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Niosome-loaded cold-set whey protein hydrogels
Food Chemistry 196 (2016) 106–113
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Niosome-loaded cold-set whey protein hydrogels Arash Abaee a, Ashkan Madadlou a,b,c,⇑ a
Department of Food Science and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Center of Excellence for Application of Modern Technologies for Producing Functional Foods and Drinks (FFDCE), University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran c Interdisciplinary Research Department of Agricultural and Natural Resources Nanotechnology (IRDANN), University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran b
a r t i c l e
i n f o
Article history: Received 20 June 2015 Received in revised form 8 September 2015 Accepted 11 September 2015 Available online 11 September 2015 Keywords: Niosome Whey protein cold-set gel Transglutaminase a-Tocopherol
a b s t r a c t The a-tocopherol-carrying niosomes with mean diameter of 5.7 lm were fabricated and charged into a transglutaminase-cross-linked whey protein solution that was subsequently gelled with glucono deltalactone. Encapsulation efficiency of a-tocopherol within niosomes was 80% and encapsulation did not influence the radical scavenging activity of a-tocopherol. Fourier transform infrared (FTIR) spectroscopy suggested formation of e-(c-glutamyl) lysine cross-linkages by transglutaminase and that enzymatic cross-linking increased proteins hydrophobicity. FTIR also proposed hydrogen bonding between niosomes and proteins. Dynamic rheometry indicated that transglutaminase cross-linking and niosomes charging of the protein solution enhanced the gelation process. However, charging the cross-linked protein solution with niosomal suspension resulted in lower elastic modulus (G0 ) of the subsequently formed gel compared with both non-cross-linked niosome-loaded and cross-linked niosome-free counterparts. Electron microscopy indicated a discontinuous network for the niosome-loaded cross-linked sample. Niosome loading into the protein gel matrix increased its swelling extent in the enzyme-free simulated gastric fluid. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Most nutraceuticals are highly susceptible to degradation by food processing operations, adverse environmental conditions, and/or gastrointestinal digestion. Thus, encapsulation within appropriately designed and fabricated delivery systems is required to protect them from destructive circumstances (McClements, Decker, Park, & Weiss, 2009). Alpha-tocopherol, an oil-soluble nutraceutical, is the most abundant and biologically active form of vitamin E which acts as antioxidant and might prevent cardiovascular diseases, atherosclerosis and cancer (Liang, Line, Remondetto, & Subirade, 2010). The utilization of a-tocopherol has been hampered due to its light, heat and oxygen sensitivity and lipophilic nature. Therefore, various delivery systems such as emulsions (Yang, Decker, Xiao, & McClements, 2015), solid-lipid nanoparticles (Trombino et al., 2009), and protein and polysaccharide matrices (Duclairoir, Orecchioni, Depraetere, & Nakache, 2002; Pierucci, Andrade, Farina, Pedrosa, & Rocha-Leão, 2007; Somchue, ⇑ Corresponding author at: Department of Food Science and Engineering, University College of Agriculture and Natural Resources, University of Tehran, P.O. Box 31587-77871, Karaj, Iran. E-mail address: [email protected] (A. Madadlou). http://dx.doi.org/10.1016/j.foodchem.2015.09.037 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
Sermsri, Shiowatana, & Siripinyanond, 2009; Song, Lee, & Lee, 2009) have been employed to encapsulate a-tocopherol. Surfactants self-assemble spontaneously into vesicular structures which can be used as model for cell membranes and, furthermore, as vehicles for drugs and bioactive materials (Liu & Guo, 2005). Non-ionic surfactant vesicles, the so-called niosomes, have found great applicability in pharmaceutical, nutraceutical and cosmetic sectors since the late 70’s when they were patented by the company L’Oreal (Handjani-Vila, Ribier, Rondot, & Vanlerberghie, 1979). Niosomes are capable of encapsulating both hydrophilic and lipophilic materials and exhibit some advantages with respect to liposomes including less production costs and higher chemical stability. Niosomes formed from sorbitan monoesters are amongst the most widely investigated vesicles (Hao & Li, 2011; Yoshioka, Sternberg, & Florence, 1994). Despite being a superb vehicle for delivery purposes, niosomes might suffer from several drawbacks including aggregation, fusion, and drug leakage (Moghassemi & Hadjizadeh, 2014). Complementary mechanisms are, therefore, required to modulate the characteristics of drug-conveying niosomes. Proniosomes were presented as powdery alternatives to circumvent the problems related to handling and storage-allowed aggregation of niosomes. In essence, proniosomes are surfactantcoated water-soluble carriers that can be hydrated by brief agitation
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in hot aqueous media immediately before use (Hu & Rhodes, 2000). Introduction of other forms of niosomal products that are edible and demonstrate stability against sedimentation, aggregation and core leakage is, however, required to widen the application of niosomes in food and animal feed sectors in addition to pharmaceutics. Whey proteins confer beneficial effects on human health and possess privileged functional properties such as surface activity, biocompatibility, biodegradability and the ability to form cold- and heat-set hydrogels. A two-step procedure that is heat denaturation of proteins followed by changing the ionic strength or acidification in the direction of the isoelectric pH is required for fabrication of whey protein cold-set gels (Ju & Kilara, 1998). These types of hydrogels are appropriate for protecting heat sensitive nutraceuticals and show good potential for designing functional food commodities (Chen, Remondetto, & Subirade, 2006). Cold-set whey protein hydrogels are, however, prone to syneresis and sensitive to mechanical abrasion and enzymatic degradation. These might limit the applicability of whey protein hydrogels for conveying and delivery of their cargo to final consumer. Eissa, Bisram, and Khan (2004) examined the rheological characteristics of transglutaminase-cross-linked cold-set whey protein gels. They observed that the elastic modulus and yield/fracture stress and strain of the enzymatically cross-linked gel was higher than those of the non-cross-linked counterparts. The enzyme transglutaminase catalyzes acyl transfer reaction between peptide-bound glutamine residues as acyl donor and primary amines as acceptor. This enzyme has been widely used for protein cross-linking in food industry (Aboumahmoud & Savello, 1990). It was of interest for the authors of the present communication to fabricate a-tocopherol-carrying niosome-loaded cold-set whey protein hydrogels reinforced by transglutaminase and characterize their mechanical and techno-functional properties. The protein gel provides an efficient protecting, orally administrable and conventionally edible matrix for niosomes whilst, the niosomal particles convey a-tocopherol within the hydrogel and can improve its bioavailability by solubilizing within the intestinal tract and promoting its permeation through epithelial cells (Song et al., 2014). The bioavailability of lipophilic nutraceuticals depends on formation of sufficient amount of mixed micelles in the upper small intestine that solubilize the core and boost their intake by enterocytes (Joye, Davidov-Pardo, & McClements, 2014). The delivery system introduced in the present article can point to a new avenue for development of functional dairy foods.
2. Materials and methods 2.1. Materials Whey protein isolate (WPI) with at least 90% protein content was kindly gifted by Arla Food Ingredients (Vibyj, Denmark). Sorbitan monostearate (span 60), a-tocopherol, pepsin, and transglu-
Encapsulation efficiency ð%Þ ¼
2.2. Niosome preparation Multilamellar niosomes were prepared using thin film hydration method. Accurately weighed quantities of sorbitan monostearate, cholesterol (molar ratio of 7:3), and a-tocopherol were dissolved in 10 mL chloroform in a 100 mL round-bottom flask. Total amount of surfactants and a-tocopherol were 200 and 11.6 lM, respectively. Organic solvent was then evaporated under vacuum and constant rotation at 60 °C using a rotary evaporator (Heidolph, Germany). The dried thin film was hydrated with deionized water in the rotary evaporator at 60 °C for 1 h under atmospheric pressure. This was followed by centrifugation (Sigma 8 k centrifuge, Germany) at 18,000g for 30 min to remove nontrapped a-tocopherol and rehydrating the niosome pellet with de-ionized water. 2.3. Niosomes characterization 2.3.1. Vesicle size measurement The size of niosomes was measured by using static light diffraction method by a Cilas 1090 particle size analyzer (Orleans, France) equipped with a 5 mw He/Ne (635 nm) laser beam. The measurement carried out 24 h after preparation at 25 °C. The particle size measurements are presented as d(0.1) lm, d(0.5) lm and d(0.9) lm on a surface weighted basis that is the size of 10%, 50% and 90% of the particles below these values. The surface area mean diameter and the volume mean diameter, known as D [3,2] and D[4,3], respectively were calculated using the software provided with the apparatus. The span, which is the distribution width of the particles in the niosome suspension was measured as follows:
Span ¼
dð0:9Þ dð0:1Þ dð0:5Þ
ð1Þ
2.3.2. Optical microscopy Niosomes were imaged by a camera (SSC-DC388, SONY, Japan) attached onto an optical microscope (BX51, OLYMPUS, Japan) at 10 40 magnification. 2.3.3. Encapsulation efficiency Encapsulation efficiency of niosomes was determined by using freshly prepared samples. Samples were centrifuged at 18,000g for 30 min at 4 °C. Pellet was diluted in 50 mL methanol so as to break niosomal membrane. The concentration of released atocopherol was determined spectrophotometrically (UNICO spectrophotometer, New Jersey, USA) in methanol at 285 nm. Each experiment was carried out in triplicate and results are expressed as mean ± standard deviation. The entrapment efficiency was defined as follows:
released tocopherol content from niosomes 100 initial tocopherol content used in niosomes preparation
taminase (activity 1500 u g1) were purchased from Sigma– Aldrich (Wicklow, Ireland). Sodium hydroxide, glucono deltalactone (GDL), phosphate buffered saline, sodium azide, hydrochloric acid, and other chemicals were procured from Merck (Darmstadt, Germany).
107
ð2Þ
2.3.4. DPPH radical scavenging activity assay Radical scavenging activity was determined according to the technique reported by Tan et al. (2014) with a slight modification. Briefly, 1 mL of free (unencapsulated) a-tocopherol ethanolic solution or a-tocopherol-loaded niosomes with same concentration of
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a-tocopherol was mixed with 1 mL of ethanolic DPPH solution (0.2 mM). Mixtures were incubated for 40 min at 25 °C in the dark. The bleaching of DPPH was determined by measuring the absorbance at 517 nm with UV–Vis spectrophotometer. The DPPH radical scavenging activity was calculated according to Eq. (3): Scavenging-activity ð%Þ ¼ ½1 ðAs AcÞ=Ab 100
ð3Þ
where As is the absorbance of sample, Ac is the absorbance of control (ethanol added to samples instead of DPPH), and Ab is the absorbance of blank (ethanol added instead of samples).
2.5.5. Swelling experiment The swelling capacity of hydrogels was measured following the method given by Maltais, Remondetto, and Subirade (2009). For this purpose, cylindrical gel specimens were weighed after dewetting their surfaces and then were immersed in an enzyme-free simulated gastric fluid at 37 °C. Periodically, the specimens were removed from the solution and re-weighed. Gel swelling percentage was calculated as follows:
Swelling ð%Þ ¼ ½mt m0 =m0 100
ð4Þ
where mt is the gel mass at time t and m0 is the initial gel mass. 2.4. Preparation of protein hydrogels WPI solution was prepared by dissolving 10 g WPI powder in 100 mL distilled water with stirring for 2 h using a magnetic stirrer. Sodium azide (50 ppm) was added for preventing microbial growth. The obtained WPI solution was kept at 4 °C for 12 h to ensure protein hydration. The solution was then heated at 80 °C for 20 min to fulfill denaturation. For cross-linking, the heat denatured WPI solution (100 mg mL1) was charged with transglutaminase solution with 1 U mL1 power (10 lL mL1 protein solution) and incubated at 37 °C for 62 h while being shaken at 150 rpm. Subsequently, 2 mL of niosomal suspension (containing 0.017 g surfactant) or distilled water was added to 3 mL of either noncross-linked or cross-linked WPI solution obtaining a final protein concentration of 60 mg mL1. Afterwards, the niosome-charged and niosome-free WPI solutions were supplemented with GDL (0.16 g g1 protein) and incubated at 40 °C for 1 h to gel. The freshly prepared gels were used for rheological, swelling and degradation analyses. 2.5. Hydrogel characterization 2.5.1. Gel chemistry Fourier transform infrared (FTIR) spectra of whey protein hydrogels and sorbitan monostearate were collected by using a FTIR spectrometer (Perkin Elmer Spectrum one, MA, USA) at 4000–500 cm1. Freeze dried (VaCo 5 freeze-dryer, Zirbus Technology, Harz, Germany) samples were pressed into KBr disks and scanned. 2.5.2. Rheological properties Rheological properties of either cross-linked or non-crosslinked niosome-charged and niosome-free whey protein dispersions were evaluated after being supplemented with GDL using a Physica MCR 301 rheometer (Anton-Paar, GmbH, Graz, Austria). For this purpose, a time ramp test at constant frequency (1 Hz) and strain (0.01%) followed immediately by a frequency sweep (0.1–50 Hz) test at 0.05% strain was carried out. All experiments were performed at 40 °C. 2.5.3. Penetration test Cylindrical gel samples (20 mm height 10 mm diameter) were subjected to penetration test using a texture analyzer apparatus (M350-10CT, Testometric, Lancashire, UK). Samples were penetrated by a probe of 5 mm diameter at the speed of 0.5 mm s1 and the maximum penetration force, defined as the force required to rupture the gel, was expressed as gel strength. 2.5.4. Microstructure Micro-morphology of whey protein hydrogels was captured with a scanning electron microscope (VEGAnnTESCAN, USA) at 30,000 magnification. Small pieces of lyophilized gels were cut and mounted on aluminum stubs and then sputter-coated with gold prior to imaging.
2.5.6. In vitro degradation of gels Degradation of hydrogels was examined based on the method by Maltais et al. (2009). Simulated gastric fluid (SGF) consisted of 0.2% sodium chloride and 0.01 N hydrochloric acid with the final pH of 1.2. The gastric degradation test was carried out with or without pepsin (0.32%). The simulated intestinal fluid (SIF) consisted of 50 mM monobasic potassium phosphate and 40 mM sodium hydroxide with the final pH of 7.4. Specified masses of the cross-linked and non-cross-linked gels were subjected to degrading fluids at 37 °C under mild shaking (100 rpm). Concentration of released protein was measured spectrophotometrically at 280 nm using a WPI standard curve. Gel degradation extent was calculated as:
Degradation ð%Þ ¼
released protein 100 total protein
ð5Þ
where total protein is the amount of protein within the gel matrix and released protein is the amount of protein which was released into the gastrointestinal fluids. 2.6. Statistical analysis All experiments were conducted with three replications. Statistical analysis was performed using SAS 9.13 software (SAS Institute Inc.). Data were subjected to analysis of variance, and any significant difference among the means was found by employing leastsignificant-difference (LSD) and Duncan’s tests at 5% significance level. 3. Results and discussion 3.1. Niosomes characteristics Encapsulation efficiency measurement indicated that 80.0 ± 9% of a-tocopherol was entrapped within the niosomal bilayers. The
d (0.1) = 0.1 d (0.5) = 2.63 d (0.9) = 13.82
D[3,2] = 5.71 D[4,3] = 21.18 Span = 5.21
Fig. 1. Light microscopy image of niosomes and the size and dispersity index of niosomes based on static light scattering measurements.
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Fig. 2. FTIR spectra of WPI powder (a) non-cross-linked gel (b), cross-linked gel (c) and non-cross-linked niosome-loaded gel (d).
Fig. 3. (A) Complex modulus development during gelation process, arrows indicate the time at which an abrupt change in the slope of diagram occurred (was considered as gelation time); (B) frequency sweep test results of hydrogels.
remarkable efficiency of a-tocopherol encapsulation is related to its lipid soluble nature which caused significant incorporation of the vitamin into niosomes structure. Alpha-tocopherol interacts
hydrophobically with the acyl chains of vesicular bilayers and thus stabilizes the structure (Quinn, 2012). Fig. 1 reports the size distribution characteristics of niosomes varying in size from below
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interface (Fryer, 1992); accordingly, the phenolic part of the head group can easily act as a hydrogen donor upon encountering free radicals. 3.3. Molecular attributes of protein hydrogels
Fig. 4. Schematic illustration of hydrophobically-driven supramolecular assembly of proteins in niosome-loaded (a), and niosome-free gel networks (b): niosomes mediate the cross-connection of protein aggregates via hydrogen bonds and also assist heat-denatured proteins refolding.
100 nm to above 13 lm with surface weighted mean diameter of 5.7 lm. Span gives a rough estimation of particles polydispersity; the lower the span value, the narrower is the particle size distribution. Span values close to unity represent narrow size distributions (Fiel, Adorne, Guterres, Netz, & Pohlmann, 2013). As expected, the multilamellar niosomes prepared in the present study were highly polydisperse. Although high pressure homogenization and ultrasonication of the niosomes suspension could yield a less polydisperse product, requirement for ultracentrifugation apparatus (P100,000g) for niosomes recovery discouraged us from carrying out subsequent monodispersing process. It is also noteworthy that ultrasonic treatment generates highly reactive radical species from water vapor molecules that might attack a-tocopherol and promote its oxidation (Makino, Mossoba, & Riesz, 1983). The optical microscopy image of niosomal suspensions (Fig. 1) confirms the high polydispersity of vesicles. Niosomes were spherically shaped objects with thick bilayer membranes. 3.2. DPPH assay The DPPH scavenging activity of free a-tocopherol and atocopherol-loaded niosomes were indifferent i.e. 30.0% and 28.7%, respectively. These results suggest that the encapsulation did not impact the radical scavenging activity of a-tocopherol. Tocopherols are amphiphilic molecules, consisting of a hydrophilic chroman head (with two rings: one phenolic and one heterocyclic) and a hydrophobic phytyl tail (Kamal-Eldin & Appelqvist, 1996). When incorporating in a bilayer membrane system, the hydrophobic part is located in the membrane, associated with the acyl chains of fatty acids whereas the polar chroman head group lies at the
FTIR analysis was conducted to find out alterations in whey proteins secondary structures as influenced by preheating, enzymatic cross-linking and niosome loading (Fig. 2). Amide I (1650 cm1), amide II (1550 cm1), and amide III (1400–1200 cm1) bands can be utilized to predict alterations in secondary structure of proteins. Amide III spectra account mainly for in-phase combination of NH bending and CN stretching vibrations (Barth, 2007). A peak displacement was observed from 1244 cm1 in native WPI to 1234 cm1 in gelled samples. The spectra in the range of 1220– 1250 cm1 is assigned to b-sheets (Cai & Singh, 1999). Accordingly, the observed peak displacement indicates alterations in the secondary structure of proteins as a result of heat denaturation prior to gelation. Beaulieu, Savoie, Paquin, and Subirade (2002) studying the retinol confining whey protein microcapsules, observed changes in the amide I spectra by heat denaturation which were attributed to formation of intermolecular antiparallel b-sheets due to proteins aggregation. The Peaks at 2800–3100 cm1 in FTIR spectra are related to CH stretching and stand for hydrophobic interactions. Transglutaminase-induced cross-linking of whey proteins resulted in shifting the band area from 2928 cm1 to 2960 cm1 which may be ascribed to increased hydrophobicity in –CH2 asymmetrical stretching regions because of the modified exposure of aliphatic amino acids and also due to formation of e-(c-glutamyl) lysine cross-linkages (Eissa, Puhl, Kadla, & Khan, 2006). In agreement Bagheri, Madadlou, Yarmand, and Mousavi (2014) observed a peak displacement from 2954 cm1 to 2959 cm1 upon enzymatic cross-linking of whey peptides. Enzyme treatment caused no remarkable change in the position of the major bands associated with the secondary structure of proteins, suggesting that crosslinking did not cause significant conformational changes in whey proteins. The Peak area at the range of 2500–3400 cm1 stands for OH stretching vibrations in a hydrogen bonded systems (Miller, 2003). The peak at 3420 cm1 for WPI and niosome-free gels became broader and shifted to 3408 cm1 in the case of niosomal gel (Fig. 2). These alterations may imply hydrogen bonding between niosomes and proteins. Niosomes loading did not cause any remarkable change in the characteristic peaks related to protein secondary structure. It is, therefore, concluded that niosome entrapment in the protein gel matrix was mainly physical. 3.4. Rheological characterization of hydrogels Acidification of heat denatured whey protein solution in the direction of its isoelectric pH causes gradual decrement in electrostatic repulsion among protein molecules. Therefore, proteins pack closer and form a gel network by attractive forces including hydrophobic interactions (Alting et al., 2004). Thus, when examining the rheological properties of GDL-injected whey protein solution, dynamic moduli were expected to increase as a function of time due to solidification of whey protein solution. Incubation temperature was of utmost importance for acidification rate by GDL; the higher the temperature, the faster was the gluconic acid generation from GDL and thus pH declined quicker (data not reported). A significantly fast acidification, although increases the gelation rate of denatured whey proteins, does not provide a long enough time scale for pH change to progress homogenous gelation throughout the sample (Eissa et al., 2004), yielding gels with susceptibility to syneresis and mechanical stress. Based on our experience and preliminary experiments, it was decided to form cold-
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Fig. 5. SEM micrographs of whey protein cold-set hydrogels: non-cross-linked (a), cross-linked (b) non-cross-linked niosome-loaded (c) and cross-linked niosome-loaded (d).
set gels at 40 °C. Gelation time was measured using a time ramp test in constant frequency and strain. The corresponding changes in the complex modulus (G⁄) of protein solutions which indicates the overall stiffness of specimen (Miri, 2011) were recorded over time. Fig. 3A demonstrates the evolution of G⁄ as a function of time. The point where an abrupt change was observed in the slope of plot was considered as gelation time (Soltani & Madadlou, unpublished data). Enzymatic cross-linking of whey proteins and charging the protein solution with niosomes, decreased significantly the time required for gelation initiation. The freshly-formed cross-linked niosome-free and the non-cross-linked niosomeloaded samples had also higher elastic modulus (G0 ) than the control (non-cross-linked niosome-free) counterpart. These are also true for the cross-linked niosome-loaded hydrogel; however, niosome loading into the cross-linked sample resulted in lower G0 values throughout the whole frequency range compared with both non-cross-linked niosome-loaded and cross-linked niosome-free samples (Fig. 3B). We hypothesize that niosomal vesicles, owing to their hydrophilic sorbitan head groups that oriented outward, interacted with neighboring protein supramolecules (based on FTIR results) and/or increased the aqueous solvent molecules ordering in their immediate vicinity through hydrogen bonding. Water molecules could, therefore, function as junction points between niosome particles and protein supramolecules, thereby facilitating gel network formation. In this view, the micro-scaled niosome particles mediate protein molecules interaction as shown schematically in Fig. 4. Niosomes might, in addition, aid denatured proteins to refold because the exposed hydrophobic patches of proteins encountered hydrophilic niosome particles. Such impact of
non-ionic surfactants (Randolph & Jones, 2002) and liposomes (Yoshimoto et al., 2006) on proteins conformation has been reported. The niosome-assisted refolding of proteins was likely of much greater value in the case of cross-linked sample since transglutaminase-catalyzed cross-linking of proteins increases their hydrophobicity (Hiller & Lorenzen, 2009). The niosomeassisted refolding of proteins in turn reduced the count of hydrophobic interactions among protein units and argues why niosome charging of the cross-linked protein decreased gel G0 value. Analysis of the texture of well-developed gels by penetration test gave results in accordance with frequency sweep data and indicated that the force required for fracturing gel network increased by either cross-linking or niosome loading alone (data not reported). 3.5. Microstructure SEM micrographs of gels are shown in Fig. 5a–d. Cross-linking increased the gel network continuity which accounts for the augmented firmness of the cross-linked sample in comparison with non-cross-linked niosome-free gel. Formation of chemical crosslinkages as well as the increased surface hydrophobicity of proteins by transglutaminase action argues the compact and less porous microstructure of this sample. The niosome-loaded crosslinked gel microstructure contained fissures and disconnections which implies the inferior textural properties of the sample and supports our hypothesis about the impact of niosome particles on hydrophobically-driven aggregation of denatured proteins. An exemplar niosome particle is observed in the SEM image of
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into the gel network and postponed its disintegration. In agreement to our findings, Monogioudi et al. (2011) reported that transglutaminase cross-linking of b-casein increased the protein stability against enzymatic digestion. Degradation of protein gels irrespective of being cross-linked or not, was much more extensive in the enzyme-free simulated intestinal fluid than corresponding gastric fluid (Fig. 6c). Therefore, although gels swelled significantly in acidic pH, they retained their integrity due likely to hydrophobic interactions. However, the gels underwent progressive disintegration with time in the presence of monopotassium phosphate (KH2PO4) added into the SIF. The anion + HPO2 4 and the cation K are early members of the Hofmeister series which strengthen remarkably the hydrophobic interactions. This at the first glance may be expected to increase the stability of hydrophobically-woven protein lattice but resulted in gel disintegration due likely to expelling water from serum pools and capillary channels of the gel matrix, thereby transforming the hydrogel to a non-integer xerogel. Niosome loading increased significantly the water uptake of protein gel (Fig. 6a) due likely to niosomes high water binding affinity.
4. Conclusion
Fig. 6. Swelling of hydrogels in enzyme-free simulated gastric fluid (SGF) over a period of 3 h at 37 °C (a), matrix degradation of cross-linked and non-cross-linked gels in pepsin-injected SGF (pH 1.2) (b), and in enzyme-free SGF (pH 1.2) and simulated intestinal fluid (SIF, pH 7.4) (c).
niosome-loaded gel network. It indicates that niosomes were successfully incorporated within the hydrogel matrix and the protein gel formation did not dismantle the vesicles integrity.
3.6. Swelling and degradation experiments Swelling is a decisive factor for controlling the release rate of active components from hydrogel matrices (Göpferich, 1996). Once a gel matrix imbibes water, it swells and the pores and voids within the gel network enlarge. This can facilitate the diffusion of entrapped bioactive materials out or digestive enzymes in. Electrostatic repulsion among the positively charged network-building protein aggregates within simulated gastric fluid triggered matrix swelling (Fig. 6a) followed after a threshold duration by degradation with pepsin (Fig. 6b). Cross-linking diminished the swelling (in the enzyme-free SGF) and degradation extents (in pepsininjected SGF) of protein gel which is ascribed to the highly packed arrangement of protein aggregates within the gel network. The close packing of proteins reduced water molecules intrusion rate
Our motivation for development of niosomal whey protein cold-set hydrogels was to fabricate a novel functional food formulation considering high nutritional value of whey proteins and superb delivery potential of niosomes and to preserve niosomes from adverse environmental and/or gastrointestinal conditions. Niosomes interact with denatured whey proteins and assist their refolding, thereby altering the acid-induced aggregation pattern of proteins. The extent of niosome-modulated gelation of proteins depends on their hydrophobicity; a higher hydrophobicity of enzymatically cross-linked proteins results in more intensive protein refolding and thus a less elastic acid gel is obtained compared with non-cross-linked proteins. Transglutaminase-catalyzed crosslinking of whey proteins decreased the simulated gastric degradability of the subsequently-formed cold-set hydrogels. The atocopherol carrying niosomes may, however, be released within the upper small intestine and facilitate vitamin adsorption through epithelial cells membranes. A more comprehensive study is required to monitor the fate and bioavailability of the niosomes embedded within whey protein gels network in the gastrointestinal tract.
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Niosome-loaded cold-set whey protein hydrogels Arash Abaee a, Ashkan Madadlou a,b,c,⇑ a
Department of Food Science and Engineering, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Center of Excellence for Application of Modern Technologies for Producing Functional Foods and Drinks (FFDCE), University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran c Interdisciplinary Research Department of Agricultural and Natural Resources Nanotechnology (IRDANN), University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran b
a r t i c l e
i n f o
Article history: Received 20 June 2015 Received in revised form 8 September 2015 Accepted 11 September 2015 Available online 11 September 2015 Keywords: Niosome Whey protein cold-set gel Transglutaminase a-Tocopherol
a b s t r a c t The a-tocopherol-carrying niosomes with mean diameter of 5.7 lm were fabricated and charged into a transglutaminase-cross-linked whey protein solution that was subsequently gelled with glucono deltalactone. Encapsulation efficiency of a-tocopherol within niosomes was 80% and encapsulation did not influence the radical scavenging activity of a-tocopherol. Fourier transform infrared (FTIR) spectroscopy suggested formation of e-(c-glutamyl) lysine cross-linkages by transglutaminase and that enzymatic cross-linking increased proteins hydrophobicity. FTIR also proposed hydrogen bonding between niosomes and proteins. Dynamic rheometry indicated that transglutaminase cross-linking and niosomes charging of the protein solution enhanced the gelation process. However, charging the cross-linked protein solution with niosomal suspension resulted in lower elastic modulus (G0 ) of the subsequently formed gel compared with both non-cross-linked niosome-loaded and cross-linked niosome-free counterparts. Electron microscopy indicated a discontinuous network for the niosome-loaded cross-linked sample. Niosome loading into the protein gel matrix increased its swelling extent in the enzyme-free simulated gastric fluid. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Most nutraceuticals are highly susceptible to degradation by food processing operations, adverse environmental conditions, and/or gastrointestinal digestion. Thus, encapsulation within appropriately designed and fabricated delivery systems is required to protect them from destructive circumstances (McClements, Decker, Park, & Weiss, 2009). Alpha-tocopherol, an oil-soluble nutraceutical, is the most abundant and biologically active form of vitamin E which acts as antioxidant and might prevent cardiovascular diseases, atherosclerosis and cancer (Liang, Line, Remondetto, & Subirade, 2010). The utilization of a-tocopherol has been hampered due to its light, heat and oxygen sensitivity and lipophilic nature. Therefore, various delivery systems such as emulsions (Yang, Decker, Xiao, & McClements, 2015), solid-lipid nanoparticles (Trombino et al., 2009), and protein and polysaccharide matrices (Duclairoir, Orecchioni, Depraetere, & Nakache, 2002; Pierucci, Andrade, Farina, Pedrosa, & Rocha-Leão, 2007; Somchue, ⇑ Corresponding author at: Department of Food Science and Engineering, University College of Agriculture and Natural Resources, University of Tehran, P.O. Box 31587-77871, Karaj, Iran. E-mail address: [email protected] (A. Madadlou). http://dx.doi.org/10.1016/j.foodchem.2015.09.037 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
Sermsri, Shiowatana, & Siripinyanond, 2009; Song, Lee, & Lee, 2009) have been employed to encapsulate a-tocopherol. Surfactants self-assemble spontaneously into vesicular structures which can be used as model for cell membranes and, furthermore, as vehicles for drugs and bioactive materials (Liu & Guo, 2005). Non-ionic surfactant vesicles, the so-called niosomes, have found great applicability in pharmaceutical, nutraceutical and cosmetic sectors since the late 70’s when they were patented by the company L’Oreal (Handjani-Vila, Ribier, Rondot, & Vanlerberghie, 1979). Niosomes are capable of encapsulating both hydrophilic and lipophilic materials and exhibit some advantages with respect to liposomes including less production costs and higher chemical stability. Niosomes formed from sorbitan monoesters are amongst the most widely investigated vesicles (Hao & Li, 2011; Yoshioka, Sternberg, & Florence, 1994). Despite being a superb vehicle for delivery purposes, niosomes might suffer from several drawbacks including aggregation, fusion, and drug leakage (Moghassemi & Hadjizadeh, 2014). Complementary mechanisms are, therefore, required to modulate the characteristics of drug-conveying niosomes. Proniosomes were presented as powdery alternatives to circumvent the problems related to handling and storage-allowed aggregation of niosomes. In essence, proniosomes are surfactantcoated water-soluble carriers that can be hydrated by brief agitation
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in hot aqueous media immediately before use (Hu & Rhodes, 2000). Introduction of other forms of niosomal products that are edible and demonstrate stability against sedimentation, aggregation and core leakage is, however, required to widen the application of niosomes in food and animal feed sectors in addition to pharmaceutics. Whey proteins confer beneficial effects on human health and possess privileged functional properties such as surface activity, biocompatibility, biodegradability and the ability to form cold- and heat-set hydrogels. A two-step procedure that is heat denaturation of proteins followed by changing the ionic strength or acidification in the direction of the isoelectric pH is required for fabrication of whey protein cold-set gels (Ju & Kilara, 1998). These types of hydrogels are appropriate for protecting heat sensitive nutraceuticals and show good potential for designing functional food commodities (Chen, Remondetto, & Subirade, 2006). Cold-set whey protein hydrogels are, however, prone to syneresis and sensitive to mechanical abrasion and enzymatic degradation. These might limit the applicability of whey protein hydrogels for conveying and delivery of their cargo to final consumer. Eissa, Bisram, and Khan (2004) examined the rheological characteristics of transglutaminase-cross-linked cold-set whey protein gels. They observed that the elastic modulus and yield/fracture stress and strain of the enzymatically cross-linked gel was higher than those of the non-cross-linked counterparts. The enzyme transglutaminase catalyzes acyl transfer reaction between peptide-bound glutamine residues as acyl donor and primary amines as acceptor. This enzyme has been widely used for protein cross-linking in food industry (Aboumahmoud & Savello, 1990). It was of interest for the authors of the present communication to fabricate a-tocopherol-carrying niosome-loaded cold-set whey protein hydrogels reinforced by transglutaminase and characterize their mechanical and techno-functional properties. The protein gel provides an efficient protecting, orally administrable and conventionally edible matrix for niosomes whilst, the niosomal particles convey a-tocopherol within the hydrogel and can improve its bioavailability by solubilizing within the intestinal tract and promoting its permeation through epithelial cells (Song et al., 2014). The bioavailability of lipophilic nutraceuticals depends on formation of sufficient amount of mixed micelles in the upper small intestine that solubilize the core and boost their intake by enterocytes (Joye, Davidov-Pardo, & McClements, 2014). The delivery system introduced in the present article can point to a new avenue for development of functional dairy foods.
2. Materials and methods 2.1. Materials Whey protein isolate (WPI) with at least 90% protein content was kindly gifted by Arla Food Ingredients (Vibyj, Denmark). Sorbitan monostearate (span 60), a-tocopherol, pepsin, and transglu-
Encapsulation efficiency ð%Þ ¼
2.2. Niosome preparation Multilamellar niosomes were prepared using thin film hydration method. Accurately weighed quantities of sorbitan monostearate, cholesterol (molar ratio of 7:3), and a-tocopherol were dissolved in 10 mL chloroform in a 100 mL round-bottom flask. Total amount of surfactants and a-tocopherol were 200 and 11.6 lM, respectively. Organic solvent was then evaporated under vacuum and constant rotation at 60 °C using a rotary evaporator (Heidolph, Germany). The dried thin film was hydrated with deionized water in the rotary evaporator at 60 °C for 1 h under atmospheric pressure. This was followed by centrifugation (Sigma 8 k centrifuge, Germany) at 18,000g for 30 min to remove nontrapped a-tocopherol and rehydrating the niosome pellet with de-ionized water. 2.3. Niosomes characterization 2.3.1. Vesicle size measurement The size of niosomes was measured by using static light diffraction method by a Cilas 1090 particle size analyzer (Orleans, France) equipped with a 5 mw He/Ne (635 nm) laser beam. The measurement carried out 24 h after preparation at 25 °C. The particle size measurements are presented as d(0.1) lm, d(0.5) lm and d(0.9) lm on a surface weighted basis that is the size of 10%, 50% and 90% of the particles below these values. The surface area mean diameter and the volume mean diameter, known as D [3,2] and D[4,3], respectively were calculated using the software provided with the apparatus. The span, which is the distribution width of the particles in the niosome suspension was measured as follows:
Span ¼
dð0:9Þ dð0:1Þ dð0:5Þ
ð1Þ
2.3.2. Optical microscopy Niosomes were imaged by a camera (SSC-DC388, SONY, Japan) attached onto an optical microscope (BX51, OLYMPUS, Japan) at 10 40 magnification. 2.3.3. Encapsulation efficiency Encapsulation efficiency of niosomes was determined by using freshly prepared samples. Samples were centrifuged at 18,000g for 30 min at 4 °C. Pellet was diluted in 50 mL methanol so as to break niosomal membrane. The concentration of released atocopherol was determined spectrophotometrically (UNICO spectrophotometer, New Jersey, USA) in methanol at 285 nm. Each experiment was carried out in triplicate and results are expressed as mean ± standard deviation. The entrapment efficiency was defined as follows:
released tocopherol content from niosomes 100 initial tocopherol content used in niosomes preparation
taminase (activity 1500 u g1) were purchased from Sigma– Aldrich (Wicklow, Ireland). Sodium hydroxide, glucono deltalactone (GDL), phosphate buffered saline, sodium azide, hydrochloric acid, and other chemicals were procured from Merck (Darmstadt, Germany).
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ð2Þ
2.3.4. DPPH radical scavenging activity assay Radical scavenging activity was determined according to the technique reported by Tan et al. (2014) with a slight modification. Briefly, 1 mL of free (unencapsulated) a-tocopherol ethanolic solution or a-tocopherol-loaded niosomes with same concentration of
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a-tocopherol was mixed with 1 mL of ethanolic DPPH solution (0.2 mM). Mixtures were incubated for 40 min at 25 °C in the dark. The bleaching of DPPH was determined by measuring the absorbance at 517 nm with UV–Vis spectrophotometer. The DPPH radical scavenging activity was calculated according to Eq. (3): Scavenging-activity ð%Þ ¼ ½1 ðAs AcÞ=Ab 100
ð3Þ
where As is the absorbance of sample, Ac is the absorbance of control (ethanol added to samples instead of DPPH), and Ab is the absorbance of blank (ethanol added instead of samples).
2.5.5. Swelling experiment The swelling capacity of hydrogels was measured following the method given by Maltais, Remondetto, and Subirade (2009). For this purpose, cylindrical gel specimens were weighed after dewetting their surfaces and then were immersed in an enzyme-free simulated gastric fluid at 37 °C. Periodically, the specimens were removed from the solution and re-weighed. Gel swelling percentage was calculated as follows:
Swelling ð%Þ ¼ ½mt m0 =m0 100
ð4Þ
where mt is the gel mass at time t and m0 is the initial gel mass. 2.4. Preparation of protein hydrogels WPI solution was prepared by dissolving 10 g WPI powder in 100 mL distilled water with stirring for 2 h using a magnetic stirrer. Sodium azide (50 ppm) was added for preventing microbial growth. The obtained WPI solution was kept at 4 °C for 12 h to ensure protein hydration. The solution was then heated at 80 °C for 20 min to fulfill denaturation. For cross-linking, the heat denatured WPI solution (100 mg mL1) was charged with transglutaminase solution with 1 U mL1 power (10 lL mL1 protein solution) and incubated at 37 °C for 62 h while being shaken at 150 rpm. Subsequently, 2 mL of niosomal suspension (containing 0.017 g surfactant) or distilled water was added to 3 mL of either noncross-linked or cross-linked WPI solution obtaining a final protein concentration of 60 mg mL1. Afterwards, the niosome-charged and niosome-free WPI solutions were supplemented with GDL (0.16 g g1 protein) and incubated at 40 °C for 1 h to gel. The freshly prepared gels were used for rheological, swelling and degradation analyses. 2.5. Hydrogel characterization 2.5.1. Gel chemistry Fourier transform infrared (FTIR) spectra of whey protein hydrogels and sorbitan monostearate were collected by using a FTIR spectrometer (Perkin Elmer Spectrum one, MA, USA) at 4000–500 cm1. Freeze dried (VaCo 5 freeze-dryer, Zirbus Technology, Harz, Germany) samples were pressed into KBr disks and scanned. 2.5.2. Rheological properties Rheological properties of either cross-linked or non-crosslinked niosome-charged and niosome-free whey protein dispersions were evaluated after being supplemented with GDL using a Physica MCR 301 rheometer (Anton-Paar, GmbH, Graz, Austria). For this purpose, a time ramp test at constant frequency (1 Hz) and strain (0.01%) followed immediately by a frequency sweep (0.1–50 Hz) test at 0.05% strain was carried out. All experiments were performed at 40 °C. 2.5.3. Penetration test Cylindrical gel samples (20 mm height 10 mm diameter) were subjected to penetration test using a texture analyzer apparatus (M350-10CT, Testometric, Lancashire, UK). Samples were penetrated by a probe of 5 mm diameter at the speed of 0.5 mm s1 and the maximum penetration force, defined as the force required to rupture the gel, was expressed as gel strength. 2.5.4. Microstructure Micro-morphology of whey protein hydrogels was captured with a scanning electron microscope (VEGAnnTESCAN, USA) at 30,000 magnification. Small pieces of lyophilized gels were cut and mounted on aluminum stubs and then sputter-coated with gold prior to imaging.
2.5.6. In vitro degradation of gels Degradation of hydrogels was examined based on the method by Maltais et al. (2009). Simulated gastric fluid (SGF) consisted of 0.2% sodium chloride and 0.01 N hydrochloric acid with the final pH of 1.2. The gastric degradation test was carried out with or without pepsin (0.32%). The simulated intestinal fluid (SIF) consisted of 50 mM monobasic potassium phosphate and 40 mM sodium hydroxide with the final pH of 7.4. Specified masses of the cross-linked and non-cross-linked gels were subjected to degrading fluids at 37 °C under mild shaking (100 rpm). Concentration of released protein was measured spectrophotometrically at 280 nm using a WPI standard curve. Gel degradation extent was calculated as:
Degradation ð%Þ ¼
released protein 100 total protein
ð5Þ
where total protein is the amount of protein within the gel matrix and released protein is the amount of protein which was released into the gastrointestinal fluids. 2.6. Statistical analysis All experiments were conducted with three replications. Statistical analysis was performed using SAS 9.13 software (SAS Institute Inc.). Data were subjected to analysis of variance, and any significant difference among the means was found by employing leastsignificant-difference (LSD) and Duncan’s tests at 5% significance level. 3. Results and discussion 3.1. Niosomes characteristics Encapsulation efficiency measurement indicated that 80.0 ± 9% of a-tocopherol was entrapped within the niosomal bilayers. The
d (0.1) = 0.1 d (0.5) = 2.63 d (0.9) = 13.82
D[3,2] = 5.71 D[4,3] = 21.18 Span = 5.21
Fig. 1. Light microscopy image of niosomes and the size and dispersity index of niosomes based on static light scattering measurements.
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Fig. 2. FTIR spectra of WPI powder (a) non-cross-linked gel (b), cross-linked gel (c) and non-cross-linked niosome-loaded gel (d).
Fig. 3. (A) Complex modulus development during gelation process, arrows indicate the time at which an abrupt change in the slope of diagram occurred (was considered as gelation time); (B) frequency sweep test results of hydrogels.
remarkable efficiency of a-tocopherol encapsulation is related to its lipid soluble nature which caused significant incorporation of the vitamin into niosomes structure. Alpha-tocopherol interacts
hydrophobically with the acyl chains of vesicular bilayers and thus stabilizes the structure (Quinn, 2012). Fig. 1 reports the size distribution characteristics of niosomes varying in size from below
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interface (Fryer, 1992); accordingly, the phenolic part of the head group can easily act as a hydrogen donor upon encountering free radicals. 3.3. Molecular attributes of protein hydrogels
Fig. 4. Schematic illustration of hydrophobically-driven supramolecular assembly of proteins in niosome-loaded (a), and niosome-free gel networks (b): niosomes mediate the cross-connection of protein aggregates via hydrogen bonds and also assist heat-denatured proteins refolding.
100 nm to above 13 lm with surface weighted mean diameter of 5.7 lm. Span gives a rough estimation of particles polydispersity; the lower the span value, the narrower is the particle size distribution. Span values close to unity represent narrow size distributions (Fiel, Adorne, Guterres, Netz, & Pohlmann, 2013). As expected, the multilamellar niosomes prepared in the present study were highly polydisperse. Although high pressure homogenization and ultrasonication of the niosomes suspension could yield a less polydisperse product, requirement for ultracentrifugation apparatus (P100,000g) for niosomes recovery discouraged us from carrying out subsequent monodispersing process. It is also noteworthy that ultrasonic treatment generates highly reactive radical species from water vapor molecules that might attack a-tocopherol and promote its oxidation (Makino, Mossoba, & Riesz, 1983). The optical microscopy image of niosomal suspensions (Fig. 1) confirms the high polydispersity of vesicles. Niosomes were spherically shaped objects with thick bilayer membranes. 3.2. DPPH assay The DPPH scavenging activity of free a-tocopherol and atocopherol-loaded niosomes were indifferent i.e. 30.0% and 28.7%, respectively. These results suggest that the encapsulation did not impact the radical scavenging activity of a-tocopherol. Tocopherols are amphiphilic molecules, consisting of a hydrophilic chroman head (with two rings: one phenolic and one heterocyclic) and a hydrophobic phytyl tail (Kamal-Eldin & Appelqvist, 1996). When incorporating in a bilayer membrane system, the hydrophobic part is located in the membrane, associated with the acyl chains of fatty acids whereas the polar chroman head group lies at the
FTIR analysis was conducted to find out alterations in whey proteins secondary structures as influenced by preheating, enzymatic cross-linking and niosome loading (Fig. 2). Amide I (1650 cm1), amide II (1550 cm1), and amide III (1400–1200 cm1) bands can be utilized to predict alterations in secondary structure of proteins. Amide III spectra account mainly for in-phase combination of NH bending and CN stretching vibrations (Barth, 2007). A peak displacement was observed from 1244 cm1 in native WPI to 1234 cm1 in gelled samples. The spectra in the range of 1220– 1250 cm1 is assigned to b-sheets (Cai & Singh, 1999). Accordingly, the observed peak displacement indicates alterations in the secondary structure of proteins as a result of heat denaturation prior to gelation. Beaulieu, Savoie, Paquin, and Subirade (2002) studying the retinol confining whey protein microcapsules, observed changes in the amide I spectra by heat denaturation which were attributed to formation of intermolecular antiparallel b-sheets due to proteins aggregation. The Peaks at 2800–3100 cm1 in FTIR spectra are related to CH stretching and stand for hydrophobic interactions. Transglutaminase-induced cross-linking of whey proteins resulted in shifting the band area from 2928 cm1 to 2960 cm1 which may be ascribed to increased hydrophobicity in –CH2 asymmetrical stretching regions because of the modified exposure of aliphatic amino acids and also due to formation of e-(c-glutamyl) lysine cross-linkages (Eissa, Puhl, Kadla, & Khan, 2006). In agreement Bagheri, Madadlou, Yarmand, and Mousavi (2014) observed a peak displacement from 2954 cm1 to 2959 cm1 upon enzymatic cross-linking of whey peptides. Enzyme treatment caused no remarkable change in the position of the major bands associated with the secondary structure of proteins, suggesting that crosslinking did not cause significant conformational changes in whey proteins. The Peak area at the range of 2500–3400 cm1 stands for OH stretching vibrations in a hydrogen bonded systems (Miller, 2003). The peak at 3420 cm1 for WPI and niosome-free gels became broader and shifted to 3408 cm1 in the case of niosomal gel (Fig. 2). These alterations may imply hydrogen bonding between niosomes and proteins. Niosomes loading did not cause any remarkable change in the characteristic peaks related to protein secondary structure. It is, therefore, concluded that niosome entrapment in the protein gel matrix was mainly physical. 3.4. Rheological characterization of hydrogels Acidification of heat denatured whey protein solution in the direction of its isoelectric pH causes gradual decrement in electrostatic repulsion among protein molecules. Therefore, proteins pack closer and form a gel network by attractive forces including hydrophobic interactions (Alting et al., 2004). Thus, when examining the rheological properties of GDL-injected whey protein solution, dynamic moduli were expected to increase as a function of time due to solidification of whey protein solution. Incubation temperature was of utmost importance for acidification rate by GDL; the higher the temperature, the faster was the gluconic acid generation from GDL and thus pH declined quicker (data not reported). A significantly fast acidification, although increases the gelation rate of denatured whey proteins, does not provide a long enough time scale for pH change to progress homogenous gelation throughout the sample (Eissa et al., 2004), yielding gels with susceptibility to syneresis and mechanical stress. Based on our experience and preliminary experiments, it was decided to form cold-
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Fig. 5. SEM micrographs of whey protein cold-set hydrogels: non-cross-linked (a), cross-linked (b) non-cross-linked niosome-loaded (c) and cross-linked niosome-loaded (d).
set gels at 40 °C. Gelation time was measured using a time ramp test in constant frequency and strain. The corresponding changes in the complex modulus (G⁄) of protein solutions which indicates the overall stiffness of specimen (Miri, 2011) were recorded over time. Fig. 3A demonstrates the evolution of G⁄ as a function of time. The point where an abrupt change was observed in the slope of plot was considered as gelation time (Soltani & Madadlou, unpublished data). Enzymatic cross-linking of whey proteins and charging the protein solution with niosomes, decreased significantly the time required for gelation initiation. The freshly-formed cross-linked niosome-free and the non-cross-linked niosomeloaded samples had also higher elastic modulus (G0 ) than the control (non-cross-linked niosome-free) counterpart. These are also true for the cross-linked niosome-loaded hydrogel; however, niosome loading into the cross-linked sample resulted in lower G0 values throughout the whole frequency range compared with both non-cross-linked niosome-loaded and cross-linked niosome-free samples (Fig. 3B). We hypothesize that niosomal vesicles, owing to their hydrophilic sorbitan head groups that oriented outward, interacted with neighboring protein supramolecules (based on FTIR results) and/or increased the aqueous solvent molecules ordering in their immediate vicinity through hydrogen bonding. Water molecules could, therefore, function as junction points between niosome particles and protein supramolecules, thereby facilitating gel network formation. In this view, the micro-scaled niosome particles mediate protein molecules interaction as shown schematically in Fig. 4. Niosomes might, in addition, aid denatured proteins to refold because the exposed hydrophobic patches of proteins encountered hydrophilic niosome particles. Such impact of
non-ionic surfactants (Randolph & Jones, 2002) and liposomes (Yoshimoto et al., 2006) on proteins conformation has been reported. The niosome-assisted refolding of proteins was likely of much greater value in the case of cross-linked sample since transglutaminase-catalyzed cross-linking of proteins increases their hydrophobicity (Hiller & Lorenzen, 2009). The niosomeassisted refolding of proteins in turn reduced the count of hydrophobic interactions among protein units and argues why niosome charging of the cross-linked protein decreased gel G0 value. Analysis of the texture of well-developed gels by penetration test gave results in accordance with frequency sweep data and indicated that the force required for fracturing gel network increased by either cross-linking or niosome loading alone (data not reported). 3.5. Microstructure SEM micrographs of gels are shown in Fig. 5a–d. Cross-linking increased the gel network continuity which accounts for the augmented firmness of the cross-linked sample in comparison with non-cross-linked niosome-free gel. Formation of chemical crosslinkages as well as the increased surface hydrophobicity of proteins by transglutaminase action argues the compact and less porous microstructure of this sample. The niosome-loaded crosslinked gel microstructure contained fissures and disconnections which implies the inferior textural properties of the sample and supports our hypothesis about the impact of niosome particles on hydrophobically-driven aggregation of denatured proteins. An exemplar niosome particle is observed in the SEM image of
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into the gel network and postponed its disintegration. In agreement to our findings, Monogioudi et al. (2011) reported that transglutaminase cross-linking of b-casein increased the protein stability against enzymatic digestion. Degradation of protein gels irrespective of being cross-linked or not, was much more extensive in the enzyme-free simulated intestinal fluid than corresponding gastric fluid (Fig. 6c). Therefore, although gels swelled significantly in acidic pH, they retained their integrity due likely to hydrophobic interactions. However, the gels underwent progressive disintegration with time in the presence of monopotassium phosphate (KH2PO4) added into the SIF. The anion + HPO2 4 and the cation K are early members of the Hofmeister series which strengthen remarkably the hydrophobic interactions. This at the first glance may be expected to increase the stability of hydrophobically-woven protein lattice but resulted in gel disintegration due likely to expelling water from serum pools and capillary channels of the gel matrix, thereby transforming the hydrogel to a non-integer xerogel. Niosome loading increased significantly the water uptake of protein gel (Fig. 6a) due likely to niosomes high water binding affinity.
4. Conclusion
Fig. 6. Swelling of hydrogels in enzyme-free simulated gastric fluid (SGF) over a period of 3 h at 37 °C (a), matrix degradation of cross-linked and non-cross-linked gels in pepsin-injected SGF (pH 1.2) (b), and in enzyme-free SGF (pH 1.2) and simulated intestinal fluid (SIF, pH 7.4) (c).
niosome-loaded gel network. It indicates that niosomes were successfully incorporated within the hydrogel matrix and the protein gel formation did not dismantle the vesicles integrity.
3.6. Swelling and degradation experiments Swelling is a decisive factor for controlling the release rate of active components from hydrogel matrices (Göpferich, 1996). Once a gel matrix imbibes water, it swells and the pores and voids within the gel network enlarge. This can facilitate the diffusion of entrapped bioactive materials out or digestive enzymes in. Electrostatic repulsion among the positively charged network-building protein aggregates within simulated gastric fluid triggered matrix swelling (Fig. 6a) followed after a threshold duration by degradation with pepsin (Fig. 6b). Cross-linking diminished the swelling (in the enzyme-free SGF) and degradation extents (in pepsininjected SGF) of protein gel which is ascribed to the highly packed arrangement of protein aggregates within the gel network. The close packing of proteins reduced water molecules intrusion rate
Our motivation for development of niosomal whey protein cold-set hydrogels was to fabricate a novel functional food formulation considering high nutritional value of whey proteins and superb delivery potential of niosomes and to preserve niosomes from adverse environmental and/or gastrointestinal conditions. Niosomes interact with denatured whey proteins and assist their refolding, thereby altering the acid-induced aggregation pattern of proteins. The extent of niosome-modulated gelation of proteins depends on their hydrophobicity; a higher hydrophobicity of enzymatically cross-linked proteins results in more intensive protein refolding and thus a less elastic acid gel is obtained compared with non-cross-linked proteins. Transglutaminase-catalyzed crosslinking of whey proteins decreased the simulated gastric degradability of the subsequently-formed cold-set hydrogels. The atocopherol carrying niosomes may, however, be released within the upper small intestine and facilitate vitamin adsorption through epithelial cells membranes. A more comprehensive study is required to monitor the fate and bioavailability of the niosomes embedded within whey protein gels network in the gastrointestinal tract.
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