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Physicochemical stability, microrheological properties and microstructure of lutein emulsions stabilized by multilayer membranes consisting of whey protein isolate, flaxseed gum and chitosan
Accepted Manuscript Physicochemical stability, microrheological properties and microstructure of lutein emulsions stabilized by multilayer membranes consisting of whey protein isolate, flaxseed gum and chitosan Duoxia Xu, Zulipiya Aihemaiti, Yanping Cao, Chao Teng, Xiuting Li PII: DOI: Reference:
S0308-8146(16)30052-8 http://dx.doi.org/10.1016/j.foodchem.2016.01.052 FOCH 18626
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
21 August 2015 2 December 2015 12 January 2016
Please cite this article as: Xu, D., Aihemaiti, Z., Cao, Y., Teng, C., Li, X., Physicochemical stability, microrheological properties and microstructure of lutein emulsions stabilized by multilayer membranes consisting of whey protein isolate, flaxseed gum and chitosan, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/ j.foodchem.2016.01.052
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Physicochemical stability, microrheological properties and microstructure of lutein emulsions stabilized by multilayer membranes consisting of whey protein isolate, flaxseed gum and chitosan Duoxia Xu, Zulipiya Aihemaiti, Yanping Cao*, Chao Teng, Xiuting Li
School of Food & Chemical Engineering, Beijing Engineering and Technology Research Center of Food Additives, Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients, Beijing Key Laboratory of Flavor Chemistry, Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University, Beijing, China
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ABSTRACT: The impact of chitosan (CTS) on the physicochemical stability, microrheological property and microstructure of whey protein isolate (WPI)-flaxseed gum (FG) stabilized lutein emulsions at pH 3.0 was studied. A layer-by-layer electrostatic deposition method was used to prepare multilayered lutein emulsions. Droplet size, zeta-potential, instability index, microstructure and microrheological behaviour of lutein emulsions were measured. The influences of interfacial layer, metal chelator and free radical scavenger on the chemical stability of lutein emulsions were also investigated. It was found that multilayer emulsions had better physical stability showing the pronounced effect of 1 wt% CTS. The mean square displacement analysis demonstrated that CTS led to increases of macroscopic viscosity and elasticity index for WPI-FG stabilized lutein emulsions due to CTS embedding in the network. CTS also helped to chemically stabilize the lutein emulsions against degradation. The combination of interfacial membrane and prooxidative metal chelator or free radical scavenger was an effective method to control lutein degradation. Keywords: Lutein emulsion; Multilayer; Whey protein isolate (WPI)-flaxseed gum (FG)-chitosan (CTS); Physicochemical stability; Microrheological property
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1. Introduction It has been reported that lutein is a yellowish pigment for improving food colour and has beneficial effects on human health, including improving vision, the prevention of skin from UV-induced damage and reducing the risk of atherosclerosis, cancer and cardiovascular disease (Bartlett & Eperjesi, 2008; Zhao, Cheng, Jiang, Yao, & Han, 2014). However, lutein is unstable in oxygen, heat and light due to its eight conjugated double bond structure. This can lead to loss of both colour and bioactivity of lutein in foods, leading to loss of product quality and consumer acceptance (Liu, Lai, Wu, Chen, Lee, & Hsu, 2015). Additionally, the enrichment of food or beverage products with lutein encounters great difficulties because lutein is insoluble in water, and only slightly soluble in oil at room temperature (Mitri, Shegokar, Gohla, Anselmib, & Müllera, 2011). Consequently, there are a number of stability issues that must be overcome before these products can be launched onto the market. Since lutein is lipid soluble, dispersing it in the oil phase of oil-in-water (O/W) emulsions might be one way of improving the delivery system to achieve better stability and bioavailability (Batista, Raymundo, Sousa, Empis, & Franco, 2006). It can be used as vehicles of nutraceutical carotenoids in food applications. Recent studies have investigated the stability of emulsions and microemulsions enriched with lutein (Amar, Aserin, & Garti, 2004). Lutein was dissolved in oil, and the lutein emulsion was formed by emulsifying the active-compound solution with the aqueous phase containing a combination of emulsifiers. It has been reported that lutein emulsion can be prepared with pea protein, whey proteins and phosphatidylglycerol 3
(Batista et al., 2006; Losso, Khachatryan, Ogawa, Godber, & Shih, 2005). The type of emulsifiers and the processing conditions used for preparing emulsions influences the stability of lutein emulsion. The selection of surface-active components significantly affected the stability of lutein emulsion (Boon, McClements, Weiss, & Decker, 2009). Numerous studies investigating multilayer emulsions formed by layer-by-layer method based on the electrostatic interaction have been published (Ettelaie & Akinshina, 2014; Shchukina & Shchukin, 2012). The multilayer emulsion is formed on a charged surface by adding polysaccharide to oppositely charged protein coated droplets (Klein, Aserin, Svitov, & Garti, 2010). The improved emulsion stability can be controlled by the interaction, composition, charge, thickness and permeability of protein-polysaccharide films. It has been reported that oil-in-water emulsions stabilized
by
β-lactoglobulin-carrageenan-gelatin,
SDS-chitosan-pectin
and
lecithin-chitosan-pectin membranes exhibited a high stability to different processing and environmental conditions, such as pH, thermal treatment, high ionic strength and freezing (Gu, Decker, & McClements, 2005; Gudipati, Sandra, McClements, & Decker, 2010; Jo, Chun, Kwon, Min, & Choi, 2015). The influence of multilayer interfacial membranes on the functional properties, physical and chemical stabilities of lutein emulsions is unclear. In our previous study, a layer-by-layer electrostatic deposition method was used to prepare multilayered β-carotene emulsions with interfacial membranes consisting of WPI and FG at pH 3.0. It was proposed that anionic FG was adsorbed to the surfaces of the positively charged droplets. 4
Chitosan (CTS) is a cationic polysaccharide, commonly obtained from deacetylated derivative of chitin. CTS structure is composed of glucosamine and N-acetyglucosamine polymers (Song, Babiker, Usui, Saito, & Kato, 2002). It is positively charged in acidic environment due to its pKa of 6.2-7.0 and the protonation of its amino residues. CTS has received “generally recognized as safe” (GRAS) status for application in dietary preparation for obesity including foods and beverages (Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012). In the present study, we intend to prepare lutein emulsions containing droplets coated with three layers interfacial membranes (WPI-FG-CTS) and to examine the influence of CTS on the physicochemical stability of WPI-FG stabilized lutein emulsion. The physicochemical properties were assessed by average droplet size, zeta-potential, instability index using novel centrifugal sedimentation technique, microstructure by confocal laser scanning microscopy, microrheological behaviour through diffusing wave spectroscopy technique and chemical stability in order to better understanding the interactions occurring during structuring of emulsion and lutein degradation during storage. Finally, the influence of metal chelator EDTA and free radical scavenger α-tocopherol on the chemical stability of single, two and three layer lutein emulsions was also investigated. Ultimately our goal is to use multilayered protein-polysaccharide to protect bioactive components and to extend the application of bioactive components in the food industry. 2. Materials and methods 2.1. Materials 5
Whey protein isolate (WPI) was purchased from Jinan SAN chemical co., LTD. The product contained 97.6% protein (dry basis), as determined by the supplier’s standard proximate analysis procedures. Flaxseed gum (FG) was purchased from Beijing pinellia technology development co., LTD. CTS (80.0-95.0 deacetylated degree) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Lutein powder (purity>97%) was purchased from Nanjing ze lang pharmaceutical technology co., LTD. Medium-chain triglyceride (MCT) oil was obtained from Lonza Inc. (Allendale, NJ, USA). Sodium azide was purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were of analytical grade. 2.2. Preparation of lutein emulsion WPI and FG were first dispersed in the 10 mM phosphate buffer at pH 7.0, separately. CTS solution (2 wt%) was prepared by dispersing CTS in 10 mM acetate buffer at pH 3.0. These solutions were kept overnight to ensure complete dispersion and dissolution, while sodium azide (0.01 wt%) was added to prevent microbial growth. WPI stabilized lutein emulsion (1 wt% WPI) was prepared with 10 wt% medium chain triacylglycerol (MCT) oil containing lutein (0.04 wt% in the final emulsion) as the dispersed phase and 90 wt% aqueous phase solution at room temperature. The lutein emulsions were prepared using an Ultra-Turrax at a speed of 10000 rpm for 3 min to form coarse emulsions, which were subsequently homogenized using a M110-PS Microfluidizer processor (Microfluidics international Corp., Newton, MA) at the operational pressure of 50 MPa for three times. After preparation, the pH was 6
adjusted to pH 3.0 using 1.0 M HCl. The WPI-FG bilayer emulsions (0.5 wt% WPI, 0.3 wt% FG, 0.02 wt% lutein and 5 wt% MCT) were prepared by slow addition of a WPI-stabilized lutein emulsion to a solution of FG (0.6 wt%) at pH 3.0, respectively. WPI-FG-CTS emulsions were prepared by diluting WPI-FG emulsion with different concentrations of CTS solution (0-2 wt%). The homogenized conditions were the same with the above preparation of WPI stabilized lutein emulsion. Final emulsion samples (0.25 wt% WPI, 0.15 wt% FG, 0-1 wt% CTS, 0.01 wt% lutein and 2.5 wt% MCT) were transferred into screw-capped brown bottles flushed with nitrogen. To evaluate the effect of transition metals and free radical scavengers on the stability of lutein in emulsions, EDTA and α-tocopherol were added directly to the diluted emulsions with the same content of lutein, respectively. EDTA was added to emulsion samples at a final concentration of 200 µM. α-Tocopherol was added at a final concentration of 200mg/kg. 2.3. Measurement of zeta-potential The WPI-FG stabilized lutein emulsions with different concentrations of CTS were diluted using buffer solution at pH 3.0 prior to analysis. Diluted emulsions were then injected into the measurement chamber of a particle electrophoresis instruments. Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK) was used to measure zeta-potential of the droplets. The zeta-potential was determined by measuring the direction and velocity of droplet movement in the applied electric field. 2.4. Determination of Droplet Size 7
The average droplet size of the WPI-stabilized lutein emulsions with different concentrations of CTS were determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK) at a fixed detector angle of 90 °. Emulsions were diluted using buffer solution at pH 3.0 to minimize multiple scattering effects prior to each measurement. The measured time correlation functions were analyzed by Automatic Program equipped with the correlator. The averaged droplets size was obtained by a CONTIN mode analysis. Results were described as cumulants mean diameter (size, nm) for droplet size. 2.5. Physical stability analyzed by Lumisizer The physical stability of lutein emulsions prepared with WPI, WPI-FG and WPI-FG-CTS at various concentrations of CTS was measured with the LUMiSizer (L.U.M. GmbH, Berlin, Germany), a novel instrument employing centrifugal sedimentation to accelerate the occurrence of instability phenomena such as sedimentation, flocculation or creaming (Sobisch & Lerche, 2008). The integration graph shows the percentage of light absorbance per hour described as the “instability index”. The instability index is correlated to the stability of the emulsion: the higher the instability index, the lower the stability. The instrumental parameters used for the measurement were as follows: volume, 1.8 ml of dispersion; 3000 rpm; time Exp, 7650 s; time interval, 30 s; temperature, 25 °C (Xu, Wang, Jiang, Yuan, & Gao, 2011). 2.6. Microstructure The microscopic observations were performed using confocal laser scanning microscopy (CLSM) to study the influence of different concentrations of CTS on the 8
droplet distribution of WPI-FG stabilized lutein emulsions at pH 3.0. A drop of sample was placed on a glass microscope slide and covered by a coverslip. Samples were scanned using the Olympus FV-inverted microscope. A 40×1.3 oil immersion objective was used. Rhodamine B fluorescence was collected, using an excitation line of 543 nm and 560-620 filter for the emission. 2.7. Microrheological behaviour The microrheolaser Lab (Formulaction, France) used for the measurements of the microrheology of lutein emulsions is based on Diffusing Wave Spectroscopy (DWS). It corresponds to dynamic light scattering and measures the particles Brownian motion which depends on the viscoelastic structure of the sample. A microrheology test of 2 h was carried out on each sample of WPI, WPI-FG and WPI-FG-CTS with different concentrations of CTS stabilized lutein emulsions. The instrument measures Brownian motion of the particle as the droplet mean square displacement (MSD) versus time. Elasticity index (EI) and macroscopic viscosity index (MVI) parameters of the samples were obtained from the software RheoSoft Master 1.4.0.0. 2.8. Chemical stability Different interfacial layers of lutein emulsion samples with the same content of lutein were transferred into screw-capped brown bottles flushed with nitrogen and were stored in the dark at 55 °C to accelerate lutein degradation. The lutein content was measured during the storage. Lutein in emulsion was extracted with ethanol and n-hexane and then the absorbance at 440 nm was measured using a Shimadzu 9
RF-5301PC UV-vis spectrophotometer. The content of lutein was calculated by referring to a standard curve of lutein prepared under the same condition. The lutein content was expressed as relative lutein C in percent: C(t)/C0, where C(t) was the lutein content after storage for a period t and C0 was the lutein content at the time of preparation. 2.9. Statistical analysis All emulsions were prepared in duplicate, and all measurements were performed three times. Data were subjected to analysis of variance (ANOVA) using the software package SPSS 12.0 for Windows (SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Zeta potential Zeta potential measurements were performed to evaluate the electrostatic deposition of CTS onto the WPI-FG interfacial film surrounding the lutein droplets. The influence of CTS on the zeta potential of lutein emulsions stabilized by WPI-FG membranes was shown in Fig. 1a. WPI, WPI-FG and WPI-FG-CTS lutein emulsions were designated as primary, secondary and tertiary emulsions surrounded by one, two and three layer interfacial membranes, respectively. All the emulsions contained the same lutein and oil content as described in materials and methods. The WPI-FG-CTS emulsions were prepared to contain a range of different CTS concentrations (0-1 wt%). As shown in Fig. 1a, in the absence of CTS, zeta-potential of the WPI-FG emulsion droplets was around -15 mV, indicating that the WPI-FG membranes exhibited a negative charge at pH 3.0. It could be explained by the fact that FG at the 10
outside interfacial layer had a net negative charge at pH 3.0. In the presence of CTS, zeta-potential of the lutein droplets became increasingly positive as the CTS concentration in the emulsion was increased. It suggested that the positively charged CTS molecules adsorbed to the surfaces of the negatively charged emulsion droplets by forming WPI-FG-CTS membranes. Zeta-potential reached a constant value of around 20 mV when CTS concentration exceeded about 1 wt%, suggesting that the lutein droplets had been saturated with CTS. 3.2. Droplet size Fig. 1b shows the dependence of the droplet size on the CTS concentration for WPI-FG stabilized lutein emulsions. Without CTS, the mean droplet size of WPI-FG stabilized lutein emulsion was relatively small showing 948.2 nm. In the presence of CTS, the mean droplet size of lutein emulsions was increased. At lower CTS concentrations (0.05 wt% and 0.1 wt%), there were significant increases of the mean droplet size. It could be explained by the fact that CTS at low concentrations interacted with WPI-FG on the surface of lutein droplets, occurring destabilization due to charge neutralization. This charge neutralization could induce droplets to come closer together, promoting the formation of aggregates of larger sizes. It was also due to insufficient CTS to cover the entire lutein droplet in the emulsion. Thus, CTS may act as a bridge to interact with WPI-FG molecules between the surfaces of the lutein droplets by attractive electrostatic forces. Therefore, the increased droplet size might be attributed to bridging flocculation of the lutein droplets induced by CTS. With the further addition of more than 0.2 wt% CTS, there was a relative 11
decrease in the mean droplet size. It can be proposed that when the concentration of CTS was increased, it would be sufficient to cover the droplets and form a thick interfacial layer on the lutein droplets. Therefore, the flocculation would be inhibited by the strong electrostatic repulsion and the steric hindrance between droplets. Similar phenomena were also found regarding CTS interaction with soybean soluble polysaccharide, and a decrease in droplet size was also noted with increasing CTS content (Hou, Gao, Yuan, Liu, Li, & Xu, 2010). The difference in droplet size between WPI-FG-CTS (1 wt% CTS) lutein emulsion (with a much smaller droplet size) and WPI-FG emulsion indicated that CTS changed the adsorbing properties of WPI and FG in the lutein droplet surface (Wooster & Augustin, 2006). 3.3. Physical stability The effect of CTS concentration on the physical stability of lutein emulsions was determined with the multisample analytical centrifuge based on the STEP technology. The creaming stability of lutein emulsion can be reflected by the instability index which is the integrated transmission profiles against the measuring time. Fig. 2 shows the instability index of WPI, WPI-FG and WPI-FG-CTS (with different concentrations of CTS) prepared lutein emulsions. As can be seen, the order of instability index was: WPI > WPI-FG-CTS (CTS less than 1 wt%) > WPI-FG > WPI-FG-CTS (1 wt% CTS) prepared lutein emulsion. It was found that the multilayer lutein emulsions had better stability. When the content of CTS was less than 1 wt%, the stability of WPI-FG-CTS lutein emulsion was obviously decreased compared with WPI-FG emulsion. It was interesting to find that when the content of CTS reached 1 12
wt%, the physical stability of lutein emulsions was greatly improved. The Lumisizer stability analysis result was agreed with droplet size and zeta-potential. The improved physical stability of WPI-FG lutein emulsion with the presence of CTS at a concentration of 1 wt% indicated that the depletion flocculation did not greatly occur at high enough CTS concentrations, probably mainly due to CTS forming gel network in the emulsion. CTS is a very good viscosity-enhancing agent in an acidic environment, due to its high molecular weight and linear unbranched structure. Therefore, with the increase of CTS concentration, the viscosity of WPI-FG lutein emulsions was increased. It was reported that CTS could form a dense elastic network in the aqueous phase (Payet & Terentjev, 2008). The rigid network of CTS reduced the lutein droplet diffusion thus improving the stability of lutein emulsions. In the remainder of the study, tertiary lutein emulsion was prepared using a CTS concentration of 1 wt% because the droplet had a relative small droplet size, high zeta-potential and small creaming/instability index. 3.4. Microstructure To further investigate the role of CTS in the stability of lutein emulsion, the CLSM images of lutein emulsions, prepared with WPI, WPI-FG and WPI-FG-CTS were shown in Fig. 3. As observed in Fig. 3, microstructure exhibited differences between these lutein emulsions. Lutein emulsions prepared with WPI alone exhibited a particular microstructure, where formation of flocculated and aggregated droplets can be clearly distinguished. The lutein emulsion prepared with WPI-FG showed a little droplet flocculation. In the presence of CTS, WPI-FG stabilized lutein emulsions 13
showed different microscopic structures depending on CTS concentrations. Lutein emulsions with CTS less than 0.5 wt% showed obviously flocculated droplets, which could be attributed to a resultant attractive interaction between the droplets. In these systems, repulsive electrostatic interaction would be smaller (Fig. 1a), mainly due to the greater charge neutralization between CTS and WPI-FG at the interfacial membrane. This phenomenon could also be attributed to the lower concentration of CTS interacting with more than one droplet surface. It was consistent with the increase of previously observed droplet size (Fig. 1b). With the further increase of CTS concentrations, CLSM images indicated that the droplet flocculation and aggregation became less pronounced, especially at 1 wt%. The lower degree of droplet flocculation and aggregation produced by increasing CTS concentration could be attributed to a greater number of CTS molecules adsorbed onto the droplet interface through layer by layer, thus promoting an increased electrostatic and steric repulsion between the droplets. It might also be explained by the viscosity increment and gel network formation in the continuous phase when increasing CTS concentration. It indicated that the high concentration of CTS was much more effective in preventing the aggregation of lutein droplets. Perrechil and Cunha (2013) reported that higher concentrations of non-adsorbing biopolymers would promote a greater degree of depletion flocculation. However, lutein emulsions prepared with higher concentrations of CTS (1 wt%) could present much less flocculation than lower concentrations of CTS, presumably because of the viscosity increment in the 14
continuous phase. It was agreed with Fioramonti, Martinez, Pilosof, Rubiolo, Santiago and Liliana (2015) that the degree of droplet aggregation decreased with increasing sodium alginate concentration. 3.5. Microrheological properties The impact of CTS on the microrheological properties of WPI-FG stabilized lutein emulsions was measured using the Rheolaser lab. The Rheolaser lab technique does not provide any kind of modification of emulsions compared with the shear rheology. It monitored the Brownian motion of droplets and interpreted it in terms of microrheology (Corredig & Alexander, 2008). The motion of emulsion droplets was measured and the particles interaction was traced by microrheology. The mean square displacement (MSD) of droplets in the lutein emulsions with different concentrations of CTS was measured as a function of time (Fig. 4A). The MSD of the droplets is a direct measure of the dynamic properties of droplets and polymers in which they are embedded. WPI stabilized lutein emulsion was purely viscous, giving a MSD that scaled linearly over the range of decorrelation times. It exhibited that with the addition of FG and CTS, the MSD curves were not linear indicating that these lutein emulsions were viscoelastic properties and the droplets were not free to move due to the droplets network interaction. For the WPI-FG stabilized lutein emulsion, the MSD curves of the embedded droplets were quite similar with different decorrelation time which indicated that the network apparently was structurally stable. It showed that the network of WPI-FG-CTS stabilized lutein emulsions apparently was structurally relaxed. It can be attributed to that CTS was embedded in the network. 15
The elasticity strength and macroscopic viscosity of the samples were represented by EI and MVI values obtained from MSD curves. EI corresponds to the inverse of the distance travelled by droplets before interacting with the network (inverse of the MSD level at the elastic plateau) and MVI corresponds to the inverse of the speed of the particles for long times (inverse of the linear slope of the MSD for long times). Fig. 4B, C exhibits EI and MVI values of WPI, WPI-FG and WPI-FG-CTS with different CTS concentrations stabilized lutein emulsions at 25 °C. The EI and MVI of lutein emulsion prepared by WPI-FG was decreased compared with the one prepared by WPI alone. There was an increase trend in the EI and MVI of lutein emulsion in the presence of CTS. When the content of CTS was 0.05 wt%, the EI and MVI values were drastically increased, which was typically attributed to the flocculation at a relatively low CTS content. In the presence of 1.0 wt% CTS, WPI-FG-CTS prepared lutein emulsions became stable; it is meaningful to compare the EI and MVI values and there existed a remarkable increase in the EI and MVI. EI and MVI values were drastically increased to 1.9 and 8.1 times compared with WPI-FG emulsion. Such increased EI and MVI was typically attributed to higher interdroplet resistance force, which could be explained by the fact that the presence of CTS resulted in dense gel network formation and spatial rearrangement of the lutein droplets. 3.6. Chemical stability The chemical stability of lutein emulsions with WPI, WPI-FG, and WPI-FG-CTS interfacial membranes was evaluated based on changes in degradation of lutein during storage for 7 d at 55 °C. All lutein emulsions showed no obvious changes in the mean 16
droplet size during the storage time (data not shown), indicating that the primary, secondary and tertiary layer lutein emulsions were physically stable during storage regardless of the number of layers on the lutein droplets. The results concerning lutein degradation in the primary, secondary, and tertiary lutein emulsions are presented in Fig. 5. In the WPI and WPI-FG coated lutein emulsions, a steep decrease in lutein content was observed during the storage, with losses of 55.3% and 64.1% after storage of 7 d. It can be concluded that there was a decrease in the chemical stability of the WPI-FG secondary lutein emulsion. The decreased chemical stability can be attributed to the negative charge around the lutein droplets by WPI-FG. Hu McClements and Decker (2003) reported that negative charged emulsion droplets could promote prooxidative metals moving towards the interface where oxidation occurred. It was found that the chemical stability of lutein emulsions was increased with the addition of CTS. In the addition of CTS, there was a steep degradation of lutein after 1 d storage, followed by a slight decrease after 2 d storage, with 30.7% loss after storage of 7 d. Therefore, lutein droplets surrounded by WPI-FG-CTS membrane clearly showed its help to increase the chemical stability of lutein emulsions. It can be attributed that the lutein droplets in the WPI-FG-CTS emulsion had a higher positive charge and a thicker interfacial membrane than those in the WPI and WPI-FG emulsions, therefore, the electrostatic and steric repulsion with prooxidative metals and free radicals was greater. CTS in the continuous phase may also prevent lutein from degradation due to their ability to chelate transition metals at negatively 17
charged sites. CTS is also likely to have antioxidant activity due to its ability to donate hydrogen with high radical-scavenging activity (Kim & Thomas, 2007). Finally, it is possible that the viscosity increment and gel network formation of the WPI-FG-CTS emulsion may prevent lutein degradation by slowing down the interaction of lutein droplets with transition metals and free radicals (Waraho, McClements, & Decker, 2011). 3.7. Influence of EDTA and α-tocopherol on the chemical stability of lutein emulsions with different interfacial layers It is reported that transition metals are naturally present in the emulsifiers, oil and water phase in the emulsion at acidic pH and therefore, it may induce the degradation of lutein (Subagio, Wakaki, & Morita, 1999). Based on our previous study (Xu, Yuan, Gao, McClements, & Decker, 2013), 200 µM EDTA as a transition metal chelator was added to emulsions stabilized by WPI, WPI-FG and WPI-FG-CTS to evaluate if metals were able to promote lutein degradation in different layers of emulsions, respectively (Fig. 6). For the primary WPI stabilized lutein emulsion, EDTA was able to decrease lutein loss at the beginning of the storage period; however, lutein degradation was increased at the end of the storage period. For the WPI-FG and WPI-FG-CTS stabilized emulsions, lutein losses were 7.2% and 22.5% lower with the addition of EDTA after storage of 7 d, respectively. It also exhibited that EDTA was more effective in protecting lutein in WPI-FG-CTS emulsion. It showed that lutein droplets coated by WPI-FG and WPI-FG-CTS were more oxidatively stable than emulsions 18
coated by WPI alone when added EDTA, demonstrating that addition of EDTA in a thicker interfacial membrane emulsion was much more effective in inhibiting lutein degradation. It was found that EDTA inhibited the loss of lutein in the three emulsions during storage indicating that transition metal was a major prooxidant in lutein emulsions. The results were similar with our previous reports about iron chelator desferoxamine inhibiting β-carotene degradation in emulsions (Xu et al., 2013). The results were also agreed to those reported by Boon and coworkers who reported that EDTA was able to inhibit lycopene degradation in oil-in-water emulsions (Boon et al., 2009). Free radicals in the emulsion may also degrade lutein (Zhao et al., 2014). Therefore, 200 mg/kg α-tocopherol as a free radical scavenger was applied to investigate the influence of free radicals on the oxidative stability of lutein in WPI, WPI-FG and WPI-FG-CTS emulsions, respectively (Fig. 6). It exhibited that α-tocopherol increased the oxidative stability of lutein in the three emulsions. After the storage of 7 d, lutein content of the three emulsions was increased by 8.8%, 8.7% and 17.2% compared with emulsions without α-tocopherol, respectively. It was revealed that α-tocopherol was the most effective in the emulsions stabilized with WPI-FG-CTS. It was interesting to find that an addition of EDTA or α-tocopherol to the WPI-FG-CTS stabilized emulsion exhibited much less lutein loss after the storage. It suggested that the combination of the tertiary interfacial membrane and prooxidative metal chelator or free radical scavenger was an effective method to control lutein 19
degradation. The greater stability of the WPI-FG-CTS emulsions with addition of EDTA or α-tocopherol can also be attributed to synergy effect of CTS with prooxidant chelator and free radical scavenger. 4. Conclusion The purpose of the study was to examine the effect of CTS on the properties of WPI-FG coated lutein emulsions. The physical and chemical stabilities of lutein emulsions containing droplets surrounded by multilayered interfacial membranes prepared by protein and polysaccharides were investigated. The major findings of the study were: Stable three layered lutein emulsions can be prepared at pH 3.0 that contain lutein droplets surrounded by WPI-FG-CTS with CTS concentration of 1 wt%. When the content of CTS was less than 1 wt%, the stability of WPI-FG-CTS lutein emulsion was obviously decreased compared with WPI-FG emulsion. The microrheological property showed that WPI stabilized lutein emulsion was purely viscous. In presence of FG and CTS, the lutein emulsions were viscoelastic properties and the droplets were not free to move due to the droplets network interaction. There was an increase trend in the EI and MVI of lutein emulsions in the presence of CTS. The order of the chemical stability was: WPI-FG-CTS > WPI > WPI-FG prepared lutein emulsion. The addition of EDTA or α-tocopherol to the WPI-FG-CTS stabilized emulsion exhibited much less lutein loss after the storage than WPI and WPI-FG emulsion. Acknowledgements This research was funded by the National Natural Science Foundation of China 20
(31501486), Beijing Natural Science - SANYUAN jointly Foundation (15S00016), Cultivation of Excellent Talents in Beijing City (2014000020124G032), National Natural Science Foundation of China (31371722) and Development of Innovative Teams and Teachers’ Occupation Advancement Project of Beijing Municipal Universities and Colleges (IDHT20130506). The authors are also grateful to Yuwei He and Guangbin Chen for the use of Rheometer LAB (Formulaction, France) and analysis of the microrheology. References Amar, I., Aserin, A., & Garti, N. (2004). Microstructure transitions derived from solubilization of lutein and lutein esters in food microemulsions. Colloids and Surfaces B: Biointerfaces, 33(3-4), 143-150. Bartlett, H. E., & Eperjesi, F. (2008). A randomised controlled trial investigating the effect of lutein and antioxidant dietary supplementation on visual function in healthy eyes. Clinical Nutrition, 27, 218-227. Batista, A. P., Raymundo, A., Sousa, I., Empis, J., & Franco, J. M. (2006). Colored food emulsions-implications of pigment addition on the rheological behaviour and microstructure. Food Biophysics, 1, 216-227. Boon, C. S., McClements, D. J., Weiss, J., & Decker, E. A. (2009). Role of iron and hydroperoxides in the degradation of lycopene in oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 57, 2993-2998. Bouyer, E., Mekhloufi, G., Rosilio, V., Grossiord, J., & Agnely, F. (2012). Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: 21
Alternatives to synthetic surfactants in the pharmaceutical field? International Journal of Pharmaceutics, 436, 359-378. Corredig, M., & Alexander, M. (2008). Food emulsions studied by DWS: recent advances. Trends in Food Science & Technology, 19, 67-75. Ettelaie, R., & Akinshina, A. (2014). Colloidal interactions induced by overlap of mixed protein+polysaccharide interfacial layers. Food Hydrocolloids, 42, 106-117. Fioramonti, S. A., Martinez, M. J., Pilosof, A. M. R., Rubiolo, A. C., Santiago, & Liliana, G. (2015). Multilayer emulsions as a strategy for linseed oil microencapsulation:
Effect
of
pH
and
alginate
concentration.
Food
Hydrocolloids, 43, 8-17. Gu, Y. S., Decker, A. E., & McClements, D. J. (2005). Production and characterization of oil-in-water emulsions containing droplets stabilized by multilayer membranes consisting of β-lactoglobulin, l-carrageenan and gelatin. Langmuir, 21, 5752-5760. Gudipati, V., Sandra, S., McClements, D. J., & Decker, E. A. (2010). Oxidative stability and intro digestibility of fish oil-in-water emulsions containing containing multilayered membranes. Journal of Agricultural and Food Chemistry, 58, 8093-8099. Hou, Z., Gao, Y., Yuan, F., Liu, Y., Li, C., & Xu, D. (2010). Investigation into the physicochemical stability and rheological properties of beta-carotene emulsion stabilized by soybean soluble polysaccharides and chitosan. Journal of 22
Agricultural and Food Chemistry, 58(15), 8604-8611. Hu, M., McClements, D. J., & Decker, E. A. (2003). Impact of whey protein emulsifiers on the oxidative stability of salmon oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 51(5), 1435-1439. Jo, Y., Chun, J., Kwon, Y., Min, S., & Choi, M. (2015). Formulation development of multilayered fish oil emulsion by using electrostatic deposition of charged biopolymers. International Journal of Food Engineering, 11(1), 31-39. Kim, K. W., & Thomas, R. L. (2007). Antioxidative activity of chitosans with varying molecular weights. Food Chemistry, 101(1), 308-313. Klein, M., Aserin, A., Svitov, I., & Garti, N. (2010). Enhanced stabilization of cloudy emulsions with gum Arabic and whey protein isolate. Colloids and Surfaces B: Biointerfaces, 77, 75-81. Liu, C., Lai, K., Wu, W., Chen, Y., Lee, W., & Hsu, C. (2015). In vitro scleral lutein distribution
by
cyclodextrin
containing
nanoemulsions.
Chemical
&
Pharmaceutical Bulletin, 63(2), 59-67. Losso, J. N., Khachatryan, A., Ogawa, M., Godber, J. S., & Shih, F. (2005). Random centroid
optimization
of
phosphatidylglycerol
stabilized
lutein-enriched
oil-in-water emulsions at acidic pH. Food Chemistry, 92, 737-744. Mitri, K., Shegokar, R., Gohla, S., Anselmib, C., & Müllera, R. H. (2011). Lutein nanocrystals as antioxidant formulation for oral and dermal delivery. International Journal of Pharmaceutics, 420, 141-146. Payet, L., & Terentjev, E. M. (2008). Emulsification and stabilization mechanisms of 23
O/W emulsions in the presence of chitosan. Langmuir, 24, 12247-12252. Perrechil, F. A., & Cunha, R. L. (2013). Stabilization of multilayered emulsions by sodium caseinate and k-carrageenan. Food Hydrocolloids, 30, 606-613. Shchukina, E. M., & Shchukin, D. G. (2012). Layer-by-layer coated emulsion microparticles as storage and delivery tool. Current Opinion in Colloid & Interface Science, 17, 281-289. Sobisch, T., & Lerche, D. (2008). Thickener performance traced by multisample analytical centrifugation. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 331, 114-118. Song, Y., Babiker, E. E., Usui, M., Saito, A., & Kato, A. (2002). Emulsifying properties and bactericidal action of chitosan-lysozyme conjugates. Food Research International, 35, 459-466. Subagio, A., Wakaki, H., & Morita, N. (1999). Stability of lutein and its myristate esters. Bioscience Biotechnology and Biochemistry, 63, 1784-1786. Waraho, T., McClements, D. J., & Decker, E. A. (2011). Mechanisms of lipid oxidation in food dispersions. Trends in Food Science & Technology, 22(1), 3-13 Wooster, T. J., & Augustin, M. A. (2006). β-Lactoglobulin-dextran maillard conjugates: their effect on interfacial thickness and emulsion stability. Journal of Colloids and Interface Science, 303, 564-572 Xu, D., Wang, X., Jiang, J., Yuan F., & Gao, Y. (2011). Impact of whey protein-beet pectin conjugation on the physicochemical stability of β-carotene emulsions. Food Hydrocolloids, 25, 258-266. 24
Xu, D., Yuan F., Gao, Y, McClements, J., & Decker, E. (2013). Influence of pH, metal chelator, free radical scavenger and interfacial characteristics on the oxidative stability of beta-carotene in conjugated whey protein-pectin stabilized emulsion. Food Chemistry, 139, 1098-1104. Zhao, C., Cheng, H., Jiang, P., Yao, Y., & Han, J. (2014). Preparation of lutein-loaded particles for improving solubility and stability by polyvinylpyrrolidone (PVP) as an emulsion-stabilizer. Food Chemistry, 156, 123-128.
25
25
Zeta-Potential(mV)
20 15 10 5 0 0.0 -5
0.2
0.4
0.6
0.8
1.0
Chitosan Concentration(wt%)
-10 -15 -20
(a)
1600 1500 1400
Size (nm)
1300 1200 1100 1000 900 800 0.0
0.2
0.4
0.6
0.8
1.0
Chitosan Concentration(wt%) Fig. 1. Dependence of the zeta-potential (a) and droplet size (b) on CTS concentration for WPI-FG stabilized lutein emulsions (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG) at pH 3.0. Some error bars lie within the data points.
26
(a)
(c) (d) (e) (f) (g)
(b) (h)
Fig. 2. Instability index of WPI, WPI-FG and WPI-FG-CTS with different concentration of CTS stabilized lutein emulsions at pH 3.0 determined by LUMiSizer (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG; a, WPI; b, WPI-FG; c-h, WPI-FG-CTS with different concentration of CTS, c, 0.05 wt%; d, 0.1 wt%; e, 0.2 wt%; f, 0.25 wt%; g, 0.5 wt%; h, 1 wt%).
27
Fig. 3. Confocal micrograph of WPI, WPI-FG, WPI-FG-CTS with different
concentration of CTS stabilized lutein emulsions at pH 3.0 (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG; a, WPI; b, WPI-FG; c-h, WPI-FG-CTS with different concentration of CTS, c, 0.05 wt%; d, 0.1 wt%; e, 0.2 wt%; f, 0.25 wt%; g, 0.5 wt%; h, 1 wt%). 28
(b)
(c)
(d)
(e)
(f)
(g)
(h)
MSD (nm2)
MSD (nm2)
MSD (nm 2)
MSD (nm2)
(a)
10-3
10-2
-3 10-1 10
Decorrelation Time (s)
A
29
10-2
10-1
(B)
(C)
Fig. 4. Typical examples of the MSD vs time curves (A) for WPI, WPI-FG, WPI-FGCTS with different concentration of CTS stabilized lutein emulsions at pH 3.0 (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG; a, WPI; b, WPI-FG; c-h, WPI-FG-CTS with different concentration of CTS, c, 0.05 wt%; d, 0.1 wt%; e, 0.2 wt%; f, 0.25 wt%; g, 0.5 wt%; h, 1 wt%). Different colours and arrows refer to different scanning time of MSD analysis. EI (B) and MVI (C) values for WPI, 30
WPI-FG, WPI-FG-CTS with different concentration of CTS stabilized lutein emulsions.
31
WPI WPI+FG WPI+FG+CTS
1.0
C /C 0
0.8
0.6
0.4
0.2
0.0 0
1
2
3
4
5
6
7
Storage time(d) Fig. 5. Lutein degradation as a function of storage time for the primary (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI), secondary (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG) and tertiary (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG, 1 wt% CTS) lutein emulsions (pH 3.0) at 70 °C. Some error bars lie within the data points.
32
control α-tocopherol EDTA
1.0
C/C0
0.8
0.6
0.4
0.2
0.0 0
1
2
3
4
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7
Storage time(d)
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0.0 0
1
2
3
4
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Storage time(d)
(b) control α-tocopherol EDTA
1.0
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0.8
0.6
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0.2
0.0 0
1
2
3
4
Storage time(d)
33
5
6
7
(c)
Fig. 6. Effect of the addition of EDTA and α-tocopherol on the oxidative stability of lutein as a function of storage time for the primary (a, 0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI), secondary (b, 0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG) and tertiary (c, 0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG, 1 wt% CTS) emulsions (pH 3.0) at 70 °C. Some error bars lie within the data points.
34
> Multilayer emulsions had better stability to droplet flocculation and aggregation. > 1 wt% CTS had a pronounced effect on the microstructure and stability of lutein emulsion. > CTS led to increases of MVI and EI for emulsions due to CTS embedding in the network. > CTS helped to chemically stabilize lutein emulsions. > Combination of WPI-FG-CTS and EDTA or α-tocopherol was an effective method to control lutein degradation.
35
S0308-8146(16)30052-8 http://dx.doi.org/10.1016/j.foodchem.2016.01.052 FOCH 18626
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
21 August 2015 2 December 2015 12 January 2016
Please cite this article as: Xu, D., Aihemaiti, Z., Cao, Y., Teng, C., Li, X., Physicochemical stability, microrheological properties and microstructure of lutein emulsions stabilized by multilayer membranes consisting of whey protein isolate, flaxseed gum and chitosan, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/ j.foodchem.2016.01.052
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Physicochemical stability, microrheological properties and microstructure of lutein emulsions stabilized by multilayer membranes consisting of whey protein isolate, flaxseed gum and chitosan Duoxia Xu, Zulipiya Aihemaiti, Yanping Cao*, Chao Teng, Xiuting Li
School of Food & Chemical Engineering, Beijing Engineering and Technology Research Center of Food Additives, Beijing Higher Institution Engineering Research Center of Food Additives and Ingredients, Beijing Key Laboratory of Flavor Chemistry, Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University, Beijing, China
*Corresponding author.
Tel.: + 86-10-68985645
Fax: + 86-10-68985645
Address: No.11, Fucheng Road, Beijing 100048, China
E-mail: [email protected]
1
ABSTRACT: The impact of chitosan (CTS) on the physicochemical stability, microrheological property and microstructure of whey protein isolate (WPI)-flaxseed gum (FG) stabilized lutein emulsions at pH 3.0 was studied. A layer-by-layer electrostatic deposition method was used to prepare multilayered lutein emulsions. Droplet size, zeta-potential, instability index, microstructure and microrheological behaviour of lutein emulsions were measured. The influences of interfacial layer, metal chelator and free radical scavenger on the chemical stability of lutein emulsions were also investigated. It was found that multilayer emulsions had better physical stability showing the pronounced effect of 1 wt% CTS. The mean square displacement analysis demonstrated that CTS led to increases of macroscopic viscosity and elasticity index for WPI-FG stabilized lutein emulsions due to CTS embedding in the network. CTS also helped to chemically stabilize the lutein emulsions against degradation. The combination of interfacial membrane and prooxidative metal chelator or free radical scavenger was an effective method to control lutein degradation. Keywords: Lutein emulsion; Multilayer; Whey protein isolate (WPI)-flaxseed gum (FG)-chitosan (CTS); Physicochemical stability; Microrheological property
2
1. Introduction It has been reported that lutein is a yellowish pigment for improving food colour and has beneficial effects on human health, including improving vision, the prevention of skin from UV-induced damage and reducing the risk of atherosclerosis, cancer and cardiovascular disease (Bartlett & Eperjesi, 2008; Zhao, Cheng, Jiang, Yao, & Han, 2014). However, lutein is unstable in oxygen, heat and light due to its eight conjugated double bond structure. This can lead to loss of both colour and bioactivity of lutein in foods, leading to loss of product quality and consumer acceptance (Liu, Lai, Wu, Chen, Lee, & Hsu, 2015). Additionally, the enrichment of food or beverage products with lutein encounters great difficulties because lutein is insoluble in water, and only slightly soluble in oil at room temperature (Mitri, Shegokar, Gohla, Anselmib, & Müllera, 2011). Consequently, there are a number of stability issues that must be overcome before these products can be launched onto the market. Since lutein is lipid soluble, dispersing it in the oil phase of oil-in-water (O/W) emulsions might be one way of improving the delivery system to achieve better stability and bioavailability (Batista, Raymundo, Sousa, Empis, & Franco, 2006). It can be used as vehicles of nutraceutical carotenoids in food applications. Recent studies have investigated the stability of emulsions and microemulsions enriched with lutein (Amar, Aserin, & Garti, 2004). Lutein was dissolved in oil, and the lutein emulsion was formed by emulsifying the active-compound solution with the aqueous phase containing a combination of emulsifiers. It has been reported that lutein emulsion can be prepared with pea protein, whey proteins and phosphatidylglycerol 3
(Batista et al., 2006; Losso, Khachatryan, Ogawa, Godber, & Shih, 2005). The type of emulsifiers and the processing conditions used for preparing emulsions influences the stability of lutein emulsion. The selection of surface-active components significantly affected the stability of lutein emulsion (Boon, McClements, Weiss, & Decker, 2009). Numerous studies investigating multilayer emulsions formed by layer-by-layer method based on the electrostatic interaction have been published (Ettelaie & Akinshina, 2014; Shchukina & Shchukin, 2012). The multilayer emulsion is formed on a charged surface by adding polysaccharide to oppositely charged protein coated droplets (Klein, Aserin, Svitov, & Garti, 2010). The improved emulsion stability can be controlled by the interaction, composition, charge, thickness and permeability of protein-polysaccharide films. It has been reported that oil-in-water emulsions stabilized
by
β-lactoglobulin-carrageenan-gelatin,
SDS-chitosan-pectin
and
lecithin-chitosan-pectin membranes exhibited a high stability to different processing and environmental conditions, such as pH, thermal treatment, high ionic strength and freezing (Gu, Decker, & McClements, 2005; Gudipati, Sandra, McClements, & Decker, 2010; Jo, Chun, Kwon, Min, & Choi, 2015). The influence of multilayer interfacial membranes on the functional properties, physical and chemical stabilities of lutein emulsions is unclear. In our previous study, a layer-by-layer electrostatic deposition method was used to prepare multilayered β-carotene emulsions with interfacial membranes consisting of WPI and FG at pH 3.0. It was proposed that anionic FG was adsorbed to the surfaces of the positively charged droplets. 4
Chitosan (CTS) is a cationic polysaccharide, commonly obtained from deacetylated derivative of chitin. CTS structure is composed of glucosamine and N-acetyglucosamine polymers (Song, Babiker, Usui, Saito, & Kato, 2002). It is positively charged in acidic environment due to its pKa of 6.2-7.0 and the protonation of its amino residues. CTS has received “generally recognized as safe” (GRAS) status for application in dietary preparation for obesity including foods and beverages (Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012). In the present study, we intend to prepare lutein emulsions containing droplets coated with three layers interfacial membranes (WPI-FG-CTS) and to examine the influence of CTS on the physicochemical stability of WPI-FG stabilized lutein emulsion. The physicochemical properties were assessed by average droplet size, zeta-potential, instability index using novel centrifugal sedimentation technique, microstructure by confocal laser scanning microscopy, microrheological behaviour through diffusing wave spectroscopy technique and chemical stability in order to better understanding the interactions occurring during structuring of emulsion and lutein degradation during storage. Finally, the influence of metal chelator EDTA and free radical scavenger α-tocopherol on the chemical stability of single, two and three layer lutein emulsions was also investigated. Ultimately our goal is to use multilayered protein-polysaccharide to protect bioactive components and to extend the application of bioactive components in the food industry. 2. Materials and methods 2.1. Materials 5
Whey protein isolate (WPI) was purchased from Jinan SAN chemical co., LTD. The product contained 97.6% protein (dry basis), as determined by the supplier’s standard proximate analysis procedures. Flaxseed gum (FG) was purchased from Beijing pinellia technology development co., LTD. CTS (80.0-95.0 deacetylated degree) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Lutein powder (purity>97%) was purchased from Nanjing ze lang pharmaceutical technology co., LTD. Medium-chain triglyceride (MCT) oil was obtained from Lonza Inc. (Allendale, NJ, USA). Sodium azide was purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were of analytical grade. 2.2. Preparation of lutein emulsion WPI and FG were first dispersed in the 10 mM phosphate buffer at pH 7.0, separately. CTS solution (2 wt%) was prepared by dispersing CTS in 10 mM acetate buffer at pH 3.0. These solutions were kept overnight to ensure complete dispersion and dissolution, while sodium azide (0.01 wt%) was added to prevent microbial growth. WPI stabilized lutein emulsion (1 wt% WPI) was prepared with 10 wt% medium chain triacylglycerol (MCT) oil containing lutein (0.04 wt% in the final emulsion) as the dispersed phase and 90 wt% aqueous phase solution at room temperature. The lutein emulsions were prepared using an Ultra-Turrax at a speed of 10000 rpm for 3 min to form coarse emulsions, which were subsequently homogenized using a M110-PS Microfluidizer processor (Microfluidics international Corp., Newton, MA) at the operational pressure of 50 MPa for three times. After preparation, the pH was 6
adjusted to pH 3.0 using 1.0 M HCl. The WPI-FG bilayer emulsions (0.5 wt% WPI, 0.3 wt% FG, 0.02 wt% lutein and 5 wt% MCT) were prepared by slow addition of a WPI-stabilized lutein emulsion to a solution of FG (0.6 wt%) at pH 3.0, respectively. WPI-FG-CTS emulsions were prepared by diluting WPI-FG emulsion with different concentrations of CTS solution (0-2 wt%). The homogenized conditions were the same with the above preparation of WPI stabilized lutein emulsion. Final emulsion samples (0.25 wt% WPI, 0.15 wt% FG, 0-1 wt% CTS, 0.01 wt% lutein and 2.5 wt% MCT) were transferred into screw-capped brown bottles flushed with nitrogen. To evaluate the effect of transition metals and free radical scavengers on the stability of lutein in emulsions, EDTA and α-tocopherol were added directly to the diluted emulsions with the same content of lutein, respectively. EDTA was added to emulsion samples at a final concentration of 200 µM. α-Tocopherol was added at a final concentration of 200mg/kg. 2.3. Measurement of zeta-potential The WPI-FG stabilized lutein emulsions with different concentrations of CTS were diluted using buffer solution at pH 3.0 prior to analysis. Diluted emulsions were then injected into the measurement chamber of a particle electrophoresis instruments. Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK) was used to measure zeta-potential of the droplets. The zeta-potential was determined by measuring the direction and velocity of droplet movement in the applied electric field. 2.4. Determination of Droplet Size 7
The average droplet size of the WPI-stabilized lutein emulsions with different concentrations of CTS were determined by dynamic light scattering (DLS) using a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK) at a fixed detector angle of 90 °. Emulsions were diluted using buffer solution at pH 3.0 to minimize multiple scattering effects prior to each measurement. The measured time correlation functions were analyzed by Automatic Program equipped with the correlator. The averaged droplets size was obtained by a CONTIN mode analysis. Results were described as cumulants mean diameter (size, nm) for droplet size. 2.5. Physical stability analyzed by Lumisizer The physical stability of lutein emulsions prepared with WPI, WPI-FG and WPI-FG-CTS at various concentrations of CTS was measured with the LUMiSizer (L.U.M. GmbH, Berlin, Germany), a novel instrument employing centrifugal sedimentation to accelerate the occurrence of instability phenomena such as sedimentation, flocculation or creaming (Sobisch & Lerche, 2008). The integration graph shows the percentage of light absorbance per hour described as the “instability index”. The instability index is correlated to the stability of the emulsion: the higher the instability index, the lower the stability. The instrumental parameters used for the measurement were as follows: volume, 1.8 ml of dispersion; 3000 rpm; time Exp, 7650 s; time interval, 30 s; temperature, 25 °C (Xu, Wang, Jiang, Yuan, & Gao, 2011). 2.6. Microstructure The microscopic observations were performed using confocal laser scanning microscopy (CLSM) to study the influence of different concentrations of CTS on the 8
droplet distribution of WPI-FG stabilized lutein emulsions at pH 3.0. A drop of sample was placed on a glass microscope slide and covered by a coverslip. Samples were scanned using the Olympus FV-inverted microscope. A 40×1.3 oil immersion objective was used. Rhodamine B fluorescence was collected, using an excitation line of 543 nm and 560-620 filter for the emission. 2.7. Microrheological behaviour The microrheolaser Lab (Formulaction, France) used for the measurements of the microrheology of lutein emulsions is based on Diffusing Wave Spectroscopy (DWS). It corresponds to dynamic light scattering and measures the particles Brownian motion which depends on the viscoelastic structure of the sample. A microrheology test of 2 h was carried out on each sample of WPI, WPI-FG and WPI-FG-CTS with different concentrations of CTS stabilized lutein emulsions. The instrument measures Brownian motion of the particle as the droplet mean square displacement (MSD) versus time. Elasticity index (EI) and macroscopic viscosity index (MVI) parameters of the samples were obtained from the software RheoSoft Master 1.4.0.0. 2.8. Chemical stability Different interfacial layers of lutein emulsion samples with the same content of lutein were transferred into screw-capped brown bottles flushed with nitrogen and were stored in the dark at 55 °C to accelerate lutein degradation. The lutein content was measured during the storage. Lutein in emulsion was extracted with ethanol and n-hexane and then the absorbance at 440 nm was measured using a Shimadzu 9
RF-5301PC UV-vis spectrophotometer. The content of lutein was calculated by referring to a standard curve of lutein prepared under the same condition. The lutein content was expressed as relative lutein C in percent: C(t)/C0, where C(t) was the lutein content after storage for a period t and C0 was the lutein content at the time of preparation. 2.9. Statistical analysis All emulsions were prepared in duplicate, and all measurements were performed three times. Data were subjected to analysis of variance (ANOVA) using the software package SPSS 12.0 for Windows (SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Zeta potential Zeta potential measurements were performed to evaluate the electrostatic deposition of CTS onto the WPI-FG interfacial film surrounding the lutein droplets. The influence of CTS on the zeta potential of lutein emulsions stabilized by WPI-FG membranes was shown in Fig. 1a. WPI, WPI-FG and WPI-FG-CTS lutein emulsions were designated as primary, secondary and tertiary emulsions surrounded by one, two and three layer interfacial membranes, respectively. All the emulsions contained the same lutein and oil content as described in materials and methods. The WPI-FG-CTS emulsions were prepared to contain a range of different CTS concentrations (0-1 wt%). As shown in Fig. 1a, in the absence of CTS, zeta-potential of the WPI-FG emulsion droplets was around -15 mV, indicating that the WPI-FG membranes exhibited a negative charge at pH 3.0. It could be explained by the fact that FG at the 10
outside interfacial layer had a net negative charge at pH 3.0. In the presence of CTS, zeta-potential of the lutein droplets became increasingly positive as the CTS concentration in the emulsion was increased. It suggested that the positively charged CTS molecules adsorbed to the surfaces of the negatively charged emulsion droplets by forming WPI-FG-CTS membranes. Zeta-potential reached a constant value of around 20 mV when CTS concentration exceeded about 1 wt%, suggesting that the lutein droplets had been saturated with CTS. 3.2. Droplet size Fig. 1b shows the dependence of the droplet size on the CTS concentration for WPI-FG stabilized lutein emulsions. Without CTS, the mean droplet size of WPI-FG stabilized lutein emulsion was relatively small showing 948.2 nm. In the presence of CTS, the mean droplet size of lutein emulsions was increased. At lower CTS concentrations (0.05 wt% and 0.1 wt%), there were significant increases of the mean droplet size. It could be explained by the fact that CTS at low concentrations interacted with WPI-FG on the surface of lutein droplets, occurring destabilization due to charge neutralization. This charge neutralization could induce droplets to come closer together, promoting the formation of aggregates of larger sizes. It was also due to insufficient CTS to cover the entire lutein droplet in the emulsion. Thus, CTS may act as a bridge to interact with WPI-FG molecules between the surfaces of the lutein droplets by attractive electrostatic forces. Therefore, the increased droplet size might be attributed to bridging flocculation of the lutein droplets induced by CTS. With the further addition of more than 0.2 wt% CTS, there was a relative 11
decrease in the mean droplet size. It can be proposed that when the concentration of CTS was increased, it would be sufficient to cover the droplets and form a thick interfacial layer on the lutein droplets. Therefore, the flocculation would be inhibited by the strong electrostatic repulsion and the steric hindrance between droplets. Similar phenomena were also found regarding CTS interaction with soybean soluble polysaccharide, and a decrease in droplet size was also noted with increasing CTS content (Hou, Gao, Yuan, Liu, Li, & Xu, 2010). The difference in droplet size between WPI-FG-CTS (1 wt% CTS) lutein emulsion (with a much smaller droplet size) and WPI-FG emulsion indicated that CTS changed the adsorbing properties of WPI and FG in the lutein droplet surface (Wooster & Augustin, 2006). 3.3. Physical stability The effect of CTS concentration on the physical stability of lutein emulsions was determined with the multisample analytical centrifuge based on the STEP technology. The creaming stability of lutein emulsion can be reflected by the instability index which is the integrated transmission profiles against the measuring time. Fig. 2 shows the instability index of WPI, WPI-FG and WPI-FG-CTS (with different concentrations of CTS) prepared lutein emulsions. As can be seen, the order of instability index was: WPI > WPI-FG-CTS (CTS less than 1 wt%) > WPI-FG > WPI-FG-CTS (1 wt% CTS) prepared lutein emulsion. It was found that the multilayer lutein emulsions had better stability. When the content of CTS was less than 1 wt%, the stability of WPI-FG-CTS lutein emulsion was obviously decreased compared with WPI-FG emulsion. It was interesting to find that when the content of CTS reached 1 12
wt%, the physical stability of lutein emulsions was greatly improved. The Lumisizer stability analysis result was agreed with droplet size and zeta-potential. The improved physical stability of WPI-FG lutein emulsion with the presence of CTS at a concentration of 1 wt% indicated that the depletion flocculation did not greatly occur at high enough CTS concentrations, probably mainly due to CTS forming gel network in the emulsion. CTS is a very good viscosity-enhancing agent in an acidic environment, due to its high molecular weight and linear unbranched structure. Therefore, with the increase of CTS concentration, the viscosity of WPI-FG lutein emulsions was increased. It was reported that CTS could form a dense elastic network in the aqueous phase (Payet & Terentjev, 2008). The rigid network of CTS reduced the lutein droplet diffusion thus improving the stability of lutein emulsions. In the remainder of the study, tertiary lutein emulsion was prepared using a CTS concentration of 1 wt% because the droplet had a relative small droplet size, high zeta-potential and small creaming/instability index. 3.4. Microstructure To further investigate the role of CTS in the stability of lutein emulsion, the CLSM images of lutein emulsions, prepared with WPI, WPI-FG and WPI-FG-CTS were shown in Fig. 3. As observed in Fig. 3, microstructure exhibited differences between these lutein emulsions. Lutein emulsions prepared with WPI alone exhibited a particular microstructure, where formation of flocculated and aggregated droplets can be clearly distinguished. The lutein emulsion prepared with WPI-FG showed a little droplet flocculation. In the presence of CTS, WPI-FG stabilized lutein emulsions 13
showed different microscopic structures depending on CTS concentrations. Lutein emulsions with CTS less than 0.5 wt% showed obviously flocculated droplets, which could be attributed to a resultant attractive interaction between the droplets. In these systems, repulsive electrostatic interaction would be smaller (Fig. 1a), mainly due to the greater charge neutralization between CTS and WPI-FG at the interfacial membrane. This phenomenon could also be attributed to the lower concentration of CTS interacting with more than one droplet surface. It was consistent with the increase of previously observed droplet size (Fig. 1b). With the further increase of CTS concentrations, CLSM images indicated that the droplet flocculation and aggregation became less pronounced, especially at 1 wt%. The lower degree of droplet flocculation and aggregation produced by increasing CTS concentration could be attributed to a greater number of CTS molecules adsorbed onto the droplet interface through layer by layer, thus promoting an increased electrostatic and steric repulsion between the droplets. It might also be explained by the viscosity increment and gel network formation in the continuous phase when increasing CTS concentration. It indicated that the high concentration of CTS was much more effective in preventing the aggregation of lutein droplets. Perrechil and Cunha (2013) reported that higher concentrations of non-adsorbing biopolymers would promote a greater degree of depletion flocculation. However, lutein emulsions prepared with higher concentrations of CTS (1 wt%) could present much less flocculation than lower concentrations of CTS, presumably because of the viscosity increment in the 14
continuous phase. It was agreed with Fioramonti, Martinez, Pilosof, Rubiolo, Santiago and Liliana (2015) that the degree of droplet aggregation decreased with increasing sodium alginate concentration. 3.5. Microrheological properties The impact of CTS on the microrheological properties of WPI-FG stabilized lutein emulsions was measured using the Rheolaser lab. The Rheolaser lab technique does not provide any kind of modification of emulsions compared with the shear rheology. It monitored the Brownian motion of droplets and interpreted it in terms of microrheology (Corredig & Alexander, 2008). The motion of emulsion droplets was measured and the particles interaction was traced by microrheology. The mean square displacement (MSD) of droplets in the lutein emulsions with different concentrations of CTS was measured as a function of time (Fig. 4A). The MSD of the droplets is a direct measure of the dynamic properties of droplets and polymers in which they are embedded. WPI stabilized lutein emulsion was purely viscous, giving a MSD that scaled linearly over the range of decorrelation times. It exhibited that with the addition of FG and CTS, the MSD curves were not linear indicating that these lutein emulsions were viscoelastic properties and the droplets were not free to move due to the droplets network interaction. For the WPI-FG stabilized lutein emulsion, the MSD curves of the embedded droplets were quite similar with different decorrelation time which indicated that the network apparently was structurally stable. It showed that the network of WPI-FG-CTS stabilized lutein emulsions apparently was structurally relaxed. It can be attributed to that CTS was embedded in the network. 15
The elasticity strength and macroscopic viscosity of the samples were represented by EI and MVI values obtained from MSD curves. EI corresponds to the inverse of the distance travelled by droplets before interacting with the network (inverse of the MSD level at the elastic plateau) and MVI corresponds to the inverse of the speed of the particles for long times (inverse of the linear slope of the MSD for long times). Fig. 4B, C exhibits EI and MVI values of WPI, WPI-FG and WPI-FG-CTS with different CTS concentrations stabilized lutein emulsions at 25 °C. The EI and MVI of lutein emulsion prepared by WPI-FG was decreased compared with the one prepared by WPI alone. There was an increase trend in the EI and MVI of lutein emulsion in the presence of CTS. When the content of CTS was 0.05 wt%, the EI and MVI values were drastically increased, which was typically attributed to the flocculation at a relatively low CTS content. In the presence of 1.0 wt% CTS, WPI-FG-CTS prepared lutein emulsions became stable; it is meaningful to compare the EI and MVI values and there existed a remarkable increase in the EI and MVI. EI and MVI values were drastically increased to 1.9 and 8.1 times compared with WPI-FG emulsion. Such increased EI and MVI was typically attributed to higher interdroplet resistance force, which could be explained by the fact that the presence of CTS resulted in dense gel network formation and spatial rearrangement of the lutein droplets. 3.6. Chemical stability The chemical stability of lutein emulsions with WPI, WPI-FG, and WPI-FG-CTS interfacial membranes was evaluated based on changes in degradation of lutein during storage for 7 d at 55 °C. All lutein emulsions showed no obvious changes in the mean 16
droplet size during the storage time (data not shown), indicating that the primary, secondary and tertiary layer lutein emulsions were physically stable during storage regardless of the number of layers on the lutein droplets. The results concerning lutein degradation in the primary, secondary, and tertiary lutein emulsions are presented in Fig. 5. In the WPI and WPI-FG coated lutein emulsions, a steep decrease in lutein content was observed during the storage, with losses of 55.3% and 64.1% after storage of 7 d. It can be concluded that there was a decrease in the chemical stability of the WPI-FG secondary lutein emulsion. The decreased chemical stability can be attributed to the negative charge around the lutein droplets by WPI-FG. Hu McClements and Decker (2003) reported that negative charged emulsion droplets could promote prooxidative metals moving towards the interface where oxidation occurred. It was found that the chemical stability of lutein emulsions was increased with the addition of CTS. In the addition of CTS, there was a steep degradation of lutein after 1 d storage, followed by a slight decrease after 2 d storage, with 30.7% loss after storage of 7 d. Therefore, lutein droplets surrounded by WPI-FG-CTS membrane clearly showed its help to increase the chemical stability of lutein emulsions. It can be attributed that the lutein droplets in the WPI-FG-CTS emulsion had a higher positive charge and a thicker interfacial membrane than those in the WPI and WPI-FG emulsions, therefore, the electrostatic and steric repulsion with prooxidative metals and free radicals was greater. CTS in the continuous phase may also prevent lutein from degradation due to their ability to chelate transition metals at negatively 17
charged sites. CTS is also likely to have antioxidant activity due to its ability to donate hydrogen with high radical-scavenging activity (Kim & Thomas, 2007). Finally, it is possible that the viscosity increment and gel network formation of the WPI-FG-CTS emulsion may prevent lutein degradation by slowing down the interaction of lutein droplets with transition metals and free radicals (Waraho, McClements, & Decker, 2011). 3.7. Influence of EDTA and α-tocopherol on the chemical stability of lutein emulsions with different interfacial layers It is reported that transition metals are naturally present in the emulsifiers, oil and water phase in the emulsion at acidic pH and therefore, it may induce the degradation of lutein (Subagio, Wakaki, & Morita, 1999). Based on our previous study (Xu, Yuan, Gao, McClements, & Decker, 2013), 200 µM EDTA as a transition metal chelator was added to emulsions stabilized by WPI, WPI-FG and WPI-FG-CTS to evaluate if metals were able to promote lutein degradation in different layers of emulsions, respectively (Fig. 6). For the primary WPI stabilized lutein emulsion, EDTA was able to decrease lutein loss at the beginning of the storage period; however, lutein degradation was increased at the end of the storage period. For the WPI-FG and WPI-FG-CTS stabilized emulsions, lutein losses were 7.2% and 22.5% lower with the addition of EDTA after storage of 7 d, respectively. It also exhibited that EDTA was more effective in protecting lutein in WPI-FG-CTS emulsion. It showed that lutein droplets coated by WPI-FG and WPI-FG-CTS were more oxidatively stable than emulsions 18
coated by WPI alone when added EDTA, demonstrating that addition of EDTA in a thicker interfacial membrane emulsion was much more effective in inhibiting lutein degradation. It was found that EDTA inhibited the loss of lutein in the three emulsions during storage indicating that transition metal was a major prooxidant in lutein emulsions. The results were similar with our previous reports about iron chelator desferoxamine inhibiting β-carotene degradation in emulsions (Xu et al., 2013). The results were also agreed to those reported by Boon and coworkers who reported that EDTA was able to inhibit lycopene degradation in oil-in-water emulsions (Boon et al., 2009). Free radicals in the emulsion may also degrade lutein (Zhao et al., 2014). Therefore, 200 mg/kg α-tocopherol as a free radical scavenger was applied to investigate the influence of free radicals on the oxidative stability of lutein in WPI, WPI-FG and WPI-FG-CTS emulsions, respectively (Fig. 6). It exhibited that α-tocopherol increased the oxidative stability of lutein in the three emulsions. After the storage of 7 d, lutein content of the three emulsions was increased by 8.8%, 8.7% and 17.2% compared with emulsions without α-tocopherol, respectively. It was revealed that α-tocopherol was the most effective in the emulsions stabilized with WPI-FG-CTS. It was interesting to find that an addition of EDTA or α-tocopherol to the WPI-FG-CTS stabilized emulsion exhibited much less lutein loss after the storage. It suggested that the combination of the tertiary interfacial membrane and prooxidative metal chelator or free radical scavenger was an effective method to control lutein 19
degradation. The greater stability of the WPI-FG-CTS emulsions with addition of EDTA or α-tocopherol can also be attributed to synergy effect of CTS with prooxidant chelator and free radical scavenger. 4. Conclusion The purpose of the study was to examine the effect of CTS on the properties of WPI-FG coated lutein emulsions. The physical and chemical stabilities of lutein emulsions containing droplets surrounded by multilayered interfacial membranes prepared by protein and polysaccharides were investigated. The major findings of the study were: Stable three layered lutein emulsions can be prepared at pH 3.0 that contain lutein droplets surrounded by WPI-FG-CTS with CTS concentration of 1 wt%. When the content of CTS was less than 1 wt%, the stability of WPI-FG-CTS lutein emulsion was obviously decreased compared with WPI-FG emulsion. The microrheological property showed that WPI stabilized lutein emulsion was purely viscous. In presence of FG and CTS, the lutein emulsions were viscoelastic properties and the droplets were not free to move due to the droplets network interaction. There was an increase trend in the EI and MVI of lutein emulsions in the presence of CTS. The order of the chemical stability was: WPI-FG-CTS > WPI > WPI-FG prepared lutein emulsion. The addition of EDTA or α-tocopherol to the WPI-FG-CTS stabilized emulsion exhibited much less lutein loss after the storage than WPI and WPI-FG emulsion. Acknowledgements This research was funded by the National Natural Science Foundation of China 20
(31501486), Beijing Natural Science - SANYUAN jointly Foundation (15S00016), Cultivation of Excellent Talents in Beijing City (2014000020124G032), National Natural Science Foundation of China (31371722) and Development of Innovative Teams and Teachers’ Occupation Advancement Project of Beijing Municipal Universities and Colleges (IDHT20130506). The authors are also grateful to Yuwei He and Guangbin Chen for the use of Rheometer LAB (Formulaction, France) and analysis of the microrheology. References Amar, I., Aserin, A., & Garti, N. (2004). Microstructure transitions derived from solubilization of lutein and lutein esters in food microemulsions. Colloids and Surfaces B: Biointerfaces, 33(3-4), 143-150. Bartlett, H. E., & Eperjesi, F. (2008). A randomised controlled trial investigating the effect of lutein and antioxidant dietary supplementation on visual function in healthy eyes. Clinical Nutrition, 27, 218-227. Batista, A. P., Raymundo, A., Sousa, I., Empis, J., & Franco, J. M. (2006). Colored food emulsions-implications of pigment addition on the rheological behaviour and microstructure. Food Biophysics, 1, 216-227. Boon, C. S., McClements, D. J., Weiss, J., & Decker, E. A. (2009). Role of iron and hydroperoxides in the degradation of lycopene in oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 57, 2993-2998. Bouyer, E., Mekhloufi, G., Rosilio, V., Grossiord, J., & Agnely, F. (2012). Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: 21
Alternatives to synthetic surfactants in the pharmaceutical field? International Journal of Pharmaceutics, 436, 359-378. Corredig, M., & Alexander, M. (2008). Food emulsions studied by DWS: recent advances. Trends in Food Science & Technology, 19, 67-75. Ettelaie, R., & Akinshina, A. (2014). Colloidal interactions induced by overlap of mixed protein+polysaccharide interfacial layers. Food Hydrocolloids, 42, 106-117. Fioramonti, S. A., Martinez, M. J., Pilosof, A. M. R., Rubiolo, A. C., Santiago, & Liliana, G. (2015). Multilayer emulsions as a strategy for linseed oil microencapsulation:
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Hydrocolloids, 43, 8-17. Gu, Y. S., Decker, A. E., & McClements, D. J. (2005). Production and characterization of oil-in-water emulsions containing droplets stabilized by multilayer membranes consisting of β-lactoglobulin, l-carrageenan and gelatin. Langmuir, 21, 5752-5760. Gudipati, V., Sandra, S., McClements, D. J., & Decker, E. A. (2010). Oxidative stability and intro digestibility of fish oil-in-water emulsions containing containing multilayered membranes. Journal of Agricultural and Food Chemistry, 58, 8093-8099. Hou, Z., Gao, Y., Yuan, F., Liu, Y., Li, C., & Xu, D. (2010). Investigation into the physicochemical stability and rheological properties of beta-carotene emulsion stabilized by soybean soluble polysaccharides and chitosan. Journal of 22
Agricultural and Food Chemistry, 58(15), 8604-8611. Hu, M., McClements, D. J., & Decker, E. A. (2003). Impact of whey protein emulsifiers on the oxidative stability of salmon oil-in-water emulsions. Journal of Agricultural and Food Chemistry, 51(5), 1435-1439. Jo, Y., Chun, J., Kwon, Y., Min, S., & Choi, M. (2015). Formulation development of multilayered fish oil emulsion by using electrostatic deposition of charged biopolymers. International Journal of Food Engineering, 11(1), 31-39. Kim, K. W., & Thomas, R. L. (2007). Antioxidative activity of chitosans with varying molecular weights. Food Chemistry, 101(1), 308-313. Klein, M., Aserin, A., Svitov, I., & Garti, N. (2010). Enhanced stabilization of cloudy emulsions with gum Arabic and whey protein isolate. Colloids and Surfaces B: Biointerfaces, 77, 75-81. Liu, C., Lai, K., Wu, W., Chen, Y., Lee, W., & Hsu, C. (2015). In vitro scleral lutein distribution
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O/W emulsions in the presence of chitosan. Langmuir, 24, 12247-12252. Perrechil, F. A., & Cunha, R. L. (2013). Stabilization of multilayered emulsions by sodium caseinate and k-carrageenan. Food Hydrocolloids, 30, 606-613. Shchukina, E. M., & Shchukin, D. G. (2012). Layer-by-layer coated emulsion microparticles as storage and delivery tool. Current Opinion in Colloid & Interface Science, 17, 281-289. Sobisch, T., & Lerche, D. (2008). Thickener performance traced by multisample analytical centrifugation. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 331, 114-118. Song, Y., Babiker, E. E., Usui, M., Saito, A., & Kato, A. (2002). Emulsifying properties and bactericidal action of chitosan-lysozyme conjugates. Food Research International, 35, 459-466. Subagio, A., Wakaki, H., & Morita, N. (1999). Stability of lutein and its myristate esters. Bioscience Biotechnology and Biochemistry, 63, 1784-1786. Waraho, T., McClements, D. J., & Decker, E. A. (2011). Mechanisms of lipid oxidation in food dispersions. Trends in Food Science & Technology, 22(1), 3-13 Wooster, T. J., & Augustin, M. A. (2006). β-Lactoglobulin-dextran maillard conjugates: their effect on interfacial thickness and emulsion stability. Journal of Colloids and Interface Science, 303, 564-572 Xu, D., Wang, X., Jiang, J., Yuan F., & Gao, Y. (2011). Impact of whey protein-beet pectin conjugation on the physicochemical stability of β-carotene emulsions. Food Hydrocolloids, 25, 258-266. 24
Xu, D., Yuan F., Gao, Y, McClements, J., & Decker, E. (2013). Influence of pH, metal chelator, free radical scavenger and interfacial characteristics on the oxidative stability of beta-carotene in conjugated whey protein-pectin stabilized emulsion. Food Chemistry, 139, 1098-1104. Zhao, C., Cheng, H., Jiang, P., Yao, Y., & Han, J. (2014). Preparation of lutein-loaded particles for improving solubility and stability by polyvinylpyrrolidone (PVP) as an emulsion-stabilizer. Food Chemistry, 156, 123-128.
25
25
Zeta-Potential(mV)
20 15 10 5 0 0.0 -5
0.2
0.4
0.6
0.8
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Chitosan Concentration(wt%)
-10 -15 -20
(a)
1600 1500 1400
Size (nm)
1300 1200 1100 1000 900 800 0.0
0.2
0.4
0.6
0.8
1.0
Chitosan Concentration(wt%) Fig. 1. Dependence of the zeta-potential (a) and droplet size (b) on CTS concentration for WPI-FG stabilized lutein emulsions (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG) at pH 3.0. Some error bars lie within the data points.
26
(a)
(c) (d) (e) (f) (g)
(b) (h)
Fig. 2. Instability index of WPI, WPI-FG and WPI-FG-CTS with different concentration of CTS stabilized lutein emulsions at pH 3.0 determined by LUMiSizer (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG; a, WPI; b, WPI-FG; c-h, WPI-FG-CTS with different concentration of CTS, c, 0.05 wt%; d, 0.1 wt%; e, 0.2 wt%; f, 0.25 wt%; g, 0.5 wt%; h, 1 wt%).
27
Fig. 3. Confocal micrograph of WPI, WPI-FG, WPI-FG-CTS with different
concentration of CTS stabilized lutein emulsions at pH 3.0 (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG; a, WPI; b, WPI-FG; c-h, WPI-FG-CTS with different concentration of CTS, c, 0.05 wt%; d, 0.1 wt%; e, 0.2 wt%; f, 0.25 wt%; g, 0.5 wt%; h, 1 wt%). 28
(b)
(c)
(d)
(e)
(f)
(g)
(h)
MSD (nm2)
MSD (nm2)
MSD (nm 2)
MSD (nm2)
(a)
10-3
10-2
-3 10-1 10
Decorrelation Time (s)
A
29
10-2
10-1
(B)
(C)
Fig. 4. Typical examples of the MSD vs time curves (A) for WPI, WPI-FG, WPI-FGCTS with different concentration of CTS stabilized lutein emulsions at pH 3.0 (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG; a, WPI; b, WPI-FG; c-h, WPI-FG-CTS with different concentration of CTS, c, 0.05 wt%; d, 0.1 wt%; e, 0.2 wt%; f, 0.25 wt%; g, 0.5 wt%; h, 1 wt%). Different colours and arrows refer to different scanning time of MSD analysis. EI (B) and MVI (C) values for WPI, 30
WPI-FG, WPI-FG-CTS with different concentration of CTS stabilized lutein emulsions.
31
WPI WPI+FG WPI+FG+CTS
1.0
C /C 0
0.8
0.6
0.4
0.2
0.0 0
1
2
3
4
5
6
7
Storage time(d) Fig. 5. Lutein degradation as a function of storage time for the primary (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI), secondary (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG) and tertiary (0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG, 1 wt% CTS) lutein emulsions (pH 3.0) at 70 °C. Some error bars lie within the data points.
32
control α-tocopherol EDTA
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0.8
0.6
0.4
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0.0 0
1
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3
4
Storage time(d)
33
5
6
7
(c)
Fig. 6. Effect of the addition of EDTA and α-tocopherol on the oxidative stability of lutein as a function of storage time for the primary (a, 0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI), secondary (b, 0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG) and tertiary (c, 0.01 wt% lutein, 2.5 wt% MCT, 0.25 wt% WPI, 0.15 wt% FG, 1 wt% CTS) emulsions (pH 3.0) at 70 °C. Some error bars lie within the data points.
34
> Multilayer emulsions had better stability to droplet flocculation and aggregation. > 1 wt% CTS had a pronounced effect on the microstructure and stability of lutein emulsion. > CTS led to increases of MVI and EI for emulsions due to CTS embedding in the network. > CTS helped to chemically stabilize lutein emulsions. > Combination of WPI-FG-CTS and EDTA or α-tocopherol was an effective method to control lutein degradation.
35