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Encapsulation of Fish Oil by an Enzymatic Gelation Process Using Transglutaminase Cross-linked Proteins
Y.-H. CHO, H.K. SHIM, AND J. PARK ABSTRACT: To improve the storage stability and achieve controlled release, fish oil containing docosahexaenoic acid was encapsulated using double emulsification and subsequent enzymatic gelation method, using microbial transglutaminase cross-linked proteins. Isolated soy protein was selected as a wall material because it showed better emulsion stability and higher reactivity with MTGase than other proteins. Microcapsules prepared by this method showed a high stability against oxygen and a low water solubility, which subsequently resulted in sustained release of fish oil. Results indicate that this microencapsulation process is suitable for preparing protein-based microcapsules containing sensitive ingredients for controlled release and stability improvement. Keywords: fish oil, microencapsulation, microbial transglutaminase, isolated soy protein, double emulsification
Introduction
here is increasing information about the nutritional and health benefits of long chain polyunsaturated fatty acids (LC-PUFA) for humans (Kitessa and others 2001). LC-PUFAs, such as arachidonic acid (AA) and docosahexaenoic acid (DHA), are structural components of cell membrane phospholipids and precursors of eicosanoids and play important roles in the development of the central nervous system, including the retina (Smit and others 2000). Fish oil is an important source of DHA and many studies have shown that fish-oil supplementation increases the DHA content of blood components (Markrides and others 1995). However, the application of fish oils in the food system has a limitation because oils are unstable against oxygen during storage. Also, the unpleasant taste and odor of fish oil can cause deterioration in the quality of food products. Research has been performed to overcome the limitations of fish oils. Ahn and others (1991) enhanced oxidative stability of EPA and DHA by adding lecithin. However, the sensory evaluation of fish oil remained a problem. Several methods have been studied for encapsulation of fish oil, and a variety of materials have been used as a wall material, including cellulose (Kantor and others 1990), gum Arabic and gelatin (Maruyama and Yamamoto 1988), agar and waxy corn starch (Chang and Ha 2000), and milk proteins (Keogh and others 2001). However, carbohydratebased microcapsules or protein-based microcapsules prepared by spray-drying are water soluble and thus not suitable for controlled-release applications. Protein films are, in general, excellent oxygen and aroma barriers and can be used in developing microcapsules for controlled core release in food applications (Lee and Rosenberg 2000a). Microcapsules using proteins as wall materials have been prepared by simple/complex coacervation (Arshady 1990) and techniques consistMS 20030315 Submitted 6/6/03, Revised 10/1/00, Accepted 10/11/00. Authors Cho, Shim, and Park are with Dept. of Biotechnology, Yonsei Univ., Seoul 120-749, South Korea. Direct inquiries to author Park (foodpro@ yonsei.ac.kr).
ing of double emulsification and subsequent cross-linking with glutaraldehyde or heat-induced gelation (Lee and Rosenberg 2000a). Both approaches provide a means for achieving high core retention and render the resulting microcapsule water insoluble (Lee and Rosenberg 2000b). However, the use of protein-based wall materials in the food industry for sensitive ingredients is limited because proteins are generally unstable with heating and damaged by organic solvents, and the cross-linking agent is usually harmful. Therefore, if a novel, mild cross-linking agent is developed, and proteins are converted into stable forms, the application of proteins would be greatly increased (Babiker 2000). Microbial transglutaminase (MTGase) is an enzyme excreted from Streptoverticillium mobaraense that catalyzes acyl-transfer reactions, resulting in formation of -( -glutaminyl) lysine intraand inter-molecular cross-links in proteins (Nielsen 1995). Several studies have been conducted on cross-linking various proteins using this enzyme to form biopolymer films (Nio and others 1986; Motoki and others 1987; Yildirim and Hettiarachchy 1998; Lim and others 1999; Liu and others 1999; Babiker 2000; Babin and Dickinson 2001). These attempts focused on the preparation of biodegradable films, while we have attempted to use MTGase crosslinked proteins as a wall material in the encapsulation process. We investigated a number of proteins for use as base materials for preparing biodegradable films using MTGase and developed a double emulsification and subsequent enzymatic gelation method using isolated soy protein as a wall material. Soy protein is an abundant byproduct of the soybean-oil industry. Soy proteins have important applications in many food products because of their high nutritional value and their contribution to food texture and emulsifying properties (Roesch and Corredig 2002). Microcapusules containing fish oil were prepared using oil-inwater-in-oil (O/W/O) double emulsification and 3 different gelation methods, including enzymatic gelation using MTGase cross-linked protein, heat gelation, and a combination of the enzyme and heat gelation methods. The physicochemical properties, microstructure, and controlled release of 3 types of fish-oil microcapsules were investigated and compared.
Vol. 68, Nr. 9, 2003JOURNAL OF FOOD SCIENCE 2717
Rheological measurement
The viscoelastic properties of MTGase-treated protein solutions were measured using a 40-mm-dia rheometer (AR1000, TA Instrument, New Castle, Del., U.S.A.) with cone steel geometry. Protein solutions were carefully poured into the rheometer cup (40-mm dia) and covered with a thin layer of vacuum oil to prevent evaporation. The temperature was maintained at 37 C for 60 min. The storage (G ) and loss (G ) moduli were recorded as a function of time at a frequency of 1 Hz and at a maximum strain of 0.05 in the linear region. Each sample was assayed in triplicate.
A value of 0 represents poor emulsion stability while a value of 1 represents high emulsion stability (Cho 2000). Each sample was assayed in triplicate.
Particle-size distribution
The microcapsules were dispersed in distilled water. The particle-size distribution, the mean particle size, and the specific surface area of microcapsules were determined using a particle-size analyzer (Model 1064, Cilas, Orleans, France).
Water solubility
The water solubility of protein capsules was determined according to the method of Lee and Rosenberg (2000a). Microcapsule samples (1.0 g) were placed in a glass vial containing 25 mL of water and incubated at 4 C, 25 C, and 37 C for 12 h. The concentration of soluble protein in the aqueous phase was determined using a Sigma diagnostic protein assay kit (Sigma Diagnostics, St. Louis, Mo., U.S.A.). Bovine serum albumin (BSA) was used as standard protein. Each sample was assayed in triplicate.
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Oxidative stability
The peroxide value of microencapsuled/nonencapsuled fish oil was estimated using a p-anisidine value (p-A.V.). Samples were stored in an incubator at 50 C and withdrawn every 2 d. One gram of sample was placed in a 25-mL volumetric flask and dissolved with n-hexane. The absorbance of this solution was measured at 350 nm. Five milliliters of the fat solution and the solvent was transferred to a test tube and 1 mL of p-anisidine reagent was added. After 10 min, the absorbance of the fat solution in the test tube was measured as a blank in the reference cell. The p-A.V. is given by the formula:
where: As = absorbance of the fat solution after reaction with the p-anisidine reagent; Ab = absorbance of the fat solution; and m = mass (in g) of the test portion.
Statistical analysis
A Pearson analysis of variance (ANOVA) correlation analysis using SAS program (SAS 1993) was performed with the 4 variables,
Figure 1Effects of different protein-based wall materials and their concentrations on emulsion stability. = 5% protein concentration without MTGase treatment; = 10% protein concentration without MTGase treatment; = 10% protein concentration with MTGase treatment.
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Figure 2Changes in storage modulus values of different protein solutions during MTGase treatment at 37 C. = whey protein isolate (WPI); = soluble wheat protein (SWP); = Na-caseinate; = isolated soy protein (ISP). Vol. 68, Nr. 9, 2003JOURNAL OF FOOD SCIENCE 2719
Figure 3Effects of the isolated soy protein (ISP) concentration on the emulsion stability and storage modulus of emulsions. - = storage modulus; = emulsion stability index 2720
Figure 4Particle size distribution of isolated soy protein (ISP) microcapsules containing fish oil. = MTGase gelled capsule; . . . . . . = MTGase/heat-gelled capsule; = heat-gelled capsule.
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Figure 6Changes in solubility of isolated soy protein (ISP) microcapsules at 3 different incubation temperatures ( = at 37 C; = at 25 C; = at 4 C). (a) MTGase-gelled capsule, (b) MTGase/heat-gelled capsule, and (c) heatgelled capsule.
Figure 5SEM micrographs of isolated soy protein (ISP) microcapsules containing fish oil. (a) MTGase-gelled capsule at 2000, (b) MTGase/heat-gelled capsule at 2000, and (c) heat-gelled capsule at 2000. = 15 m.
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Figure 7Cumulative release of fish oil from isolated soy protein (ISP) microcapsules during incubation at 37 C. = MTGase-gelled capsule; = MTGase/heat-gelled capsule; = heat-gelled capsule. Vol. 68, Nr. 9, 2003JOURNAL OF FOOD SCIENCE 2721
Conclusions
For gel formation of a double emulsion, a 4-h incubation time was required. There were no significant differences in the mean diameter of microcapsules prepared with different gelation methods. The size of capsules manufactured by the O/W/O double emulsification method was not influenced by the gelation method. However, the capsule morphology was highly influenced by the gelation method. Microcapsules prepared by enzyme (MTGase)-induced gelation were spherical in shape with a dent-free surface, while heat-gelled capsules were wrinkled with a rough surface. MTGase-gelled capsules exhibited lower water solubility and sustained release in a pepsin solution. However, the total oil retention for MTGase-gelled capsules was lower than for heat-gelled capsules. Regardless of the gelation method, encapsulated fish oil was more stable than nonencapsulated fish oil for each microcapsule type, indicating that the encapsulation method presented herein is effective for protection of the core material from oxidation. A microencapsulation process consisting of double emulsification and subsequent gelation is suitable for protecting sensitive ingredients and for controlled release in the food industry.
TGase modified intra- and inter-molecular interactions with protein-based wall materials increasing the emulsion stability and rheological properties of proteins. ISP exhibited the highest emulsion stability and gelling capacity. A CIWE (primary O/W emulsion) prepared with 10% ISP was stable without any emulsifier.
References
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Figure 8Changes in the p-anisidine concentration in fish oil in isolated soy protein (ISP) microcapsules during storage at 50 C. = nonencapsulated fish oil; = MTGasegelled capsule; = MTGase/heat-gelled capsule; = heatgelled capsule. 2722
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