Iron microencapsulation with blend of gum arabic, maltodextrin and modified starch using modified solvent evaporation method – Milk fortification

Food Hydrocolloids xxx (2014) 1e7

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Iron microencapsulation with blend of gum arabic, maltodextrin and modified starch using modified solvent evaporation method e Milk fortification Chitra Gupta a, Prince Chawla a, Sumit Arora a, *, S.K. Tomar b, A.K. Singh c a b c

Dairy Chemistry Division, National Dairy Research Institute, Karnal, Haryana, 132001, India Dairy Microbiology Division, National Dairy Research Institute, Karnal, Haryana, 132001, India Dairy Technology Division, National Dairy Research Institute, Karnal, Haryana, 132001, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 May 2014 Accepted 19 July 2014 Available online xxx

Iron microcapsules were prepared with blend of gum arabic, maltodextrin and modified starch using modified solvent evaporation method. Process parameters were optimized for obtaining maximum encapsulation efficiency and stability of microcapsules. Effect of different concentration of alcohol, different ratio of mixture to absolute alcohol, different composition of wall material and different amount of iron salt on the encapsulation efficiency (EE) of iron microcapsules were evaluated. Microcapsules prepared with gum arabic, maltodextrin and modified starch in the ratio of 4:1:1 and mixture to absolute alcohol ratio 1:10 showed maximum encapsulation efficiency (91.58%) and stability. External morphology of iron microcapsules revealed slightly circular structure with minimum cracks and dents on the surface. Particle size as analysed by inverted light microscope was in the range of 6.84e33.42 mm. Iron microcapsules were added to milk and evaluated for sensory characteristics and oxidative stability. Sensory scores of iron salt fortified milk were significantly lower (P < 0.05) as compared to iron microcapsules fortified milk during storage, however, this difference could be observed only upto the 5th day of storage. Iron microcapsules fortified milk showed significantly higher (P < 0.05) in-vitro bioavailability of iron as compared to control (unfortified) and iron salt fortified milk. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Microencapsulation Gum arabic Maltodextrin Modified starch (HiCap 100) Iron fortification Milk

1. Introduction Iron is an essential trace element, naturally present in the structure of cytochrome, enzymes, haemoglobin and myoglobin (Abbasi & Azari, 2011) and stored in the form of ferritin and haemosiderin in liver (Allen, Benoist, Dary, & Hurrell, 2006). Its deficiency causes lower growth rate, impaired cognitive scores in children, poor pregnancy outcomes and lower working capacity in adults (Derbyshire, Brennan, Li, & Bokhari, 2010). In India, inadequate dietary intake of iron and low bioavailability of iron from foods are identified as major cause of anaemia. Among multiple strategies to control iron deficiency, food fortification is an effective measure to increase the intake of iron without causing a change in the existing dietary patterns (Tripathi & Platel, 2011). Milk and milk products are always good candidates for iron

* Corresponding author. Tel.: þ91 (0) 184 2259156 (O), þ91 9896054444 (M). E-mail addresses: [email protected] (C. Gupta), princefoodtech@gmail. com (P. Chawla), [email protected], [email protected] (S. Arora), [email protected] (S.K. Tomar), [email protected] (A.K. Singh).

fortification, not only due to world-wide consumption by all groups at risk of deficiency, but also because of their high nutritional value, absorption processes and positive effect on growth, cognition and morbidity (Boccio & Montiero, 2004). Iron fortification of milk seems to be an effective nutritional strategy to correct dietary iron deficiency (Bhawana et al., 2011). Adding iron directly to milk results in reaction with milk components (lipids and protein) and decreased bioavailability, along with development of organoleptic problems such as colour, odour and taste (Gaucheron, 2000). Microencapsulation technique has multitude applications and has been widely used to protect iron from oxidation by forming an impermeable membrane as barrier to oxygen diffusion, to mask the unacceptable flavour caused by iron salt and to increase bioavailability (De souza et al., 2013). Selection of an appropriate wall material and physicochemical properties of wall material are critical issues in governing the functionality of microcapsule systems (Sarkar, Gupta, Variyar, Sharma, & Singhal, 2013; Wang, Wang, Li, Adhikari, & Shi, 2011). Gum arabic, commercial exudate gum obtained from Acacia Senegal, is one of the most common wall material used in microencapsulation due to its low viscosity, good emulsifying, high

http://dx.doi.org/10.1016/j.foodhyd.2014.07.021 0268-005X/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Gupta, C., et al., Iron microencapsulation with blend of gum arabic, maltodextrin and modified starch using modified solvent evaporation method e Milk fortification, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.021

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stabilizing and film forming properties (Ali, Zaida, & Blunden, 2009; Sarkar et al., 2013). It has the ability of better retention of volatile substances and effective protection against oxidation (Gabas, Telis, Sobral, & Telis, 2007; Righetto & Netto, 2005). Gum arabic has a highly branched structure which results in compact spheroidal conformation (Tombs & Harding, 1998). Other wall materials used are modified starch (HiCap 100) and maltodextrin. Maltodextrins are acid or enzyme hydrolysed starch used for encapsulation of oils and flavouring compounds (Carneiro, Tonon, Grosso, & Hubinger, 2013). Maltodextrins have numerous properties that allow them to be used for diverse purposes in both the food and pharmaceutical industries (Toure, Zhang, Jia, & Dong, 2007). Their ability to retain water and form gels explains their choice as efficient food stabilizers (Chronakis, 1998). It has also reported that maltodextrins have been used as stabilizers in the microencapsulation of vitamins, minerals, colourants as well as fat and oils (Toure et al., 2007). However, the poor film-forming ability, hygroscopicity and turbidity of maltodextrins account for their inability to protect volatile compounds during spray drying (Raja, Sankarikutty, Sreekumar, Jayalekshmy, & Narayanan, 1989). Modified starch (HiCap 100) has good film forming properties. It has been used for microencapsulation of ascorbic acid and showed high retention of ascorbic acid during storage (Wijaya, Small, & Bui, 2011). Gums, starches, polysaccharides and proteins show good compatibility with gum arabic (Krishnan, Bhosale, & Singhal, 2005). Therefore, maltodextrin and modified starch (HiCap100) are often used as microencapsulating material with gum arabic to improve the encapsulation efficiency and binding property (Carneiro et al., 2013; Kanakdande, Bhosale, & Singhal, 2007; Krishnan et al., 2005; Shaikh, Bhosale, & Singhal, 2006; Vaidya, Bhosale, & Singhal, 2006). Zilberboim, Kopelman, and Talmon (1986) reported cold dehydration as an alternative to spray drying for encapsulation of highly volatile materials and microencapsulate paprika oleoresin and aromatic esters using alcohol as dehydrating liquid. Iron was microencapsulated using modified solvent evaporation method which involved alcohol as dehydrating medium. As soon as coat material comes in contact with the alcohol, gets dehydrated and resulted in the formation of microcapsules. Microcapsules were separated from the alcohol or dehydrating medium and residual alcohol was evaporated at low temperature (4e7  C). Higher temperature resulted in the disruption of coat material and leaking of the encapsulated material (Richmond & Moss, 1983). Limited attempts have been made so far to access the stability and encapsulation efficiency of iron microcapsules and in-vitro bioavailability of iron from these microcapsules prepared by modified solvent evaporation method using plant based exudate gums such as gum arabic, maltodextrin and modified starch as a coating. Therefore, present work was designed to evaluate the ability of gum arabic in combination with maltodextrin and modified starch (HiCap 100) as alternative materials for iron (ferrous sulphate) microencapsulation by modified solvent evaporation method and to minimize the iron induced oxidation and safe delivery of iron to consumers via milk fortification. Iron content, ratio of wall materials, concentration of dehydrating medium and ratio of mixture to dehydrating medium were also standardized for obtaining stable microcapsules. 2. Materials and methods 2.1. Materials Gum arabic (spray dried) (GA), maltodextrin (DE 16.5e19.5) (MD), iron salt (Ferrous sulphate hepta hydrate), cyclohexanone, thiobarbituric acid (TBA), ammonium sulphate, citric acid, sodium

hydroxide, phenolphthalein, ethanol and L-ascorbic acid were procured from Sigma Aldrich, St. Louis, USA. Chemicals used were of AR grade. Modified Starch (HiCap 100) (MS) was obtained from National Starch Chemicals Corporation, Mumbai, India. Fresh cow and buffalo milk were collected from cattle yard of National Dairy Research Institute, Karnal, India. a-amylase (EC 3.2.1.1), human pancreatic lipase (EC 3.1.1.3), colipase, cholesterol esterase (EC 3.1.1.13), phospholipase A2 (EC 3.1.1.4), mucin, bovine serum albumin, pepsin (2080 units mg1 of protein), pancreatin and taurocholate salts were purchased from Sigma Chemical Co (Madrid, Spain). Cellulose dialysis membranes (flat width, 25 mm; internal diameter, 16 mm; molecular weight cut-off, 12,000e14,000 Da) were procured from Himedia Laboratories Pvt., Ltd., Mumbai, India. Milli Q water (10 mS) and acid washed glassware were used throughout the experiments. 2.2. Methods 2.2.1. Preparation of microcapsules Microcapsules were prepared by dissolving a blend of GA, MD and MS in the ratio of 4:1:1 (6 g) in 10 mL deionized water at 60  C and kept it for rehydration under refrigerated condition (4e7  C) for 12 h. Iron salt (ferrous sulphate) and ascorbic acid (used as antioxidant) in the ratio of 15:1 were dissolved in 10 mL deionized water and added to rehydrated blend solution. Ferrous sulphate hepta hydrate has been used as core material for preparation of iron microcapsules as it is the cheapest iron source with high iron bioavailability. Solution of core and coat materials were mixed well and kept in water bath maintained at 5  C, followed by sonication using a probe sonicator (Model VCx750, Sonics and Materials Inc., New Town, USA) at 5  C with 5.0 s pulse rate for 15 min. During sonication, proper mixing of core and coat materials occur. Iron may interact with the carboxylic group of gum arabic and forms the ionic/electrostatic bond. This mixture was then sprayed in chilled alcohol. It was kept on a magnetic stirrer and stirred at 500 rpm. Airless paint sprayer (Wagema Professional Quality, Pretoria, South Africa) was used for spraying of the mixture in chilled alcohol which was operated at 1.5 kg/cm2. After spraying, it was left undisturbed for 5 min. Finally, filtration was carried out with filter paper (Whatman No. 1) using vacuum filtration assembly. Microcapsules retained on the filter paper had residual amount of alcohol which was easy to evaporate at low temperature. The retentate was spread in a petridish and stored at 4e7  C for 12e14 h for complete removal of alcohol. Powdered microcapsules were stored in air tight glass containers at room temperature. This method has an advantage over other methods, since the solvent can be reused by distillation of filtered alcohol. 2.2.1.1. Optimization of process parameters. Effect of different concentration of alcohol, different ratio of mixture to absolute alcohol, different composition of wall material and different amount of iron salt on the encapsulation efficiency (EE) of iron microcapsules were evaluated. 2.2.1.1.1. Concentration of alcohol. Different concentrations of chilled alcohol (80%, 90% and absolute alcohol) were used for the spraying of mixture and its effect on EE of iron microcapsules was evaluated. 2.2.1.1.2. Ratio of mixture to absolute alcohol. Mixture prepared after sonication was sprayed in different amount of alcohol. Amount of chilled alcohol was varied according to the ratio of mixture to absolute alcohol. Different ratios of mixture to absolute alcohol (1:5, 1:7.5 and 1:10) were used for preparation of iron microcapsules and its effect on the EE of iron microcapsules was evaluated.

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2.2.1.1.3. Composition of wall material. Various compositions of wall materials were used for the preparation of microcapsules. GA, MD and MS in the ratio of 4:1:1, 1:4:1, 1:1:4, 2:2:2, 3:2:1, 3:1:2, 6:0:0 were used and evaluated for its effect on EE. 2.2.1.1.4. Amounts of iron salt. Different amounts of iron salt (300, 500, 800 and 1000 mg) were used for the preparation of iron microcapsules and its effect on EE was evaluated. 2.2.2. Estimation of iron content Iron content of microcapsules was estimated by atomic absorption spectrophotometer (AAS) (AA-7000, Shimadzu, Tokyo, Japan) using the method of AOAC (2005). Microcapsules were subjected to ashing (550  C for 8 h), solubilized in tri acid mixture (HNO3:HClO4:H2SO4 in 3:2:1 ratio) and heated for complete dissolution. Samples were diluted suitably for analysis by AAS at lmax 248.3 nm. 2.2.3. Encapsulation efficiency (EE) During preparation, iron microcapsules were separated from alcohol by filtration. It was assumed that iron in the retentate was in encapsulated/bound form. Non encapsulated iron was present in the filtrate. Retentate was dried under refrigerated conditions (4e7  C) for 12e14 h. Encapsulated iron in retentate was estimated using AAS. Iron content which was added initially for the preparation of iron microcapsules was considered as total iron content and bound iron was estimated in the retentate. Filtrate contained majorly alcohol which was volatile and difficult to measure volumetrically and gravimetrically, therefore, bound iron was measured in the retentate. EE was calculated as follows:

Encapsulation efficiency ¼

Bound iron  100 Total iron

2.2.4. Scanning electron microscopy The external structure of powdered microcapsules was examined by scanning electron microscopy (Carl Zeiss EV018, 18th edition, Cambridge, UK). Microcapsules were attached on scanning electron microscopy stub by double coated adhesive tape. Mounted samples were coated with gold (20 nm thickness) on ion coater (Eiko IB3, Tokyo, Japan) at 0.05e0.07 torr for 4 min maintaining the ion current at 6 mA. Samples were finally examined by scanning electron microscopy at an acceleration voltage of 15 KV under high vacuum (9.0*105 torr) and micrographs were recorded. 2.2.5. Size analysis of iron microcapsules Microcapsules were kept on the glass slides and dispersed in one or two drops of distilled water. Distilled water is used for proper spreading of the microcapsules on the glass slide. These were observed (magnification 400-fold) under an inverted light microscope (Nikon Eclipse Ti, Tokyo, Japan) and photographed using a fitted digital camera (Nikon Digital Sight, Tokyo, Japan). Size of the microcapsules was measured with the help of inbuilt software (Nikon Basic Research Imaging Software (v 3.1)) with microscope. 2.2.6. Fortification in milk Fresh cow and buffalo milk were mixed in the ratio of 1:1 and standardized to the fat and solid not fat (SNF) percent of toned milk. Toned milk (3% fat and 8.5% SNF) was fortified with iron salt (ferrous sulphate at 25 ppm iron) and iron microcapsules (@25 ppm iron). Addition of iron salt and iron microcapsules was accompanied by mixing for 10 min with the help of magnetic stirrer for

3

complete dissolution of fortificants. After mixing, the milk samples were pasteurized at 63  C for 30 min followed by cooling to 4  C. 2.2.7. Sensory evaluation of fortified milk Prepared milk samples were evaluated by the sensory panelists. Sensory panel of ten trained judges from the institute were asked to grade fortified milk samples for changes in colour and appearance, odour, taste and mouthfeel. Composite score card for sensory analysis of milk as approved by BIS (IS: 7768, 1975) was used. In taste characteristics, the main focus was on metallic, rancid and oxidized taste. Other mentioned taste characteristics e.g. cowy, acidic, astringent, bitter, cooked, flat, foreign, malty, salty, barny etc. were excluded. The sensory booth environment was held at a constant temperature (20  C), red lighting was used to obscure any colour differences between the samples and a positive airflow removed any odour from the testing area. Saline water (0.89% sodium chloride solution) (at room temperature) was provided as palate cleanser for rinsing mouth and cleaning the tongue before sensory evaluation of each sample. 2.2.8. Thiobarbituric acid (TBA) value of fortified milk TBA value of milk samples were evaluated by the method described by Hegenauer, Saltman, Ludwig, Ripley, and Bajo (1979). In this method, TBA reagent was prepared immediately before use by mixing equal volumes of freshly prepared 0.025 M TBA (neutralized with NaOH) and 2 M H3PO4/2 M citric acid. Reaction was terminated by mixing 5.0 mL of milk sample with 2.5 mL TBA reagent into a glass centrifuge tube. The mixture was heated immediately in boiling water bath for exactly 10 min and then cooled on ice. 10 mL cyclohexanone and 1 mL of 4 M ammonium sulphate were then added and centrifuged (Kubota Corporation, Gyeonggi-do, South Korea) at 5000 g for 5 min at room temperature. The orange-red cyclohexanone supernatant was decanted and its absorbance was measured at 532 nm using spectrophotometer (SPECORD 200 Analytik Jena, Jena, Germany) in a 1 cm light path. 2.2.9. In-vitro bioavailability of iron In-vitro bioavailability of iron was determined by simulated gastro-intestinal model as described by Herrero-Barbudo, Granado-Lorencio, Blanco-Navarro, and Perez-Sacristan (2009) with slight modification. Compositions and concentrations of inorganic and organic solutions, saliva, gastric juice, duodenal juices and bile constituents were carefully duplicated as described by (Granado-Lorencio et al., 2007). Control (unfortified), iron salt and iron microcapsules fortified milk were assessed for bioavailability of iron. 2.2.9.1. Preparation of membrane. Cellulose dialysis membrane were poured in deionized water and boiled for 5 min in deionized water before use. 2.2.9.2. In-vitro digestion model. Approximately, 15 ml milk samples (control, iron salt and iron microcapsules fortified) were transferred to a flask and saliva solution (9 ml, pH 6.5) containing organic and inorganic components and a-amylase (700 mg/L of saliva solution) was added after which the samples were incubated in a shaking water bath at 37  C, 95 rpm for 5 min. Gastric juice (13.5 ml) with organic and inorganic solutions, mucin (6 g/L of gastric juice), bovine serum albumin (2 g/L of gastric juice), and pepsin (2 g/L of gastric juice) from porcine stomach was added. pH was adjusted to 1.1 with HCl and the solution was incubated for 1 h at 37  C. Duodenal juice (25 ml, organic plus inorganic solutions containing porcine pancreatin 6 g/L of duodenal juice) and bile solutions (9 ml, containing bile

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salt 12 g/L of bile solution), prepared on the day of assay, were introduced after neutralization of the pH (7.8) and the human pancreatic lipase (5 units), colipase (25 mg), cholesterol esterase (10 units), phospholipase A2 (50 ml) and taurocholate salts (39.8 mg) were added. Final volume was approximately 70 ml, the mixture was then incubated up to 3 h at 37  C. Dialysis sac was formed by closing the one end of dialysis tube with membrane closure, the digested contents thus obtained were poured in this dialysis sac and sac was closed from opposite side also. The contents along with this dialysis sac were dipped in distilled water kept in a 1000 ml beaker for 16 h at 37  C. Iron content in the retentate was determined by AAS to estimate the digestibility of the added nutrient under simulated gastro-intestinal conditions. Bioavailability of nutrient (iron) was calculated from the amount of the nutrient (iron) that had passed the dialysis membrane proportional to the total nutrient (iron) content of the sample. Bioavailability was calculated as:

Bioavailability ð%Þ ¼ D=C  100 where, D ¼ Iron content in the dialysate and C ¼ Iron content of sample 2.2.10. Statistical analysis Means and standard error mean (SEM) were calculated using Microsoft Excel, 2007 (Microsoft Corp., Redmond, WA). Significant

difference between values was verified by one way or two way analysis of variance and comparison between means was made by critical difference value (Snedecor & Cochran, 1994).

3. Results and discussion 3.1. Optimization of preparation process Process was optimized by varying one parameter and keeping the others constant. The ratio of GA, MD, MS as 4:1:1, iron salt (300 mg) and ascorbic acid (20 mg) in the ratio of 15:1 were dissolved in 20 mL deionized water and absolute alcohol was used as a dehydrating medium in the ratio of mixture to absolute alcohol 1:10 for optimization of process parameters. Different concentrations of alcohol, 80%, 90% and absolute alcohol were used as dehydrating medium (Fig. 1A). Absolute alcohol showed maximum EE as compared to 80% and 90% alcohol and hence was the most suitable dehydrating medium required for dehydration of microcapsules. Our results were in agreement with Zilberboim et al. (1986) who observed that the reduction in alcohol concentration reduced the retention capacity of the microcapsules, as lower concentration resulted in slow release of water from the microcapsules and longer dehydration time. Lower concentration of alcohol also resulted in particle agglomeration into sticky mass which was difficult to filter. The ratio of mixture to absolute alcohol was varied as 1:5, 1:7.5 and 1:10 for the preparation of microcapsules with maximum EE (Fig. 1B). 1:10 resulted in highest EE as compared to 1:5 and 1:7.5.

Fig. 1. Encapsulation efficiency of microcapsules as affected by (A) Different concentration of alcohol, (B) Different ratio of mixture to alcohol ratio, (C) Composition of gum arabic (GA), maltodextrin (MD) and modified starch (MS) & (D) Iron concentration. aebSamples represented with different letters are significantly different (P < 0.05) from each other. Error bars show the variations of three determinations in terms of standard error of mean.

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Fig. 2. (A) Scanning electron microscopy (500) and (B) light microscopic (400) observation of 300 mg iron salt containing microcapsules.

EE significantly decreased (P < 0.05) with decreasing concentration of alcohol. 1:10 mixture to alcohol ratio was the most suitable for microencapsulation of iron. Our results were in agreement with Zilberboim et al. (1986) who reported that alcohol to emulsion ratio below 10:1 reduced the retention efficiency of the microcapsules. However, at higher concentration it reduced the water content without affecting the retention efficiency as higher ratio of alcohol to emulsion resulted in rapid drying of microcapsules and formed a protective crust that prevented the extensive leaching of core material. Cho and Park (2003) also evaluated the process parameters for oil in water in oil (O/W/O) multiple emulsion method for flavour encapsulation and reported that increase in the GA content resulted in more stable emulsion and highest flavour retention (71%) was observed with ethanol to mixture ratio of 9:1 as a dehydrating agent. Absolute alcohol and ratio of mixture to absolute alcohol 1:10 showed highest EE. Therefore, keeping these two conditions constant, iron microcapsules were prepared with different ratios of GA, MD and MS and evaluated for EE (Fig. 1C). Ratio of 4:1:1 showed highest EE as compared to all other microcapsules and a ratio of 6:0:0 showed significantly lower (P < 0.05) EE as compared to 4:1:1. Hence, 4:1:1 ratio of GA, MD, and MS was most suitable for microencapsulation of iron. EE significantly decreased (P < 0.05) with decreasing content of GA. Our results were in accordance with Kanakdande et al. (2007) and Krishnan et al. (2005) who observed that a blend of GA, MD and MS in the ratio of 4:1:1 gave better results as compared to 100% GA. Microcapsules prepared with GA, MD and MS in the ratio of 1:4:1 and 1:1:4 form a sticky mass after spraying in alcohol, therefore, which was difficult to separate from alcohol as they blocked the pores of filter paper (Whatman No. 1). This might be due to the high water holding capacity of these two wall materials. The resulting microcapsules showed lower EE as compared to others, therefore, 0:6:0 and 0:0:6 ratio of GA, MD and MS were not tried for preparation of iron microcapsules. It was also evident from the above results that MS enhanced the EE more as compared to the MD. Finally, iron content was optimized by adding 300 (60 mg iron), 500 (100 mg iron), 800 (160 mg iron) and 1000 (200 mg iron) mg iron salt to the selected blend of GA/MD/MS (4:1:1) and mixture to absolute alcohol ratio (1:10) for stable microencapsulation (Fig. 1D). Iron microcapsules containing 300 mg showed highest EE as compared to all other microcapsules. EE significantly decreased (P < 0.05) with increasing iron concentration. Iron microcapsules prepared by optimized process containing 300 mg iron was further subjected for scanning electron

microscopy and particle size analysis and also used for fortification of milk. 3.2. Scanning electron microscopy and particle size analysis Scanning electron microscopy was used to evaluate morphological structures of microcapsules obtained from GA/MD/MS (4:1:1) and mixture to absolute alcohol ratio (1:10) showed slightly circular, uniform and minimum cracks and dents on the surface of microcapsules (Fig. 2A). Spherical shape results in maximum stability due to minimum surface area to volume ratio. Microcapsules were not as spherical as we obtain in the spray drying technique. In spray drying, immediate dehydration of microcapsules occur which resulted in proper spherical shape of microcapsules. However, in modified solvent evaporation method slow dehydration of microcapsules occur which resulted in the slight disruption of the spherical structure. Kanakdande et al. (2007), Krishnan et al.

Table 1 Effect of iron fortification (@25 ppm of iron) on sensory scores of milk during storage at 4e7  C. Characteristics

Storage Control (unfortified) time (days)

Colour and 0 appearance (10) 3 5 7 Odour (20) 0 3 5 7 Taste (40) 0 3 5 7 Mouthfeel (30) 0 3 5 7 Total (100) 0 3 5 7

9.18 8.86 8.56 8.40 18.24 17.30 16.30 16.50 36.00 36.00 34.20 32.80 27.50 27.20 26.60 25.90 90.92 89.36 85.66 83.60

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.111dB 0.098cB 0.040bC 0.100aB 0.112cC 0.122bC 0.374aC 0.224aB 0.632cA 0.447cC 0.374bC 0.374aC 0.224cA 0.374cB 0.245bB 0.245aB 0.637dC 0.806cC 0.398bC 0.660aC

Iron salt fortified 8.96 8.60 8.22 8.28 15.70 15.30 15.40 15.10 35.80 31.80 30.20 27.40 27.20 26.40 25.20 24.10 87.66 83.10 79.02 74.88

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Iron microcapsules fortified 0.040cA 0.100bA 0.136aA 0.116aA 0.255cA 0.122bA 0.292bA 0.332aA 0.663dA 0.374cA 0.374bA 0.678aA 0.123dA 0.400cA 0.374bA 0.400aA 0.430dA 0.400cA 0.510bA 0.441aA

9.10 8.66 8.42 8.30 17.50 16.60 15.90 15.20 36.20 34.20 31.20 27.80 27.20 26.90 25.00 24.40 90.00 86.36 80.52 75.80

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.100dB 0.093cA 0.107bB 0.122aA 0.158dB 0.187cB 0.292bB 0.255aA 0.735dA 0.374cB 0.374bB 0.663aA 0.123cA 0.100cB 0.548bA 0.224aA 0.837dB 0.427cB 0.648bB 0.490aB

Data are presented as means ± SEM (n ¼ 10). aeb Means within columns with different lowercase superscript are significantly different (P < 0.05) from each other. AeB Means within rows with different uppercase superscript are significantly different (P < 0.05) from each other.

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Table 2 Effect of iron fortification on TBA value of milk during storage at 4e7  C. Milk samples

0th day

3rd day

5th day

7th day

Control (unfortified) Iron salt fortified Iron microcapsules fortified

0.016 ± 0.0006aA 0.017 ± 0.0007aA 0.017 ± 0.0003aA

0.029 ± 0.0009aB 0.061 ± 0.0012cB 0.043 ± 0.0012bB

0.049 ± 0.0009aC 0.083 ± 0.0009cC 0.076 ± 0.0012bC

0.062 ± 0.0009aD 0.103 ± 0.0009bD 0.101 ± 0.0009bD

Data are presented as means ± SEM (n ¼ 3). aeb Means within columns with different lowercase superscript are significantly different (P < 0.05) from each other. AeB Means within rows with different uppercase superscript are significantly different (P < 0.05) from each other.

(2005) and Vaidya et al. (2006) observed the microcapsules prepared by spray drying method using scanning electron microscopy and found that GA, MD and MS in the ratio of 4:1:1 gave spherical microcapsules with smooth surface. Microcapsules from GA alone were found to be nearly spherical but had many dents on the surface, whereas the microcapsules obtained from MD and MS were partially disrupted. Average particle size of microcapsules was observed as 15.54 mm (range 6.84e33.42 mm) by light microscope (Fig. 2B). 3.3. Milk fortification, sensory evaluation and oxidative changes Three type of milk samples i.e. control (unfortified), iron salt and iron microcapsules fortified (@25 ppm of iron) milk were prepared. Colour and appearance, odour and taste scores of iron salt and iron microcapsules fortified milk samples were significantly different (P < 0.05) from each other upto the 5th day of storage whereas mouthfeel scores were significantly different (P < 0.05) upto the 3rd day of storage (Table 1). Total sensory scores of iron salt and iron microcapsules fortified milk samples were slightly but significantly different (P < 0.05) from each other upto the 7th day of storage. TBA value of iron microcapsules fortified milk was significantly lower (P < 0.05) than iron salt fortified milk upto the 5th day of storage (Table 2). Iron salt fortified milk showed highest TBA value due to the presence of free iron, however, iron microcapsules fortified milk showed lower value due to presence of bound iron. These microcapsules slowly release the iron in the milk, therefore, TBA value of milk increases.

microcapsules fortified milk. Iron microcapsules are less affected by the presence of inhibitors, therefore resulting in high iron bioavailability (Olivares, 2002). 4. Conclusion Stable iron microcapsules were successfully prepared with a blend of GA/MD/MS using modified solvent evaporation method. GA/MD/MS (4:1:1) and mixture to absolute alcohol ratio (1:10) proved to be a superior microcapsules composition for iron microencapsulation with EE of 91.58%. External morphological characteristics revealed circular and uniform structure with minimum cracks and dents on the surface of iron microcapsules. The average particle size of the microcapsules was 15.54 mm. These iron microcapsules were added to milk and showed significant difference in total sensory scores from iron salt fortified milk during storage. TBA value of iron microcapsules fortified milk was significantly lower (P < 0.05) than milk fortified with iron salt upto the fifth day of storage. Iron microcapsules fortified milk showed significantly higher (P < 0.05) in-vitro bioavailability of iron as compared to control (unfortified) and iron salt fortified milk. The results of this study revealed the potential of GA, MD and MS for use as a superior and convenient wall material for iron microencapsulation. Acknowledgement The author would like to acknowledge National Starch Chemicals Corporation, Mumbai, India for providing modified starch (HiCap 100) used during the study.

3.4. In-vitro bioavailability of iron In-vitro bioavailability of iron was determined for three types of milk i.e. control (unfortified), iron salt and iron microcapsules fortified (@25 ppm of iron) milk under simulated gastro-intestinal conditions. All the three samples showed significant difference (P < 0.05) in iron bioavailability (Table 3). Iron microcapsules fortified milk showed significantly higher (P < 0.05) in-vitro bioavailability of iron as compared to control (unfortified) and iron salt fortified milk. Control (unfortified) milk showed lowest iron bioavailability (%), might be due to the strong binding of iron with casein which makes it insoluble in the gastro-intestinal conditions (Jackson & Lee, 1992). Iron salt fortified milk also showed significantly lower (P < 0.05) iron bioavailability as compare to iron Table 3 In-vitro bioavailability of iron from control (unfortified), Iron salt and iron microcapsules fortified milk. Milk samples

% Bioavailability

Control (unfortified) Iron salt fortified Iron microcapsules fortified

19.86 ± 0.23a 54.31 ± 0.36b 63.78 ± 0.23c

Data are presented as means ± SEM (n ¼ 3). aeb Means within columns with different lowercase superscript are significantly different (P < 0.05) from each other.

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Please cite this article in press as: Gupta, C., et al., Iron microencapsulation with blend of gum arabic, maltodextrin and modified starch using modified solvent evaporation method e Milk fortification, Food Hydrocolloids (2014), http://dx.doi.org/10.1016/j.foodhyd.2014.07.021