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Gelation characteristics of the sugar beet pectin solution charged with fish oil-loaded zein nanoparticles
Food Hydrocolloids xxx (2014) 1e6
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Gelation characteristics of the sugar beet pectin solution charged with fish oil-loaded zein nanoparticles Sahar Soltani a, Ashkan Madadlou a, b, * a
Department of Food Science and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Centre of Excellence for Application of Modern Technologies for Producing Functional Foods and Drinks, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2014 Accepted 27 July 2014 Available online xxx
It is of interest to fabricate chemical- and sugar-free pectin gels by which nutraceuticals or nutraceuticalloaded particles are delivered to consumers. Fish oil, a well known source of omega-3 fatty acids, was encapsulated in zein via alcohol evaporation. The mean size of the core-free and fish oil-loaded zein particles was 69 and 83 nm, respectively. The oil-loaded zein nanoparticles were then inoculated into the sugar beet pectin solution that gelified subsequently by the action of the oxidative enzyme, laccase. It was found that nanoparticle charging and enzymatic oxidation of pectin solution changed the flow model of the solution from Newtonian to Power law. An initial gelation rate was estimated for laccaseinjected pectin solutions by using the data obtained from small amplitude rheometry tests. Nanoparticle charging of pectin solution slowed down the gelation rate ~26 folds and resulted in a much less firm final gel. Fourier transform infrared spectroscopy confirmed that ferulic acid residues of sugar beet pectin were oxidized by laccase, resulting in gel formation. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Nutraceutical Pectin Zein Nanoparticle Gel Laccase
1. Introduction Sugar beet pectin (SBP) is a co-processing product of sucrose production from sugar beets. This type of pectin has received an increasing attention by the food industry because of its specific functional properties including oxidative gelability (Norsker, Jensen, & Adler-Nissen, 2000). Some of the arabinose and galactose residues in SBP are esterified with ferulic acid (Takei, Sugihara, Ijima, & Kawakami, 2011) and provide a route for cross-linking of arabinan chains by the oxidative chemicals or enzymes (Zaidel, Chronakis, & Mayer, 2012). The enzyme laccase is a polyphenol oxidase that oxidizes and cross-links the intra- and inter-molecular ferulic acid units (Kuuva, Lantto, Reinikainen, Buchert, & Autio, 2003). Cross-linked arabinan chains in high enough concentrations form a thermo-irreversible gel which can be heated while maintaining integrity (Norsker et al., 2000). Laccase gels pectin through a green mechanism meeting both consumers' and
* Corresponding author. Department of Food Science and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. Tel.: þ98 26 32248804; fax: þ98 26 32249453. E-mail address: [email protected] (A. Madadlou).
environmental concerns about chemicals. The sugar-free pectin gel may be regarded as a low-calorie healthy product. Omega-3 polyunsaturated fatty acids such as a-linolenic, eicosapentaenoic and docosahexaenoic acids are health-promoting and physiologically active compounds. These fatty acids play vital roles in the prevention and treatment of cardiovascular diseases, hypertension, arthritis, immune response disorders, and some types of cancers including colon, breast, and prostate (Duan, Jiang, & Zhao, 2011). The ratio of u-6 to u-3 fatty acids in the diet is an important determinant of health. The optimal ratio varies from 1/1 to 4/1 depending on the disease under consideration (Simopoulos, 2002). The dietary intake of u-3 fatty acids of most populations is however considerably low (Mahan, Escott-Stump, & Raymond, 2012). Therefore an increased consumption of u-3 fatty acids is recommended (Kolanowski, 2006). Fish oil is an excellent source of polyunsaturated fatty acids (Nakanishi, Iitsuka, & Tsukamoto, 2013). Enrichment of foods with fish oil is however, limited due to the insolubility of oil in most food systems and extensive sensitivity of long chain polyunsaturated fatty acids to oxidation. The latter is of particular importance when the enriched food undergoes a complementary heat treatment for microbial inactivation purposes. Encapsulation of the oil may retard or even prevent the thermo-oxidation reactions and widen the range of food commodities intended for enrichment purposes.
http://dx.doi.org/10.1016/j.foodhyd.2014.07.030 0268-005X/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Soltani, S., & Madadlou, A., Gelation characteristics of the sugar beet pectin solution charged with fish oilloaded zein nanoparticles, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.030
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Zein, the prolamin fraction of the corn protein, is insoluble in water but is readily dispersed in binary mixtures of alcohols and water. It has long been recognized for application in food industry (Quispe-Condori, Saldana, & Temelli, 2011). This potential is mostly arisen from the self-assembly property of zein because of its amphiphilic character (Wang & Padua, 2010). The protein is assembled into micro/nanoscaled particles in along with the increasing polarity of solvent, which is achieved through alcohol evaporation or water addition. As the solvent polarity increase, hydrophobic domains of zein molecules are buried inwards to escape from the polar medium (Wang & Padua, 2010). Fat soluble compounds are enclosed successfully within zein particles for encapsulation and controlled release purposes (Parris, Cooke, & Hicks, 2005). It is of interest to fabricate SBP gels charged with fish-oil loaded zein nanoparticles as the delivery vehicles of u-3 fatty acids and lipid-soluble drugs to consumers. There was no report in literature on the gelation behavior and gel characteristics of the pectin gels carrying zein nanoparticles. The objective of the present study was therefore to investigate the influence of charging of SBP solution with fish-oil loaded zein nanoparticles on laccase-induced gelation and gel characteristics of pectin. 2. Materials and methods 2.1. Materials Maize zein, ethanol and laccase obtained from Trametes versicolor (optimum pH ¼ 5.0; optimum temperature ¼ 40 C) were purchased from SigmaeAldrich (Taufkirchen, Germany). Sugar beet pectin (Betapec Ru 301) was a kind gift by Herbstreith and Fox (Werder/Havel, Germany). Fish oil with the commercial name of “omega 3 fish oil” (Ho 307-Batch VO 10045) was supplied kindly by LYSI (Reykjavik, Iceland). 2.2. Zein nanoparticles preparation Fish oil-loaded zein nanoparticles were produced by solvent evaporation (Wang & Padua, 2010). Zein (0.2 gr 100 mL1) and fish oil (0.04 gr 100 mL1) were dissolved in 80% ethanol (w/w) while stirring at 700 rpm for 15 min at 25 C. The beaker containing 100 mL zein solution was placed under the hood at 50 C for 80 min to allow evaporation until the solution volume diminished to 20 mL. Removal of ethanol through evaporation resulted in formation of zein nanoparticles enveloping the fish oil. 2.3. Entrapment of nanoparticles in pectin gel Sugar beet pectin solution was prepared via dispersing pectin powder in distilled water (7.5 g 100 mL1), stirring at 500 rpm for 60 min and then storing at 25 C for 10 h in order to fully hydrate the polysaccharide. Sodium azide (50 mg L1) was added to the freshly prepared pectin solution to suppress the microbial activities. Authors emphasize that sodium azide is highly toxic for human being and very dangerous for environment and would never be added to a consumer product. The zein nanoparticles aqueous dispersion was added into the pectin solution at ratio of 2:1 so that the final concentration of pectin became 2.5 g 100 mL1. Subsequently, pH value of the mixed solution was adjusted to 5.0 and then inoculated with laccase (5 unit g1 pectin). The solution was incubated at 40 C for 5 h to warrant gelation. This resulted in a fully developed pectin gel containing fish oil-loaded zein nanoparticles. The fish oil content of the pectin gel was estimated to be 0.88 mg mL1. Current recommended intake of u-3 long chain fatty acids is at the range of 130e260 mg day1 (Hibbeln, Nieminen,
Blasbalg, Riggs, & Lands, 2006). Hence, an approximate volume of 147e295 mL of the pectin gel carrying fish oil-loaded zein nanoparticles would be required to meet the daily recommended intake. A control nanoparticle-free counterpart gel was also fabricated. 2.4. Size measurement of zein nanoparticles Aqueous fresh dispersion of zein nanoparticles was employed for particle size measurement by using a dynamic light scattering particle size analyzer (ZetaPlus, Brookhaven Instruments Co., NY, USA). The number-averaged diameter was selected to express the particle size of nanoparticles. Sample was read three times. 2.5. Atomic force microscopy The topographic images of pectin gel specimens were acquired by an atomic force microscope (Nanowizard II instrument, JPK, Germany) in the intermittent contact mode using ACTA cantilever with resonance frequency of 1 Hz and force constant of 13e77 N m1. For this purpose, a thin slice was cut from the produced pectin gel carrying fish oil-loaded zein nanoparticles with a surgery blade, placed on a lamella and let air dried before microscopic imaging. 2.6. Fourier transform infrared (FTIR) spectroscopy FTIR spectra of zein nanoparticles and pectin gels were characterized by means of a Perkin Elmer FT-IR spectrometer (Perkin Elmer Co., MA, USA) using the potassium bromide disk method in 4000e500 cm1 range with resolution of 4 cm1. 2.7. Rheological analysis 2.7.1. Viscosity of pectin solutions The viscosity of the pectin solution impregnated with fish oilloaded zein nanoparticles and its nanoparticle-free counterpart either immediately after laccase injection or before the injection was measured by using a Brookfield viscometer (LVDV-II Pro, Brookfield Engineering Inc., USA) equipped with the LV spindle at 25 C. In each test, about 20 mL sample was poured into the measuring cylinder and shear rate was adjusted to increase from 12 to 110 s1 within 12 s intervals. The Newtonian and power law models were used to analyze the rheological behavior of samples. 2.7.2. Gelation rate determination Gelation rate of the pectin solution charged with fish oil-loaded zein nanoparticles and its nanoparticle-free counterpart was determined through measuring the complex modulus (G*) immediately after being injected with laccase. A controlled rate Bohlin Gemini 2 rheometer (Malvern Instruments Ltd., Worcestershire, UK) fitted with a cup and vane geometry was employed in the experiment. Specimens were placed within the cup and measurements were carried out at frequency of 1 Hz and strain of 0.5% at 40 C. A frequency sweep (0.01e100 Hz) test at 2% strain was also performed on the formed gels 110 min after laccase injection to determine and compare the storage and loss moduli (G' and G00 , respectively) of the nanoparticle-loaded and nanoparticle-free pectin gels. 2.8. Statistical analysis The data were subjected to one-way analysis of variance (ANOVA) by SPSS (ver. 16, IBM software, NY, USA) software. Any
Please cite this article in press as: Soltani, S., & Madadlou, A., Gelation characteristics of the sugar beet pectin solution charged with fish oilloaded zein nanoparticles, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.030
S. Soltani, A. Madadlou / Food Hydrocolloids xxx (2014) 1e6
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significant difference among the means was found by employing Duncan's test at p level of 0.05. 3. Results and discussion 3.1. Size of zein nanoparticles Table 1 reports the particle size distribution of core-free and fish oil-loaded zein nanoparticles obtained in the present study through solvent evaporation technique. The mean size of both kinds of particles was nano-scaled though a number of sub-micron sized particles present in samples. Zou, Li, Percival, Bonard, and Gu (2012) fabricated cranberry procyanidins-loaded zein nanoparticles through antisolvent addition method with mean size of 392 nm. Zhong, Tian, and Zivanovic (2009) by antisolvent addition procedure produced fish oil-loaded zein nanoparticles with size of 350e450 nm. The smaller size of nanoparticles produced in the present study in comparison with those reported by Zou et al. (2012) and Zhong et al. (2009) is partly due to the difference in alcohol removal procedure. It is argued that a more gradual removal of alcohol via evaporation lets more thermodynamically favorable conformational changes of molecules and establishment of higher number of intermolecular interactions compared with the considerable faster method of antisolvent addition. The difference in the initial concentration of zein solutions, as well as, the alcohol content of solvent employed in various studies could influence the solubility of zein molecules (Kim & Xu, 2008; Wang & Padua, 2010) and play a notable role in the particle size variation of different studies. Fish oil encapsulation within zein increased the mean size of particles in agreement with the results obtained by Wu, Luo, and Wang (2012) for encapsulation of essential oils in zein nanoparticles. The fish oil-loaded zein particles were also more polydisperse than the oil-free counterparts (Table 1). 3.2. Rheological characteristics In Fig. 1, the changes in shear stress against shear rate are demonstrated for the pectin solution charged with fish oil-loaded zein nanoparticles and its nanoparticle-free counterpart. The shear stress vs. shear rate behavior of core-free zein nanoparticles dispersion is also demonstrated in Fig. 1C. The viscosity of the particle-free pectin solution was constant throughout the shear rate range, providing a flow behavior index (n) of 1.0 (Table 2). An identical behavior is also observed for the zein nanoparticles dispersion. These indicate that the particle-free pectin solution and zein nanoparticles dispersion were Newtonian fluids. In contrast, the pectin solution impregnated with zein particles demonstrated shear-thinning character (n < 1) due most probably to the progressive alignment of the electrostatically-set zein particles/pectin molecules supra-assemblies in direction of shear field. Positively charged zein particles at pH of 5.0 (Casella & Whitaker, 1990) could interact electrostatically with the negatively charged pectin and form supra-assemblies in which zein particles surrounded by pectin. Fig. 1B and Table 2 show that the viscosity of both types of pectin solutions (charged with zein nanoparticles or not) and their corresponding consistency coefficients (K) increased after laccase
Table 1 Size and polydispersity of core-free and fish oil-loaded zein nanoparticles. Samples
Mean diameter (nm)
Diameter range (nm)
Polydispersity
Core-free Fish oil-loaded
69 83
60e263 73e265
0.165 0.249
Fig. 1. Shear stress as function of shear rate for (A): particle-free pectin solution and particle-charged pectin solution; (B): laccase-injected particle-free pectin solution and the laccase-injected particle-charged pectin solution; (C) core-free zein nanoparticles dispersion.
injection. This viscosifying influence of laccase arose from the cross-linking function of the enzyme by which feruloyl-bearing arabinan chains of galacturonan backbone inter-connected. Jung & Wicker (2008) confirmed the laccase-induced conjugation of sugar beet pectin by reduced ferulic acid concentration and increased Table 2 Rheological models fitted to the pectin solution and the pectin solution charged with fish oil-loaded zein nanoparticles. Sample
Model
K (pa sn)
n
Confidence of fit (%)
Particle-free pectin solution Particle-loaded pectin solution Enzymatically oxidized particle-free pectin solution Enzymatically oxidized particle-loaded pectin solution
Newtonian Power law Power law
0.013d 0.3b 0.03c
1a 0.51c 0.87b
99.6 99.4 99.2
Power law
0.46a
0.4d
99.1
Means with different superscripts in the same column differ significantly (p < 0.05).
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Fig. 2. Changes in the complex modulus (G*) versus t of (A): laccase-injected particlefree pectin solution; (B): laccase-injected particle-charged pectin solution.
particle size results. The enzymatically oxidized pectin solutions either being charged with zein particles or not were shear thinning (Table 2) which implies that a shear-sensitive network of pectin molecules formed by the immediate action of enzyme. It is noteworthy that sodium azide used as the antimicrobe agent (see Section 2.3) is an inhibitor of laccase (Johannes & Majcherczyk, 2000) and could interfere with the cross-linking action of enzyme. The laccase-induced gelation rate of the nanoparticle-loaded pectin solution and its particle-free counterpart was determined via plotting G* versus time (t) (Fig. 2). The Initial rate of gelation was measured as the changes in G* value (delta G*) at the early linear region of G* vs. time curve (Zaidel et al., 2012). Gelation rates of 0.8 Pa s1 and 0.03 Pa s1 are calculated for the particle-free and particle-charged pectin solutions, respectively. It is clear that charging of the pectin solution with fish oil-loaded zein nanoparticles slowed down the gelation rates ~26 folds. Littoz and McClements (2008) reported that laccase injection of pectin solution causes a steep decrease in the number of ferulic acid groups
during the first 250e2500 s (depending on enzyme concentration). We argue that a number of feruloyl groups of pectin were not accessible to the oxidative action of laccase in particle-charged pectin solution due to being wrapped around the surrounded zein nanoparticles. It has been similarly hypothesized that the less availability of feruloyl groups for enzymatic cross-linking in an emulsion in which sugar beet pectin had been homogenized together with other ingredients resulted in slower gelation compared with the emulsion charged with sugar beet pectin after homogenization (Zaidel et al., 2012). The G* values of both samples continued to increase over time until reached to maximum constant values when almost completely stable gels formed. In is noteworthy that the final G* (relative firmness) value of the particle-free sample was several times greater than the particleloaded counterpart, which emphasizes that oxidative gelation process was weakened due to zein nanoparticle loading into pectin solution. The frequency dependence of G' and G00 of the well-established pectin gels either impregnated with fish oil-loaded zein nanoparticles or not is indicated in Fig. 3. The frequency sweep test illustrated that gels G' value was greater than that of G00 throughout the entire frequency range. This result is in agreement with the report of Zaidel et al. (2012) and indicates that the samples were nezmore elastic than viscous (Lucero, Claro, Casas, & Jime Castellanos, 2011). Small amplitude oscillatory shear analysis indicated that zein nanoparticles diminished the elastic component of pectin gel (Fig. 3). Dynamic moduli cast light into the molecular aspects of biosystems and provide fundamental knowledge about the interactions of bi-component systems in their equilibrium state rather than bulk rheological properties. It is argued that the presence of zein particles decreased the integrity of the energy-storing pectin network (i.e. smaller G' values) and let more dissipation of the energy through system (i.e. greater G00 values). An AFM image of the pectin gel carrying zein nanoparticles is represented in Fig. 4. It is observed that the integrity of the gel network was disrupted to some extent by the zein nanoparticles resulting in less inter-locked elasticity contributing pectin network. 3.3. Fourier transform infrared spectroscopy In Fig. 5, upper panel shows the FTIR spectra of fish oil, core-free and fish oil-loaded zein nanoparticles. In the infrared spectrum of zein nanoparticles, the peak at 1648 cm1 shows the C]O stretching vibration of Amide I and the peak at 1538 cm1 is attributed to the NeH bending vibration (Stuart, 2004). These
Fig. 3. Storage (G') and loss (G00 ) moduli of the enzyme-induced nanoparticle-free and nanoparticle-loaded pectin gels.
Please cite this article in press as: Soltani, S., & Madadlou, A., Gelation characteristics of the sugar beet pectin solution charged with fish oilloaded zein nanoparticles, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.030
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Fig. 4. AFM images of zein nanoparticles-loaded pectin gel.
absorptions are important in identifying proteins. These peaks were displaced in the spectrum of fish oil-loaded zein nanoparticles to 1687 cm1 and 1541 cm1, respectively. The displacement of the C]O indicator peak to a higher wavenumber in the FTIR spectrum of oil-loaded zein particles implies that fish oil interacted most probably with the hydrophobic domains of protein molecules, resulting in weakening of the C]O resonance. The presence of two bands at 880 and 925 cm1 in the spectrum of oil-loaded particles which are attributed to eCeC/CeO (mixture of stretches) and outof-plane bending of ¼ CeH groups (Zhang, 2009) in fish oil, as well as, of the peak at 1742 cm1 which is related to carbonyl stretching (eC]O) suggest the entrapment of fish oil within zein nanoparticles. The peak at 1520 cm1 in the FTIR spectrum of pectin (Fig. 5, lower panel) indicates C]C bond in the aromatic ring of feruloyl groups. The peaks appeared at 760 cm1 and 830 cm1 represent the out-of-plane CeH bending and the peak at 918 cm1 shows the in-plane CeH bending of feruloyl rings (Stuart, 2004). The oxidative cross-linking of feruloyl groups by the laccase resulted in disappearance of these indicatory peaks in the spectrum of pectin gel in agreement to the results already reported by Gazme and Madadlou (2014). 4. Conclusions Fish-oil was successfully loaded into zein particles via solvent evaporation method. The generated zein particles were nano-scalar irrespective of being loaded with fish oil or not. Charging of pectin solution with fish oil-loaded zein nanoparticles influenced the flow behavior index (n) and consistency coefficient (K) of the solution, resulting in transformation of flow model from Newtonian to Power law. It was hypothesized that zein nanoparticles are covered electrostatically with pectin molecules. Small amplitude rheometry revealed that the enzyme-induced gelation rate of the pectin solution charged with fish oil-loaded zein nanoparticles was significantly slower than that of the particle-free counterpart. The zein particles-carrying pectin gel was also of smaller G* value in comparison with particle-free counterpart. Acknowledgment
Fig. 5. FTIR spectra of (A) fish oil, core-free and fish oil-loaded zein nanoparticles; (B) pectin powder and laccase-induced pectin gel.
The authors are thankful to Iranian National Science Foundation (INSF), grant number 90003665; and the Center of Excellence for Application of Modern technologies for Producing Functional Foods
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and Drinks; University of Tehran for the financial support of the research. References Casella, M. L., & Whitaker, J. R. (1990). Enzymatically and chemically modified zein for improvement of functional properties. Journal of Food Biochemistry, 14(6), 453e475. Duan, J., Jiang, Y., & Zhao, Y. (2011). Chitosanewhey protein isolate composite films for encapsulation and stabilization of fish oil containing ultra-pure omega-3 fatty acids. Journal of Food Science, 76(1), C133eC141. Gazme, B., & Madadlou, A. (2014). Fabrication of whey proteinepectin conjugate particles through laccase-induced gelation of microemulsified nanodroplets. Food Hydrocolloids, 40, 189e195. Hibbeln, J. R., Nieminen, L. R. G., Blasbalg, T. L., Riggs, J. A., & Lands, W. E. M. (2006). Healthy intakes of ne3 and ne6 fatty acids: estimations considering worldwide diversity. American Journal of Clinical Nutrition, 83(Suppl.), 1483Se1493S. Johannes, C., & Majcherczyk, A. (2000). Laccase activity tests and laccase inhibitors. Journal of Biotechnology, 78, 193e199. Jung, J., & Wicker, L. (2008). Laccase mediated conjugation of sugar beet pectin and the effect on emulsion stability. Food Hydrocolloids, 28, 168e173. Kim, S., & Xu, J. (2008). Aggregate formation of zein and its structural inversion in aqueous ethanol. Journal of Cereal Science, 47(1), 1e5. Kolanowski, W., Ziolkowski, M., Weißbrodt, J., Kunz, B., & Laufenberg, G. (2006). Microencapsulation of fish oil by spray drying:impact on oxidative stability. Part 1. European Food Research and Technology, 222(3e4), 336e342. Kuuva, T., Lantto, R., Reinikainen, T., Buchert, J., & Autio, K. (2003). Rheological properties of laccase-induced sugar beet pectin gels. Food Hydrocolloids, 17(5), 679e684. Littoz, F., & McClements, D. J. (2008). Bio-mimetic approach to improving emulsion stability: cross-linking adsorbed beet pectin layers using laccase. Food Hydrocolloids, 22, 1203e1211. nez-Castellanos, M. R. (2011). Rheological Lucero, M. J., Claro, C., Casas, M., & Jime and mechanical properties of hydrophilic dispersions using a new hydrophobically modified polymer and Igepal® CO520. Carbohydrate Polymers, 86(2), 891e896.
Mahan, L. K., Escott-Stump, S., & Raymond, J. L. (2012). Krause's food and the nutrition care process (13th ed.). St. Louis, Missouri: Elsevier (Chapter 2). Nakanishi, A., Iitsuka, N., & Tsukamoto, I. (2013). Fish oil suppresses bone resorption by inhibiting osteoclastogenesis through decreased expression of M-CSF, PU.1, MITF and RANK in ovariectomized rats. Molecular Medicine Reports, 7, 1896e1903. Norsker, M., Jensen, M., & Adler-Nissen, J. (2000). Enzymatic gelation of sugar beet pectin in food products. Food Hydrocolloids, 14(3), 237e243. Parris, N., Cooke, P. H., & Hicks, K. B. (2005). Encapsulation of essential oils in zein nanospherical particles. Journal of Agricultural and Food Chemistry, 53(12), 4788e4792. ~ a, M. D., & Temelli, F. (2011). Microencapsulation of flax Quispe-Condori, S., Saldan oil with zein using spray and freeze drying. LWT e Food Science and Technology, 44(9), 1880e1887. Simopoulos, A. P. (2002). The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomedicine & Pharmacotherapy, 56(8), 365e379. Stuart, B. (2004). Infrared spectroscopy. New York: John Wiley & Sons, Inc. Takei, T., Sugihara, K., Ijima, H., & Kawakami, K. (2011). In situ gellable sugar beet pectin via enzyme-catalyzed coupling reaction of feruloyl groups for biomedical applications. Journal of Bioscience and Bioengineering, 112(5), 491e494. Wang, Y., & Padua, G. W. (2010). Formation of zein microphases in ethanol-water. Langmuir, 26(15), 12897e12901. Wu, Y., Luo, Y., & Wang, Q. (2012). Antioxidant and antimicrobial properties of essential oils encapsulated in zein nanoparticles prepared by liquideliquid dispersion method. LWT e Food Science and Technology, 48(2), 283e290. Zaidel, D. N. A., Chronakis, I. S., & Meyer, A. S. (2012). Enzyme catalyzed oxidative gelation of sugar beet pectin: kinetics and rheology. Food Hydrocolloids, 28(1), 130e140. Zhang, J. (2009). Analysis and characterization of consumer products by FTIR, Raman, Chemometrics, and two dimensional ATR-FTIR correlation spectroscopy. Ph.D thesis. New Brunswick, New Jersey, United States: Rutgers University. Zhong, Q., Tian, H., & Zivanovic, S. (2009). Encapsulation of fish oil in solid zein particles by liquid-liquid dispersion. Journal of Food Processing and Preservation, 33(2), 255e270. Zou, T., Li, Z., Percival, S. S., Bonard, S., & Gu, L. (2012). Fabrication, characterization, and cytotoxicity evaluation of cranberry procyanidins-zein nanoparticles. Food Hydrocolloids, 27(2), 293e300.
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Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Gelation characteristics of the sugar beet pectin solution charged with fish oil-loaded zein nanoparticles Sahar Soltani a, Ashkan Madadlou a, b, * a
Department of Food Science and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Centre of Excellence for Application of Modern Technologies for Producing Functional Foods and Drinks, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2014 Accepted 27 July 2014 Available online xxx
It is of interest to fabricate chemical- and sugar-free pectin gels by which nutraceuticals or nutraceuticalloaded particles are delivered to consumers. Fish oil, a well known source of omega-3 fatty acids, was encapsulated in zein via alcohol evaporation. The mean size of the core-free and fish oil-loaded zein particles was 69 and 83 nm, respectively. The oil-loaded zein nanoparticles were then inoculated into the sugar beet pectin solution that gelified subsequently by the action of the oxidative enzyme, laccase. It was found that nanoparticle charging and enzymatic oxidation of pectin solution changed the flow model of the solution from Newtonian to Power law. An initial gelation rate was estimated for laccaseinjected pectin solutions by using the data obtained from small amplitude rheometry tests. Nanoparticle charging of pectin solution slowed down the gelation rate ~26 folds and resulted in a much less firm final gel. Fourier transform infrared spectroscopy confirmed that ferulic acid residues of sugar beet pectin were oxidized by laccase, resulting in gel formation. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Nutraceutical Pectin Zein Nanoparticle Gel Laccase
1. Introduction Sugar beet pectin (SBP) is a co-processing product of sucrose production from sugar beets. This type of pectin has received an increasing attention by the food industry because of its specific functional properties including oxidative gelability (Norsker, Jensen, & Adler-Nissen, 2000). Some of the arabinose and galactose residues in SBP are esterified with ferulic acid (Takei, Sugihara, Ijima, & Kawakami, 2011) and provide a route for cross-linking of arabinan chains by the oxidative chemicals or enzymes (Zaidel, Chronakis, & Mayer, 2012). The enzyme laccase is a polyphenol oxidase that oxidizes and cross-links the intra- and inter-molecular ferulic acid units (Kuuva, Lantto, Reinikainen, Buchert, & Autio, 2003). Cross-linked arabinan chains in high enough concentrations form a thermo-irreversible gel which can be heated while maintaining integrity (Norsker et al., 2000). Laccase gels pectin through a green mechanism meeting both consumers' and
* Corresponding author. Department of Food Science and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran. Tel.: þ98 26 32248804; fax: þ98 26 32249453. E-mail address: [email protected] (A. Madadlou).
environmental concerns about chemicals. The sugar-free pectin gel may be regarded as a low-calorie healthy product. Omega-3 polyunsaturated fatty acids such as a-linolenic, eicosapentaenoic and docosahexaenoic acids are health-promoting and physiologically active compounds. These fatty acids play vital roles in the prevention and treatment of cardiovascular diseases, hypertension, arthritis, immune response disorders, and some types of cancers including colon, breast, and prostate (Duan, Jiang, & Zhao, 2011). The ratio of u-6 to u-3 fatty acids in the diet is an important determinant of health. The optimal ratio varies from 1/1 to 4/1 depending on the disease under consideration (Simopoulos, 2002). The dietary intake of u-3 fatty acids of most populations is however considerably low (Mahan, Escott-Stump, & Raymond, 2012). Therefore an increased consumption of u-3 fatty acids is recommended (Kolanowski, 2006). Fish oil is an excellent source of polyunsaturated fatty acids (Nakanishi, Iitsuka, & Tsukamoto, 2013). Enrichment of foods with fish oil is however, limited due to the insolubility of oil in most food systems and extensive sensitivity of long chain polyunsaturated fatty acids to oxidation. The latter is of particular importance when the enriched food undergoes a complementary heat treatment for microbial inactivation purposes. Encapsulation of the oil may retard or even prevent the thermo-oxidation reactions and widen the range of food commodities intended for enrichment purposes.
http://dx.doi.org/10.1016/j.foodhyd.2014.07.030 0268-005X/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Soltani, S., & Madadlou, A., Gelation characteristics of the sugar beet pectin solution charged with fish oilloaded zein nanoparticles, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.030
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Zein, the prolamin fraction of the corn protein, is insoluble in water but is readily dispersed in binary mixtures of alcohols and water. It has long been recognized for application in food industry (Quispe-Condori, Saldana, & Temelli, 2011). This potential is mostly arisen from the self-assembly property of zein because of its amphiphilic character (Wang & Padua, 2010). The protein is assembled into micro/nanoscaled particles in along with the increasing polarity of solvent, which is achieved through alcohol evaporation or water addition. As the solvent polarity increase, hydrophobic domains of zein molecules are buried inwards to escape from the polar medium (Wang & Padua, 2010). Fat soluble compounds are enclosed successfully within zein particles for encapsulation and controlled release purposes (Parris, Cooke, & Hicks, 2005). It is of interest to fabricate SBP gels charged with fish-oil loaded zein nanoparticles as the delivery vehicles of u-3 fatty acids and lipid-soluble drugs to consumers. There was no report in literature on the gelation behavior and gel characteristics of the pectin gels carrying zein nanoparticles. The objective of the present study was therefore to investigate the influence of charging of SBP solution with fish-oil loaded zein nanoparticles on laccase-induced gelation and gel characteristics of pectin. 2. Materials and methods 2.1. Materials Maize zein, ethanol and laccase obtained from Trametes versicolor (optimum pH ¼ 5.0; optimum temperature ¼ 40 C) were purchased from SigmaeAldrich (Taufkirchen, Germany). Sugar beet pectin (Betapec Ru 301) was a kind gift by Herbstreith and Fox (Werder/Havel, Germany). Fish oil with the commercial name of “omega 3 fish oil” (Ho 307-Batch VO 10045) was supplied kindly by LYSI (Reykjavik, Iceland). 2.2. Zein nanoparticles preparation Fish oil-loaded zein nanoparticles were produced by solvent evaporation (Wang & Padua, 2010). Zein (0.2 gr 100 mL1) and fish oil (0.04 gr 100 mL1) were dissolved in 80% ethanol (w/w) while stirring at 700 rpm for 15 min at 25 C. The beaker containing 100 mL zein solution was placed under the hood at 50 C for 80 min to allow evaporation until the solution volume diminished to 20 mL. Removal of ethanol through evaporation resulted in formation of zein nanoparticles enveloping the fish oil. 2.3. Entrapment of nanoparticles in pectin gel Sugar beet pectin solution was prepared via dispersing pectin powder in distilled water (7.5 g 100 mL1), stirring at 500 rpm for 60 min and then storing at 25 C for 10 h in order to fully hydrate the polysaccharide. Sodium azide (50 mg L1) was added to the freshly prepared pectin solution to suppress the microbial activities. Authors emphasize that sodium azide is highly toxic for human being and very dangerous for environment and would never be added to a consumer product. The zein nanoparticles aqueous dispersion was added into the pectin solution at ratio of 2:1 so that the final concentration of pectin became 2.5 g 100 mL1. Subsequently, pH value of the mixed solution was adjusted to 5.0 and then inoculated with laccase (5 unit g1 pectin). The solution was incubated at 40 C for 5 h to warrant gelation. This resulted in a fully developed pectin gel containing fish oil-loaded zein nanoparticles. The fish oil content of the pectin gel was estimated to be 0.88 mg mL1. Current recommended intake of u-3 long chain fatty acids is at the range of 130e260 mg day1 (Hibbeln, Nieminen,
Blasbalg, Riggs, & Lands, 2006). Hence, an approximate volume of 147e295 mL of the pectin gel carrying fish oil-loaded zein nanoparticles would be required to meet the daily recommended intake. A control nanoparticle-free counterpart gel was also fabricated. 2.4. Size measurement of zein nanoparticles Aqueous fresh dispersion of zein nanoparticles was employed for particle size measurement by using a dynamic light scattering particle size analyzer (ZetaPlus, Brookhaven Instruments Co., NY, USA). The number-averaged diameter was selected to express the particle size of nanoparticles. Sample was read three times. 2.5. Atomic force microscopy The topographic images of pectin gel specimens were acquired by an atomic force microscope (Nanowizard II instrument, JPK, Germany) in the intermittent contact mode using ACTA cantilever with resonance frequency of 1 Hz and force constant of 13e77 N m1. For this purpose, a thin slice was cut from the produced pectin gel carrying fish oil-loaded zein nanoparticles with a surgery blade, placed on a lamella and let air dried before microscopic imaging. 2.6. Fourier transform infrared (FTIR) spectroscopy FTIR spectra of zein nanoparticles and pectin gels were characterized by means of a Perkin Elmer FT-IR spectrometer (Perkin Elmer Co., MA, USA) using the potassium bromide disk method in 4000e500 cm1 range with resolution of 4 cm1. 2.7. Rheological analysis 2.7.1. Viscosity of pectin solutions The viscosity of the pectin solution impregnated with fish oilloaded zein nanoparticles and its nanoparticle-free counterpart either immediately after laccase injection or before the injection was measured by using a Brookfield viscometer (LVDV-II Pro, Brookfield Engineering Inc., USA) equipped with the LV spindle at 25 C. In each test, about 20 mL sample was poured into the measuring cylinder and shear rate was adjusted to increase from 12 to 110 s1 within 12 s intervals. The Newtonian and power law models were used to analyze the rheological behavior of samples. 2.7.2. Gelation rate determination Gelation rate of the pectin solution charged with fish oil-loaded zein nanoparticles and its nanoparticle-free counterpart was determined through measuring the complex modulus (G*) immediately after being injected with laccase. A controlled rate Bohlin Gemini 2 rheometer (Malvern Instruments Ltd., Worcestershire, UK) fitted with a cup and vane geometry was employed in the experiment. Specimens were placed within the cup and measurements were carried out at frequency of 1 Hz and strain of 0.5% at 40 C. A frequency sweep (0.01e100 Hz) test at 2% strain was also performed on the formed gels 110 min after laccase injection to determine and compare the storage and loss moduli (G' and G00 , respectively) of the nanoparticle-loaded and nanoparticle-free pectin gels. 2.8. Statistical analysis The data were subjected to one-way analysis of variance (ANOVA) by SPSS (ver. 16, IBM software, NY, USA) software. Any
Please cite this article in press as: Soltani, S., & Madadlou, A., Gelation characteristics of the sugar beet pectin solution charged with fish oilloaded zein nanoparticles, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.030
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significant difference among the means was found by employing Duncan's test at p level of 0.05. 3. Results and discussion 3.1. Size of zein nanoparticles Table 1 reports the particle size distribution of core-free and fish oil-loaded zein nanoparticles obtained in the present study through solvent evaporation technique. The mean size of both kinds of particles was nano-scaled though a number of sub-micron sized particles present in samples. Zou, Li, Percival, Bonard, and Gu (2012) fabricated cranberry procyanidins-loaded zein nanoparticles through antisolvent addition method with mean size of 392 nm. Zhong, Tian, and Zivanovic (2009) by antisolvent addition procedure produced fish oil-loaded zein nanoparticles with size of 350e450 nm. The smaller size of nanoparticles produced in the present study in comparison with those reported by Zou et al. (2012) and Zhong et al. (2009) is partly due to the difference in alcohol removal procedure. It is argued that a more gradual removal of alcohol via evaporation lets more thermodynamically favorable conformational changes of molecules and establishment of higher number of intermolecular interactions compared with the considerable faster method of antisolvent addition. The difference in the initial concentration of zein solutions, as well as, the alcohol content of solvent employed in various studies could influence the solubility of zein molecules (Kim & Xu, 2008; Wang & Padua, 2010) and play a notable role in the particle size variation of different studies. Fish oil encapsulation within zein increased the mean size of particles in agreement with the results obtained by Wu, Luo, and Wang (2012) for encapsulation of essential oils in zein nanoparticles. The fish oil-loaded zein particles were also more polydisperse than the oil-free counterparts (Table 1). 3.2. Rheological characteristics In Fig. 1, the changes in shear stress against shear rate are demonstrated for the pectin solution charged with fish oil-loaded zein nanoparticles and its nanoparticle-free counterpart. The shear stress vs. shear rate behavior of core-free zein nanoparticles dispersion is also demonstrated in Fig. 1C. The viscosity of the particle-free pectin solution was constant throughout the shear rate range, providing a flow behavior index (n) of 1.0 (Table 2). An identical behavior is also observed for the zein nanoparticles dispersion. These indicate that the particle-free pectin solution and zein nanoparticles dispersion were Newtonian fluids. In contrast, the pectin solution impregnated with zein particles demonstrated shear-thinning character (n < 1) due most probably to the progressive alignment of the electrostatically-set zein particles/pectin molecules supra-assemblies in direction of shear field. Positively charged zein particles at pH of 5.0 (Casella & Whitaker, 1990) could interact electrostatically with the negatively charged pectin and form supra-assemblies in which zein particles surrounded by pectin. Fig. 1B and Table 2 show that the viscosity of both types of pectin solutions (charged with zein nanoparticles or not) and their corresponding consistency coefficients (K) increased after laccase
Table 1 Size and polydispersity of core-free and fish oil-loaded zein nanoparticles. Samples
Mean diameter (nm)
Diameter range (nm)
Polydispersity
Core-free Fish oil-loaded
69 83
60e263 73e265
0.165 0.249
Fig. 1. Shear stress as function of shear rate for (A): particle-free pectin solution and particle-charged pectin solution; (B): laccase-injected particle-free pectin solution and the laccase-injected particle-charged pectin solution; (C) core-free zein nanoparticles dispersion.
injection. This viscosifying influence of laccase arose from the cross-linking function of the enzyme by which feruloyl-bearing arabinan chains of galacturonan backbone inter-connected. Jung & Wicker (2008) confirmed the laccase-induced conjugation of sugar beet pectin by reduced ferulic acid concentration and increased Table 2 Rheological models fitted to the pectin solution and the pectin solution charged with fish oil-loaded zein nanoparticles. Sample
Model
K (pa sn)
n
Confidence of fit (%)
Particle-free pectin solution Particle-loaded pectin solution Enzymatically oxidized particle-free pectin solution Enzymatically oxidized particle-loaded pectin solution
Newtonian Power law Power law
0.013d 0.3b 0.03c
1a 0.51c 0.87b
99.6 99.4 99.2
Power law
0.46a
0.4d
99.1
Means with different superscripts in the same column differ significantly (p < 0.05).
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Fig. 2. Changes in the complex modulus (G*) versus t of (A): laccase-injected particlefree pectin solution; (B): laccase-injected particle-charged pectin solution.
particle size results. The enzymatically oxidized pectin solutions either being charged with zein particles or not were shear thinning (Table 2) which implies that a shear-sensitive network of pectin molecules formed by the immediate action of enzyme. It is noteworthy that sodium azide used as the antimicrobe agent (see Section 2.3) is an inhibitor of laccase (Johannes & Majcherczyk, 2000) and could interfere with the cross-linking action of enzyme. The laccase-induced gelation rate of the nanoparticle-loaded pectin solution and its particle-free counterpart was determined via plotting G* versus time (t) (Fig. 2). The Initial rate of gelation was measured as the changes in G* value (delta G*) at the early linear region of G* vs. time curve (Zaidel et al., 2012). Gelation rates of 0.8 Pa s1 and 0.03 Pa s1 are calculated for the particle-free and particle-charged pectin solutions, respectively. It is clear that charging of the pectin solution with fish oil-loaded zein nanoparticles slowed down the gelation rates ~26 folds. Littoz and McClements (2008) reported that laccase injection of pectin solution causes a steep decrease in the number of ferulic acid groups
during the first 250e2500 s (depending on enzyme concentration). We argue that a number of feruloyl groups of pectin were not accessible to the oxidative action of laccase in particle-charged pectin solution due to being wrapped around the surrounded zein nanoparticles. It has been similarly hypothesized that the less availability of feruloyl groups for enzymatic cross-linking in an emulsion in which sugar beet pectin had been homogenized together with other ingredients resulted in slower gelation compared with the emulsion charged with sugar beet pectin after homogenization (Zaidel et al., 2012). The G* values of both samples continued to increase over time until reached to maximum constant values when almost completely stable gels formed. In is noteworthy that the final G* (relative firmness) value of the particle-free sample was several times greater than the particleloaded counterpart, which emphasizes that oxidative gelation process was weakened due to zein nanoparticle loading into pectin solution. The frequency dependence of G' and G00 of the well-established pectin gels either impregnated with fish oil-loaded zein nanoparticles or not is indicated in Fig. 3. The frequency sweep test illustrated that gels G' value was greater than that of G00 throughout the entire frequency range. This result is in agreement with the report of Zaidel et al. (2012) and indicates that the samples were nezmore elastic than viscous (Lucero, Claro, Casas, & Jime Castellanos, 2011). Small amplitude oscillatory shear analysis indicated that zein nanoparticles diminished the elastic component of pectin gel (Fig. 3). Dynamic moduli cast light into the molecular aspects of biosystems and provide fundamental knowledge about the interactions of bi-component systems in their equilibrium state rather than bulk rheological properties. It is argued that the presence of zein particles decreased the integrity of the energy-storing pectin network (i.e. smaller G' values) and let more dissipation of the energy through system (i.e. greater G00 values). An AFM image of the pectin gel carrying zein nanoparticles is represented in Fig. 4. It is observed that the integrity of the gel network was disrupted to some extent by the zein nanoparticles resulting in less inter-locked elasticity contributing pectin network. 3.3. Fourier transform infrared spectroscopy In Fig. 5, upper panel shows the FTIR spectra of fish oil, core-free and fish oil-loaded zein nanoparticles. In the infrared spectrum of zein nanoparticles, the peak at 1648 cm1 shows the C]O stretching vibration of Amide I and the peak at 1538 cm1 is attributed to the NeH bending vibration (Stuart, 2004). These
Fig. 3. Storage (G') and loss (G00 ) moduli of the enzyme-induced nanoparticle-free and nanoparticle-loaded pectin gels.
Please cite this article in press as: Soltani, S., & Madadlou, A., Gelation characteristics of the sugar beet pectin solution charged with fish oilloaded zein nanoparticles, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.030
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Fig. 4. AFM images of zein nanoparticles-loaded pectin gel.
absorptions are important in identifying proteins. These peaks were displaced in the spectrum of fish oil-loaded zein nanoparticles to 1687 cm1 and 1541 cm1, respectively. The displacement of the C]O indicator peak to a higher wavenumber in the FTIR spectrum of oil-loaded zein particles implies that fish oil interacted most probably with the hydrophobic domains of protein molecules, resulting in weakening of the C]O resonance. The presence of two bands at 880 and 925 cm1 in the spectrum of oil-loaded particles which are attributed to eCeC/CeO (mixture of stretches) and outof-plane bending of ¼ CeH groups (Zhang, 2009) in fish oil, as well as, of the peak at 1742 cm1 which is related to carbonyl stretching (eC]O) suggest the entrapment of fish oil within zein nanoparticles. The peak at 1520 cm1 in the FTIR spectrum of pectin (Fig. 5, lower panel) indicates C]C bond in the aromatic ring of feruloyl groups. The peaks appeared at 760 cm1 and 830 cm1 represent the out-of-plane CeH bending and the peak at 918 cm1 shows the in-plane CeH bending of feruloyl rings (Stuart, 2004). The oxidative cross-linking of feruloyl groups by the laccase resulted in disappearance of these indicatory peaks in the spectrum of pectin gel in agreement to the results already reported by Gazme and Madadlou (2014). 4. Conclusions Fish-oil was successfully loaded into zein particles via solvent evaporation method. The generated zein particles were nano-scalar irrespective of being loaded with fish oil or not. Charging of pectin solution with fish oil-loaded zein nanoparticles influenced the flow behavior index (n) and consistency coefficient (K) of the solution, resulting in transformation of flow model from Newtonian to Power law. It was hypothesized that zein nanoparticles are covered electrostatically with pectin molecules. Small amplitude rheometry revealed that the enzyme-induced gelation rate of the pectin solution charged with fish oil-loaded zein nanoparticles was significantly slower than that of the particle-free counterpart. The zein particles-carrying pectin gel was also of smaller G* value in comparison with particle-free counterpart. Acknowledgment
Fig. 5. FTIR spectra of (A) fish oil, core-free and fish oil-loaded zein nanoparticles; (B) pectin powder and laccase-induced pectin gel.
The authors are thankful to Iranian National Science Foundation (INSF), grant number 90003665; and the Center of Excellence for Application of Modern technologies for Producing Functional Foods
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