Preparation, physical characterization, and stability of

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Preparation, physical characterization, and stability of Ferrous-Chitosan microcapsules using different iron sources Noer Abyor Handayani, M. Luthfansyah, Elsa Krisanti, Sutrasno Kartohardjono, and Kamarza Mulia

Citation: AIP Conference Proceedings 1904, 020053 (2017); View online: https://doi.org/10.1063/1.5011910 View Table of Contents: http://aip.scitation.org/toc/apc/1904/1 Published by the American Institute of Physics

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Preparation, Physical Characterization, and Stability of Ferrous-Chitosan Microcapsules Using Different Iron Sources Noer Abyor Handayani a), M. Luthfansyahb), Elsa Krisanti c), Sutrasno Kartohardjono d), and Kamarza Muliae), Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Indonesia Kampus UI Depok, Depok 16424, West Java, Indonesia. a)

[email protected]; b) [email protected]; c) [email protected]; d) [email protected]; e) [email protected]

Abstract. Dietary modification, supplementation and food fortification are common strategies to alleviate iron deficiencies. Fortification of food is an effective long-term approach to improve iron status of populations. Fortification by adding iron directly to food will cause sensory problems and decrease its bioavailability. The purpose of iron encapsulation is: (1) to improve iron bioavailability, by preventing oxidation and contact with inhibitors and competitors; and (2) to disguise the rancid aroma and flavor of iron. A microcapsule formulation of two suitable iron compounds (iron II fumarate and iron II gluconate) using chitosan as a biodegradable polymer will be very important. Freeze dryer was also used for completing the iron microencapsulation process. The main objective of the present study was to prepare and characterize the ironchitosan microcapsules. Physical characterization, i.e. encapsulation efficiency, iron loading capacity, and SEM, were also discussed in this paper. The stability of microencapsulated iron under simulated gastrointestinal conditions was also investigated, as well. Both iron sources were highly encapsulated, ranging from 71.5% to 98.5%. Furthermore, the highest ferrous fumarate and ferrous gluconate loaded were 1.9% and 4.8%, respectively. About 1.04% to 9.17% and 45.17% to 75.19% of Fe II and total Fe, were released in simulated gastric fluid for two hours and in simulated intestinal fluid for six hours, respectively.

INTRODUCTION Micronutrient deficiencies are a common health problems, which are rife in both low- and medium-income countries with large number of population, the absence of natural resources, and lack of government supervision [1]. Among these, iron deficiency (ID) is the most prevalent nutritional problem. ID is also considered as the main cause of anemia, called Iron Deficiency Anemia (IDA) [2,3]. In fact, ID brings about more than 20 thousand deaths per year in the case of young children and maternal mortality [4]. IDA has some negative impacts, such as lower growth rate, impaired cognitive scores, lower school performances, reduced metal development and declined immune function in children under 5 years age [1,5]. In adults, IDA can cause weakness, exhaustion, decreased work capacity and physical capacities [6,7]. IDA among adolescent girls will bring poor consequences on growth, capability to study, decease status and reproductive performance in the future [7]. Maternal consequences of anemia in pregnant women tend to low birth weight infants, perinatal mortality, and premature birth [8-10]. IDA in pregnant women is generally caused by increasing iron requirements during gestation and low iron reserves in the body of prospective mother [11]. Given these serious consequences, preventions of IDA are considered as the main nation priority by Government, Ministry

Proceedings of the 3rd International Symposium on Applied Chemistry 2017 AIP Conf. Proc. 1904, 020053-1–020053-8; https://doi.org/10.1063/1.5011910 Published by AIP Publishing. 978-0-7354-1594-2/$30.00

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of Health, and others Health Organization. IDA alleviation is also included in one of its six WHO Global Nutrition Targets for 2025 [12]. Food-based approach around the needs and activities of women can be more effective; since the greatest anemia prevalence is women. However, adding un-encapsulated iron directly to food will cause sensory problems due to their susceptibility on oxidation reaction [13]. Iron is easily influenced by the presence of inhibitors (phytic acid, tannins), and competitors (calcium, zinc, etc.), therefore, its bioavailability might be decreased [5,8,14,15]. Recent year, microcapsules formulations of suitable iron compounds encapsulated in biodegradable polymers will be very important to solve these problems [16]. The purposes of encapsulation are: (1) to improve iron bioavailability and (2) to mask unpleasant aroma, flavor, color, and the taste of iron [5]. Some previous studies were conducted to investigate iron microencapsulation on difference polymers. Perez-Moral et al. (2013) prepared alginate gel beads with the iron content of 275-460 mg Fe/g dried beads using a different route [14]. They also stated that the presence of calcium may compete on iron loading. The used of niosome, liposome, sodium alginate and polyglycerol monostearate were succeed to encapsulate iron up to 84%, 36%, 75%, and 95% efficiency, respectively [17-20]. Most of the research utilized polymers, which are coming from vegetable carbohydrate, protein, and fat rather than animal carbohydrate. Chitosan is an animal carbohydrate and natural polycationic polymer, obtained by alkaline deacetylation of chitin. It has been attracting many researchers due to its biodegradable and biocompatible properties. The interest application of chitosan for food industries is caused by antioxidant, antimicrobial, and anticholesterol characteristics [21]. Chitosan was commonly used as a material to encapsulate many kinds of vitamin, such as ascorbic acid and folate [22]. However, information on preparation of microcapsules using chitosan as biopolymer for iron encapsulation are still lacking. In addition, iron type selection is also critical to determine the physical, stability, and sensory properties, indeed bioavailibility of the final product, as well [1,8]. Ferrous sulphate is the most common type used due to its high bioavailability and affordable price. However, it also provides oxidation and organoleptic problems while unencapsulated [5,14,17]. Ferrous fumarate and ferrous gluconate are the second alternative because of their high stability to oxidation [15,23-25]. The aims of this work was to evaluate the suitability of microcapsule formulation using chitosan as a biodegradable polymer to encapsulate iron compounds (ferrous fumarate and ferrous gluconate). The physical (encapsulation efficiency, iron loading, morphology) and stability properties (in simulated gastrointestinal fluid) were also investigated in this paper. In addition, the result would be discussed on both Fe total and Fe2+ point of view.

MATERIALS AND METHODS Materials 1,10 phenanthroline, sodium acetate, hydroxylamine hydrochloride, acetic acid, ferrous ammonium sulfate, hydrochloric acid, potassium chloride, sodium hydroxide, and monopotassium phosphate were supplied by Merck (Darmstadt, Germany). Ferrous fumarate and ferrous gluconate pharmacy grade were obtained from Jost Chemical. Co (Lackland, USA) and Dalian Chem Import and Export Group Co. (China), respectively. Chitosan-medium molecular weight and sodium tripolyphosphate were purchased from CV. Chimultiguna (Cirebon-Indonesia), respectively. α-Amylase enzyme was obtained from Sigma Aldrich (Saint Louis, USA).

Preparation of iron microcapsules For the preparation of chitosan loaded iron microcapsules, the following procedure was followed: firstly a solution iron-chitosan were prepared by dispersing 0.5 g of iron (ferrous fumarate and ferrous gluconate) in 0.5; 0.75; and 1 g of chitosan and 25 ml acetate acid 2.5% (v/v), left to dissolve for 15 minutes under magnetic stirring at room temperature. The iron-chitosan solution prior to extrusion into a sodium tripolyphosphate cross-linking bath to create a stream of beads and allowed to harden for 30 minutes. The obtained beads were vacuum filtered, rinsed with 25 ml distilled water, and then freeze dried. The dried-beads were shredded into microcapsules using mortar and pestle.

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Determination of iron content Both total Fe and Fe II were analyzed by the 1,10-phenanthroline method using UV/VIS Spectrophotometer at 510 nm. The maximum wavelength (510 nm) was determined by measuring the absorbance solution of a solution over a large range of wavelengths. Furthermore, a standard curve of absorbance versus iron concentration (R2 = 0.9994) was obtained using ferrous ammonium sulfate as an iron standard solution. The five known concentrations of Fe II in the range 0.5-3 mg/L were applied to prepare it. For total Fe (Fe II and Fe III) determination, the sample was treated with hydroxylamine hydrochloride (10% w/v) to reduce any Fe III present to Fe II. For increasing chemical reaction rate, then the mixture was boiled. Sodium acetate (10% w/v) as pH buffer and 1,10-phenanthroline (0.2% w/v) as Fe II chelating agent were added to form an orangered complex ion. This colored solution is directly proportional to absorbance and Fe II concentration. Distilled water was transferred and diluted into the desired volume (25 ml). Let the solution stand for at least 10 minutes before spectrophotometry measurement. It was necessary to slightly modify the analytical procedure for Fe II. Hydroxylamine hydrochloride was not added and the sample was not heated to avoid oxidation. The mixture should be measured immediately to prevent its oxidation to Fe III.

Determination of iron loading and encapsulation efficiency While the iron microcapsules preparation, the filtrate was determined as iron loss using a UV-Vis spectrophotometer at 510 nm. The iron loading content and encapsulation efficiency are calculated using the equation (1) and (2). iron in microcapsu les iron loading content (%)   100% microcapsu les recovered (1) (iron fed - iron loss) iron encapsulation efficiency (%)   100% iron fed (2)

Iron microcapsules morphology Scanning electron microscopy (SEM) was used to evaluate morphological structures of iron microcapsules from different iron sources.

Iron microcapsules stability in the simulated gastrointestinal fluid The stability of iron microcapsules was conducted using simulated gastric fluid (SGF) which is integrated with simulated intestinal fluid (SIF). SGF was prepared by dissolving 2.2 g of potassium chloride and hydrochloric acid 0.2 M in a total volume of 150 ml. On the other hand, SIF was made by dissolving 4.1 g of monopotassium phosphate, and 0.7 g of sodium hydroxide in deionized water in a total volume 150 ml. 4.5 mg of α-amylase was added to the solution. The pH solution of SGF and SIF were kept at 1.2 and 7.4, respectively. Dialysis method was used to determine the release profile of iron. The sample of known iron concentration was transferred into a dialysis bag and immersed in a place containing SGF. It was stirred for 2 hours, the average time of gastric digestion, and maintained at 37˚C. The liquid solution was taken at 1 and 2 h. At the second hour, the SGF solution was replaced with the SIF, while the same sample was kept in the dialysis membrane for 6 hours later. Solution were determined the iron content at 4, 6, 7 and 8 hours. Fe total and Fe II analysis were conducted using 1,10phenanthroline method. Both data in SGF and SIF were used to calculate the iron release profile.

RESULTS AND DISCUSSIONS Iron loaded chitosan microcapsules was prepared by extrusion method using sodium tripolyphosphate as a cross linking agent. The samples were characterized on its physical and stability properties. Physical characterizations, i.e. includes encapsulation efficiency (% EE), iron loading, and morphology of iron microcapsules, were discussed in this paper. In addition, stability of iron microcapsules under simulated gastrointestinal fluid was also presented.

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Physical Characterization Encapsulation efficiency (% EE)

100.0% 80.0% 60.0% 40.0% 20.0% 0.0% Fumarate Gluconate

1:1.0 83.2% 98.5%

1:1.5 92.2% 95.7%

1:2.0 79.6% 97.6%

100.0%

Encapsulation Efficiency (%) of total Fe

Encapsulation Efficiency (%) of Fe II

Encapsulation efficiency is one of the important properties of a delivery system [18]. The influence of the ratio core to wall of iron-chitosan microcapsules on the % EE of Fe II and total Fe is shown in Figure 1. Commonly, both iron sources were highly encapsulated, ranging from 71.5% to 98.5%. The highest encapsulation efficiency of Fe II and total Fe were 98.5% and 83%, respectively, in the ratio ferrous gluconate to chitosan 1:1.0 (% wt). It was higher than that the values reported in the literature [18,19,26] and similar to the results reported by Gupta et al. (2015) who was successful in encapsulating 91.58% of iron with a blend of gum arabic, maltodextrin and modified starch [5]. Both % EE of Fe II and total Fe were investigated in this work. Ferrous form (Fe II) is better absorbed than ferric form (Fe III) [12]. While Fe III is encapsulated and release in the intestinal, then the ferric will never be absorbed. Thus, it is very important to ensure the state of iron which is encapsulated. As shown in Figure 1, there is indication that Fe II has been oxidized to Fe III, which was in agreement with [17]. The % EE of Fe II, for both iron sources and core-to-wall ratios, were always higher than %EE of total Fe. It might be due to the sum of total Fe loss was greater that Fe II loss because some of Fe II have been already transformed into Fe III state.

80.0% 60.0% 40.0% 20.0% 0.0% Fumarate Gluconate

(a)

1:1.0 71.5% 83.0%

1:1.5 72.2% 80.0%

1:2.0 66.5% 82.8%

(b)

FIGURE 1. The influence of the core-to-wall ratios of iron: chitosan in %-wt (1:1.0; 1:1.5; 1:2.0) for both ferrous fumarate and ferrous gluconate on the encapsulation efficiency of Fe (II) (a) and total Fe (b).

Iron loading (%) The iron microcapsules with some formulations represented the different of Fe II and total Fe loading (Figure 2). Iron loading indicates of iron content (g) on microcapsules per total weight (g) of dried microcapsules. This information is needed to determine the appropriate dosage for the application in the field. Iron loading of Fe II were ranging from 0.3% to 1.9% and 1.4% to 4.8% for ferrous fumarate and ferrous gluconate, respectively. The use of core-to-wall ratio of 1:1 produced the highest loading of Fe II and total Fe for both iron sources. This outputs were in agreement with Yu et al (2008) who prepared the composite microparticle drug (bovine serum albumin) delivery system based on chitosan, alginate, and pectin [27]. The iron loading of Fe II and total Fe was relatively low, in the range of 0.8% to 4.8%. This can probably be attributed to the similarity of electrically charge between chitosan and iron. Since both iron and chitosan are electrically positive thus polyelectrolyte complex could not be formed. Furthermore, iron was not able to entrap effectively in the matrices. Polyelectrolyte complex will be created between positive and negatively charged compounds [27].

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Loading of total Fe (%)

Loading of Fe II (%)

6.0% 5.0% 4.0% 3.0% 2.0% 1.0% 0.0% Fumarate Gluconate

1: 1 1.3% 4.8%

1:1.5 1.9% 2.6%

1:2.0 1.7% 2.6%

3.5% 3.0% 2.5% 2.0% 1.5% 1.0% 0.5% 0.0%

Fumarate Gluconate

(a)

1: 1 0.8% 3.0%

1:1.5 1.0% 1.4%

1:2.0 0.3% 2.0%

(b)

FIGURE 2. Loading of Fe II (a) and total Fe (b) of iron-chitosan microcapsules in the ratio core to wall 1:1.0; 1:1.5; and 1:2.0 (%wt) for both ferrous fumarate and ferrous gluconate

Iron microcapsules morphology The effect of core-to-wall ratios of 1:1 (a), 1:1.5 (b), and 1:2 (c) on microcapsules morphology are depicted in Figure 3. In addition, Figure 4 represents the images of two different iron sources in the ratio core to wall 1:2. All the images are in the same magnification (1000x). Preparation of chitosan-iron microcapsules were conducted using a modified procedure described previously [27]. The dried-beads, the final product of the freeze drying step, were shredded into microcapsules using mortar and pestle. Contact between the pestle and iron beads create a mechanical force to form microparticles. Thus the surface of iron microcapsules is rough and rugged. The morphology of microcapsules is smoother using core-to-wall ratio of 1:2, due to the greater sum of chitosan, as shown in Figure 3. The effect of two different iron sources on the microcapsules morphology was also investigated in this work. The use of ferrous gluconate induced the surface of microcapsules to become more delicate in comparison with ferrous fumarate (Figure 4).

(a) (b) (c) FIGURE 3. Scanning electron microscopy (SEM) images of ferrous gluconate microcapsule with different ratio core to wall 1:1 (a); 1:1.5 (b); and 1:2 (c)

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(a) (b) FIGURE 4. Scanning electron microscopy (SEM) images of iron microcapsule with ratio core to wall 1:2 for ferrous fumarate (a) and ferrous gluconate (b), respectively.

Stability of iron microcapsules

Release cumulative of Fe II (%)

In this work, the iron release profile of the microparticles in the absence and in the presence of the enzyme was investigated. The pH conditions of SGF and SIF were 1.2 and 7.4, respectively. The stability of iron-chitosan microcapsules on the simulated gastrointestinal fluid for 8 hours digestion with the different core-to-wall ratios are depicted in Figure 5 and 6. The interest application of chitosan for drug or food delivery systems due to its biodegradable and biocompatible properties [21]. For this reason, it allows the burst release of Fe II and total Fe, in the first two hours, as shown in Figure 5 and 6. However, there was no significant Fe II release in the six hours later (Figure 5). In contrast with it, release cumulative of total Fe tends to increase (Figure 6). It indicates that the total of Fe III content in the SIF raised.

Gluconate (Fe II): Chitosan= 1:1 Gluconate (Fe II): Chitosan= 1:1.5 Gluconate (Fe II): Chitosan= 1:2

16.00% 12.00% 8.00% 4.00% 0.00% 0

1

2

3

4

5

6

7

8

9

Hours

FIGURE 5. The release profile of Fe II in the simulated gastrointestinal fluid for 8 hour digestion

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Release comulative of total Fe (%)

80.00% 60.00% 40.00% Gluconate (Fe total): Chitosan= 1:1 Gluconate (Fe total): Chitosan= 1:1.5 Gluconate (Fe total): Chitosan= 1:2

20.00% 0.00% 0

1

2

3

4

5

6

7

8

9

Hours

FIGURE 6. The release profile of total Fe in the simulated gastrointestinal fluid for 8 hour digestion

CONCLUSIONS Iron loaded chitosan microcapsules was prepared by extrusion method using sodium tripolyphosphate as a cross linking agent. The samples were characterized on its physical and stability properties. Both iron sources were highly encapsulated, ranging from 71.5% to 98.5%. Furthermore, the highest ferrous fumarate and ferrous gluconate loaded were 1.9% and 4.8%, respectively. About 1.04% to 9.17% and 45.17% to 75.19% of Fe II and total Fe, were released in simulated gastric fluid for two hours and in simulated intestinal fluid for six hours, respectively.

ACKNOWLEDGMENTS This research work was financially supported by Universitas Indonesia through the PITTA research scheme.

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