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Encapsulation efficiency of coenzyme Q10-liposomes in alginate

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Phornsinee Sakchareonkeat Food Science and Technology, Kasetsart University, Bangkok, Thailand

Tzou-Chi Huang Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan

Prisana Suwannaporn Food Science and Technology, Kasetsart University, Bangkok, Thailand

Yu Hsuan Chiang and Jue Liang Hsu Biological Science and Technology, National Pingtung University of Science and Technology, Pingtung, Taiwan, and

Yong Han Hong Department of Nutrition, I-Shou University, Kaohsiung City, Taiwan Abstract Purpose – The purpose of this study is to evaluate the effectiveness of alginate as a vehicle to protect coenzyme Q10 in liposomes. Design/methodology/approach – Encapsulation efficiency and stability were conducted at varying temperatures (20, 30, 408C) for 5 d and at exposure to simulated gastric conditions (pH 2) for 2 h. The content of coenzyme Q10 was determined using HPLC (LC/MS). Cytotoxicity and phagocytosis of mouse macrophages (RAW264.7) was determined. Findings – Results showed that thermostability was strongly improved by alginate complex formation with liposomes. Moreover, alginate could maintain coenzyme Q10 at a significantly higher level in simulated gastric pH for at least 2 h ( p , 0.00). Practical implications – This allowed a higher amount of coenzyme Q10 remaining to be absorbed in the small intestine. Alginate not only showed no toxic effect on mouse macrophages but also activated their proliferation and phagocytosis ability. Originality/value – As a consequence, alginate could be applied as an aid to encapsulation stability and immunostimulating potency. Keywords Alginate, Liposome, Coenzyme Q10, Encapsulation, Cytotoxicity, Phagocytosis, Nutrition, Heat Paper type Research paper

Nutrition & Food Science Vol. 43 No. 2, 2013 pp. 150-160 q Emerald Group Publishing Limited 0034-6659 DOI 10.1108/00346651311313463

The authors would like to thank the Department of Biological Science and Technology, National Pingtung University of Science and Technology (NPUST), Taiwan, for research facilities and advisory support. Many thanks also to the International Affairs Division, Kasetsart University, Thailand, for additional research funding to support international cooperation. The authors are also grateful to Mr Chang-Kao Tsai, Nanosome Enterprise Co., Ltd, Taiwan, for providing liposome samples.

Introduction There has been a growing interest in the development of food-grade colloidal delivery systems to encapsulate lipophilic functional ingredients using biopolymers, such as proteins and polysaccharides, as building blocks. Ionotropic gelation of some polysaccharides will self-associate upon heating, which attributed to an increase in the strength of hydrophobic attraction (Burey et al., 2008). Alginate self-associates when cooled below thermal transition temperature due to helix formation and association through hydrogen bonding (Minussi et al., 2002). Encapsulation used to stabilize bioactive compounds during processing and storage, protect them from pH, degradation from enzyme, and control the release (Smith et al., 2010). Emulsion-based delivery systems are developed to encapsulate highly lipophilic active agents. These particles must be carefully designed – such as controlled aggregation, segregation, and/or disruption – to be able to exhibit the required functional attributes within the final product (Matalanis et al., 2011). Moreover, the control of digestibility of emulsified lipids in the human gastrointestinal tract is also of interest (Li and McClements, 2011). Coenzyme Q10 is hydrophobic-like vitamin E. It could stabilize cell membranes, in addition to acting as a potent antioxidant to protect mitochondria from free radical damage and preventing programmed cell death or apoptosis (Crane and Navas, 1997; Lass and Sohal, 2000). Coenzyme Q10 is a fat-soluble compound with relatively large molecular weight (863.34 g/mol). It is poorly and slowly absorbed by the gastrointestinal tract (Greenberg and Frishman, 1990). The absorption into tissues is often slow and limited. To maximize coenzyme Q10 absorption would not only improve its uptake into the plasma, but also into the skeletal muscle. Encapsulating the bioactive compounds in liposomes for controlled release has been widely studied in pharmaceuticals (Choi and Maibach, 2005), foods and cosmetics (Taylor et al., 2005) and food antimicrobials (Were et al., 2003). Lipid-based systems generally suffer from instability, as opposed to polymer-based systems which are usually more stable. However, lipid-based systems are usually more biocompatible. For this reason, research has been directed toward improved applications of liposomes. Several studies have reported on the encapsulation of liposomes into microcapsules made of different polymers: for example, dextran (Dhoot and Wheatley, 2003), chitosan (Hong et al., 2008); and alginate (Dai et al., 2005, 2006; Liu et al., 2003; Huang, 1989). In order to protect coenzyme Q10 for oral delivery purposes, alginate was selected as it is insoluble in acid. The cross-linking process was applied to enhance its stability under simulated gastric pH conditions. Materials and methods Preparation of coenzyme Q10-liposomes in alginate gel beads A solvent injection method was used to prepare coenzyme Q10 liposomes. Soybean phosphatidylcholine (SPC . 95.5 percent) and coenzyme Q10 were dissolved in ethanol (1 ml) and injected through a 23-gauge needle into deionized water (10 ml). During injection into the aqueous solution, an ultrasonic processor (XL2210, Misonix, Inc., CT, USA) was used for dispersion. Sonication was carried out for 5 min with cycle 0.5 (0.5 s pulse, 0.5 s pause). The mixture was then stirred at 1,000 rpm overnight to vaporize ethanol. The mixture volume was replenished with deionized water to 10 ml (Lee and Tsai, 2010) the final mixture consisted of SPC liposomes

Efficiency of coenzyme Q10

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containing 1 mg/ml of coenzyme Q10. These conventionally prepared liposomes were then stored at 48C for further use. Sodium alginate (Sigma-Aldrich, Germany) was dissolved in distilled water. Previously prepared coenzyme Q10 liposomes were then added into the alginate solution by magnetic stirring. The solution was then dropped into CaCl2 solution. Different proportions of sodium alginate to CaCl2 – at 0.5-1 percent, 1-0.5 percent, 1-1 percent, 1-1.5 percent, and 1.5-1 percent – were studied for their encapsulation efficiency. Spherical calcium-alginate gel beads were formed and were further cured in CaCl2 solution at room temperature for 5 min (Arica et al., 2003; Elcin, 1995; Bodmeier and Paeratakul, 1991). Samples were then taken from the CaCl2 solution to investigate the release of coenzyme Q10. Encapsulation efficiency of coenzyme Q10-liposomes The contents of coenzyme Q10 released from the liposomes were quantified using HPLC (Karpin´ska et al., 2006). The HPLC column was a silica gel RP-18 GP, 250 mm £ 4.6 mm i.d., (5 mm); mobile phase was methanol/n-hexane in a ratio of 72:28 (v/v), with a flow rate 0.5 ml/min, and UV detection wavelength of 278 nm. The solutions were applied to the ion-trap LC/MS/MS system. Peaks of product ions were monitored using liquid chromatography-mass spectrometry (LC/MS). Encapsulation efficiency was calculated as a percentage of releasing coenzyme Q10 in CaCl2 solution compared to the control (CaCl2 solution): % Relative encapsulation efficiency ¼

Peak area of control £ 100 Peak area of sample

Thermostability of coenzyme Q10-liposomes Samples of coenzyme Q10 liposomes in alginate gel beads, conventional coenzyme Q10 liposomes, and free coenzyme Q10 were prepared and maintained at temperatures of 20, 30 and 408C. Then, 5.50 g of each liposome sample were then soaked in 20 ml deionized water for 5 min. Samples were then taken from the soaked water to quantify the release of coenzyme Q10 using the method stated in previous section. Coenzyme Q10 contents of each sample were investigated at 24 h intervals for up to five days. Stability in simulated gastric pH of coenzyme Q10-liposomes Simulated gastric juice was prepared by adjusting the pH of hydrochloric acid to 2.0. A test was performed to compare the stability of coenzyme Q10 liposomes in alginate gel beads, conventional coenzyme Q10 liposomes, and free coenzyme Q10 when exposed to simulated gastric pH conditions. Samples (5.50 g) of each liposome were then soaked in 20 ml of simulated gastric juice for 120 min at body temperature (378C). Samples were then removed from the soaked gastric juice for quantification of the release of coenzyme Q10, using the method stated in previous section. Coenzyme Q10 contents of each sample were investigated at 30 min intervals for up to 2 h. Cytotoxicity determination A mouse macrophage-like cell line (RAW264.7) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/ml of penicillin, 100 mg/ml of streptomycin, and 10 percent fetal bovine serum. Cells were grown at 378C under 5 percent CO2 atmosphere. To assess cytotoxicity of sodium alginate on macrophages,

MTT 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed at different concentrations of alginate. Cultured cells were then grown in 96-well plates at a density of 1 £ 105 cells/well at 378C under 5 percent CO2 atmosphere. After 24 h, cells were washed with fresh medium and then treated with different concentrations of sodium alginate (100, 200, 300 and 400 mg/ml). After incubation for 24 h, cells were washed again and 50 ml of MTT (1 mg/ml) was added, followed by further incubation for another 4 h. Finally, 50 ml dimethyl sulfoxide (DMSO) was added to solubilize the formazan salt formed. The amount of formazan salt was determined by measuring the optical density (OD) at 570 nm using an ELISA plate reader. Relative cell viability was determined by the amount of MTT converted into formazan salt. Macrophage activation assay by fluorescence microscopy Activation of macrophages by sodium alginate was studied using fluorescence assay. Activation of macrophages (1 £ 105 cells/ml) treated with 100, 200, 300 and 400 mg/ml sodium alginate was compared to 100 mg/ml of lipopolysaccharide (LPS) as a positive control for macrophage activation markers. Fluorescence microscopy assay was performed on glass culture dishes using directly labeled antibodies (phalloidin, 1:300 dilution). Intensity changes in the actin cytoskeleton were observed under a fluorescence microscope (model BX51 with U-LH100HG mercury lamphouse; Olympus, Japan). Statistical analysis Comparison of means was conducted using one-way analysis of variance with a post hoc Tukey test to identify significant differences ( p , 0.05) between data sets. Results/discussion Coenzyme Q10 liposomes in alginate gel beads were produced by the typical external gelation (i.e. ionotropic gelation) method. Coenzyme Q10 liposomes were mixed in sodium alginate solution; then the solution was dropped into a calcium chloride bath. The complex of coenzyme Q10 in liposomes entrapped in alginate gel beads appeared yellow in color due to the presence of coenzyme Q10. Liposomes can entrap a lipid bilayer, a water layer, and a layer between the lipid and water bilayers. Components for entrapment can be polar or non-polar within the same molecule. Entrapment efficiency of lipids is 90-100 percent. Liposomes were composed of a structural part, phospholipids and cholesterol, and a non-structural part, a charged surfactant or charged lipids. The charged group can improve the performance of a drug or bioactive compound, causing increased water volume which could entrap the soluble bioactive compound in the liposome structure (Betageri and Burrell, 1993). Alginate was applied on the surface of the liposomes, and then became entangled with adjacent liposomes once cross-linking and gelation occurred, causing adhesion of the particles (Smith et al., 2010). Quantification of coenzyme Q10 in liposome Ionization of coenzyme Q10 in liposome alginate gel beads prior to LC/MS analysis was performed by electrospray ionization (ESI) both in positive and negative mode. The possible ionization combinations were tested in order to obtain the most efficient analytical ESI. Figure 1 shows the ESI mass spectrum of coenzyme Q10 liposomes in alginate gel beads which were dissolved in isopropanol. In positive mode, a single ion m/z ¼ 864.33 [M þ H]þ with high intensity was produced, and in negative mode the


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(a)

Figure 1. LC-MS analysis of different ionization modes of coenzyme Q10-liposomes

(b)

Notes: (a) ESI +; (b) ESI –

radical anion m/z ¼ 862.39 [M þ H]2 was generated. Confirmation of this result was obtained, since the most common m/z of coenzyme Q10 in human serum dissolved in methanol with 10 mM ammonium acetate is 863.8 [M þ H]þ in positive mode and 862.8 [M þ H]2 in negative mode (Hansen et al., 2003). Encapsulation efficiency of coenzyme Q10-liposomes In an effort to protect coenzyme Q10, this mixture was encapsulated in liposomes and then cross-linked with alginate by the addition of calcium chloride. Alginate gel beads, prepared from different proportions of sodium alginate and calcium chloride, were determined for their encapsulation efficiency of releasing coenzyme Q10. Proportions of sodium alginate to calcium chloride at 0.5-1 percent, 1-0.5 percent, 1-1 percent, 1-1.5 percent, and 1.5-1 percent gave encapsulation efficiencies of 64.7, 60.7, 85.5, 71.6 and 63.3 percent, respectively, (Figure 2). Encapsulation efficiency showed significant difference between proportions ( p , 0.001). The 1-1 percent proportion gave the best encapsulation efficiency of 85.5 percent. Thermostability of coenzyme Q10-liposome Coenzyme Q10 is a thermosensitive compound; therefore, the effect of temperature on coenzyme Q10 degradation was of interest. Results showed that after five days of heating at 20, 30 and 408C, the degradation of coenzyme Q10 protected with alginate gel


Encapsulation Efficiency (%)

100 90 80 70 60 50

155

40 30 20

Figure 2. Effect of proportion of sodium alginate and calcium chloride on encapsulation efficiency of coenzyme Q10 liposomes

10 0 0.5 to 1 1 to 0.5 1 to 1 1 to 1.5 1.5 to 1 Proportion of Sodium alginate and Calcium chloride

% Coenzyme Q10 Remaining

Notes: Each value represents the mean ± SD; n = 3 Coenzyme Q10-liposomes in alginate gel beads Conventional CoenzymeQ10-liposomes

100 80

FRee CoenzymeQ10

60 40 20 0 1

2

3 Day (a)

4

5

1

2

3 Day

4

5

4

5


100 80 60 40 20 0


(b) 100 80 60 40 20 0 1

2

3 Day (c)

Notes: (a) 20°C; (b) 30°C; (c) 40°C

Figure 3. Stability of coenzyme Q10-liposomes encapsulated in alginate gel bead at temperatures

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was less than 50 percent, while the degradation of pure coenzyme Q10 and liposome coenzyme Q10 was nearly 100 percent (Figure 3). The complex of coenzyme Q10 with alginate showed significantly improved stability compared to pure coenzyme Q10 and conventionally prepared coenzyme Q10 liposomes. The results suggested a temperature-dependent nature of alginate. The rigidity of alginate gel decreased with increased temperature (Andresen et al., 1997). At temperatures below the boiling point of water, and under small-deformation oscillatory conditions, the non-covalent bonding between contiguous polymeric segments kept the alginate intact. However, the equilibrium was disrupted by steady shear under thermal agitation (Zheng, 1997).

Figure 4. Stability of coenzyme Q10-liposomes encapsulated in alginate gel bead in simulated gastric pH conditions (pH 2) for 120 min at body temperature (378C)

% Coenzyme Q10 Recovery

Stability in simulated gastric pH of coenzyme Q10-liposomes To determine the protective and controlled release effect of coenzyme Q10 liposomes, samples were exposed to simulated gastric pH (pH 2) for 2 h. The quantities of coenzyme Q10 released from free coenzyme Q10, conventional coenzyme Q10 liposomes and coenzyme Q10-liposomes in alginate gel beads were monitored. The release curves in Figure 4 reveal that coenzyme Q10 liposomes in alginate gel beads released half of their entrapped coenzyme Q10 over a period of 1 h, while free coenzyme Q10 drastically dropped to almost zero, and less than 10 percent was left in conventional liposomes. Coenzyme Q10-liposomes in alginate gel beads released coenzyme Q10 at a slower rate, and were almost stable after 1.5 h in simulated gastric pH. Significantly, a higher amount of coenzyme Q10 (16 percent) was left over after 2 h compared with conventional coenzyme Q10 liposomes ( p , 0.00). Encapsulated coenzyme Q10 was released from alginate pellets by diffusional processes through pores, and was facilitated by polymeric network degradation. The release of water-insoluble coenzyme Q10 was largely dependent on gel erosion (Mi et al., 2002; Sriamornsak et al., 2007). The movement of encapsulated coenzyme Q10 in an alginate matrix was governed by the properties of coenzyme Q10 and the chemical composition of the alginate polymer. The differences in sustaining the release from alginate matrices was reported to be related to the molecular weight of entrapped coenzyme Q10, and ionic interaction between liposomes and negatively charged alginates (Mumper et al., 1994). Alginate gel, which acted as an acid and heat shield, could improve stability of coenzyme Q10 in liposomes when exposed to simulated gastric pH for at least 2 h. This allowed a higher amount of coenzyme Q10 left to be absorbed in the small intestine. 100

Coenzyme Q10-liposomes in alginate gel beads Conventional Coenzyme Q10 liposome

80

Free Coenzyme Q10

60 40 20 0 30

60

90 Time (min)

120

Cytotoxicity determination The toxicity effect of sodium alginate concentrations on cell proliferation through MTT assay was studied. Figure 5 shows that there was a higher percentage of cell viability in every alginate concentration compared to control (no alginate). This result demonstrated that sodium alginate had no toxic effect on mouse macrophages and was in a better state than those in the control. Cultures under sodium alginate stimulation could proliferate at both low and high concentrations. Sodium alginate concentrations between 200 and 400 mg/ml showed proliferation similar to the positive control (LPS 100 mg/ml).


157

Cell viability (% of control)

Macrophage activation assay To address whether sodium alginate was able to stimulate the functional activation of macrophages, the morphological change of cells was evaluated. The morphological changes of macrophages were associated with marked phagocytosis of mouse macrophages in the actin cytoskeleton, as determined by fluorescence microscopy. Sodium alginate demonstrated an effective activation of macrophage functions. It could induce morphological changes, as shown by numerous pseudopodia. Figure 6 shows pseudopodia expressed in the actin cytoskeleton of mouse macrophages. Phagocytosis of these mouse macrophages was also consistently observed. The result was confirmed by comparison with the macrophage activation marker LPS, which also effectively induced the activation of macrophages. 250 200 150 100 50 0 Control

Lps 100 µg/ml

100

200 300 Concentration (µg/ml)

400

Notes: Each value represents the mean ± SD; n = 5

(a)

(b)

Notes: (a) Control; (b) LPS100 mg/ml; (c) sodium alginate 100 mg/ml

(c)

Figure 5. Effect of sodium alginate on the macrophage cell viability via MTT assay

Figure 6. Fluorescence microscope detection actin cytoskeleton during macrophage phagocytosis in different concentration of sodium alginate directly labeled with phalloidin antibodies (1:300 dilutions)

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Conclusions The stability of coenzyme Q10 entrapped in liposomes and shielded by alginate gel was significantly improved. Without alginate coating, almost all coenzyme Q10 was degraded within five days at 408C, which makes its use commercially impossible. Moreover, the slow release of coenzyme Q10 in simulated gastric pH at body temperature was also beneficial for gastrointestinal absorption. Sodium alginate concentrations of 100-400 mg/ml had no toxic effect on mouse macrophages. The best encapsulation efficiency of coenzyme Q10, up to 85.5 percent, was achieved by a proportion of sodium alginate to calcium chloride of 1-1 percent. Interestingly, sodium alginate could also activate macrophage proliferation and function. Sodium alginate did not only aid in encapsulation stability but also exhibited immunostimulating potency. References Andresen, I.L., Painter, T. and Smidsrod, O. (1997), “Concerning the effect of periodate oxidation upon the intrinsic viscosity of alginate”, Carbohydrate Research, Vol. 59 No. 2, pp. 563-6. Arica, M.Y., Arpa, C., Ergene, A., Bayramoglu, G. and Genc, O. (2003), “Ca-alginate as a support for Pb(II) and Zn(II) biosorption with immobilized Phanerochaete chrysosporium”, Carbohydrate Polymers, Vol. 52 No. 2, pp. 167-74. Betageri, G.V. and Burrell, L.S. (1993), “Stability of antibody-bearing liposomes containing dideoxyinosine triphosphate”, International Journal of Pharmaceutics, Vol. 98 Nos 1-3, pp. 149-55. Bodmeier, R. and Paeratakul, O. (1991), “A novel multiple-unit sustained release indomethacin-hydroxypropyl methylcellulose delivery system prepared by ionotropic gelation of sodium alginate at elevated temperatures”, Carbohydrate Polymers, Vol. 16 No. 4, pp. 399-408. Burey, P., Bhandari, B.R., Howes, T. and Gidley, M. (2008), “Hydrocolloid gel particles: formation, characterization, and application”, Critical Reviews in Food Science and Nutrition, Vol. 48 No. 5, pp. 361-77. Choi, M.J. and Maibach, H.I. (2005), “Liposomes and niosomes as topical drug delivery systems”, Skin Pharmacology and Physiology, Vol. 18 No. 5, pp. 209-19. Crane, F. and Navas, P. (1997), “The diversity of coenzyme Q function”, Molecular Aspects of Medicine, Vol. 18 No. 1, pp. 1-6. Dai, C., Wang, B., Zhao, H. and Li, B. (2005), “Factors affecting protein release from microcapsule prepared by liposome in alginate”, Colloids and Surfaces B: Biointerfaces, Vol. 42 Nos 3/4, pp. 253-8. Dai, C., Wang, B., Zhao, H., Li, B. and Wang, J. (2006), “Preparation and characterization of liposomes-in-alginate (LIA) for protein delivery system”, Colloids and Surfaces B: Biointerfaces, Vol. 47 No. 2, pp. 205-10. Dhoot, N.O. and Wheatley, M.A. (2003), “Microencapsulated liposomes in controlled drug delivery: strategies to modulate drug release and eliminate the burst effect”, Journal of Pharmaceutical Sciences, Vol. 92 No. 3, pp. 679-89. Elcin, Y.M. (1995), “Encapsulation of urease enzyme in xanthan-alginate spheres”, Biomaterials, Vol. 16 No. 15, pp. 1157-61. Greenberg, S. and Frishman, W.H. (1990), “Co-enzyme Q10: a new drug for cardiovascular disease”, Journal of Clinical Pharmacology, Vol. 30 No. 7, pp. 596-608.

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Were, L.M., Bruce, B.D., Davidson, P.M. and Weiss, J. (2003), “Size, stability, and entrapment efficiency of phospholipid nanocapsules containing polypeptide antimicrobials”, Journal of Agricultural and Food Chemistry, Vol. 51 No. 27, pp. 8073-9. Zheng, H. (1997), “Interaction mechanism in sol-gel transition of alginate solutions by addition of divalent cations”, Carbohydrate Research, Vol. 302 Nos 1/2, pp. 97-101. Corresponding author Prisana Suwannaporn can be contacted at: [email protected]

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