Food Hydrocolloids 25 (2011) 1596e1603

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Preparation of lutein microencapsulation by complex coacervation method and its physicochemical properties and stability
Xiao-Ying Qv, Zhi-Ping Zeng, Jian-Guo Jiang*
College of Food and Bioengineering, South China University of Technology, Guangzhou 510640, China

a r t i c l e i n f o
Article history: Received 21 September 2010 Accepted 14 January 2011 Keywords: Lutein Microcapsule Complex coacervation method Stability

a b s t r a c t
Although lutein possesses multiple valuable physiological functions, its application in food industry is limited due to the instability in adverse conditions. Using the complex coacervation method, the work is aimed to optimize the encapsulation process, investigate physicochemical properties of microcapsules and nally appraise the extent of stability improvement. The optimum process conditions determined by response surface analysis were as follows: concentration of wall materials 1.0%, ratio of core material to wall 1.25:1 and pH value 4.2, where the theoretical and practical encapsulation efciency were 86.41% and 85.32% 0.63%. The particles had a conned distribution in the range of 0e30 mm, indicating a relatively homogeneous distribution. Moreover, the lutein in particles presented an improvement of ability against light, humidity, temperature. Especially, the retention rate of lutein incorporated in products reached 92.86% at 4  C, 90.16% at 25  C, 90.16% with the relative humidity of 33%, and 90.25% under the aerobic condition. 2011 Elsevier Ltd. All rights reserved.

1. Introduction Lutein is one type of carotenoid without bioactivity of vitamin A but holding many other signicant physiological functions. As reported, the strong antioxidation inactivating singlet oxygen enables lutein to promote the enhancement of body immunity against arteriosclerosis, cataract (Calvo, 2005), and improve food and beverage color due to its powerful coloration ability. However, its application in food industry is limited by the instability towards oxygen, light and temperature due to the eight conjugated double bond structure. Taking lutein properties into account, the methodology of microencapsulation is adoptable for its multiple advantages. Generally, microencapsulation referring to a methodology of enveloping one or several materials into microcapsules (Champagne & Fustier, 2007; Li et al., 2009) has acquired broad applications in food industry for the protection of vitamins, minerals and other sensitive components from the external inuences, improvement of material physical properties, isolating product components, masking unfavorable taste, and controlling the release of core materials considered as the most important function (Champagne & Fustier, 2007; Gouin, 2004; Im & Sah, 2009). The most commonly applied technologies are emulsication, coacervation, spray drying (Teixeira, Andrade, Farina, & Rocha, 2004), spray cooling, freeze drying, uid

* Corresponding author. Tel.: 86 20 87113849; fax: 86 20 87113843. E-mail address: jgjiang@scut.edu.cn (J.-G. Jiang). 0268-005X/$ e see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2011.01.006

bed coating and extrusion technologies, etc. (de Vos, Faas, Spasojevic, & Sikkema, 2010). As a development of coacervation, complex coacervation method necessitates two or more linear and irregular polymers with opposite charges served as wall materials. The common used material compositions include gelatin/gum arabic (GA), alginate/ polylysine, gelatin/carboxymethylcellulose, albumin/gum arabic, etc. After the dissolution of wall materials and then the dispersion of core materials into water, coating materials electrostatically coagulate from the solution and encapsulate the core materials by adjusting pH and temperature or adding inorganic salt electrolyte (Huang, Cheng, Yu, Tsai, & Cham, 2007; Schmitt et al., 2001). The preparation is performed under gentle conditions applicable for live cells and instable materials vulnerable to severe condition changes. From an experimental and practical point of view, gelatin/gum arabic (GA) system was extensively utilized (de Kruif, Weinbreck, & de Vries, 2004; Malay, Bayraktar, & Batgn, 2007). Gum arabic (or acacia gum) is a complex polysaccharide containing a protein fraction responsible for its efcient surface property that is attributed to the structure of the gum (Connolly, Fenyo, & Vandevelde, 1987; Menzies, Osman, Malik, & Baldwin, 1996). Acacia gum is an arabinogalactan type polysaccharide composed of three distinct fractions with different protein contents and different molecular weights (Osman, Williams, Menzies, & Phillips, 1993; Phillips, Takigami, & Takigami, 1996; Randall, Phillips, & Williams, 1989). The highly efcient and practical approach can produce rigorously

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wrapped microcapsules with coating thickness in a wide adjustable range, which is also nontoxic and degradable. For that reason, gelatin/gum arabic (GA) system was adopted in our experiment for exploring the optimization of process conditions by response surface analysis, assessing physicochemical properties and stability of products. 2. Materials and methods 2.1. Materials and apparatus Gelatin (type B, 240 Bloom) was purchased from Tianjin Fu Yu chemical Co., Ltd and prepared for use with the isoelectric point at 5.21. Gum arabic of food grades in spray dried from was also purchased from Tianjin Fu Yu chemical Co., Ltd. Lutein and its standard were both purchased from Shanxi Sciphar Biotechnology Co., Ltd. WFJ2100 visible-infrared spectrometer was bought from unique instrument Co., Ltd (Shanghai). S-3D pH meter was the product of Shanghai Precision and Scientic Instrument Co., Ltd. Diamond-I differential scanning calorimeter was purchased from USA Perkin Elmer company. OLYMPUS Scanning electron microscope, Mastersizer 2000 laser granulometer and BX51 polarizing microscope were from Hitachi Ltd. (Japan), Malvern Instruments Ltd. (England), OLYMPUS Ltd. (Japan), respectively. 2.2. Optimization of lutein microencapsulation process 2.2.1. Designs of single factor experiments Single factor experiments were performed to determine optimal conditions for single factors by analyzing their inuences to microencapsulation effect, which were concentration of wall materials (CWM) (gelatin/gum arabic, ratio 1:1), ratio of core material to wall (RCW), temperature and pH value. For studying CWM effects on encapsulation, wall materials were prepared into 0.5% (w/w), 1.0% (w/w), 1.5% (w/w), 2.0% (w/w), 2.5% (w/w) solutions, respectively. Then core materials were added according to the 1:1 RCW and emulsied in water bath (45  C, 30 min) at 550r/ min stirring speed. Next, pH value of emulsion was adjusted to 4.4 causing gelatin and gum arabic coagulate (at 550r/min stirring speed, in 45  C water bath for 15 min). After that, the pH value was readjusted to about 7.0 and then certain glycerin was added for immobilization (at 350r/min stirring speed, in 0e10  C water bath for 30 min). Lastly, products were obtained after ltration and drying. The other three single factor tests RCW, temperature, pH value took the similar process as the above. Their individual conditions were that (1) RCW: CWM 1% (w/w), ratios of core material to wall 3:1, 2:1, 1:1, 1:2, 1:3, temperature 45  C, pH value 4.4; (2) temperature: CWM 1% (w/w), RCW 1:1, temperatures 30  C, 35  C, 40  C, 45  C, 50  C, 55  C, pH value 4.4; (3) pH value: CWM 1% (w/w), RCW 1:1, temperature 45  C, pH values 3.5, 3.8, 4.1, 4.4, 4.7. 2.2.2. Determination of lutein and encapsulation efciency The lutein weighed precisely was dissolved and diluted with anhydrous ethanol, and then formulated into 20, 40, 60, 80, 100, 120 mg/ml standard solution, the absorption of which were surveyed at 446 nm with ethanol as the blank, respectively. The linear regression of absorption (A) on concentration (C) was made and the regression equation was calculated. The standard curve was drawn and its equation is y 0.011x 0.0408, R2 0.9998. A certain amount of sample weighed with precision was put into a brown capacity bottle, added with anhydrous ethanol, treated in ultrasonic water bath for 15 min, cooled into the room temperature, xed to the calibration with anhydrous ethanol, mixed and ltered to measure its absorption and calculate its content.

Encapsulation efciency is a signicant indicator to appraise the quality of the prepared microcapsule products. The equitation is as follows (Saravanan & Rao, 2010):

E%

w1 100%; w2

(1)

where E is encapsulation efciency, w1 and w2 represent the weight of lutein loaded in capsules and consumed for the encapsulation respectively. 2.2.3. Response surface analysis of lutein microcapsule process On the basis of the results of single factor experiments, the experiments for optimization of microencapsulation process was designed and carried out by response surface analysis taking encapsulation efciency (Y) as response value and CWM (A), RCW (B), pH value (C) as factors.

2.3. Physicochemical properties of lutein microcapsules 2.3.1. Determination of water content The rate of water content in capsules was determined using the vacuum oven to dry products, and gured out by the formula as follows (Mendanha et al., 2009):

C%

w1 w2 100%; w2

(2)

where C is the water content rate, w1 is the weight of products before dry treatment, and w2 is the weight after that operation. 2.3.2. Size distribution of microcapsules A certain amount of microcapsule dispersion prepared under the optimal process conditions and mixed homogenously, was taken by pipette and diluted with water, and then assayed for size distribution by Mastersizer 2000 laser particle analyzer. The average particle diameter formula is as follows:

Pn i 1 n di Dn ; Pn i i 1 ni

(3)

where Dn represents average diameter, ni refers to the number of microcapsules and di is diameter of single microcapsule. 2.3.3. Scanning electron microscopy Prior to scanning electron microscopy (SEM) analysis, the samples were sprinkled on one side of double-side adhesive stuck on the stub and then was coated with gold. The SEM analysis of the microspheres was carried out by using S3700 scanning electronic microscope (Hitachi Japan). The microspheres were observed at an accelerating voltage of 10 kV. 2.3.4. Determination of product liquidity The repose angle method was taken to assess the liquidity of microparticles. The product powder fell on the center of the disc with a certain diameter through the funnel placed on the iron stand until the powder of the formed bulk automatically ew out along the disc edge. The included angle between the product-formed accumulation and the plane is so-called the angle of repose. 2.3.5. Differential scanning calorimetry Differential scanning calorimetry (DSC) was applied to detect the glass transition temperature of products. The freeze dried samples were added into the DSC sample box with the blank as reference (pans are sealed), and heated from 20  C to 120  C at the rate of 5  C/min.

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X.-Y. Qv et al. / Food Hydrocolloids 25 (2011) 1596e1603 Table 1 Levels of three variables, BoxeBenhnkens central composite design and response results for the study of microencapsulation. Numbers Level A 0.5% 1.0% 1.5% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0.5 1 1 0.5 1 1.5 0.5 0.5 1.5 1 1.5 1 1 1.5 1 1 1 B 2:1 1:1 1:2 1.25 1.25 0.5 1.25 1.25 0.5 2 0.5 2 0.5 1.25 1.25 1.25 1.25 2 2 1.25 C 4.1 4.4 4.7 4.7 4.4 4.7 4.1 4.4 4.4 4.4 4.4 4.4 4.1 4.7 4.4 4.4 4.1 4.7 4.1 4.4 0.734 0.817 0.83 0.763 0.804 0.735 0.692 0.714 0.704 0.783 0.833 0.805 0.811 0.773 0.84 0.795 0.79 Encapsulation rate

2.3.6. Infrared atlas analysis of microcapsules and wall materials Chlorine potassium was grinded with gelatin, gum arabic and freeze dried products, respectively, to obtain three powders. Then the three prepared powders were made into transparent plates using the presser. Finally, these plates were scanned by infrared instruments at 500e4000 wavenumber. 2.4. Stability of lutein microcapsule 2.4.1. Determination of lutein retention rate The lutein retention rate can be calculated by the following formula:

Y%

CA 100%; CB

(4)

where Y is lutein retention rate, CA and CB are lutein content existing in microcapsules after and before a time of storage, respectively. 2.4.2. Effects of relative humidity on lutein stability A certain amount of lutein and its microcapsule (freeze dried) were separately kept in dark and 25  C constant temperature incubator with different relative humidity of 33% and 80% respectively. Samples were taken every ve days to examine lutein content for the calculation of retention rate. The experiment period was thirty days. Relative humidity and its corresponding saturated solution were that saturated magnesium chloride solution (relative humidity 33%), and saturated potassium iodide solution (relative humidity 80%). 2.4.3. Effects of light on lutein stability 3.5 g of lutein and freeze dried microcapsules were preserved in brown jars respectively at room temperature and avoiding light, while other equivalent samples were kept in transparent jars and light. The preserved lutein and microcapsules were sampled 0.5 g to determine lutein content and gure out their retention rate every ve days in the consequent thirty days. 2.4.4. Effects of temperature on lutein stability Freeze dried microcapsules were weighed out 3.5 g, put in petri dishes and kept at 4  C, 25  C, 50  C without light, respectively, and sampled 0.5 g for examination of retention rate. The experiment lasted for thirty days. 2.5. Statistical analysis The data were presented as mean standard deviation (SD). Statistically signicant differences between groups were evaluated
Table 2 Analysis of variance of the regression parameters. Source Model A B C AB AC BC A2 B2 C2 Residual Lack of t Pure error Total R2
a b

using Students test. Statistical signicance was set at P < 0.05, P values < 0.05 were regarded as signicant and P values < 0.01 as extremely signicant. 3. Results and discussion 3.1. Process optimization The good levels of single factors concluded from single factor experiments were as follows: CWM 0.5e1.5%, RCW 1:2e2:1 and pH value 4.1e4.7. On the basis of these results, taking encapsulation efciency (Y) as response value of CWM (A), RCW (B) and pH value (C), experiments were arranged according to BoxeBenhnkens central composite design (Table 1). The levels of three variables were listed in Table 1. The experimental results shown in Table 1 were analyzed by Design the expert 7.0, the regression equation is:

Y% 0:81 0:018A 3:875B 0:015C 2:250AB 0:022AC 5:000BC 0:065A2 0:029B2 0:036C 2 :

Sum of squares 0.032753 0.002521 0.00012 0.001891 2.02E05 0.00198 1E06 0.017899 0.003529 0.005321 0.002295 0.00189 0.000405 0.035048

Df 9 1 1 1 1 1 1 1 1 1 7 3 4 16

Mean square 0.003639 0.002521 0.00012 0.001891 2.02E05 0.00198 1E06 0.017899 0.003529 0.005321 0.000328 0.00063 0.000101

F-value 11.09786 7.686292 0.366323 5.767006 0.061753 6.038794 0.00305 54.58355 10.76127 16.22728 6.219974

Prob > F 0.0022 0.0276 0.5641 0.0474 0.8109 0.0436 0.9575 0.0002 0.0135 0.0050 0.0549

Signicance **a *b * * ** * **

0.9345

Shows a highly prominence, P < 0.01. Shows prominence, P < 0.05.

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The P value (lower than 0.01) signies a highly remarkable linear relationship between independent variable and dependent variables. It can be concluded from Table 2 that the A, C, B2 and AC are prominent factors, and the inuences of A2 and C2 are highly prominent. The wall material to core ratio is a meaningful

parameter in mixed biopolymer systems. It controls the balance of macromolecules charges and therefore, the intensity of the electrostatic interactions driving the formation of complexes between the two biopolymers (Schmitt et al., 2000; Schmitt, Sancheza, Thomas, & Hardy, 1999).

Fig. 1. The contour plots of response surface methodology. (a) The response surface and contour plot of the effect of the wall material concentration and wall core/material material ratio; (b) the response surface and contour plot of the effect of the wall material concentration and the pH value; (c) the response surface and contour plot of the effect of the wall core/material material ratio and the pH value.

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Fig. 2. (a) Particle size distribution of freeze dried microcapsules prepared under the optimal conditions; (b) particle size distribution of spray-dried microcapsules prepared under the optimal conditions. A certain amount of microcapsule dispersion mixed homogenously was diluted with water, and then assayed with Mastersizer 2000 laser particle analyzer.

The response surfaces and contour plots for encapsulation efciency as the response value were manifested in Fig. 1. The optimum process conditions identied by the results of response surface analysis (Fig. 1) and the aid of Design the expert 7.0 were that the wall material concentration 1.0%, the rate of core and wall material 1.25:1 and pH 4.2. The actual embedding rate is proved to be 85.32% 0.63%, while the theoretical value is 86.41%.

3.2. Results of physicochemical property study The assessment of physicochemical properties of microcapsules including size distribution, water content, uidity appearance, thermodynamics properties, etc. is a basic method to appraise product quality, an auxiliary means for technique optimization as well as an important theoretical foundation for choice of storage conditions.

Fig. 3. SEM micrographs of lutein microcapsule products obtained by two treatments spray drying and freeze drying. After the dry procedure, the surface morphology of product powder samples was observed at 3000 and 6000 magnication times. The surfaces of spray-dried products were smooth while the freeze dried product surfaces were wrinkled.

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3.2.1. Water content rate, size distribution and morphology The rate of water content is 3.12% in a relative low level, advantageous to the prevention against mildewing and oxidation. The results shown in Fig. 2(a) demonstrated that freeze dried particles had a broad distribution in the size range of 0e30 mm and had the biggest distributional proportion in the range of 10e20 mm, indicating homogeneous microcapsules produced under the optimum condition. Their mean diameter was 14.198 mm. Fig. 2(b) is the size distribution of spray-dried products. By contrast, spraydried products had a better homogeneity and integrity. Besides, Fig. 3 demonstrated that products treated by spray drying had a spherical appearance and a smooth surface, while the surface of freeze-drying capsules had typical folds and slight cracks due to the loss of water content during the freeze-drying process (Kim et al., 2008; Kwok, Groves, & Burgess, 1991). 3.2.2. Determination of product liquidity The criteria suggested by Carr (1970) for assessing ow function by static repose angle is that the angle lower than 30 is considered good uidity, 30e45 some cohesiveness, 45e55 true cohesiveness, while the angle beyond 55 indicates high cohesiveness and poor owability. The angle of repose examined in our experiment is 37.1 (between 30 and 45 ) signifying a good uidity. The application value of microcapsules can be decreased by the poor uidity that is closely associated with coating material properties. Most of lm materials used in the preparation are colloids with a strong hygroscopic ability which affects product liquidity combined with the usual chinks on the unsmoothed microspherical surface due to the increase of friction. Additionally, the small-sized particles will also reduce the liquidity because of more surfaces offered for particle-particle interaction (Xu, Yao, Han, & Shao, 2007). 3.2.3. DSC analysis of lutein product DSC thermogram (shown in Fig. 4) demonstrated a quite obvious turning point occurred at the starting of phase change with T0 (characteristic temperature) at 31.96  C, Tc (nal temperature) at 47.15  C, and Tg (peak temperature) at 40.52  C (DH 226.3 J/g). The glass transition relates to the phenomena observed when a supercooled, malleable liquid or rubbery material is changed into a disordered solid glass upon cooling, or conversely when a brittle glass is changed upon heating into a supercooled liquid or a rubbery material (Roudaut, Simatos, Champion, Contreras-Lopez, & Le Meste, 2004). The product may be shelf stable when stored

below glass transition temperature since deterioration due to microbial growth and chemical reaction is greatly reduced (Sablani, Kasapis, & Rahman, 2007). Wall materials at vitrication temperature possess small permeability, which is helpful for preventing oxygen entry and benecial to the preservation of core materials. If the storage temperature is set higher than the vitrication temperature, because of the increase in internal mobility of reactants and diffusivity to oxygen, various chemical reactions are also accelerated in dried products (Bhandari & Howes, 1999). The vitrication temperature assayed was 40.52  C, higher than room temperature. Consequently, products appeared stable when kept at normal temperature.

a
absorption intensity

1.2 1 0.8 0.6 0.4 0.2

0 5000

4000

3000

2000
-1

1000

wavenumber/cm

b
absorption intensit

1.2 1 0.8 0.6 0.4 0.2 0 4500 3500 2500 1500

-1

500

-500

wavenumber/cm

c
absorption intensit

1.2 1 0.8 0.6 0.4 0.2 0 4500 3500 2500 1500

-1

500

-500

wavenumber/cm

Fig. 4. DSC analysis of microcapsules containing lutein for the determination of glass transition temperature. The DSC curves were recorded during the heating process with the rate of 5  C/min. The temperature 40.52  C at the turning point is the vitrication temperature higher than room temperature.

Fig. 5. Comparison analysis of the infrared spectroscopy of (a) gelatin, (b) gum arabic and (c) microcapsule to reveal if there exist newly generated chemical bonds between gelatin and gum arabic. Before the analysis by using the infrared instrument at the wavenumber 500e4000, the three samples were separately grinded with a certain amount of potassium chloride. By comparison, it is veried that the interaction between gelatin and gum arabic was based on electrostatic force.

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3.2.4. Infrared spectrum analysis of gelatin, gum arabic and microcapsule The infrared spectrums of gelatin, gum arabic and lutein loaded microcapsule were shown in Fig. 5. The peak values of particles from complex coacervation approximated those peak values of gum arabic at the wave numbers of 3032.82, 2825.15, 1587.51, 1475.52, 1081.03, 627.21, respectively, showing gum arabic present in capsules. Also, gelatin existed in products as the peak values are approximate at the wave numbers of 3082.57, 2822.34, 1627.21,

1512.33, 1465.54, and 1237.58, respectively. No generation of new chemical bond evidenced by no specic peak value found between spectrums of microcapsules and wall materials, further conrms the formation of complexes promoted by physical interaction such as electrostatic interaction rather than chemical reactions (Yan, Ke, Dong, Ding, & Li, 2007).

3.3. Appraisal of lutein microcapsule stability 3.3.1. Effect of relative humidity Relative humidity is taken as a signicant factor inuencing product storage. Both of gelatin and gum arabic have strong hygroscopicity, when placed in high humility environment, promoting the increase of water content in products and the consequent gradual loss of lutein and structural collapse of capsules. The results (Fig. 6(a)) showed that after 30 days storage with 33% relative humidity, the lutein retention rate of products was 90.16% whereas the proportion of the non-encapsulated lutein was just 68.18%. When kept in 80% relative humility for 30 days, the retention rates of the encapsulated and non-encapsulated lutein were 68.18% and 30.15 respectively. It demonstrated that the products should be preserved in low relative humidity conditions. 3.3.2. Effect of light As is known, the light exerts destructive inuences on lutein. The results (Fig. 6(b)) suggested microencapsulation was an adoptable method to protect lutein against light effects. The retention rates of lutein and its product were 71.16% and 80.16% when preserved in dark for 30 days, and 63.16% and 74.16% when stored with light for 30 days. 3.3.3. Effect of temperature The temperature has remarkable effects on those heat-sensitive bioactive substances. The retention rate of lutein decreases with the rise of temperature. As is shown in Fig. 6(c), at the end of preservation for 30 days, the retention rates of lutein in products were 92.86% at 4  C, 90.16% at 25  C and 73.63  C at 50  C. There was an obvious decline of stability at 50  C. The main reason is that the temperature 50  C beyond the vitrication temperature 40.5  C makes the system lose the glass state and promotes molecular chains to act drastically, reducing the stability.

0.9 0.8 retention rate (%) 0.7 0.6 0.5 0.4 0.3 0.2 0 5 10 15 time (day) 20 25 30
microcapsule at 33% relative humidity lutein at 33% relative humidity microcapsule at 80% relative lutein at 80% relative humidity

B
retention rate (%)

1 0.9 0.8 0.7 0.6 0.5 0 5 10 15 time (day) 20 25 30

microcapsule in dark lutein in dark microcapsule in light lutein in light

4. Conclusion The optimum process conditions determined by response surface analysis were as follows: CWM 1.0%, RCW 1.25:1 and pH 4.2. Under the condition, the theoretical encapsulation efciency is 86.41%, while the veried practical value is 85.32% 0.63%. The particles had a broad distribution in the range of 0e30 mm, and held the biggest proportion in the range of 10e20 mm. The encapsulation effectively enhanced the product stability from four aspects. The products stored in normal temperature can sustain stability due to the vitrication temperature 40.5 higher than the normal, and had the retention rate of 92.86% at 4  C, 90.16% at 25  C after 30 day preservation. The ability to resist the impacts of light and temperature had also been improved. The retention rates were 90.16% with the relative humidity of 33%, 90.25% with the preservation in oxygen, and 74.16% with the storage in light, respectively.

C
retention rate (%)

1 0.95 0.9 0.85 0.8

microcapsule at 4C

0.75 0.7 0 5

microcapsule at 25C microcapsule at 50C

10

15 time (day)

20

25

30

Fig. 6. The stability assessment of lutein in microcapsule product with relative humidity, light, temperature and oxygen as four inuential factors. (A) Effect of relative humidity on the stability of lutein microcapsules. (B) Effect of light on the stability of lutein microcapsules. (C) Effect of temperature on the stability of lutein microcapsules. (D) Effect of oxygen on the stability of lutein microcapsules.

Acknowledgments This project was supported by National Key Technology R&D Program 2006BAD27B04.

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