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Journal of Microencapsulation, 2009; 26(3): 263271

Alginate/starch composites as wall material to achieve microencapsulation with high oil loading
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Lay Hui Tan, Lai Wah Chan and Paul Wan Sia Heng*
Department of Pharmacy, National University of Singapore, Singapore
Abstract Alginate/starch blends were used as wall material to encapsulate fish oil by spray-drying. The effects of alginate type and content on microsphere morphology, yield and microencapsulation efficiency were investigated. The ability of microspheres to confer protection to the microencapsulated fish oil on storage under stress was also determined. The results showed that the addition of alginate to the wall component resulted in rounder microspheres with higher oil encapsulation efficiencies. Microencapsulated oil was found to be more stable to degradation compared to unencapsulated oil. Key words: Alginate; fish oil; spray-drying

Introduction
Microencapsulation has been carried out on oils for reasons including protection, controlled release and/or taste masking12. Fish oils, in particular, have received much attention in recent years. Studies have shown that the !-3 polyunsaturated acids, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in fish oils have beneficial effects on human physiology and health36. However, due to their high degrees of unsaturation, fish oils are prone to oxidative degradation and rancidity. Commercially available fish oil products are predominantly in the emulsion or oil-containing gelatin capsule forms. The former dosage form is multi-dose, bulky and requires careful administration to avoid spillage, while the latter is associated with issues concerning production cost and efficiency, as well as patient acceptability due to dietary and religious reasons. As such, it is advantageous to develop a microencapsulated, powder form of fish oil for increased ease of processing, storage, delivery and administration. Such a product can also be used in combination with other dry bioactive ingredients. Different methods and polymers have been used for fish oil microencapsulation with the primary objective

of achieving oxidative protection of the core components711. Spray-drying is a one-step, continuous drying process which involves the transformation of a fluid feed into a dried particulate form by spraying the feed into a hot drying medium12. Although the initial capital outlays for commercial microsphere production using spray-drying may be high due to equipment costs, this is mitigated by the relative ease of scale-up compared to other encapsulation methods like freeze-drying or emulsification. This method also does not require the use of organic solvents and is relatively simple. The potential to operate continuously also offers commercial advantage in terms of reduction in time and product losses during process start-up and shut-down. In addition, the integrity of the ingredient to be encapsulated is preserved as product exposure to heat is limited due to rapid drying. Polymers used as wall materials include carbohydrates, milk proteins and starches, alone or in blends. These studies were able to demonstrate the reduction in oxidation of fish oil by microencapsulation. The oil-to-wall weight ratios usually ranged from 0.4 : 11 : 1. However, it was difficult to compare and quantify the merits of the different wall systems used due to variations in compositions of the microspheres studied.

Address for correspondence: Lay Hui Tan, Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543. Tel: 65-65162930. Fax: 65-67752265. E-mail: phapaulh@nus.edu.sg (Received 14 Feb 2008; accepted 26 Jun 2008) ISSN 0265-2048 print/ISSN 1464-5246 online 2009 Informa UK Ltd DOI: 10.1080/02652040802305519 http://www.informapharmascience.com/mnc

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Preparation of fish oil-containing microspheres Emulsions were prepared according to the formulae shown in Table 1. The amount of oil used was 150% of the dry weight of the total wall material used. Except for formulation C, the alginate solutions were left to hydrate overnight before they were subjected to autoclaving at 121 C for 45 min. This was carried out to reduce their viscosity and facilitate droplet formation during the atomization step of the spray-drying process. The required amounts of modified starch were subsequently added and allowed to hydrate. ROPUFA was then homogenized (L4RT, Silverson Machines, Waterside, UK) with the wall material solutions. The homogenization conditions used were 4500 rpm for 3 min, followed by 5000 rpm for 2 min. The emulsions were then spray-dried using a pilot-scale spray-dryer (Mobile Minor, Niro A/S, Soeborg, Denmark) equipped with a rotary atomizer. The operational conditions used were: air inlet temperature 150 C, air outlet temperature 80 C and atomizer wheel speed 25 000 rpm. The emulsions were subjected to gentle stirring during the spraying process to minimize oil droplet coalescence. The powders collected were sealed in plastic bags and stored in a freezer while awaiting further tests.

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Alginates are naturally occurring polymers that are widely regarded as biocompatible and non-toxic. They are readily available, relatively inexpensive and commonly used in food and pharmaceutical preparations. Chen et al.13 used alginate as wall material for oil encapsulation by the emulsification method. Although high oil encapsulation was achieved, the method was limited by small batch size, difficulty in scale-up and involved the use of organic solvents. Limited studies have been carried out on the use of alginates for oil encapsulation by other methods. Hence, this study aimed to investigate the use of alginate as a wall material for microencapsulation of fish oil by spray-drying. The capacity of the microspheres to encapsulate a high oil load was studied. In addition, the ability of the microspheres to preserve the integrity of the encapsulated fish oil on storage under stress was also determined.

Materials and methods


Materials ROPUFA, a marine oil, was supplied by Roche Vitamins (Basel, Switzerland). Modified food starch, Capsul, was obtained from National Starch and Chemical Company (New Jersey, USA). The alginates used, Manucol LB (low guluronic acid content) and Manugel LBB (high guluronic acid content), were supplied by ISP Alginates (UK) Limited (Surrey, UK). All other chemicals used were of analytical grade.

Microsphere characterization The roundness, size, yield and microencapsulation efficiency (ME) of the microspheres were determined according to the methods mentioned in a previous paper17. Briefly, scanning electron microscopy was used for morphological characterization. Light microscopy interfaced with an image analysis system was used for size and roundness determination. Roundness was calculated by the following equation: Roundness P2 4A 1

NMR of alginates Acid hydrolysis of the alginates was carried out according to the procedure described by Hang and Larsen14 and modified by Schurks et al.15. Briefly, alginate was dissolved in water to form a 1% solution and the pH adjusted to 3.0. Hydrolysis was then carried out at 100 C for 1 h, after which the solution was cooled down and neutralized. The resulting solution was dialysed against deionized water for 24 h and dried under reduced pressure. For the acquisition of the 13C NMR spectra, the samples were first dissolved in D2O to a concentration of $80100 mg ml1. The solutions were then incubated at 60 C for 5 h to maintain their solubility and to de-gas them. The spectra were recorded using a Bruker DPX-300 NMR spectrometer and analysed. The internal reference was sodium 3-(trimethylsilyl)propionate. Major signals were assigned according to Grasdalen et al.16.

Table 1. Composition of different microsphere formulations studied. Wall material (g) Formulation Capsul Manucol LB Manugel LBB Core (g) ROPUFA 337.5 337.5 337.5 337.5 337.5 337.5 337.5

C LB1 LB5 LB10 LBB1 LBB5 LBB10

225 210 150 75 210 150 75

15 75 150 15 75 150

Alginate/starch composites as wall material to achieve microencapsulation with high oil loading
where P is the perimeter of microsphere and A is the crosssectional area of the microsphere. Roundness values closer to 1 were indicative of rounder microspheres. ME was calculated by taking the difference between total oil and surface oil of a known constant weight of microspheres. Total oil was determined by taking the difference in microsphere weight before and after Soxhlet extraction (Buchi Extraction System B-811, Buchi Labortechnik AG, Flawil, Switzerland) with n-hexane, while surface oil was the weight loss of microspheres after quick rinsing with n-hexane and subsequent drying. The specific surface area of microspheres was determined using the Brunauer, Emmett and Teller (BET) technique (SA 3100TM Surface Area and Pore Size Analyser, Beckman Coulter, California, USA). Samples were de-gassed under vacuum for $16 h at room temperature. Triplicate experiments were performed for each formulation produced.

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Results and discussion


Most studies conducted for oil encapsulation for spraydrying have used oil loadings of less than 100% of the weight of the wall component. Therefore, in this study, higher oil loadings (150%) were used to allow evaluation of the ability of alginate to achieve superior oil holding capacities when used as a wall material component for microsphere production by spray-drying. From preliminary studies, this was also the threshold value above which microspheres with excessive amounts of surface oil resulting in significant agglomeration with greatly reduced yields were formed. However, the viscosities of the alginate solutions were too high to allow effective atomization of the feed into small droplets during the spray-drying process. Instead of obtaining spherical products, filamentous particles were formed. Alginate solutions were thus autoclaved to reduce their viscosities, as well as to sterilize the solutions. Sterilization treatments of alginate, either in hydrated or dry powder form, have been conducted for cell immobilization1819. It was reported that depolymerization with resultant decrease in solution viscosity occurred when alginates were subjected to high heat or irradiation. An NMR study on the alginate solutions before and after autoclaving was performed to assess the effect of autoclaving on alginate, especially the possibility of other degradation processes besides depolymerization. It was found that the frequency of M and G fragments remained unchanged with autoclaving for both grades of alginate. Similarly, the M/G ratio was unaffected by the autoclaving process (Table 2). This finding was evident that macromolecules of alginate were broken down by depolymerization into smaller sub-units and not appreciably affected chemically. Microspheres produced generally had a spherical shape with no obvious surface cracks. They had the typical appearance of spray-dried products. However, different degrees of surface indentations were observed for microspheres made from the different formulations. This was more apparent for microspheres produced with a high proportion of alginate. It appeared that microspheres

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Oil content on storage For each batch of microspheres produced, samples of 30 mg each were weighed into amber-coloured wide mouthed jars (3 cm i.d. and 4 cm height) with screw cap closures. The jars were placed in an environment-controlled chamber at 70% relative humidity and 40 C. At intervals of 0, 3, 7, 14, 28, 42 and 56 days, samples were removed from the chamber. Four millilitres of n-hexane was added to each sample, which was then placed in a shaker bath at room temperature for 24 h to allow complete extraction of oil to take place. The samples were then filtered and the residue rinsed with fresh solvent. The filtrate was collected and made up to 10 ml for analysis. The EPA and DHA contents were determined by gas chromatography equipped with a flame ionization detector (HP 5890 II, Agilent Technologies, California, USA). The inlet and outlet temperatures were 190 and 240 C respectively, and heating rate was 5 C min1 to 220 C followed by 3 C min1 to 240 C. This final temperature was held for 5 min. One microlitre of each sample was injected for each analysis. Triplicates were performed.

Table 2. Compositional data of the alginates before and after autoclaving.

Statistical analysis Results were reported as means of triplicate experiments. Treatments with three or more groups were analysed by one-way analysis of variance, while those with two groups were analysed by the Students t-test (SPSS 11.5, SPSS Inc., Chicago, USA). Tukeys and LSD tests were applied to determine significance of differences between means.

Alginate Manucol LB Manucol LB (autoclaved) Manugel LBB Manugel LBB (autoclaved)


a

FM

FG

FMM

FGG

M/G ratio 2.22 2.20 0.67 0.65

0.69 0.69 0.40 0.39

0.31 0.31 0.60 0.61

0.49 0.45 0.22 0.21

0.17 0.20 0.54 0.55

Mannuronic acid fraction; bGuluronic acid fraction; cMM doublet fraction; dGG doublet fraction.

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produced using a greater proportion of alginate had a lower degree of surface indentations. The type of alginate used also affected the microsphere appearance. At the same amount of alginate added, microspheres produced using Manugel LBB (Figure 1b) appeared to have a greater degree of surface indentation than those made using Manucol LB (Figure 1c). Microspheres produced using purely starch as wall material (Formulation C) had the highest degree of surface indentations (Figure 1a). Morphological studies by other workers have identified three distinct forms of spray-dried product: crystalline, agglomerate and skin-forming20. It was reported that inorganic materials formed spray-dried products that are mainly composed of crystalline structures, whereas agglomerates were formed from insoluble or partially soluble materials. Polymeric materials like the starch and alginates used in this study produced skin-forming particles with a continuous non-liquid phase. Single droplet drying experiments demonstrated characteristic drying behaviours for each of the morphological types. For polymeric materials, it was observed that at 200 C, a skin was formed on the droplet surface almost immediately, followed by several cycles of internal bubble nucleation, particle collapse and re-inflation. The skin finally dried up and hardened, forming inflated hollow particles with smooth surfaces or collapsed particles with surface indentations as seen in this study. The ability of the particles to inflate, collapse and re-inflate to eventually form a continuous skin was due to the flexible nature of the polymeric membrane. Other researchers postulated that the formation of indentations was due to uneven particle shrinkage caused by rapid droplet drying during the spray-drying process21. Indentations could also have been formed from inter-particle collisions or particle impact with the walls of the spray dryer. From the appearance of the microspheres produced using the different wall materials employed in this study, it could be deduced that those containing alginate, especially Manucol LB, were less prone to irregular shrinkage or collapse during drying. Particle morphology is also affected by spray-drying process variables including atomization conditions, drying temperatures, feed properties and the chemical and physical nature of the material being dried. Since spray-drying conditions were kept constant for the different microspheres produced in this study, the likely cause for the differences in microsphere morphology was the nature of the material being spray-dried. Although they were all polymeric materials, they differed in chemical composition. Wandrey et al.22 have studied the effect of alginate composition on the mechanical properties of alginate microbeads and microcapsules made by gelation with calcium chloride. It was observed that less shrinkage was observed with the high G alginates, which was the reverse of what was observed in this study.

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Figure 1. SEM of spray-dried microspheres using formulation (a) C, (b) LBB10 and (c) LB10.

High G alginates, due to the stronger affinity of G residues for calcium ions, formed stronger beads and gels than high M alginates. However, in this case, no ionic cross-linking was involved. Manucol LB could have formed a less flexible microsphere wall resulting in fewer surface indentations or it could be inferred that the addition of Manucol LB resulted in the formation of a stronger microsphere matrix that was more resistant to collapse.

Alginate/starch composites as wall material to achieve microencapsulation with high oil loading
Table 3. Mean particle size, roundness, BET surface area, yield and ME of microspheres prepared using the different formulations. Formulation C LB1 LB5 LB10 LBB1 LBB5 LBB10
a

267

Diameter (mm) 18.9 0.4 18.6 0.3 19.1 0.2 19.8 0.3 18.8 0.3 19.0 0.4 19.2 0.3

Roundness 1.13 1.12 1.10 1.08 1.12 1.12 1.10

BET surface area (m2 g1) 0.66 0.04 0.64 0.02 0.54 0.05 0.51 0.05 0.66 0.03 0.65 0.00 0.63 0.02

Yield (%) 47.8 7.5 51.3 5.8 65.5 4.3 72.6 4.5 49.2 5.2 58.7 4.9 68.6 4.7

ME (%) 57.4 2.9 59.4 2.0 67.3 2.7 76.6 2.1 60.8 3.4 66.2 2.8 72.2 1.1

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Standard deviation 50.001.

Table 3 gives the results of microsphere size, roundness, BET surface area, spray-drying yield and ME for the different microspheres produced. Microsphere size was the least affected by the composition of wall materials used in this study (p 4 0.05). As the proportion of alginate increased, the differences in the other parameters became more apparent. For both types of alginate used, microspheres became rounder with increasing amounts of alginate (p 5 0.05). The BET surface areas were also lower when more Manucol LB was used. This suggested less porous particles or integrally better fused microspheres. Yield and microencapsulation efficiencies, on the other hand, were significantly higher. The differences in these properties were more marked when Manucol LB was used. The effect of alginate incorporation on microsphere roundness was most likely due to the phenomenon of the formation of a more resistant microsphere wall matrix as mentioned in the earlier section. This also had an effect on yield, which was the total amount of powder collected at the end of the process stream. Rounder particles would tend to flow better and would be less likely to stick on the internal surfaces of the spray-dryer, allowing more product to be collected. This could be one of the factors contributing to the higher yields obtained with formulations LB5, LB10, LBB5 and LBB10. However, the yield was also affected by the degree of stickiness of the microspheres, which was in turn dependent on the amount of surface oil present. ME values were higher for the formulations with higher yields, implying that the lower amount of surface oil reduced microsphere tackiness which facilitated their collection. The ME results were also related to microsphere shrinkage, as oil could have been squeezed out of the microspheres during the drying process. In short, substitution of Capsul with the alginates used in this study allowed the formation of rounder microspheres and higher microencapsulation efficiencies, giving rise to greater yields. Manucol LB appeared to be superior to Manugel LBB in these aspects.

The specific surface area of particles can be affected by factors such as shape, size and surface roughness. The microspheres produced from formulations LB5 and LB10 had significantly lower specific surface areas than microspheres formed from the other formulations. This corresponded to the SEM observations, as these were the batches with lower degrees of surface indentations and thus smoother surfaces. It could also be related to microsphere roundness, as these particular batches were found to have roundness values closest to 1. However, the effect of Manucol LBB addition on microsphere specific surface area was less apparent, although SEM studies showed that this batch has a lower degree of surface indentations than the control. This implied that microspheres formed from this particular formulation, although having smoother surfaces, could have more porous walls compared to the control, thereby balancing out the effects on specific surface area. After the microspheres were successfully produced, it became pertinent to determine the ability of the microsphere wall to preserve the integrity of the encapsulated components of interest. As EPA and DHA are widely considered to be the main active components in fish oil, their levels were monitored. Since polyunsaturated fatty acids are sensitive to the effects of heat and humidity, the microspheres were stored at 40 C and 70% RH to determine if they could protect the microencapsulated components. As a control, bulk unencapsulated oil was also subjected to the same storage conditions and analysed at the appropriate intervals. Most studies have focused on analysing the oxidative products formed910 but did not quantify the residual amount of polyunsaturated fatty acids, which was also an important indicator of microsphere functionality. Figures 2a and 2b show the change in EPA and DHA amounts over time respectively for the different formulations studied. For all the microspheres produced, the greatest decrease in EPA and DHA content occurred over the first 3 days of storage. This was likely due to the

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100 90 80 70 EPA (%) 60 50 40 30 20 10 0 0 10 20 30 Time (days) 100 90 80 70 DHA (%) 60 50 40 30 20 10 0 0 10 20 30 Time (days) 40 50 60 40 50 60

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Figure 2. EPA and DHA content on storage for ^ unencapsulated oil, ^ C, * LB1, LB5, 4 LB10, f LBB1, g LBB5, m LBB10.

degradation of oil present on the surface of the microspheres. Due to their high degrees of unsaturation, degradation of polyunsaturated fatty acids usually occurred via oxidation7 which was affected by light, temperature and humidity. Since physical contact between the oil and oxygen had to take place before degradation could occur, the surface area of oil exposed to oxygen would thus affect the rate and degree of degradation, especially in the initial stages. The film of oil present on the microsphere surface was subject to a high rate of oxidation due to its large effective surface area for interaction with the external environment. The unencapsulated oil (control) was presented in the bulk form and effectively presented a much lower surface area than the oil on the microsphere surface. This resulted in the lower initial reduction in EPA and DHA amounts for the control.

The decrease in EPA and DHA contents became more gradual after 4 weeks to almost levelling around 6 weeks. The subsequent gradual decline could be due to the slow diffusion of oxygen into the encapsulated oil situated nearer to the microsphere surface, while levelling indicated the inaccessibility of oxygen to the interior oil reservoir of the microspheres. Microspheres stored at elevated temperature and humidity showed susceptibility to caking and similar findings were also reported by other workers2324. This could have also contributed to the decreased degradation of the encapsulated oil as exposure became increasingly restricted. As for the unencapsulated oil, without the protective or insulating barrier present, almost complete degradation of EPA and DHA took place at the end of the storage duration and less than 10% each of EPA and DHA remained.

Alginate/starch composites as wall material to achieve microencapsulation with high oil loading
EPA 0.25

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ln [-ln(C/C0)] Journal of Microencapsulation Downloaded from informahealthcare.com by Memorial University of Newfoundland on 06/21/12 For personal use only.

0.75

1.25

1.75

2.25 1 1.5 2 2.5 ln (t) 0 0.2 0.4 ln [-ln(C/C0)] 0.6 0.8 1 1.2 1.4 1.6 1.8 1 1.5 2 2.5 ln (t)
Figure 3. Application of the Weibull model to DHA and EPA content on storage for ^ C, * LB1, LB5, 4 LB10, f LBB1, g LBB5, m LBB10.

3.5

DHA

3.5

Microspheres prepared using Formulations C, LB1 and LBB1 had generally lower levels of EPA and DHA on storage compared to microspheres made using higher proportions of alginate in the wall material composition. This could be a consequence of the differences in the morphology of microspheres made using the different formulations. These microspheres were less round as a result of a higher degree of surface indentations, which translated to increased effective surface area (also shown by BET studies) for oxygen exposure and diffusion. This resulted in a greater extent of deterioration of EPA and DHA. As the proportion of alginate increased, the extent of indentations observed on microspheres was lower and the more spherical microspheres ensured that the encapsulated oil was better protected by the presence of lower diffusion areas. The differences could also be related to the porosity of the wall material, although further studies are needed to confirm these.

Nevertheless, effective protection of microencapsulated oil was achieved for all formulations studied, as $50% of EPA and DHA were preserved under the harsh storage conditions compared to the unencapsulated bulk oil (10%). It is known that the deterioration of !-3 polyunsaturated acids occurs mainly via an autocatalytic process which involves the formation of free radicals2526. However, for microencapsulated oil, the process is complicated by other factors including the presence of the protective microsphere matrix shielding the encapsulated oil from the external environment. The Weibull model (Equation 2) was found to adequately describe shelf-life failures2728 and had been used to express the oxidation kinetics of microencapsulated polyunsaturated fatty acids29, C exp ktn 2

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oil holding capacities. Microspheres prepared using Manucol LB performed relatively better than those made using Manugel LBB in terms of yield and ME. Microencapsulation of fish oil brought about increased protection with lower loss of unsaturated components on storage compared to unencapsulated oil. The degree of protection of encapsulated oil increased as the alginate content within the microsphere matrix increased. The spraydried, microencapsulated form of fish oil using alginate blend as wall material can potentially have widespread commercial and industrial applications due to the ease of production and relatively high ME and oxidative protection achieved.

Table 4. Parameters derived from the Weibull model for EPA and DHA. Formulation n EPA C LB1 LB5 LB10 LBB1 LBB5 LBB10 0.422 0.459 0.505 0.524 0.422 0.429 0.485 DHA 0.435 0.430 0.433 0.432 0.434 0.440 0.406 EPA 19.7 103 14.3 103 10.2 103 7.59 103 10.9 103 8.65 103 8.63 103 Parameters k DHA 18.0 103 14.1 103 8.73 103 6.93 103 15.2 103 10.2 103 7.19 103 EPA 0.969 0.982 0.976 0.975 0.961 0.961 0.967 r2 DHA 0.969 0.986 0.985 0.980 0.969 0.972 0.970

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Acknowledgements
where C is the fraction of unoxidized EPA or DHA at time t, k is the rate constant and n is the shape constant. The shape constant describes an increasing or decreasing degradation rate, depending on its magnitude. A shape constant of greater than 1 indicates that the degradation rate increases with time while n 5 1 indicates the opposite27. Equation (2) can be rearranged into a linear model as follows:  ! C ln ln nln t ln k 3 C0 Figures 3a and 3b show the plot of ln [ln(C/C0)] against ln t for EPA and DHA respectively in the microspheres produced. Table 4 shows the values of k, n and correlation coefficients derived from the plots. The r2 values showed linear correlations for the formulations produced, indicating that the Weibull model was suitable for describing the degradation of EPA and DHA in the microspheres produced. From the rate constants, it was evident that as the alginate content within the microspheres increased, the rate of degradation of EPA and DHA decreased (p 5 0.05). This was seen for both Manucol LB and Manugel LBB and corresponded with the discussions stated earlier. The shape constants were less than 1, indicating that the degradation rates of EPA and DHA decreased over time. This could be attributed to the increasing impedence to oxygen penetration into the microsphere matrix or exhaustion of the surface oil component as discussed in the earlier section. This study was made possible with the research scholarship from the National University of Singapore. The authors would like to thank Dr Anton Dolzhenko for performing the NMR studies. The authors would also like to thank Roche Vitamins, UK, National Starch & Chemical, USA and ISP Alginates Inc., USA for samples used during the study. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Conclusions
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Alginate-composite microspheres were successfully produced by spray-drying, with high oil loadings achieved. The use of alginate as a wall material component enabled the production of rounder microspheres with higher

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Alginate/starch composites as wall material to achieve microencapsulation with high oil loading
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