- Email: [email protected]
Nanoencapsulation of date palm pit extract in whey protein particles generated via desolvation method
Food Research International 51 (2013) 866–871
Contents lists available at SciVerse ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Short communication
Nanoencapsulation of date palm pit extract in whey protein particles generated via desolvation method Leila Bagheri, Ashkan Madadlou ⁎, Mohammadsaeed Yarmand, Mohammad E. Mousavi Department of Food Science, Technology and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karadj, Iran
a r t i c l e
i n f o
Article history: Received 18 November 2012 Accepted 29 January 2013 Keywords: Desolvation Date palm Encapsulation Whey protein nanoparticles
a b s t r a c t An alkaline solution of whey protein isolate was charged with absolute ethanol resulting in precipitation of whey protein particles. The vacuum-dried particles were then dispersed either in water or aqueous ethanol. Heat-treatment of whey proteins before desolvation process decreased the mean size of particles when dispersed in aqueous ethanol from 280 nm to 183 nm. The range and mean size of particles prepared from heat-treated protein solution when dispersed in water were 41–212 nm and 103 nm, respectively. Date palm pit aqueous extract was encapsulated inside the particulating heat-treated whey proteins during the desolvation stage with encapsulation efficiency of ~78%. Extract-loaded particles had mean size of 163 nm in alcoholic dispersion and 92 nm in water dispersion. Scanning electron microscopy imaging showed spherical nanoparticles aggregated in dry state. Fourier transform infrared spectroscopy suggested that extract and whey proteins did not covalently bind. Heat-treatment of whey proteins before desolvation resulted in the absence of denaturation endotherm in differential scanning calorimetry curve of extract-free particles. Extract loading in particles interrupted the continuity of protein matrix causing the occurrence of mild glass transition phenomenon in extract-loaded particles when heated. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Nanoparticles when used in biological and food systems may provide superior characteristics to microparticles including unique quality, improved sensory properties, extended flavor perception, better mouthfeel, transparent appearance and enhanced processability (Moraru et al., 2003). Therefore, nanobioparticles prepared from generally recognized as safe (GRAS) biopolymers are increasingly nominated for various applications in biomedical, pharmaceutical and nutraceutical industries remembering their acknowledged biocompatibility. Polyphenols act as metal scavengers, antimutagenes, and antimicrobial agents (Proestos et al., 2005). Their consumption may reduce the risk of some chronic diseases such as cancer, cardiovascular disease, chronic respiratory disease and diabetes (Arts, Van de Putte, & Hollman, 2000; Scalbert & Williamson, 2000). However, incorporation of polyphenols into foods may cause quality defects such as astringent taste and increased haze in beverages. They can act as substrates for browning reactions (Lesschaeve & Noble, 2005) resulting in undesirable color changes in food products. Also, polyphenols and other bioactive substances may undergo degradation and/or deterioration by food processing operations and storage (e.g. heating, acidification, light and oxygen) or in the gastrointestinal tract (acidic pH, enzymes, presence of other nutrients). These drawbacks impose limits for application ⁎ Corresponding author. E-mail address: [email protected] (A. Madadlou). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.01.058
and potential benefits of these nutraceuticals (Bell, 2001). Nanoencapsulation of bioactive molecules within appropriately structured carriers may overcome these problems without adverse effect on sensory characteristics and appearance of final product. Desolvation technique is a straightforward, rapid and easily applicable method to carry out the whole nanoparticle preparation procedure in one pot. This technique requires only two miscible solvents without involvement of destructing factors such as high shear rate, heating and sonication that damage the tertiary structure of proteins. As well, the procedure does not include toxic reagents and surfactants (Bilati, Allémann, & Doelker, 2005). Gunasekaran, Ko, and Xiao (2007) used acetone to desolvate β-lactoglobulin and then crosslinked the generated nanoparticles by glutaraldehyde–ethanol mixture. Recently, fish oil was encapsulated in zein nanoparticles desolvated by water from aqueous alcoholic solution (Zhong, Tian, & Zivanovic, 2009). Gülseren, Fang, and Corredig (2012a) generated protein nanoparticles by adding ethanol as antisolvent to an alkaline solution of whey proteins, followed by resolvation of particles through diluting the suspension by aqueous buffers. The procedure was later used to encapsulate zinc chloride within whey protein particles (Gülseren, Fang, & Corredig, 2012b). To the best of authors' knowledge there is no report in the literature on encapsulation of polyphenols in protein nanoparticles prepared by antisolvent (desolvation) method. The objective of the present study was therefore to encapsulate the aqueous extract of date palm pit as a rich source of phenolic compounds within whey protein nanoparticles obtained through desolvating by ethanol. The obtained extract-loaded
L. Bagheri et al. / Food Research International 51 (2013) 866–871
particles are expected to be easily added to beverages with minimum influence on sensory characteristics of food product.
867
2.5. Particle size measurement
Sodium chloride (NaCl), sodium bicarbonate (NaHCO3), sodium hydroxide (NaOH), hydrochloric acid (HCl), ethanol and Folin and Ciocalteau's phenol reagent (mixture of phosphomolybdate and phosphotungstate) were purchased from Merck (Darmstadt, Germany). Lactose- and fat-free whey protein isolate (WPI) with 92% protein content was a kind gift from Arla Food Ingredients (Viby J, Denmark). Bi-distilled water was used throughout the study.
Size and polydispersity of particles and capsules were determined by using a dynamic light scattering particle size analyzer (ZetaPALS, Brookhaven Instruments Co., NY, USA). For this purpose, dried samples were either dispersed in 10 mL ethanol at pH 9.0 or bi-distilled water with pH 9.0 at ratio of 1:200 (w/v) and shaken continuously at room temperature for 12 h using orbital incubator (Stuart®, S150, Guill Bern Corporation Inc., Philippines) to allow complete hydration. To investigate the influence of heat-treatment of WPI on particle size, a sample was also prepared from non-heated WPI following the same procedure as described. Particle size measurements were carried out at 25 °C with laser beam operated at 657 nm and scattering angle of 90°. Each sample was read three times. Average sizes reported are the volume-averaged diameters.
2.2. Preparation of date palm pit extract
2.6. Scanning electron microscopy
Date palm fruit (Kabkab variety) pits were washed and air-dried at 50 °C for 4 h. The dried pits were milled using a heavy-duty grinder to pass 1 mm screen, afterwards, 1 L boiling water was added to 50 g pit powder and extraction was carried out at 30 °C for 7 h while shaken at 100 rpm. The mixture was filtered through a series of Whatman filter papers in order to remove all suspended materials. The extract was freeze-dried and kept in dark glass bottles at −80 °C until use.
The morphology of dry extract-free and extract-loaded nanoparticles was observed with a scanning electron microscope (SEM, KYKYEM3200, KYKY Technology Development Ltd, China) operated at 24 kV. The surfaces of particles were sputtered with gold, observed and photographed.
2. Materials and methods 2.1. Materials
2.3. Measurement of phenolic content Total phenolic content was measured using the Folin–Ciocalteau method (Singleton & Rossi, 1965). Pit extract (30 μL) was mixed with 2.37 mL deionized water and 150 μL Folin–Ciocalteau's phenol reagent and allowed to stand at room temperature for 7 min, then 450 μL sodium bicarbonate (20% w/v) was added to the mixture. After standing for 70 min at room temperature, absorbance was measured (Spectrophotometer BioQuest CE2502, Cecil Instruments Ltd., UK) at 760 nm. Results were expressed as mg gallic acid equivalents (GAE)/100 g sample (Shui & Leong, 2006). Polyphenol content of extract was 1582 mg gallic acid equivalent per 100 g dry weight at pH 6.25.
2.7. Particle yield and encapsulation efficiency The weight of dried pellet of extract-free and extract-loaded particles was used for calculating the particle yield as follows:
Particle Yieldð%Þ ¼
weight of dry pellet 100: total weight of extract and WPI used for particles preparation
The efficiency of extract entrapment in particles was calculated as the difference between phenolic content added to WPI solution before desolvation stage and the content remained in the centrifugal supernatant. To determine the holding degree of encapsulated polyphenols within particles, precipitated pellet was dispersed both in 70% ethanol and bi-distilled water, mildly shaken for 30 min and re-precipitated through centrifugation after which the supernatant was used for polyphenol measurement.
2.4. Preparation of extract-free and extract-loaded particles WPI was dissolved (3% w/v) in 10 mM NaCl solution by stirring at 500 rpm at room temperature for 2 h; sodium azide (50 mg/L) was added to prevent microbial growth. The solution was stored at 4 °C for 12 h and filtered through 0.45 μm PVDF syringe filter (Whatman, Germany) prior to use. Protein solution was heat-treated at 60 °C for 30 min (Qi & Onwulata, 2011) and its pH was adjusted to 9.0 with 2 M NaOH. The pH adjustment led to smaller particles based on preliminary experiments. The solution was then charged with ethanol at a rate of 1 mL min −1 while stirring at 500 rpm until became turbid. The rate of ethanol addition was controlled carefully since it influences the size of generated particles (Langer et al., 2003). The amount of ethanol added was approximately 3.3 mL per mL protein solution. Nanoparticle suspension was centrifuged at 18,000 ×g (refrigerated centrifuge model RS-20IV, Tomy Seiko Co., Ltd., Tokyo, Japan) for 10 min and obtained supernatant was used in measurement of encapsulation efficiency. The resulting nanoparticles were then vacuum dried at 60 °C and stored at − 80 °C until analyses. For preparation of pit extract-loaded particles, WPI solution was supplemented before pH adjustment with 0.045 g or 0.06 g extract powder to obtain 1:20 or 1:15 mass ratio of extract to WPI, respectively. The whole procedure for preparation and separation of nanocapsules was performed the same as particles.
2.8. Fourier transform infrared (FTIR) spectroscopy and thermal analysis FTIR spectra of pit extract, WPI, extract-free and extract-loaded particles were obtained with a Perkin Elmer 2000 FT-IR spectrometer (Perkin Elmer Co., MA, USA) using the KBr disk method. Spectra were obtained in transmission mode from 450 to 4500 cm −1 wavenumber range. The thermal analysis of samples was performed by using a calibrated differential scanning calorimeter (Star System DSC1, Mettler Toledo, OH, USA). For this purpose, each specimen (5 mg) was heated under nitrogen stream (20 mL/min) from 25 to 150 °C at rate of 10 °C min −1. 2.9. Statistical analysis The data, reported as mean± standard deviation, are from experiments conducted in triplicate. One-way analysis of variance (ANOVA) was performed using SPSS (ver. 16) software. ANOVA was used to check the assumptions of variance homogeneity and normality and compare the treatment means. Differences among mean values were examined by the least significant difference (LSD) and Duncan's test at P b 0.05 significance level.
868
L. Bagheri et al. / Food Research International 51 (2013) 866–871
Table 1 Size and polydispersity of WPI nanoparticles and date pit extract-loaded nanocapsules. Polydispersity
Size distribution (nm)
Diameter (nm)
Sample
0.423 ± 0.016a 0.326 ± 0.022b 0.232 ± 0.011c 0. 366 ± 0.006d 0.296 ± 0.007e
77–443 70–353 60–366 41–212 20–241
280.88 ± 29.61a 183.98 ± 20.66b 163.1 ± 10.43c 103.78 ± 15.09d 92.89 ± 17.31e
Particles in ethanol (from non-heated WPI) Particles in ethanol (from heated WPI) Capsules in ethanol (from heated WPI) Particles in water (from heated WPI) Capsules in water (from heated WPI)
Data are expressed as mean ± standard deviation for triplicate tests. Different superscripts in the same column indicate significant differences at P b 0.05.
3. Results and discussion 3.1. Particle size The mean particle size, polydispersity index and size distribution range of extract-free and extract-loaded particles are reported in Table 1. All samples were of nanoscalar size indicating the successful generation of protein nano-associations via alcoholic desolvation. The repulsive forces among negatively charged protein particles at alkaline pH (Papiz et al., 1986) of dispersing medium that was aqueous ethanol or water prevented from particle aggregation. As well, the alkalinity of protein solution before desolvation stage might unfold the whey protein native assemblies exposing their hidden thiol groups to undergo thiol– disulfide interchanges. This led to enhancement of particulation and inhibition of large aggregate formation (Gunasekaran et al., 2007). Heat treatment of WPI before desolvation stage resulted in significantly smaller particles (Table 1). It is argued that heat-treating of WPI solution at low temperature i.e. 60 °C might favor the unfolding of whey proteins (Qi & Onwulata, 2011) into a state referred to as molten globule (Nicolai, Britten, & Schmitt, 2011). This resulted in extensive exposure of hidden groups and more interconnection of protein molecules during precipitation, leading to generation of tinier assemblies. Eissa (2012) found that the size of whey protein assemblies decreased with increasing temperature in the range of 30–65 °C. The type of dispersing medium influenced the size of extract-free and extract-loaded particles significantly. Water-dispersed particles and capsules were smaller than their aqueous ethanol-dispersed counterparts (Table 1). Water probably well hydrated the desolvated protein nanoassemblies causing the solubilization of a population of whey proteins and thus fragmented the originally formed particles to smaller offsprings. Gülseren et al. (2012a) reported sizes as small as ~ 8.8–36 nm for WPI particles desolvated by ethanol and dispersed in pH 3.0 buffer. The majority of amino groups (over 90%) are protonated at low pH value (pH b 3) (Kumar, 2000) to form an extended molecular chain which probably accounts for the extremely small size of particles reported by Gülseren et al. (2012a). The polydispersity of all samples was less than 0.4 except for particles prepared from non-heated WPI (Table 1). This manifests the importance of preheating in obtaining more homogenous particles from desolvated WPI. Encapsulation of date palm pit extract resulted in generation of smaller and more monodisperse particles (Table 1) due either to some chemical interactions between core and whey proteins resulting in denser assemblies or action of extract ingredients namely phenolic compounds as base for arrangement of protein molecules around those during precipitation.
assemblies by desolvation method. Zou, Li, Percival, Bonard and Gu (2012) produced zein nanocapsules with particle yield of 76% by desolvation method. Langer et al. (2003) fabricated human serum albumin nanoparticles with yield of 65–95% by desolvation procedure. It is clear from the results that heat-treating of whey protein isolate solution before desolvation increased the particle yield significantly (Table 2). As discussed earlier, partially denatured whey proteins by heat are extensively interconnected, which boosted the particulation of molecules and thus a higher yield of precipitated pellet was recovered. Encapsulation of pit extract in WPI particles resulted in higher particle yields (Table 2) due probably to action of extract ingredients as base for protein arrangement during desolvation. However, a higher mass ratio of core to matrix-forming protein i.e. 1:15 caused a lower yield of particulation in comparison to a ratio of 1:20. A similar trend was
3.2. Morphology, particle yield and encapsulation efficiency Fig. 1 shows the exemplar SEM images of nanoparticles and extract-loaded particles. Samples had spherical morphology and in dry state were clumped. A similar connection/aggregation phenomenon was observed by Zhong and Jin (2009) in SEM images of freezedried zein particles. The yield of nanoparticles was higher than 50% and ~76% for samples prepared from non-heated and heat-treated WPI, respectively (Table 2) implying in proper mass generation of associated protein
Fig. 1. SEM images of WPI extract-free (S1) and pit extract-loaded (S2) particles from heat-treated WPI.
L. Bagheri et al. / Food Research International 51 (2013) 866–871
869
Table 2 Particle yield and encapsulation efficiency of WPI extract-free and date pit extract-loaded particles. Encapsulation efficiency (%)
Particle yield (%)
Extract-to-WPI ratio
Sample
– – 78.33 ± 2.3a 70.26 ± 1.3b
52.23 ± 1.6a 77.45 ± 1.3b 95.43 ± 1.5c 90.23 ± 1.1d
– – 1:20 1:15
Nanoparticles (from non-heated WPI) Nanoparticles (from heated WPI) Nanocapsules Nanocapsules
Data are expressed as mean±standard deviation for triplicate tests. Different superscripts in the same column indicate significant differences at Pb 0.05.
observed for encapsulation efficiency at higher extract-to-WPI ratio. It is argued that protein–protein and polyphenol–protein interactions were weakened with increasing extract-to-WPI ratio resulting in remaining of more extract and protein in the supernatant instead of being particulated and/or encapsulated. These results are consistent with those of Zou et al. (2012). It has been similarly reported for other polymeric matrices that over-loading of core may cause a decrease in encapsulation efficiency (Liu & Park, 2009; Shah, Pal, Kaushik, & Devi, 2009). Antisolvent precipitation of whey proteins to enclose polyphenols was significantly efficient as an encapsulation efficiency of >70% was obtained (Table 2). It is remembered that pit powder was extracted by sole water and thus water-soluble phenolics present in the extract were efficiently enclosed inside the associating protein assemblies during the desolvation stage by alcohol. Wu, Luo, and Wang (2012) encapsulated volatile essential oils in desolvated zein and obtained an efficiency of 50%. The amount of zinc attached to WPI nanoparticles was higher than 80% (Gülseren et al., 2012b). When capsules were dispersed in bi-distilled water and shaken for relatively short time (30 min), a minor portion of phenolic compounds i.e. 3.5% ± 0.5 of total phenolics was recovered in centrifugal supernatant; while, no trace of polyphenols was detected in aqueous alcohol used for extract-loaded particle dispersion. This result confirms our hypothesis based on the particle size measurements that WPI desolvated particles unpack and fragment when dispersed in water. 3.3. FTIR spectrum FTIR spectra of pit extract, WPI and WPI extract-free and extract-loaded particles are shown in Fig. 2. The band at 3400 cm−1 for pit extract may assign to O\H stretching of the hydroxyl group of acidic carboxyl group (COOH) on the benzene ring of phenolic acids. Peaks observed at 1611, 1524, and 1450 cm−1 correlate to C_C stretching of aromatic rings which are the typical functional groups of phenolic compounds. Peak observed at 1285 cm−1 attributed to the C\O in flavonoids of extract (Yazaki & Hillis, 1977). Peak observed at 1062 cm−1 attributed to the asymmetrical C\O vibration. The peaks appeared at 778 and 617 cm−1attributed to out-of-plane bending of the ring C\H bonds in the benzene rings (Silverstein, Webster, & Kiemle, 2005) Peaks at 1535 and 1653 cm−1 in the FTIR spectra of native WPI, extract-free and extract-loaded particles are attributed to C_O stretching of Amide I band in α-helical component of α-lactalbumin and β-lactoglobulin secondary structures (Bayler & Purcell, 1986; Geara, 1999; Kretschmer, 1957) and Amide II band of both C\N stretching and C\N\H in plane bending (Sessa, Mohamed, & Byars, 2008), respectively. The appearance of a band at 1238 or 1242 cm−1 in spectra of extract-free and extract-loaded nanoparticles in comparison to native WPI was assigned to β-sheet structure in protein secondary structures (Seo et al., 2010). Heat-treatment of whey proteins before desolvation stage therefore modified the spatial conformation of proteins resulting in the enhanced β-sheet structure. Two peaks appeared at 1450 cm −1 and 1400 cm −1 in spectra of native and particulated WPI due to C\H bending and C\N stretching, respectively. Peaks due to N\H stretching, C\H stretching and O\H bending vibrations of deionized carboxylic acid were observed at 3300, 2963 and 1396 cm −1, respectively. Peaks at 3300 cm −1 and
2963 cm −1 in spectra of extract-free and extract-loaded particles were because of O\H and C\H stretching, respectively. In the FTIR spectrum of extract-loaded particle, the peak of WPI's hydroxyl group (3298 cm − 1) merges with that of phenolic hydroxyl groups (3400 cm − 1). The sharp peaks at 2963 cm − 1 representing C\H stretching of WPI's CH3 and CH2 functional groups overlaid with the C\H stretching peak of extract's phenolic rings methyl and isopropyl groups at 2962 cm − 1. The three peaks specific to the phenolic rings of extract at wavenumbers ranging from 1611 to 1443 cm − 1 disappeared with the overlaying effect of Amide I, Amide II and C\H bending of WPI at similar positions i.e. 1653 cm − 1, 1535 cm − 1 and 1450 cm − 1, respectively. The peak due to polyflavonoids at 1285 cm − 1 for extract disappeared by the overlaying effect of protein β-sheet structure at a similar position (1242 cm − 1). Peaks observed at 1062 cm − 1 for extract due to asymmetrical C\O vibration merged with the peak at 1083 cm − 1 for C\OH in WPI. Peaks due to out-of-plane bending of C\H bonds in the benzene rings at 778 and 617 cm − 1 disappeared by the overlaying effect of out-of-plane N\H wagging vibration in WPI at similar positions (817 and 667 cm − 1). All information reflects that extract ingredients were physically entrapped in the particle matrix without covalent interactions. Hydrogen bonds, van der Waals and hydrophobic interactions might contribute in extract inclusion (He et al., 2011). Our results are in accordance with previous observations that polyphenols interact with proteins via hydrophobic interactions and hydrogen bonding (Emmambux & Taylor, 2003; Taylor, Taylor, Belton, & Minnaar, 2009).
Fig. 2. FTIR spectra of date palm pit extract, native WPI, and WPI extract-free and extract-loaded nanoparticles.
870
L. Bagheri et al. / Food Research International 51 (2013) 866–871
interactions were weakened at the higher extract-to-WPI ratio. FTIR results evidenced no covalent attachment between pit extract and WPI in extract-loaded particles. Acknowledgements The authors would like to thank Zam Zam Co. (Tehran, Iran) for providing the financial support for this project. References
Fig. 3. DSC thermograms of native WPI (…), WPI extract-free (− − −) and extract-loaded (22)nanoparticles.
3.4. Thermal behavior The DSC scan (Fig. 3) of native WPI exhibited a large endothermic peak between 39 and 110 °C centered at 74 °C which is attributed to heat-induced transitions occurring in α-lactalbumin and β-lactoglobulin. Fitzsimmons, Mulvihilla, and Morris (2007) by using a microcalorimeter observed an exotherm following the denaturation endotherm for 3.0 wt.% WPI in 100 mM NaCl due to aggregation of protein molecules. In conventional fast scanning calorimeters, only endothermic transitions are observed (Fitzsimmons et al., 2007) as they occurred in the present study. It is also worthy to note that aggregation of denatured protein molecules can occur solely in protein solutions; while, the WPI powder analyzed in the present study could merely undergo some heat-induced transitions in the conformational topology of protein molecules. Interestingly, no endotherm occurred during heat scanning of particulated proteins most probably because of the heat treatment already applied to proteins during particle preparation. A similar result has been reported in preparation of WPI nanoparticles via microemulsification of native WPI followed by heat gelation (Zhang & Zhong, 2010). Extract-free nanoparticles showed no apparent endothermic peak when heated in DSC apparatus; however, several mild endoand exothermic peaks were recorded for extract-loaded particles. The endotherms suggest that extract-loaded particles underwent some glass transition phenomena during which the glassy particles became rubbery by the thermal energy supplied. Particulation of heat-treated whey proteins by antisolvent resulted in a homogenous amorphous assembly. However, the matrix of assembled particles was interrupted by the presence of core biomaterials including polyphenols. Therefore, the less organized assemblies of extract-loaded particles tended to mobilize by lower thermal energy when thermally scanned during DSC analysis. The recorded small exotherms may refer to a number of heat-induced complicated transitions in extract ingredients. 4. Conclusions WPI extract-free and pit extract-loaded particles were successfully prepared at alkaline conditions. Heat-treatment of whey proteins before desolvation process decreased the mean size and increased the monodispersity and particle yield of extract-free and extractloaded particles. A noticeable efficiency for pit extract entrapment inside the WPI particles was achieved. However, a higher mass ratio of core to matrix-forming protein produced a lower yield of particulation and encapsulation efficiency. It was argued that because of over-loading protein–protein and polyphenol–protein
Arts, I. C. W., Van de Putte, B., & Hollman, P. C. H. (2000). Catechin contents of foods commonly consumed in The Netherlands. 1. Fruits, vegetables, staple foods, and processed foods. Journal of Agricultural and Food Chemistry, 48, 1746–1751. Bayler, D. M., & Purcell, J. M. (1986). FTIR examination of thermal denaturation and gel formation in whey proteins. SPIE Fourier Transform Spectroscopy, 1145, 415–417. Bell, L. N. (2001). Stability testing of nutraceuticals and functional foods. In R. E. C. Wildman (Ed.), Handbook of nutraceuticals and functional foods (pp. 501–516). New York: CRC Press. Bilati, U., Allémann, E., & Doelker, E. (2005). Development of a nanoprecipitation method intended for the entrapment of hydrophilic drugs into nanoparticles. European Journal of Pharmaceutical Sciences, 24, 67–75. Eissa, A. S. (2012). Newtonian viscosity behavior of dilute solutions of polymerized whey proteins. Would viscosity measurements reveal more detailed molecular properties? Food Hydrocolloids, 30, 200–205. Emmambux, N. M., & Taylor, J. R. N. (2003). Sorghum kafirin interaction with various phenolic compounds. Journal of the Science of Food and Agriculture, 83(5), 402–407. Fitzsimmons, S. M., Mulvihilla, D. M., & Morris, E. R. (2007). Large enhancements in thermogelation of whey protein isolate by incorporation of very low concentration of guar gum. Food Hydrocolloids, 22, 575–586. Geara, C. (1999). Study of the gelation of whey protein isolate by FTIR spectroscopy and rheological measurements. M.Sc. thesis, Montreal, Canada: McGill University. Gülseren, I., Fang, Y., & Corredig, M. (2012a). Whey protein nanoparticles prepared with desolvation with ethanol: Characterization, thermal stability and interfacial behavior. Food Hydrocolloids, 29, 258–264. Gülseren, I., Fang, Y., & Corredig, M. (2012b). Zinc corporation capacity of whey protein nanoparticles prepared with desolvation with ethanol. Food Chemistry, 135, 770–774. Gunasekaran, S., Ko, S., & Xiao, L. (2007). Use of whey protein for encapsulation and controlled delivery applications. Journal of Food Engineering, 83, 31–40. He, L., Mu, C., Shi, J., Zhang, Q., Shi, B., & Lin, W. (2011). Modification of collagen with a natural cross-linker, procyanidin. International Journal of Biological Macromolecules, 48(2), 354–359. Kretschmer, C. B. (1957). Infrared spectroscopy and optical rotatory dispersion of zein, wheat gluten and gliadin. The Journal of Physical Chemistry, 61(12), 1627–1631. Kumar, M. N. V. R. (2000). Nano and microparticles as controlled drug delivery devices. Journal of Pharmacy & Pharmaceutical Science, 3(2), 234–258. Langer, K., Balthasar, S., Vogel, V., Dinauer, N., Briesen, H. V., & Schubert, D. (2003). Optimization of the preparation process for human serum albumin (HSA) nanoparticles. International Journal of Pharmaceutics, 257, 169–180. Lesschaeve, I., & Noble, A. C. (2005). Polyphenols: Factors influencing their sensory properties and their effects on food and beverage preferences. The American Journal of Clinical Nutrition, 81(Suppl.), 330S–335S. Liu, N., & Park, H. J. (2009). Chitosan-coated nanoliposome as vitamin E carrier. Journal of Microencapsulation, 26, 235–242. Moraru, C. I., Panchapakesan, C. P., Huang, Q., Takhistov, P., Liu, S., & Kokini, J. L. (2003). Nanotechnology: A new frontier in food science. Food Technology, 57, 24–29. Nicolai, T., Britten, M., & Schmitt, C. (2011). β-Lactoglobulin and WPI aggregates: Formation, structure and applications. Food Hydrocolloids, 25, 1945–1962. Papiz, M. Z., Sawyer, L., Eliopoulos, E. E., North, A. C. T., Findlay, J. B. C., Sivaprasadarao, R., et al. (1986). The structure of beta-lactoglobulin and its similarity to plasma retinol-binding protein. Nature, 324, 383–385. Proestos, C., Bakogiannis, A., Psarianos, C., Koutinas, A. A., Kanellaki, M., & Komaitis, M. (2005). High performance liquid chromatography analysis of phenolic substances in Greek wines. Food Control, 16, 319–323. Qi, P. X., & Onwulata, C. I. (2011). Physical properties, molecular structures, and protein quality of texturized whey protein isolate: Effect of extrusion temperature. Journal of Agricultural and Food Chemistry, 59, 4668–4675. Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of polyphenols. Journal of Nutrition, 130, 2073S–2085S. Seo, J. A., Hédoux, A., Guinet, Y., Paccou, L., Affouard, F., Lerbret, A., et al. (2010). Thermal denaturation of beta-lactoglobulin and stabilization mechanism by trehalose analyzed from Raman spectroscopy investigations. The Journal of Physical Chemistry. B, 114(19), 6675–6684. Sessa, D. J., Mohamed, A., & Byars, J. A. (2008). Chemistry and physical properties of melt-processed and solution-cross-linked corn zein. Journal of Agricultural and Food Chemistry, 56(16), 7067–7075. Shah, S., Pal, A., Kaushik, V. K., & Devi, S. (2009). Preparation and characterization of venlafaxine hydrochloride-loaded chitosan nanoparticles and in vitro release of drug. Journal of Applied Polymer Science, 112(5), 2876–2887. Shui, G., & Leong, L. P. (2006). Residue from star fruit as valuable source for functional food ingredients and antioxidant nutraceuticals. Food Chemistry, 97, 277–284.
L. Bagheri et al. / Food Research International 51 (2013) 866–871 Silverstein, R. M., Webster, F. X., & Kiemle, D. J. (2005). Spectrometric identification of organic compounds (7th ed.). New York: John Wiley & Sons Inc. Singleton, V. L., & Rossi, J. A., Jr. (1965). Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158. Taylor, J., Taylor, J. R. N., Belton, P. S., & Minnaar, A. (2009). Kafirin microparticle encapsulation of catechin and sorghum condensed tannins. Journal of Agricultural and Food Chemistry, 57(16), 7523–7528. Wu, Y., Luo, Y., & Wang, Q. (2012). Antioxidant and antimicrobial properties of essential oils encapsulated in zein nanoparticles prepared by liquid–liquid dispersion method. LWT — Food Science and Technology, 48, 283–290. Yazaki, Y., & Hillis, W. E. (1977). Components of the extractives from Callitris columellaris F. Muell. heartwood which affect termites. Holzforschung, 31(6), 188–191.
871
Zhang, W., & Zhong, Q. (2010). Microemulsions as nanoreactors to produce whey protein nanoparticles with enhanced heat stability by thermal pretreatment. Food Chemistry, 119, 1318–1325. Zhong, Q., & Jin, M. (2009). Zein nanoparticles produced by liquid–liquid dispersion. Food Hydrocolloids, 23, 2380–2387. Zhong, Q., Tian, H., & Zivanovic, S. (2009). Encapsulation fish oil in solid zein particles by liquid–liquid dispersion. Journal of Food Processing and Preservation, 33, 255–270. Zou, T., Li, Z., Percival, S. S., Bonard, S., & Gu, L. (2012). Fabrication, characterization, and cytotoxicity evaluation of cranberry procyanidins-zein. Food Hydrocolloids, 27, 293–300.
Contents lists available at SciVerse ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Short communication
Nanoencapsulation of date palm pit extract in whey protein particles generated via desolvation method Leila Bagheri, Ashkan Madadlou ⁎, Mohammadsaeed Yarmand, Mohammad E. Mousavi Department of Food Science, Technology and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karadj, Iran
a r t i c l e
i n f o
Article history: Received 18 November 2012 Accepted 29 January 2013 Keywords: Desolvation Date palm Encapsulation Whey protein nanoparticles
a b s t r a c t An alkaline solution of whey protein isolate was charged with absolute ethanol resulting in precipitation of whey protein particles. The vacuum-dried particles were then dispersed either in water or aqueous ethanol. Heat-treatment of whey proteins before desolvation process decreased the mean size of particles when dispersed in aqueous ethanol from 280 nm to 183 nm. The range and mean size of particles prepared from heat-treated protein solution when dispersed in water were 41–212 nm and 103 nm, respectively. Date palm pit aqueous extract was encapsulated inside the particulating heat-treated whey proteins during the desolvation stage with encapsulation efficiency of ~78%. Extract-loaded particles had mean size of 163 nm in alcoholic dispersion and 92 nm in water dispersion. Scanning electron microscopy imaging showed spherical nanoparticles aggregated in dry state. Fourier transform infrared spectroscopy suggested that extract and whey proteins did not covalently bind. Heat-treatment of whey proteins before desolvation resulted in the absence of denaturation endotherm in differential scanning calorimetry curve of extract-free particles. Extract loading in particles interrupted the continuity of protein matrix causing the occurrence of mild glass transition phenomenon in extract-loaded particles when heated. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Nanoparticles when used in biological and food systems may provide superior characteristics to microparticles including unique quality, improved sensory properties, extended flavor perception, better mouthfeel, transparent appearance and enhanced processability (Moraru et al., 2003). Therefore, nanobioparticles prepared from generally recognized as safe (GRAS) biopolymers are increasingly nominated for various applications in biomedical, pharmaceutical and nutraceutical industries remembering their acknowledged biocompatibility. Polyphenols act as metal scavengers, antimutagenes, and antimicrobial agents (Proestos et al., 2005). Their consumption may reduce the risk of some chronic diseases such as cancer, cardiovascular disease, chronic respiratory disease and diabetes (Arts, Van de Putte, & Hollman, 2000; Scalbert & Williamson, 2000). However, incorporation of polyphenols into foods may cause quality defects such as astringent taste and increased haze in beverages. They can act as substrates for browning reactions (Lesschaeve & Noble, 2005) resulting in undesirable color changes in food products. Also, polyphenols and other bioactive substances may undergo degradation and/or deterioration by food processing operations and storage (e.g. heating, acidification, light and oxygen) or in the gastrointestinal tract (acidic pH, enzymes, presence of other nutrients). These drawbacks impose limits for application ⁎ Corresponding author. E-mail address: [email protected] (A. Madadlou). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.01.058
and potential benefits of these nutraceuticals (Bell, 2001). Nanoencapsulation of bioactive molecules within appropriately structured carriers may overcome these problems without adverse effect on sensory characteristics and appearance of final product. Desolvation technique is a straightforward, rapid and easily applicable method to carry out the whole nanoparticle preparation procedure in one pot. This technique requires only two miscible solvents without involvement of destructing factors such as high shear rate, heating and sonication that damage the tertiary structure of proteins. As well, the procedure does not include toxic reagents and surfactants (Bilati, Allémann, & Doelker, 2005). Gunasekaran, Ko, and Xiao (2007) used acetone to desolvate β-lactoglobulin and then crosslinked the generated nanoparticles by glutaraldehyde–ethanol mixture. Recently, fish oil was encapsulated in zein nanoparticles desolvated by water from aqueous alcoholic solution (Zhong, Tian, & Zivanovic, 2009). Gülseren, Fang, and Corredig (2012a) generated protein nanoparticles by adding ethanol as antisolvent to an alkaline solution of whey proteins, followed by resolvation of particles through diluting the suspension by aqueous buffers. The procedure was later used to encapsulate zinc chloride within whey protein particles (Gülseren, Fang, & Corredig, 2012b). To the best of authors' knowledge there is no report in the literature on encapsulation of polyphenols in protein nanoparticles prepared by antisolvent (desolvation) method. The objective of the present study was therefore to encapsulate the aqueous extract of date palm pit as a rich source of phenolic compounds within whey protein nanoparticles obtained through desolvating by ethanol. The obtained extract-loaded
L. Bagheri et al. / Food Research International 51 (2013) 866–871
particles are expected to be easily added to beverages with minimum influence on sensory characteristics of food product.
867
2.5. Particle size measurement
Sodium chloride (NaCl), sodium bicarbonate (NaHCO3), sodium hydroxide (NaOH), hydrochloric acid (HCl), ethanol and Folin and Ciocalteau's phenol reagent (mixture of phosphomolybdate and phosphotungstate) were purchased from Merck (Darmstadt, Germany). Lactose- and fat-free whey protein isolate (WPI) with 92% protein content was a kind gift from Arla Food Ingredients (Viby J, Denmark). Bi-distilled water was used throughout the study.
Size and polydispersity of particles and capsules were determined by using a dynamic light scattering particle size analyzer (ZetaPALS, Brookhaven Instruments Co., NY, USA). For this purpose, dried samples were either dispersed in 10 mL ethanol at pH 9.0 or bi-distilled water with pH 9.0 at ratio of 1:200 (w/v) and shaken continuously at room temperature for 12 h using orbital incubator (Stuart®, S150, Guill Bern Corporation Inc., Philippines) to allow complete hydration. To investigate the influence of heat-treatment of WPI on particle size, a sample was also prepared from non-heated WPI following the same procedure as described. Particle size measurements were carried out at 25 °C with laser beam operated at 657 nm and scattering angle of 90°. Each sample was read three times. Average sizes reported are the volume-averaged diameters.
2.2. Preparation of date palm pit extract
2.6. Scanning electron microscopy
Date palm fruit (Kabkab variety) pits were washed and air-dried at 50 °C for 4 h. The dried pits were milled using a heavy-duty grinder to pass 1 mm screen, afterwards, 1 L boiling water was added to 50 g pit powder and extraction was carried out at 30 °C for 7 h while shaken at 100 rpm. The mixture was filtered through a series of Whatman filter papers in order to remove all suspended materials. The extract was freeze-dried and kept in dark glass bottles at −80 °C until use.
The morphology of dry extract-free and extract-loaded nanoparticles was observed with a scanning electron microscope (SEM, KYKYEM3200, KYKY Technology Development Ltd, China) operated at 24 kV. The surfaces of particles were sputtered with gold, observed and photographed.
2. Materials and methods 2.1. Materials
2.3. Measurement of phenolic content Total phenolic content was measured using the Folin–Ciocalteau method (Singleton & Rossi, 1965). Pit extract (30 μL) was mixed with 2.37 mL deionized water and 150 μL Folin–Ciocalteau's phenol reagent and allowed to stand at room temperature for 7 min, then 450 μL sodium bicarbonate (20% w/v) was added to the mixture. After standing for 70 min at room temperature, absorbance was measured (Spectrophotometer BioQuest CE2502, Cecil Instruments Ltd., UK) at 760 nm. Results were expressed as mg gallic acid equivalents (GAE)/100 g sample (Shui & Leong, 2006). Polyphenol content of extract was 1582 mg gallic acid equivalent per 100 g dry weight at pH 6.25.
2.7. Particle yield and encapsulation efficiency The weight of dried pellet of extract-free and extract-loaded particles was used for calculating the particle yield as follows:
Particle Yieldð%Þ ¼
weight of dry pellet 100: total weight of extract and WPI used for particles preparation
The efficiency of extract entrapment in particles was calculated as the difference between phenolic content added to WPI solution before desolvation stage and the content remained in the centrifugal supernatant. To determine the holding degree of encapsulated polyphenols within particles, precipitated pellet was dispersed both in 70% ethanol and bi-distilled water, mildly shaken for 30 min and re-precipitated through centrifugation after which the supernatant was used for polyphenol measurement.
2.4. Preparation of extract-free and extract-loaded particles WPI was dissolved (3% w/v) in 10 mM NaCl solution by stirring at 500 rpm at room temperature for 2 h; sodium azide (50 mg/L) was added to prevent microbial growth. The solution was stored at 4 °C for 12 h and filtered through 0.45 μm PVDF syringe filter (Whatman, Germany) prior to use. Protein solution was heat-treated at 60 °C for 30 min (Qi & Onwulata, 2011) and its pH was adjusted to 9.0 with 2 M NaOH. The pH adjustment led to smaller particles based on preliminary experiments. The solution was then charged with ethanol at a rate of 1 mL min −1 while stirring at 500 rpm until became turbid. The rate of ethanol addition was controlled carefully since it influences the size of generated particles (Langer et al., 2003). The amount of ethanol added was approximately 3.3 mL per mL protein solution. Nanoparticle suspension was centrifuged at 18,000 ×g (refrigerated centrifuge model RS-20IV, Tomy Seiko Co., Ltd., Tokyo, Japan) for 10 min and obtained supernatant was used in measurement of encapsulation efficiency. The resulting nanoparticles were then vacuum dried at 60 °C and stored at − 80 °C until analyses. For preparation of pit extract-loaded particles, WPI solution was supplemented before pH adjustment with 0.045 g or 0.06 g extract powder to obtain 1:20 or 1:15 mass ratio of extract to WPI, respectively. The whole procedure for preparation and separation of nanocapsules was performed the same as particles.
2.8. Fourier transform infrared (FTIR) spectroscopy and thermal analysis FTIR spectra of pit extract, WPI, extract-free and extract-loaded particles were obtained with a Perkin Elmer 2000 FT-IR spectrometer (Perkin Elmer Co., MA, USA) using the KBr disk method. Spectra were obtained in transmission mode from 450 to 4500 cm −1 wavenumber range. The thermal analysis of samples was performed by using a calibrated differential scanning calorimeter (Star System DSC1, Mettler Toledo, OH, USA). For this purpose, each specimen (5 mg) was heated under nitrogen stream (20 mL/min) from 25 to 150 °C at rate of 10 °C min −1. 2.9. Statistical analysis The data, reported as mean± standard deviation, are from experiments conducted in triplicate. One-way analysis of variance (ANOVA) was performed using SPSS (ver. 16) software. ANOVA was used to check the assumptions of variance homogeneity and normality and compare the treatment means. Differences among mean values were examined by the least significant difference (LSD) and Duncan's test at P b 0.05 significance level.
868
L. Bagheri et al. / Food Research International 51 (2013) 866–871
Table 1 Size and polydispersity of WPI nanoparticles and date pit extract-loaded nanocapsules. Polydispersity
Size distribution (nm)
Diameter (nm)
Sample
0.423 ± 0.016a 0.326 ± 0.022b 0.232 ± 0.011c 0. 366 ± 0.006d 0.296 ± 0.007e
77–443 70–353 60–366 41–212 20–241
280.88 ± 29.61a 183.98 ± 20.66b 163.1 ± 10.43c 103.78 ± 15.09d 92.89 ± 17.31e
Particles in ethanol (from non-heated WPI) Particles in ethanol (from heated WPI) Capsules in ethanol (from heated WPI) Particles in water (from heated WPI) Capsules in water (from heated WPI)
Data are expressed as mean ± standard deviation for triplicate tests. Different superscripts in the same column indicate significant differences at P b 0.05.
3. Results and discussion 3.1. Particle size The mean particle size, polydispersity index and size distribution range of extract-free and extract-loaded particles are reported in Table 1. All samples were of nanoscalar size indicating the successful generation of protein nano-associations via alcoholic desolvation. The repulsive forces among negatively charged protein particles at alkaline pH (Papiz et al., 1986) of dispersing medium that was aqueous ethanol or water prevented from particle aggregation. As well, the alkalinity of protein solution before desolvation stage might unfold the whey protein native assemblies exposing their hidden thiol groups to undergo thiol– disulfide interchanges. This led to enhancement of particulation and inhibition of large aggregate formation (Gunasekaran et al., 2007). Heat treatment of WPI before desolvation stage resulted in significantly smaller particles (Table 1). It is argued that heat-treating of WPI solution at low temperature i.e. 60 °C might favor the unfolding of whey proteins (Qi & Onwulata, 2011) into a state referred to as molten globule (Nicolai, Britten, & Schmitt, 2011). This resulted in extensive exposure of hidden groups and more interconnection of protein molecules during precipitation, leading to generation of tinier assemblies. Eissa (2012) found that the size of whey protein assemblies decreased with increasing temperature in the range of 30–65 °C. The type of dispersing medium influenced the size of extract-free and extract-loaded particles significantly. Water-dispersed particles and capsules were smaller than their aqueous ethanol-dispersed counterparts (Table 1). Water probably well hydrated the desolvated protein nanoassemblies causing the solubilization of a population of whey proteins and thus fragmented the originally formed particles to smaller offsprings. Gülseren et al. (2012a) reported sizes as small as ~ 8.8–36 nm for WPI particles desolvated by ethanol and dispersed in pH 3.0 buffer. The majority of amino groups (over 90%) are protonated at low pH value (pH b 3) (Kumar, 2000) to form an extended molecular chain which probably accounts for the extremely small size of particles reported by Gülseren et al. (2012a). The polydispersity of all samples was less than 0.4 except for particles prepared from non-heated WPI (Table 1). This manifests the importance of preheating in obtaining more homogenous particles from desolvated WPI. Encapsulation of date palm pit extract resulted in generation of smaller and more monodisperse particles (Table 1) due either to some chemical interactions between core and whey proteins resulting in denser assemblies or action of extract ingredients namely phenolic compounds as base for arrangement of protein molecules around those during precipitation.
assemblies by desolvation method. Zou, Li, Percival, Bonard and Gu (2012) produced zein nanocapsules with particle yield of 76% by desolvation method. Langer et al. (2003) fabricated human serum albumin nanoparticles with yield of 65–95% by desolvation procedure. It is clear from the results that heat-treating of whey protein isolate solution before desolvation increased the particle yield significantly (Table 2). As discussed earlier, partially denatured whey proteins by heat are extensively interconnected, which boosted the particulation of molecules and thus a higher yield of precipitated pellet was recovered. Encapsulation of pit extract in WPI particles resulted in higher particle yields (Table 2) due probably to action of extract ingredients as base for protein arrangement during desolvation. However, a higher mass ratio of core to matrix-forming protein i.e. 1:15 caused a lower yield of particulation in comparison to a ratio of 1:20. A similar trend was
3.2. Morphology, particle yield and encapsulation efficiency Fig. 1 shows the exemplar SEM images of nanoparticles and extract-loaded particles. Samples had spherical morphology and in dry state were clumped. A similar connection/aggregation phenomenon was observed by Zhong and Jin (2009) in SEM images of freezedried zein particles. The yield of nanoparticles was higher than 50% and ~76% for samples prepared from non-heated and heat-treated WPI, respectively (Table 2) implying in proper mass generation of associated protein
Fig. 1. SEM images of WPI extract-free (S1) and pit extract-loaded (S2) particles from heat-treated WPI.
L. Bagheri et al. / Food Research International 51 (2013) 866–871
869
Table 2 Particle yield and encapsulation efficiency of WPI extract-free and date pit extract-loaded particles. Encapsulation efficiency (%)
Particle yield (%)
Extract-to-WPI ratio
Sample
– – 78.33 ± 2.3a 70.26 ± 1.3b
52.23 ± 1.6a 77.45 ± 1.3b 95.43 ± 1.5c 90.23 ± 1.1d
– – 1:20 1:15
Nanoparticles (from non-heated WPI) Nanoparticles (from heated WPI) Nanocapsules Nanocapsules
Data are expressed as mean±standard deviation for triplicate tests. Different superscripts in the same column indicate significant differences at Pb 0.05.
observed for encapsulation efficiency at higher extract-to-WPI ratio. It is argued that protein–protein and polyphenol–protein interactions were weakened with increasing extract-to-WPI ratio resulting in remaining of more extract and protein in the supernatant instead of being particulated and/or encapsulated. These results are consistent with those of Zou et al. (2012). It has been similarly reported for other polymeric matrices that over-loading of core may cause a decrease in encapsulation efficiency (Liu & Park, 2009; Shah, Pal, Kaushik, & Devi, 2009). Antisolvent precipitation of whey proteins to enclose polyphenols was significantly efficient as an encapsulation efficiency of >70% was obtained (Table 2). It is remembered that pit powder was extracted by sole water and thus water-soluble phenolics present in the extract were efficiently enclosed inside the associating protein assemblies during the desolvation stage by alcohol. Wu, Luo, and Wang (2012) encapsulated volatile essential oils in desolvated zein and obtained an efficiency of 50%. The amount of zinc attached to WPI nanoparticles was higher than 80% (Gülseren et al., 2012b). When capsules were dispersed in bi-distilled water and shaken for relatively short time (30 min), a minor portion of phenolic compounds i.e. 3.5% ± 0.5 of total phenolics was recovered in centrifugal supernatant; while, no trace of polyphenols was detected in aqueous alcohol used for extract-loaded particle dispersion. This result confirms our hypothesis based on the particle size measurements that WPI desolvated particles unpack and fragment when dispersed in water. 3.3. FTIR spectrum FTIR spectra of pit extract, WPI and WPI extract-free and extract-loaded particles are shown in Fig. 2. The band at 3400 cm−1 for pit extract may assign to O\H stretching of the hydroxyl group of acidic carboxyl group (COOH) on the benzene ring of phenolic acids. Peaks observed at 1611, 1524, and 1450 cm−1 correlate to C_C stretching of aromatic rings which are the typical functional groups of phenolic compounds. Peak observed at 1285 cm−1 attributed to the C\O in flavonoids of extract (Yazaki & Hillis, 1977). Peak observed at 1062 cm−1 attributed to the asymmetrical C\O vibration. The peaks appeared at 778 and 617 cm−1attributed to out-of-plane bending of the ring C\H bonds in the benzene rings (Silverstein, Webster, & Kiemle, 2005) Peaks at 1535 and 1653 cm−1 in the FTIR spectra of native WPI, extract-free and extract-loaded particles are attributed to C_O stretching of Amide I band in α-helical component of α-lactalbumin and β-lactoglobulin secondary structures (Bayler & Purcell, 1986; Geara, 1999; Kretschmer, 1957) and Amide II band of both C\N stretching and C\N\H in plane bending (Sessa, Mohamed, & Byars, 2008), respectively. The appearance of a band at 1238 or 1242 cm−1 in spectra of extract-free and extract-loaded nanoparticles in comparison to native WPI was assigned to β-sheet structure in protein secondary structures (Seo et al., 2010). Heat-treatment of whey proteins before desolvation stage therefore modified the spatial conformation of proteins resulting in the enhanced β-sheet structure. Two peaks appeared at 1450 cm −1 and 1400 cm −1 in spectra of native and particulated WPI due to C\H bending and C\N stretching, respectively. Peaks due to N\H stretching, C\H stretching and O\H bending vibrations of deionized carboxylic acid were observed at 3300, 2963 and 1396 cm −1, respectively. Peaks at 3300 cm −1 and
2963 cm −1 in spectra of extract-free and extract-loaded particles were because of O\H and C\H stretching, respectively. In the FTIR spectrum of extract-loaded particle, the peak of WPI's hydroxyl group (3298 cm − 1) merges with that of phenolic hydroxyl groups (3400 cm − 1). The sharp peaks at 2963 cm − 1 representing C\H stretching of WPI's CH3 and CH2 functional groups overlaid with the C\H stretching peak of extract's phenolic rings methyl and isopropyl groups at 2962 cm − 1. The three peaks specific to the phenolic rings of extract at wavenumbers ranging from 1611 to 1443 cm − 1 disappeared with the overlaying effect of Amide I, Amide II and C\H bending of WPI at similar positions i.e. 1653 cm − 1, 1535 cm − 1 and 1450 cm − 1, respectively. The peak due to polyflavonoids at 1285 cm − 1 for extract disappeared by the overlaying effect of protein β-sheet structure at a similar position (1242 cm − 1). Peaks observed at 1062 cm − 1 for extract due to asymmetrical C\O vibration merged with the peak at 1083 cm − 1 for C\OH in WPI. Peaks due to out-of-plane bending of C\H bonds in the benzene rings at 778 and 617 cm − 1 disappeared by the overlaying effect of out-of-plane N\H wagging vibration in WPI at similar positions (817 and 667 cm − 1). All information reflects that extract ingredients were physically entrapped in the particle matrix without covalent interactions. Hydrogen bonds, van der Waals and hydrophobic interactions might contribute in extract inclusion (He et al., 2011). Our results are in accordance with previous observations that polyphenols interact with proteins via hydrophobic interactions and hydrogen bonding (Emmambux & Taylor, 2003; Taylor, Taylor, Belton, & Minnaar, 2009).
Fig. 2. FTIR spectra of date palm pit extract, native WPI, and WPI extract-free and extract-loaded nanoparticles.
870
L. Bagheri et al. / Food Research International 51 (2013) 866–871
interactions were weakened at the higher extract-to-WPI ratio. FTIR results evidenced no covalent attachment between pit extract and WPI in extract-loaded particles. Acknowledgements The authors would like to thank Zam Zam Co. (Tehran, Iran) for providing the financial support for this project. References
Fig. 3. DSC thermograms of native WPI (…), WPI extract-free (− − −) and extract-loaded (22)nanoparticles.
3.4. Thermal behavior The DSC scan (Fig. 3) of native WPI exhibited a large endothermic peak between 39 and 110 °C centered at 74 °C which is attributed to heat-induced transitions occurring in α-lactalbumin and β-lactoglobulin. Fitzsimmons, Mulvihilla, and Morris (2007) by using a microcalorimeter observed an exotherm following the denaturation endotherm for 3.0 wt.% WPI in 100 mM NaCl due to aggregation of protein molecules. In conventional fast scanning calorimeters, only endothermic transitions are observed (Fitzsimmons et al., 2007) as they occurred in the present study. It is also worthy to note that aggregation of denatured protein molecules can occur solely in protein solutions; while, the WPI powder analyzed in the present study could merely undergo some heat-induced transitions in the conformational topology of protein molecules. Interestingly, no endotherm occurred during heat scanning of particulated proteins most probably because of the heat treatment already applied to proteins during particle preparation. A similar result has been reported in preparation of WPI nanoparticles via microemulsification of native WPI followed by heat gelation (Zhang & Zhong, 2010). Extract-free nanoparticles showed no apparent endothermic peak when heated in DSC apparatus; however, several mild endoand exothermic peaks were recorded for extract-loaded particles. The endotherms suggest that extract-loaded particles underwent some glass transition phenomena during which the glassy particles became rubbery by the thermal energy supplied. Particulation of heat-treated whey proteins by antisolvent resulted in a homogenous amorphous assembly. However, the matrix of assembled particles was interrupted by the presence of core biomaterials including polyphenols. Therefore, the less organized assemblies of extract-loaded particles tended to mobilize by lower thermal energy when thermally scanned during DSC analysis. The recorded small exotherms may refer to a number of heat-induced complicated transitions in extract ingredients. 4. Conclusions WPI extract-free and pit extract-loaded particles were successfully prepared at alkaline conditions. Heat-treatment of whey proteins before desolvation process decreased the mean size and increased the monodispersity and particle yield of extract-free and extractloaded particles. A noticeable efficiency for pit extract entrapment inside the WPI particles was achieved. However, a higher mass ratio of core to matrix-forming protein produced a lower yield of particulation and encapsulation efficiency. It was argued that because of over-loading protein–protein and polyphenol–protein
Arts, I. C. W., Van de Putte, B., & Hollman, P. C. H. (2000). Catechin contents of foods commonly consumed in The Netherlands. 1. Fruits, vegetables, staple foods, and processed foods. Journal of Agricultural and Food Chemistry, 48, 1746–1751. Bayler, D. M., & Purcell, J. M. (1986). FTIR examination of thermal denaturation and gel formation in whey proteins. SPIE Fourier Transform Spectroscopy, 1145, 415–417. Bell, L. N. (2001). Stability testing of nutraceuticals and functional foods. In R. E. C. Wildman (Ed.), Handbook of nutraceuticals and functional foods (pp. 501–516). New York: CRC Press. Bilati, U., Allémann, E., & Doelker, E. (2005). Development of a nanoprecipitation method intended for the entrapment of hydrophilic drugs into nanoparticles. European Journal of Pharmaceutical Sciences, 24, 67–75. Eissa, A. S. (2012). Newtonian viscosity behavior of dilute solutions of polymerized whey proteins. Would viscosity measurements reveal more detailed molecular properties? Food Hydrocolloids, 30, 200–205. Emmambux, N. M., & Taylor, J. R. N. (2003). Sorghum kafirin interaction with various phenolic compounds. Journal of the Science of Food and Agriculture, 83(5), 402–407. Fitzsimmons, S. M., Mulvihilla, D. M., & Morris, E. R. (2007). Large enhancements in thermogelation of whey protein isolate by incorporation of very low concentration of guar gum. Food Hydrocolloids, 22, 575–586. Geara, C. (1999). Study of the gelation of whey protein isolate by FTIR spectroscopy and rheological measurements. M.Sc. thesis, Montreal, Canada: McGill University. Gülseren, I., Fang, Y., & Corredig, M. (2012a). Whey protein nanoparticles prepared with desolvation with ethanol: Characterization, thermal stability and interfacial behavior. Food Hydrocolloids, 29, 258–264. Gülseren, I., Fang, Y., & Corredig, M. (2012b). Zinc corporation capacity of whey protein nanoparticles prepared with desolvation with ethanol. Food Chemistry, 135, 770–774. Gunasekaran, S., Ko, S., & Xiao, L. (2007). Use of whey protein for encapsulation and controlled delivery applications. Journal of Food Engineering, 83, 31–40. He, L., Mu, C., Shi, J., Zhang, Q., Shi, B., & Lin, W. (2011). Modification of collagen with a natural cross-linker, procyanidin. International Journal of Biological Macromolecules, 48(2), 354–359. Kretschmer, C. B. (1957). Infrared spectroscopy and optical rotatory dispersion of zein, wheat gluten and gliadin. The Journal of Physical Chemistry, 61(12), 1627–1631. Kumar, M. N. V. R. (2000). Nano and microparticles as controlled drug delivery devices. Journal of Pharmacy & Pharmaceutical Science, 3(2), 234–258. Langer, K., Balthasar, S., Vogel, V., Dinauer, N., Briesen, H. V., & Schubert, D. (2003). Optimization of the preparation process for human serum albumin (HSA) nanoparticles. International Journal of Pharmaceutics, 257, 169–180. Lesschaeve, I., & Noble, A. C. (2005). Polyphenols: Factors influencing their sensory properties and their effects on food and beverage preferences. The American Journal of Clinical Nutrition, 81(Suppl.), 330S–335S. Liu, N., & Park, H. J. (2009). Chitosan-coated nanoliposome as vitamin E carrier. Journal of Microencapsulation, 26, 235–242. Moraru, C. I., Panchapakesan, C. P., Huang, Q., Takhistov, P., Liu, S., & Kokini, J. L. (2003). Nanotechnology: A new frontier in food science. Food Technology, 57, 24–29. Nicolai, T., Britten, M., & Schmitt, C. (2011). β-Lactoglobulin and WPI aggregates: Formation, structure and applications. Food Hydrocolloids, 25, 1945–1962. Papiz, M. Z., Sawyer, L., Eliopoulos, E. E., North, A. C. T., Findlay, J. B. C., Sivaprasadarao, R., et al. (1986). The structure of beta-lactoglobulin and its similarity to plasma retinol-binding protein. Nature, 324, 383–385. Proestos, C., Bakogiannis, A., Psarianos, C., Koutinas, A. A., Kanellaki, M., & Komaitis, M. (2005). High performance liquid chromatography analysis of phenolic substances in Greek wines. Food Control, 16, 319–323. Qi, P. X., & Onwulata, C. I. (2011). Physical properties, molecular structures, and protein quality of texturized whey protein isolate: Effect of extrusion temperature. Journal of Agricultural and Food Chemistry, 59, 4668–4675. Scalbert, A., & Williamson, G. (2000). Dietary intake and bioavailability of polyphenols. Journal of Nutrition, 130, 2073S–2085S. Seo, J. A., Hédoux, A., Guinet, Y., Paccou, L., Affouard, F., Lerbret, A., et al. (2010). Thermal denaturation of beta-lactoglobulin and stabilization mechanism by trehalose analyzed from Raman spectroscopy investigations. The Journal of Physical Chemistry. B, 114(19), 6675–6684. Sessa, D. J., Mohamed, A., & Byars, J. A. (2008). Chemistry and physical properties of melt-processed and solution-cross-linked corn zein. Journal of Agricultural and Food Chemistry, 56(16), 7067–7075. Shah, S., Pal, A., Kaushik, V. K., & Devi, S. (2009). Preparation and characterization of venlafaxine hydrochloride-loaded chitosan nanoparticles and in vitro release of drug. Journal of Applied Polymer Science, 112(5), 2876–2887. Shui, G., & Leong, L. P. (2006). Residue from star fruit as valuable source for functional food ingredients and antioxidant nutraceuticals. Food Chemistry, 97, 277–284.
L. Bagheri et al. / Food Research International 51 (2013) 866–871 Silverstein, R. M., Webster, F. X., & Kiemle, D. J. (2005). Spectrometric identification of organic compounds (7th ed.). New York: John Wiley & Sons Inc. Singleton, V. L., & Rossi, J. A., Jr. (1965). Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158. Taylor, J., Taylor, J. R. N., Belton, P. S., & Minnaar, A. (2009). Kafirin microparticle encapsulation of catechin and sorghum condensed tannins. Journal of Agricultural and Food Chemistry, 57(16), 7523–7528. Wu, Y., Luo, Y., & Wang, Q. (2012). Antioxidant and antimicrobial properties of essential oils encapsulated in zein nanoparticles prepared by liquid–liquid dispersion method. LWT — Food Science and Technology, 48, 283–290. Yazaki, Y., & Hillis, W. E. (1977). Components of the extractives from Callitris columellaris F. Muell. heartwood which affect termites. Holzforschung, 31(6), 188–191.
871
Zhang, W., & Zhong, Q. (2010). Microemulsions as nanoreactors to produce whey protein nanoparticles with enhanced heat stability by thermal pretreatment. Food Chemistry, 119, 1318–1325. Zhong, Q., & Jin, M. (2009). Zein nanoparticles produced by liquid–liquid dispersion. Food Hydrocolloids, 23, 2380–2387. Zhong, Q., Tian, H., & Zivanovic, S. (2009). Encapsulation fish oil in solid zein particles by liquid–liquid dispersion. Journal of Food Processing and Preservation, 33, 255–270. Zou, T., Li, Z., Percival, S. S., Bonard, S., & Gu, L. (2012). Fabrication, characterization, and cytotoxicity evaluation of cranberry procyanidins-zein. Food Hydrocolloids, 27, 293–300.