pH 7), in the same manner as for the measurements of the dilatational modulus. Of these solutions, 20 mL was poured into a foam tube with a glass grid at the bottom. The tube diameter was 2.5 cm. Both solutions were aerated from below with nitrogen gas at a flow rate of 16.7 mL/s.
Results and Discussion The esters of lauric acid and Raftiline LS were synthesized using lipase as a catalyst, in a solvent mixture containing 20%/ 80% v/v DMSO/tert-butanol at 80 °C with vinyl laurate as the acyl donor (Experimental Section). The formation of ester bonds on the substrate molecules after the enzymatic reaction was investigated using infrared spectroscopy (FTIR). Infrared spectra of the isolated products clearly show the characteristic absorption of the lauryl ester group at 1716-1731 cm-1 as well as the absorption due to the C-H stretching of CH2 groups of the alkyl chain at 2920 cm-1 (symmetric) and 2856 cm-1 (asymmetric). For illustration, the FTIR spectra of Raftiline LS and the lauryl ester of Raftiline are shown in Figure 1. From NMR spectroscopy, the presence of the lauryl ester group could also be confirmed. 1H NMR (300 MHz, DMSO-d ): δ 0.8-0.9 (3H, br t, CH ); 6 3 1.1-1.3 (16H, br s, (CH2)8; 1.4-1.6 (br s, CH2CH2CO); 2.2-
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Figure 2. Dilatational modulus Ed as a function of surface pressure, Π, for Raftilin LS (O), sucrose monolaurate [(Sucr)1C12] (4), and (Fruc)n(Glc)1CO-C11H23 (9).
2.4 (br t, CH2CO); signals from 3.3 to 5.5 ppm (10H) belong to CH, CH2, and OH of the carbohydrate moiety. 13C NMR (75 MHz, DMSO-d6): δ 14.2 (CH3); 22.4 and 28.8-31.5 (CH2 of the lauryl group); 61.7-61.9, 72-79, 81.8-82.1 (CH and CH2 of the carbohydrate moiety); 103.2-104.2 (Cquat at position 2); 170.7 (CdO). A lauryl ester content of approximately 5.8% was determined using the GC-FAME method.18 A degree of substitution (DS) of 0.1 ( 0.009 mol/mol was calculated, which implies that on average we have one lauric acid coupled to one Raftiline LS molecule. The modified oligosaccharide product therefore can be assigned the structure (Fruc)n(Glc)1CO-C11H23. From the spectroscopic data, however, no conclusions could be drawn regarding the regioselectivity of the substitution toward the primary hydroxyl group of fructose or the terminal glucose residues of the inulin. To test the functionality of this new functionalized fructan oligomer, we first determined the surface dilatational elastic modulus Ed of the air-water interface of a 0.1% w/w solution of the product and compared it to the dilatational elastic modulus of a 0.1% w/w Raftiline LS solution and a 0.1% w/w sucrose monolaurate solution (Figure 2). The sucrose monolaurate sample was a commercially available emulsifier. The dilatational elastic modulus was determined using an automated drop tensiometer (ADT). We clearly see that pure Raftiline LS is only mildly surface-active. Sucrose monolaurate is surface-active but has a negligible dilatational elastic modulus. The new amphiphilic inulin-based oligomer has a surface pressure comparable to that of sucrose monolaurate but has a much higher dilatational modulus. It should therefore have a significantly increased performance as a foam stabilizer. Note that for the modified sample the data points on the Π-Ed curve (measured with increasing time from left to right) do not coincide as in the case of inulin and sucrose monolaurate. The adsorption of the modified sample appears to be relatively slow when compared to that of the other two samples. To determine if the modified sample has improved foamstabilizing properties, we prepared a new solution containing 0.3% w/w (Fruc)n(Glc)1CO-C11H23 and a solution containing 0.66% w/w sucrose monolaurate in 10 mM phosphate buffer at a pH of 7. The sucrose monolaurate produced about 15 cm of foam during aeration, which collapsed within 8 s after the nitrogen flow was stopped. The (Fruc)n(Glc)1CO-C11H23 solution produced 25 cm of foam that was significantly more stable. The foam drained relatively quickly, within 6 min, but the remaining dried-out foam stayed stable for more than an hour. No coarsening could be observed in the time span of the experiment. Figure 3a shows a picture of the foam after 55 min. The lower 3 cm of the foam still contained fluid, as a result of capillary forces. After 1 h, the foam tube was slowly tilted to bring the dried-out foam into contact with the fluid. The foam rehydrated very rapidly, as a result of capillary forces (Figure 3b).
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Figure 3. Pictures of the foam produced with a modified Raftilin sample: (A) after 55 min, (B) immediately after the first rehydration at 60 min, and (C) immediately after the second rehydration at 75 min.
The mechanical disturbance of the foam induced by the tilting caused some minor coalescence in the foam, resulting in a slight local coarsening of the foam, but the foam height did not change. After rehydration, the foam drains again in about 5 min, resulting in stable dried-out foam. The tilting process was repeated after another 15 min (total time 75 min), and again the foam rehydrated (Figure 3c). In the process, a few holes are created in the foam, but the foam height remains constant and no further coarsening is observed during the next 30 min. The films in the foam stabilized with modified Raftiline are apparently very strong and have a high resistance to mechanical disturbances. These molecules are linear and fairly stiff, and a possible explanation for the high stability of the films is that the modified Raftiline molecules crystallize at the air-solution interface. However, this is only a hypothesis, and the exact structure of the interface should be tested using grazing angle X-ray reflectivity measurements.19 The results clearly show that this component is a very good foam stabilizer. Drainage is rather fast, but this is a result of the low viscosity of the solution (equal to the viscosity of water). In an actual product, the viscosity of the continuous phase would probably be a factor 100 to 1000 times higher, which would mean that the foam stays hydrated for 10 to 100 h. In view of the high stability of the interface, these components also have potential as emulsifiers. Their linear and fairly stiff structure may also allow them to form vesicles, but additional research will be needed to confirm this.
Conclusions The results of this study show that enzymatic coupling of oligosaccharides with fatty acids produces novel derivatives with enhanced surface-active properties that could find application in the stabilization of foams and emulsions,20 in encapsulation systems, and in structuring applications. Using Raftiline, the approach resulted in acylated inulin oligosaccharides having functional properties comparable to those of synthetic block copolymers. The results of this work clearly show that it is possible to create food-grade block copolymers by the enzymatic coupling of polysaccharides and/or fatty acids and create an entirely new generation of structuring agents for application in food products. Because these components are also biotolerable, they could also be used in pharmaceutical applications, medical diagnostics, and cosmetic products. LA7030494 (19) Kaganer, V. M.; Mo¨hwald, H.; Dutta, P. ReV. Mod. Phys. 1999, 71, 779819. (20) Dalgleish, D. G. Food Hydrocolloids 2006, 20, 415-422.
