Powder Technology 286 (2015) 527–537

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Development and physico-chemical characterization ofmicroencapsulated flaxseed oil powder: A functional ingredient foromega-3 fortification

Ankit Goyal a,⁎, Vivek Sharma a, Manvesh Kumar Sihag a, S.K. Tomar b, Sumit Arora a, Latha Sabikhi c, A.K. Singh c

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

⁎ Corresponding author.E-mail address: ankit.ndri@gmail.com (A. Goyal).

http://dx.doi.org/10.1016/j.powtec.2015.08.0500032-5910/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 December 2014Received in revised form 20 August 2015Accepted 31 August 2015Available online 2 September 2015

Keywords:Flaxseed oilMicroencapsulationPhysico-chemical characterizationMicroencapsulation efficiencyWhey protein concentrateOxidative stability

The objective of the study was to develop and characterize highly polyunsaturated flaxseed oil powder whichcould serve as a potential delivery system of omega-3 fatty acids in vegan diet. Three formulations of oil-in-water emulsions containing flaxseed oil (≥35% on dry basis), whey protein concentrate (WPC)/sodium caseinate(NaCas) and lactose were prepared, homogenized and spray dried for further physico-chemical analyses. Devel-oped flaxseed oil powder was characterized for moisture content, water activity, particle size distribution,flowing characteristics, dissolution behavior, surface characteristics, oxidative stability and oil release behaviorunder simulated gastro-intestinal conditions. The results revealed that moisture content and water activitywere in the range of 3–4% and 0.346–0.358, respectively, which is suitable for long term storage of powders.Particle size distribution profile showed poly-dispersed nature with mean droplet diameter (d4,3) in the rangeof 5.82 to 10.01 μm. Scanning electronmicrograph ofmicrocapsules showed spherical shapeswithout any appar-ent fissures on surfaces. Peroxide value (PV) indicated high oxidative stability ofmicroencapsulated oil at the endof sixmonths storage at room temperature (35±1 °C). Preparedflaxseedoil powderwas fortified (at 1% level) inmarket milk, which showed sensory characteristics comparable to control (p b 0.05) for up to 5 days of storage.It can be concluded that flaxseed oil could be stabilized at higher concentration which can also be used as afortifying agent inmilk formeeting the nutritional requirements ofω−3 fatty acids in vegan and non-fish eatingmeat eaters' diet.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

Omega-3 (ω−3) fatty acids are essential fatty acids, which are pri-marily restricted to sea foods and some vegetable oils (such as flaxseedoil, canola oil). Several previous investigations have suggested thatω−3 fatty acids are critical for appropriate growth and developmentin humans. As such, it is not surprising that ω−3 fatty acids have alsobeen implicated in the prevention of systemic diseases and syndromesincluding coronary heart diseases, hypertension, hypercholesterolemia,cancer (including colon, breast and prostate), inflammatory boweldiseases, diabetes and neurodegenerative disorders [1–3]. However, de-spite their essential roles in human growth, dietaryω−3 fatty acids aremostly limited to sea-food consuming population only. Therefore, bothvegetarians and non-fish eating populations are apparently at a disad-vantage of not receiving appropriate levels of ω−3 in their generaldiet. On the contrary, the consumption of ω−6 rich refined vegetableoils (such as soybean, sunflower and groundnut oil) among general

population has gradually increased in the last two decades resulting indisruption of ω−6:ω−3 metabolic homeostasis. Furthermore, it isalso speculated that higher levels of ω−6 in regular diet could be oneof the major factors responsible for increased risk of cardiovascularand inflammatory disorders. According to a report by World HealthOrganization, cardiovascular diseases were responsible for over 17.3million deaths in the year 2008, representing approximately 30% ofglobal mortality in the world [4]. Therefore, it is strongly believed thatadequate ω−6:ω−3 ratio could be beneficial to health because of in-creased antioxidant, anti-inflammatory and anti-arrhythmic functions.This scenario further gains importance asω−3 fatty acids have limitedaccessibility as well as suitability owing to diverse socio-geographicalconstraints of the consumers. Thus it is not surprising that nutritionalstrategies aiming at development of ω−3 rich novel functional foodshave gained prime importance among nutritionists worldwide.

Flaxseed (Linum usitatissimum) oil is the richest vegetarian source ofω−3 fatty acid, comprising 50–60% α-linolenic acid (ALA, C18:3).Although available widely, flaxseed is not preferred as it is prone tooxidation due to high levels (~75%) of polyunsaturated fatty acids(PUFAs), resulting in production of off-flavors and toxic peroxides on

528 A. Goyal et al. / Powder Technology 286 (2015) 527–537

heating [5,6]. Therefore, despite being the richest and prevalent sourceof ω−3 fatty acids, flaxseed oil continues to remain potentially un-tapped for fulfilling the ω−3 nutritional requirements in humans.Thus, strategies aimed at stabilization and fortification of flaxseed oilcould have profound influence in developing novelω−3 rich functionalingredients and foods. Previous studies have attemptedmicroencapsula-tion of flaxseed oil (9–20%) which however, could not yield satisfactorylevels of ω−3 in the final product [7–9]. Other researchers have alsoworked on the microencapsulation of omega-3 oils (flaxseed, chia andfish oil) but from a different point of view. Tontul and Topuz [10] studiedthe influence of emulsion composition and ultrasonication time on flax-seed oil powder properties and concluded that maltodextrin-WPC wasthe most successful combination for microencapsulation of flaxseed oil.Ixtaina et al. [11] worked onmicroencapsulation of chia oil using sodiumcaseinate and lactose and investigated the influence of operatingconditions (homogenization pressure and spray-drying inlet/outlettemperatures) on physico-chemical characteristics of chia oil powder.Goyal et al. [12] studied the effect ofmicroencapsulation on the oxidativestability and in vitro release behavior of flaxseed oil and concluded thatmilk proteins could successfully protected the highly polyunsaturatedflaxseed oil. By keeping all these factors in consideration, themain objec-tive of the study was to microencapsulate and stabilize high amount offlaxseed oil (≥35% on dry basis) using whey protein concentrate-80,sodium caseinate and lactose so as to develop a potentially efficientdelivery system of ω−3 fatty acids in vegetarian and non-fish eatingmeat eaters' diet. The other objective was to investigate in-vitro releasebehavior of flaxseed oil from microcapsules under simulated gastro-intestinal conditions. The stabilized flaxseed oil powder was furtherutilized for fortification of milk followed by its organoleptic evaluation.A milk based delivery system was preferred owing to its widespreadavailability among general population.

2. Materials and methods

2.1. Materials

Refined flaxseed oil was procured from Kamani Oil Industries Pvt.Ltd., Khopoli, Maharashtra, India. Sodium caseinate and whey proteinconcentrate (WPC)-80 (Davisco, USA) were purchased from Ace Inter-national LLP, New Delhi, India. Whey protein concentrate (WPC) wasclaimed to contain 82.5% protein (on dry basis), 6.4% fat, 0.2% moisture,7.5% lactose and 2.4% ash content. Lactose was purchased from FischerScientific, Mumbai, India; and antioxidants were purchased fromSigma-Aldrich, Germany. Other chemicals were of analytical grade andwere purchased from Sigma-Aldrich, Germany and Himedia, Mumbai,India.

2.1.1. Qualitative determination for the presence of antioxidant in flaxseedoil by thin layer chromatographic (TLC) method

Before starting the experiments, flaxseed oil was qualitativelyanalyzed for the presence/absence of any synthetic antioxidants (BHA,BHT and TBHQ) through thin-layer chromatographic method. Tocheck the presence of antioxidants, first each of the synthetic antioxi-dants was added in flaxseed oil at 100 ppm concentration. Then theantioxidants were extracted from the flaxseed oil (antioxidants addedand sample) by AOAC [13] method. The developing glass chamber wassaturated with petroleum ether:benzene:glacial acetic acid::2:2:1solvent system. Six microliter extract solution was applied along withstandards on TLC plate coated with silica gel G (Merck, Germany). Theplate was developed to a distance of 15 cm, then left for air dryingand finally sprayed with Gibb's reagent. The plate was re-dried at103 ± 2 °C for 5 min. Color and position of spots (bands) were com-pared with the standards. Chromatogram indicated no bands in flax-seed oil used corresponding to the bands of BHA, BHT and TBHQ(chromatogram is not shown here). This TLC profile of the flaxseed oil

clearly suggested that there was no synthetic antioxidant present inthe oil used in the study.

2.1.2. Preparation of emulsions followed by spray dryingThree formulations of emulsions were used to encapsulate flaxseed

oil (Table 1). These formulations were made to obtain a high solid con-tent in O/W emulsions (32.5% on wet basis) and to accommodate highcontent of flaxseed oil (~35% w/w, on dry basis) in the final spraydried powder. In the first formulation (FO/WPC), lactose and WPCwere mixed in distilled water followed by addition of flaxseed oil. Inthe second formulation (FO-AO/WPC), ascorbyl palmitate was mixedin flaxseed oil. This mixture was further added to the pre-mixed solu-tion of the dry-ingredients. In the third formulation (FO/NaCas), sodiumcaseinatewasdissolved inwarmwater (55±5 °C) followedby additionof lactose and flaxseed oil.

All the formulations were mixed well and homogenized at lowpressure [69 bar @ 20 l per hour (LPH)] to obtain coarse emulsions,followed by high pressure homogenization (241.31 bar @ 20 LPH;Goma Engineering Pvt. Ltd., Thane, India). The emulsions were spraydried by single stage spray drier with 10 kg/h capacity (SSP Pvt. Ltd,Faridabad, India) equipped with rotary atomizer. The rotational speedand diameter of the wheel were ~15,000 revolutions per minute (rpm)and 12 cm, respectively. Spray dryer was operated in co-current mannerwith airflow rate of 450m3/h. The tower height and inner diameter of thespray dryer were 4 and 2m, respectively. Emulsions were pumped to thespray drier at a flow rate of 40 mL/min at room temperature (30–35 °C).The inlet and outlet temperatures of spray dryer were maintained at170 ± 1 °C and 75 ± 1 °C, respectively. The developed spray dried prep-arations [microencapsulated flaxseed oil powder (MFOP)] were packedseparately in an aluminum foil pack (thickness: 80–100 μm) and storedat room temperature (30–35 °C) for further analysis.

2.1.3. Physico-chemical characterization of flaxseed oil microcapsules

2.1.3.1. Moisture content andwater activity (aw). Themoisture content offlaxseed oil powder was determined by a Halogen moisture analyzer(WENSAR, HMB 100, Bengaluru, India) standardized at 108 °C for5 min. Each analysis was performed in triplicates. Water activity (aw)of MFOP was measured using water activity analyzer (AQUA Lab Pre,Decagon Devices, WA, USA) at a temperature of 35 °C. The instrumentwas calibrated first with charcoal powder at 35 °C.

2.1.3.2. Bulk (ρB) and tapped (ρT) density. For bulk (ρB) density, eachpowder sample (2 g) was filled in 25 mL measuring glass cylinder(diameter 2.5 cm) and the cylinder was slightly tapped to remove thepowder sticking to the walls. The volume (Vo) was read directly fromthe cylinder and bulk density was calculated by using following formula(Eq. (1)). For tapped density (ρT), the cylinder was tapped manuallyapproximately 50 times on marble solid surface from a height of10 cm to measure final volume (Vn) of the powder (Eq. (2)):

Bulk density ρBð Þ ¼ m=Vo ð1Þ

Tapped density ρTð Þ ¼ m=Vn: ð2Þ

2.1.3.3. Flowing properties. The flowing characteristics of MFOP wereevaluated by using Carr's index (Compressibility Index: C) and Hausnerratio (HR) by themethod given by Turchiuli et al. [15]. Carr's index indi-cates the compressibility or free-flowing property; while HR indicatesthe cohesiveness of powder. The Carr's index (C) and Hausner ratio(HR) were calculated using bulk density and tapped density by thefollowing equations (Eqs. (3) and (4)):

Carr0s index Cð Þ ¼ Tapped density ρTð Þ−Bulk density ρBð ÞTapped density ρTð Þ � 100 ð3Þ

Table 1Composition of fresh emulsions prepared for spray drying.

Formulation (emulsion) Flaxseed oil (%) WPC-80 (%) Sodium caseinate (%) Lactose (%) Antioxidant (ascorbyl palmitate) (ppm) Distilled water (%)

FO/WPC 12.5 10.0 – 10.0 – 68.5FO-AO/WPC 12.5 10.0 – 10.0 200 68.5FO/NaCas 12.5 – 10.0 10.0 – 68.5

FO/WPC: flaxseed oil powdermicroencapsulatedwith whey protein concentrate-80 (WPC-80); FO-AO/WPC: antioxidant added flaxseed oil microencapsulatedwithWPC-80; FO/NaCas:flaxseed oil powder microencapsulated with sodium caseinate.

529A. Goyal et al. / Powder Technology 286 (2015) 527–537

Hausner ratio HRð Þ ¼ Tapped density ρTð ÞBulk density ρBð Þ : ð4Þ

2.1.3.4. Particle size distribution and average particle size of powder. Parti-cle size distribution profile wasmeasured byMalvern Mastersizer 2000(Malvern Instruments Ltd., UK). Powder sample (20 mg) was added to15 mL of distilled water at 25 °C and vortex mixed for 5 min to preparea homogeneous emulsion. Mean droplet size of reconstituted emulsionis expressed as d4, 3 and the particle size distribution curves areexpressed as % intensity vs particle size diameter.

2.1.3.5. Dissolution behavior. The dissolution behaviorwas spectrophoto-metrically measured on a UV–VIS spectrophotometer according to themethod of Millqvist-Fureby et al. [16]. Powder sample (30mg)was lay-ered on top of 3.0 mL water in a cuvette (5 × 1 × 1 cm3), and the in-crease in absorbance at 620 nm was recorded every min until theconstant reading was obtained.

2.1.3.6. Color (L*, a* and b* values). The color of spray dried MFOP wasmeasured using a “Colorflex” colorimeter and the results were expressedin terms of the CIELAB system. The light source was dual beam xenonflash lamp. Data is expressed in terms of L* (lightness): ranging from 0(black) to 100 (white), a* (redness): ranging from +60 (red) to −60(green), and b* (yellowness) ranging from+60 (yellow) to−60 (blue)values. Powder sample (50 g) was spread homogeneously into a cleanand dry glass beaker (provided with the instrument: 6 cm height and6 cm diameter) and evaluated for the color values.

2.1.3.7. Morphology of microcapsules by scanning electron microscopy(SEM).Microstructure of spray dried flaxseed oil powder was evaluatedby Scanning Electron Microscopy (SEM) (ZEISS, EVO 18 Special Edition,Cambridge, UK) operating at 15 kV. Powder samples were subjected tometallization (sputtering) for 5 min with a thin layer of gold nano-particles (25 Å) in a sputter coater (EIKO, IB3 Ion coater, Japan) under0.07 Torr (mm Hg) vacuum and 7 mA current. After metallization, thesamples were observed with magnification of 1000, 2000 and 5000×.

2.1.3.8. Total oil & surface (free) oil. Total oil (TO) content was calculatedusing Soxhlet extraction method given by AOCS [17] with slightmodifications. In brief, 5 g powder was filled in extraction thimble(30 × 80 mm) (Merck, China) and oil was extracted by chloroform at50 °C for 2 h. After extraction of oil, the solvent was evaporated usingrotary flash evaporator under vacuum at 40 °C temperature and theextracted oil was weighed. Free or surface oil was calculated by themethod given by Hogan [40]. Hundred milliliters of petroleum etherwas added to 5 g of spray dried powder in a conical flask and mixedwell at room temperature for 15 min to extract the surface oil.The solvent mixture was filtered through Whatman filter paper 41.The powder collected on the filter paper was again rinsed with 50 mLpetroleum ether, which was mixed with previous filtrate. The filtratewas evaporated using rotary flash evaporator under vacuum at 60 °C.The free oil was weighed by the following formula (Eq. (5.0)):

Free oil %ð Þ ¼ wt: of beaker withoilð Þ−wt: of empty beakerwt: of sample gð Þ � 100:ð5:0Þ

2.1.3.9. Microencapsulation efficiency (ME %). Microencapsulationefficiency was calculated by the method given by Hogan [40] usingthe following Equation (Eq. (6.0)):

Microencapsulation efficiency ME%ð Þ ¼ 100� Totaloil−SurfaceoilTotaloil

:

ð6:0Þ

2.1.3.10. Peroxide value (oxidative stability). Flaxseed oil was extractedfrom the microcapsules using Soxhlet extraction method given byAOCS [17]. Peroxide value of extracted oil was measured spectrophoto-metrically by IDF (International Dairy Federation) standard method74A:1991 [18]. To determine PV, 0.3 g of extracted oil was mixed in aglass test tube with 9.8 mL chloroform:methanol (7:3, v/v) solution ona vortex mixer for 2–4 s. Fifty microliter mixture of ammonium thiocy-anate solution and Fe (II) solution (1:1) was added to the resulting so-lution and mixed on a vortex shaker for 2–4 s. After 5 min incubationat room temperature, the absorbance of the sample was determinedat 500 nm against a blank using a spectrophotometer. The entire proce-dure was conducted in subdued light and completed within 10 min.Peroxide value was calculated by the following equation (Eq. (7.0)):

Peroxide value meq:peroxides=kgoilð Þ ¼ As−Abð Þ �m55:84�m0� 2

ð7:0Þ

where, As: absorbance of the sample; Ab: absorbance of the blank;m = slope, obtained from the calibration curve (in this experiment,m was 41.52 for the IDF method); m0: mass in grams of the oiltaken; 55.84 = atomic weight of iron; The division by factor 2 is toexpress the peroxide value as milliequivalents of peroxide insteadof milliequivalents of oxygen.

2.1.3.11. In vitro release behavior of flaxseed oil microcapsules. In vitrorelease behavior of microencapsulated flaxseed oil was investigatedusing a simulated gastrointestinal model according to the methodgiven by Burgar et al. [19] with slight modifications. Simulated gastricfluid (SGF) was prepared by dissolving 2.0 g of NaCl and 7.0 mL of 36%HCl in 900mL of water. After the addition of 5.0 g of pepsin, the solutionpH was adjusted to 1.2 with 0.1 M HCl and the final volume was madeup to 1000mLwithwater. Simulated intestinal fluid (SIF) was preparedby dissolving 6.8 g of K2HPO4 in 800mL of water. To this solution, 77mLof 0.2MNaOH and 100.0 g of pancreatinwas added followed by stirringovernight at 4 °C. Solution pHwas adjusted to 6.8with 1MNaOHor 1MHCl and the final volume was made up to 1000 mL with water.

2.1.3.11.1. In-vitro release behavior of flaxseed oil from microcapsulesexposed to SGF conditions only. Flaxseed oil powder (5 g) was mixedwith 50 mL of distilled water followed by the addition of 50 mL SGFand incubated at 37 °C for 2 h. The contents were continuously mixedby using magnetic beads and magnetic stirrer. Released flaxseed oilwas extracted using 30 mL petroleum ether and 30 mL di-ethyl etherin a separating funnel. The extraction of oil was repeated two timeswith 40 mL of mixture of petroleum ether and di-ethyl ether (1:1).Solvent was evaporated at 80 °C and extracted oil was dried in hot airoven maintained at 100 ± 2 °C for 30 min and the quantity of releasedoil was determined gravimetrically. Total oil in the prepared flaxseed oil

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powder was 35% (measured by the chemical method). By taking this asa reference value, percent released oil was calculated by simple unitarymethod.

2.1.3.11.2. In-vitro release behavior of flaxseed oil from microcapsulesexposed to SGF+SIF. For sequential exposure to SGF and SIF, 5 g flaxseedoil powderwasmixedwith 50mL of SGF and incubated under the sameconditions as mentioned above (Section 2.1.3.11.1). After 2 h, pH of thesample was adjusted to 6.8 using 1 M NaOH, followed by addition of50 mL of SIF and the sample was incubated under the same conditions(Section 2.1.3.11.1.) for 3 h. The amount of flaxseed oil released fromthe microcapsules was extracted in petroleum ether and di-ethylether as described above (Section 2.1.3.11.1.) and determined by gravi-metric analysis.

2.1.3.12. Effect of MFOP fortification on sensory characteristics of marketmilk. Buffalo whole milk was standardized by mixing skimmed milk insuch a way that it contained total 3% fat and 8.5% SNF after the additionof MFOP at 1% level. Fresh standardized milk was warmed to 45–50 °Cand then homogenized at 172 bar (~2500 psi) pressure. The whole ho-mogenizedmilk was divided in to 3 batches. All of the batches were for-tified with three different MFOP formulations separately. Milk samplecontaining 3% fatwas used as a control. All the batcheswere pasteurizedat 74 °C for 15 s and then cooled to 4 °C, followed by packaging and stor-age at refrigerated temperature (4–7 °C) for further sensory evaluation(color and appearance, odor, taste and mouthfeel). Sensory evaluationwas performed by a panel of ten judges of Dairy Chemistry and DairyTechnology Division of National Dairy Research Institute, Karnal, India.Composite scoring card for sensory analysis of pasteurized milk asgiven by BIS [20] was used, with slight modifications. In flavor charac-teristics, the main focus was on rancid and oxidized flavor in fortifiedmilk. Milk was served at 35 ± 2 °C temperature for sensory evaluation.

3. Statistical analyses

All measurements were performed in triplicate and reported asthe mean ± standard deviation (SD). One-way analysis of variance(ANOVA) with Tukey's Multiple Comparison Test was performed todetermine the effect of wall material in different formulations. Whiletwo-way ANOVA with Bonferroni post-tests was performed to see thesignificant effect in peroxide value during storage. All statistical analyseswere performed at 95% confidence interval with GraphPad Prism soft-ware (version 5.01 for Windows, San Diego California, USA).

4. Results and discussion

4.1. Moisture content and water activity (aw)

Moisture content plays a significant role in establishing the shelf-lifeof powders. Highermoisture content leads to fungal growth and caking;thus affecting it's physical, chemical stability and overall acceptability.In general, moisture content of 3–4% is the minimum specification formost dried powders used in the food industry [9]. Moisture content ofdifferent flaxseed oil powders (at zero day) ranged from 3.88 ± 0.09to 3.98 ± 0.13% (Table 2). No significant difference (p b 0.05) was ob-served in the moisture content for all the formulations after spray

Table 2Physical characteristics of differentMFOP preparations (at zero day). MFOP: microencapsulatedconcentrate-80 (WPC-80); FO-AO/WPC: antioxidant added flaxseed oil microencapsulated wValues are mean ± SD (n = 3). Values with different superscripts letters within a column indi

MFOP preparation(Powder)

Moisturecontent (%)

Water activity(aw)

Bulk density (ρB)(g/cm3)

Tapped den(g/cm3)

FO/WPC 3.98 ± 0.13a 0.358 ± 0.016a 0.328 ± 0.006a 0.491 ± 0.0FO-AO/WPC 3.95 ± 0.13a 0.354 ± 0.006a 0.321 ± 0.001a 0.498 ± 0.0FO/NaCas 3.88 ± 0.09a 0.346 ± 0.007a 0.297 ± 0.003b 0.454 ± 0.0

drying as well as during the storage of six months. In general, lipids ox-idation is lowest at the water activity of 0.2–0.3 for most of the driedfoods [18] due to decrease in the catalytic effect of transition metals,quenching of free radicals and singlet oxygen and/or retardation of hy-droperoxide decomposition. In the present investigation, aw of differentMFOP formulationswas in the range of 0.346–0.358 on zero day of stor-age (Table 2), which did not change significantly (p b 0.05) throughoutthe storage period of 6 months (values are not shown). Generally,moisture content and aw depend upon the compositional changes ofwall & core materials, inlet/outlet temperature & flow rate of spraydrier, drier design, etc. In the present work, all the operating conditionsand amount of the ingredients were kept constant except the nature ofwall material. The results are in agreement with the finding of Quispe-Condori et al. [14] and Aghbashlo et al. [21], who reported 3.88–5.06%and 1.41–4.36% moisture content in flaxseed and fish oil powder,respectively.

4.2. Bulk density and tapped density

Physical parameters such as bulk density, tapped density and com-pressibility affect the powder's flowability and storage stability. In thepresent study, bulk density of different MFOP formulations rangedfrom 0.297 to 0.328 g/cm3 (Table 2), which is the typical range ofbulk density for microencapsulated powders [22,23]. It is evidentfrom the data that FO/NaCas powder showed the lowest bulk density(p b 0.05); while there was no significant difference between the bulkdensities of other MFOP formulations (i.e., FO/WPC and FO-AO/WPC).This indicates thatwall material had a significant effect on the bulk den-sity of powders. Lower bulk density in case of FO/NaCas powder mightbe explained by the spongy nature of microcapsule's wall, which wasnot observed in rest of the microcapsules' SEM micrographs. Anotherreason for lowest bulk density of FO/NaCas powder could be attributedto highest particle size among different types of microcapsules, which isalso suggested by other researchers [24,25]. By definition, density de-creases as volume increases for a constant mass, and we therefore, ex-pect a similar relationship between the bulk density of the powderand the diameter of the particles. The observations of the presentstudy are in good agreement with the findings of Quispe-Condori et al.[14] and Tonon et al. [26] who reported the bulk density of microencap-sulatedflaxseed oil powder in the range of 0.174–0.350 g/cm3 and 0.289to 0.458 g/cm3, respectively.

Tapped density of a material influences the factors such as the pack-aging, transport, and commercialization of powders [27]. It is evidentfrom Table 2 that tapped density of different MFOP formulationswas found to be in the range of 0.454–0.498 g/cm3. The perusal of thedata also revealed that the tapped density was found to be highest forFO/WPC and FO-AO/WPC powders and lowest for FO/NaCas powder(p b 0.01). The data showed that the type of wall material affected thetapped density of the powders significantly (p b 0.01). No significantcorrelation was observed between bulk density and tapped density(R2 = 0.0058). The obtained results are in agreement with thefindings reported by Finney et al. [28], who observed similar values oftapped density for microencapsulated orange essential oil powder(0.48–0.65 g/mL).

flaxseed oil powder; FO/WPC: flaxseed oil powder microencapsulatedwith whey proteinith WPC-80; FO/NaCas: flaxseed oil powder microencapsulated with sodium caseinate.cate significant difference from each other (p b 0.05) (Tukey's test).

sity (ρT) Carr's index(C)

Hausner ratio(HR)

Mean droplet size[d4,3 (μm)]

Polydispersityindex (PDI)

09a 33.94 ± 0.589a 1.50 ± 0.013a 6.68 ± 0.72a 0.465 ± 0.036a

14a 33.82 ± 0.514a 1.55 ± 0.027a 5.82 ± 0.50a 0.478 ± 0.015a

07b 34.57 ± 0.898a 1.53 ± 0.006a 10.01 ± 0.58b 0.847 ± 0.056b

Fig. 1. Particle size distributions profile of flaxseed oil powders after reconstitution.FO/WPC: flaxseed oil powder microencapsulated with whey protein concentrate-80(WPC-80); FO-AO/WPC: antioxidant added flaxseed oil microencapsulated with WPC-80; FO/NaCas: flaxseed oil powder microencapsulated with sodium caseinate. Values aremean ± SD (n = 3).

531A. Goyal et al. / Powder Technology 286 (2015) 527–537

4.3. Flowing properties

Flowing properties of dry powders are generally assessed by Carr'sindex (% compressibility) and Hausner ratio. The borderline betweenfree-flowing (granular) and non-free-flowing (powder) is about20–25% compressibility [27]. In the present investigation, it was foundthat the Carr's index for different MFOP formulations was in the rangeof 33.82 to 34.57 (Table 2), which is corresponding to the referencevalues of 32–37 (Table 3) indicating the very poor flowability of thedeveloped MFOP formulations. It is also evident from the table thatthere was no significant difference among the Carr's index among theformulations of flaxseed oil powder (p b 0.05). It is reported that theoil content of the powder negatively influences the bulk density ofthe powder, reducing its flowability [29]. In the present study, all ofthe formulations of flaxseed oil powder contained very high oil content(~36% w/w, dry basis), which contributes towards the poor flowabilityof prepared formulations. Quispe-Condori et al. [14] reported verypoor flowing properties with high Carr's index values ranging from33.72 to 48.65 in flaxseed oil microcapsules. Similarly, several other re-searchers have also reported poor flowability of microencapsulatedpowders containing different types of oil [15,30]. Hausner ratio (HR) isanother parameter which is used to characterize the flowability ofpowders. The higher Hausner ratio indicates that the powder is morecohesive and less able to flow freely. Hausner ratio of more than 1.34generally indicates the poor flowing characteristics of the dry powders[15]. In present study, HR for different MFOP formulations was in therange of 1.50–1.55.

4.4. Particle size distribution (After reconstitution)

Particle size of microcapsules influences the textural as well as sen-sory properties of food products. In the present study, typical particlesize distribution profile of reconstituted flaxseed oil powder was deter-mined and results are presented in Fig. 1. It is evident from the figurethat the microcapsules had a wide range of particle size varying from0.54 to 70.60 μm. Particle size distribution profiles indicated nearly a bi-modal and almost similar poly-dispersed distribution in WPC encapsu-lated formulations. Contrary to the FO/WPC and FO-AO/WPC, FO/NaCasmicrocapsules showed a clear bimodal distribution, having ~82% parti-cles in a wide range of 4.78–70.60 μm. The remaining 18% of FO/NaCasmicrocapsules were found to be in the range of 0.54–3.32 μm, whichcould be microcapsules, free oil droplets or single or aggregated caseinmicelles [31]. Our results are in agreement with the findings of otherresearchers, who reported similar bimodal and poly-dispersed distribu-tion profile of fish oil powder encapsulated by milk proteins [32],chia essential oil powder prepared by using WPC-polysaccharides [33]and D-limonene containing powder using modified starch [34]. In thepresent study, mean droplet size (d4,3) in reconstituted MFOP formula-tions was statistically non-significant (p b 0.05) between FO/WPC andFO-AO/WPC formulations (Table 2). This non-significant differencemay be due to the fact that in both the cases the wall material wasWPC-80, except the presence of antioxidant in the later. The dropletparticle size also depends on the microencapsulation efficiency andemulsifying capacity of the wall material. In the mixtures of proteinand lactose, it is the protein component that will move to an interface

Table 3Powder flowability from the Carr's index and Hausner ratio [15].

Carr's index Flowability Hausner ratio

≤10 Excellent 1.00–1.1111.0–15.0 Good 1.12–1.1816–20 Fair 1.19–1.2521–25 Passable 1.26–1.3426–31 Poor 1.35–1.4532–37 Very poor 1.46–1.59N38 Awful N1.60

of the newly created oil droplet surfaces during homogenization, asthe protein is surface active component. It is reported that lactose,when present in the wall materials, acts as a sealant (filler) [35], facili-tates crust formation during spray drying and enhances thewall hydro-philicity; thus, limits the diffusion of encapsulated oil through surface ofthemicrocapsules. Limited diffusion of the oil droplets favors the homo-geneity of the particles after reconstitution. A wide range of literaturesuggested that particle size, and thus homogeneity among the particlesis also affected by the formation of protein–carbohydrate conjugates(melanoidins) formed in theMaillard reaction. Melanoidins are expect-ed to be better emulsifiers than the unreacted proteins [36] and hencewould be expected to be involved in the stabilization of oil droplets inthe emulsion [21]. There are several factors that can contribute to theimproved emulsifying properties of conjugates. The covalent attach-ment of a carbohydrate to a protein is expected to increase the solubilityof the protein. Solubility is essential for good emulsifying properties. Inthe present study, therewas no significant difference in the particle sizeof the reconstituted emulsion consisting of WPC because, firstly, therewas adequate and same surfactant (i.e., WPC) and secondly, the amountof lactosewas constant in all the formulations. Themicrocapsules of FO/NaCas formulation showed the maximum d4,3 (10.01 ± 0.58 μm)among all the formulations. This phenomenon can also be attributedto the aggregative properties of caseinmicelles. Reports suggest that ca-sein monomers cannot sufficiently remove their hydrophobic surfacesfrom water at oil–water interface, and thus tend to associate witheach other [31]. Data with respect to Polydispersity Index (PDI) hasbeen depicted in Table 2. PDI of reconstitutedMFOP formulations variedfrom 0.465–0.847. In general, PDI N0.7 indicates more heterogeneousnature and wide distribution of particles. Therefore, it can be concludedthat the reconstitution of MFOP prepared using WPC-80 resulted intohomogenous emulsions, whereas emulsion of MFOP prepared usingNaCas was heterogeneous in nature.

4.5. Dissolution behavior

Dissolution behavior of powders in water affects the structure aswell as sensory quality of the food/system in which it is added. It de-pends on the wall material, solvent/food matrices and temperature,etc. It is evident from Fig. 2 that different MFOP formulations preparedby WPC-80 showed similar dissolution behavior with higher absor-bance and were completely dissolved in less than 15 min. On theother hand, flaxseed oil powder prepared with sodium caseinateshowed the lowest dissolution rate. It is well known that sodium casein-ate is not easily soluble in water at room temperature because caseinmolecules, in aqueous dispersion of sodium caseinate, exist as amixtureof monomers, complexes and aggregates [36] and these monomers

Fig. 2. Dissolution behavior of flaxseed oil microcapsules in water at room temperature(30–35 °C). WPC: Whey protein concentrate-80; FO/WPC: flaxseed oil powder microen-capsulated with whey protein concentrate-80 (WPC-80); FO-AO/WPC: antioxidantadded flaxseed oil microencapsulated with WPC-80; FO/NaCas: flaxseed oil powdermicroencapsulated with sodium caseinate.

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cannot sufficiently remove their hydrophobic surfaces from coming intocontact with water [31], which limits its dissolution.

4.6. Color (L*, a* and b* values)

Color of any food product is affected by the ingredients used in itsformulation. Therefore, during the formulation of any new food productutmost care is taken while selecting the ingredients, so that the overallacceptability of the food product is not affected negatively. In thepresent study, the color of the MFOP formulations ranged from creamto off white. Data revealed that color values (L* = 88.60 to 88.93,a* = 0.06 to 0.08 and b* = 13.56 to 13.63) for FO/WPC and FO-AO/WPC powder were almost similar (Table 4) and the difference wasstatistically non-significant (p b 0.05). However, MFOP prepared usingsodium caseinate (FO/NaCas) showed the lightest color among all theformulations (Table 4), which can be attributed to the bright whitecolor of sodium caseinate. Thus, it could be inferred from the data thatthe wall material significantly (p b 0.05) affected L*, a* and b* valuesof all theMFOP formulations. Our results are in agreement with the ob-servations reported by Karaca et al. [10], who found L* values rangedfrom 87.3 to 90.6, a* values from −0.5 to 0.3 and b* values from 11.2to 20.3 for microencapsulated flaxseed oil employing chickpea proteinisolates.

4.7. Morphology of microcapsules by Scanning Electron Microscopy

The scanning electron microscopy, a commonly used technique forimaging and characterization of microstructures, was employed to

Table 4Color (L*, a*, b*) values of different formulations of flaxseed oil powder.

MFOP preparation (powder) Color values

L* a* b*

WPC-80 78.68 ± 0.146a 2.00 ± 0.010a 25.75 ± 0.070a

FO/WPC 88.60 ± 0.049b 0.08 ± 0.009b 13.56 ± 0.113b

FO-AO/WPC 88.93 ± 0.185b 0.06 ± 0.004b 13.63 ± 0.004b

FO/NaCas 91.62 ± 0.163d −0.66 ± 0.014d 8.05 ± 0.033c

MFOP:microencapsulatedflaxseedoil powder; FO/WPC:flaxseed oil powdermicroencap-sulated with whey protein concentrate-80 (WPC-80); FO-AO/WPC: antioxidant addedflaxseed oilmicroencapsulatedwithWPC-80; FO/NaCas:flaxseed oil powdermicroencap-sulated with sodium caseinate. Values are mean ± SD (n = 3). Values with differentsuperscripts letters within a column indicate significant difference from each other(p b 0.05) (Tukey's test). L*: lightness; a*: redness; b*: yellowness.

observe the external (surface) as well as internal microstructures ofdifferent MFOP microcapsules.

4.7.1. External morphologyIt is evident from Fig. 3 that in the FO/WPC and FO-AO/WPC formu-

lations, most of themicrocapsules had spherical shape and smooth sur-face with no apparent fissure or crack, which is important to providelower permeability to gases, better protection and core retention [25].The electron micrographs also indicated a significant proportion ofparticles showing dents on the surface, which is typical to themicrocap-sules produced by spray drying [9]. Presence of dents on surface ofmicrocapsules could be attributed to the high total solids content inthe formulations as well as uneven drying and subsequent cooling asreported in other studies [9]. Several researchers observed sphericalshaped microcapsules with no cracks and pore on the surface ofthe fish oil powder particles prepared by using chia/fish oil, sodiumcaseinate, lactose, and whey proteins [11,21,38]. In FO/NaCas powder,a significant proportion of microcapsules were spherical in shape withsmoother surface. These smooth powder particles appeared to be com-paratively more agglomerated, indicating higher surface fat levels andthus, lower microencapsulation efficiency of sodium caseinate; whichalso helps to explain the large powder particle sizes of FO/NaCas formu-lation as observed by Malvern analysis. It can also be observed thatFO/NaCas microcapsules showed comparatively fewer dents and teeth(imperfections) due to faster film formation at drying stage as statedby Tonon et al. [26]. Fig. 3 also indicates that surface dents were moreprevalent in the smaller particles than in the larger particles in all theformulations. This prevalence of surface indentation in smaller particlescould be attributed to the effect of drying rate. It has been demonstratedthat high drying rates, associated with small particles, usually lead tomore rapid wall solidification, consequently forming less smooth sur-face [39]. Imperfections or dents are generally formed due to unevendrying or droplet collapse/shrinkage during the initial stages of drying,when there is a slow process of film formation [40]. Another character-istic feature observed in different formulations of MFOP was the aggre-gation of themicrocapsules as evident from the figure. This aggregationbehavior of themicrocapsulesmight be due to presence of surface (free)fat and presence and/or absorption of moisture [41]. Our results are inline with the findings of earlier researchers who reported a similarkind of aggregation in microencapsulated oil powders [22,40,42].

4.7.2. Internal morphologyInternalmorphology of rupturedflaxseedoilmicrocapsules revealed

a hollow structure, which is another characteristic of particles obtainedby spray drying. Microcapsules of FO/WPC and FO-AO/WPC formula-tions showed a smooth internal surface without cracks, fissures orpores. The smooth internal surface could be attributed to the presenceof lactose in all the formulations. It has been previously discussed thatlactose acts as a sealant (filler) in wall material, facilitates crust forma-tion during spray drying and limits the diffusion of encapsulated oilthrough surface of themicrocapsules. Tang and Li [37] studied the effectof lactose and heating on the microstructure of soy oil microcapsulesand reported that powder particles preparedwith unheated soy proteinisolate without lactose, showed pores on the internal aswell as externalsurfaces. On the contrary, they found smooth, pores-free surface whenmicrocapsules were prepared with lactose. It can also be hypothesizedthat lactose will react with the proteins and form melanoidins(protein–carbohydrate conjugates), which have been shown betteremulsifying properties as well as solubility; and thus, reducing thechances of pores formation or retention on the surface of themicrocap-sules. Similarly, Moreau and Rosenberg [43,44] studied the effect of lac-tose on the gas permeability of whey protein isolate (WPI)-based wallmatrix and found that the WPI-based wall matrix with lactose hadlower gas permeability than that of WPI-only wall matrix. It is clearfrom Fig. 3 that in FO/NaCas microcapsules, a continuous sponge likestructure was seen within the wall, which might be due to gel forming

Fig. 3. Scanning electron micrograph of flaxseed oil microcapsules in group and single at different resolutions. FO/WPC: flaxseed oil powder microencapsulated with whey proteinconcentrate-80 (WPC-80); FO-AO/WPC: antioxidant added flaxseed oil microencapsulated with WPC-80; FO/NaCas: flaxseed oil powder microencapsulated with sodium caseinate.

Table 5Surface fats and microencapsulation efficiency (ME%) of different MFOP preparations.

MFOP preparation Surface fats (%) Microencapsulation efficiency (ME %)

FO/WPC 4.73 ± 0.16a 86.77 ± 0.51a

FO-AO/WPC 4.42 ± 0.05a 87.70 ± 0.16a

FO/NaCas 5.57 ± 0.09b 84.51 ± 0.25b

MFOP: microencapsulated flaxseed oil powder; FO/WPC: flaxseed oil powder encapsulat-ed with WPC; FO-AO/WPC: antioxidant added flaxseed oil powder encapsulatedwith WPC; FO/NaCas: flaxseed oil powder with sodium caseinate. Values are mean ±S.E. (n = 3). Values with different superscripts letters are significantly different withinthe column at p b 0.05.

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nature of caseins. These findings are in good agreementwith the resultsof Schoonman et al. [45], who reported similar spongy wall structurein the microcapsules prepared by using sodium caseinate and malto-dextrin. It can be inferred from the morphology of flaxseed oil micro-capsules that WPC and sodium caseinate, along with carbohydrate(lactose), both are suitable encapsulating agent for themicroencapsula-tion of flaxseed oil, which provide lesser surface area of microcapsules,reducing the diffusion of gases (like O2 and air) and thus are able toprotect the core oil from oxidation.

4.8. Surface (free) oil and Microencapsulation efficiency (ME)

Presence of surface (free) oil and thus,microencapsulation efficiency(ME) of wall materials affect the physico-chemical stability of dry pow-ders. Free oil leads to the aggregation of the powder particles and in-creases the rate of oxidation. In the present study, amount of surfaceoil in different MFOP preparations and microencapsulation efficiency(ME) of different wall materials were measured; and are representedin Table 5. In FO/WPC and FO-AO/WPC formulations, the percentageof free oil was in the range of 4.42–4.73%, and consequently, ME rangedfrom86.70 to 86.77%, which can be considered adequate for oil powders[46]. There was no significant difference between the FO/WPC andFO-AO/WPC formulations in terms of surface oil as well as ME(p b 0.05). However, FO/NaCas powder showed the minimum ME(84.51%), which resulted in the maximum free oil content (5.57%)among the powders.

Lower surface fats and higher ME in first two formulations might beattributed to the more formation of Maillard reaction products in themixtures due to the presence of higher lysine content in WPC-80 thanthat of sodium caseinate. Maillard reaction conjugates have betteremulsifying and encapsulating properties, suggesting better ME andlower surface fats in FO/WPC and FO-AO/WPC formulations. Augustinet al. [47] reported positive influence on encapsulating propertiesof protein–carbohydrate conjugates formed by Maillard reaction.Similarly, Tontul and Topuz [48] reported that out of WPC and sodiumcaseinate, in combinationwith otherwallmaterials,WPC showedbettermicroencapsulating properties and protection to flaxseed oil; whichis in the agreement with our findings. It can be noticed here that inthe present study, microencapsulation efficiency was higher (84.51–93.25%) as compared to ME of 62.3% reported by Carneiro et al. [9] for

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flaxseed oil microencapsulation and 82.16% reported by Karthik andAnandharmakrishnan [49] for algal DHA, wherein only whey proteinswere used as encapsulating agent. Highermicroencapsulation efficiencyor lower free fats observed in our study can be attributed to the pres-ence of lactose in all the formulations. It is reported that lactose helpsin early formation of crust of microcapsule during the spray drying,which hinders the diffusion of fat globules to the surface of the particles[24]. Thefindings of the present study are in accordancewith the resultsreported by Young et al. [50], who found improved microencapsulationefficiency by partial (50%) replacement of whey proteins with lactosesealant.

4.9. Peroxide value (oxidative stability)

Peroxide value (PV) indicates the extent of oxidation and formationof primary oxidation products. Although, the oxidation products partic-ularly hydroperoxides are colorless & odorless and produce no off-flavors. However, these products are highly toxic and reduce the bio-availability of fatty acids. Flaxseed oil is highly polyunsaturated (~75%PUFAs) and thus, highly susceptible to oxidation by the atmosphericoxygen, high temperature and metal ions. It is reported that ALA is 20times more susceptible to oxidation as compared to oleic acid [51]. Inthe present study, peroxide value of different MFOP formulationswere measured at an interval of one month up to the 6 months of stor-age at 35±1 °C. The peroxide value of refined flaxseed oil was observedto be ~0.78meq peroxides/kg at zero day,which continuously increasedand reached to 1.59 meq peroxides/kg at the end of six months of stor-age at room temperature (Fig. 4). It is evident from the figure that therewas a significant difference (p b 0.05) among the PV of different MFOPformulations and free flaxseed oil during the storage. The PV of refinedflaxseed oil on zero day was almost similar to the PV of MFOP formula-tions and no statistically significant difference (p b 0.05) was observedbetween the two values. The data suggested that the microencapsula-tion followed by spray drying had not caused any oxidative damage tothe flaxseed oil.

All the formulations showed a gradual but significant increase inPV during the storage of six months, varying from 0.81 to 0.99 meqperoxides/kg. It is also evident from the figure that the PV of free oil in-creased at a faster rate as compared to the encapsulated oil. Peroxide

Fig. 4. Peroxide value of free andmicroencapsulated flaxseed oil at onemonth interval up to sixflaxseed oil powder microencapsulated with whey protein concentrate-80 (WPC-80); FO-flaxseedoil powdermicroencapsulatedwith sodium caseinate. Values aremean±SD (n=3). D(p b 0.05).

value of MFOP formulations was about 62.68% less as compared tofree oil at the end of storage period. There was no significant effect ofantioxidant or the wall material on the oxidative stability of microen-capsulatedflaxseed oil, which could be due to very low initial PV of flax-seed oil, wherein no measurable effects of antioxidant were observed.Another possible reason of lower PV of microencapsulated oil couldbe the protection provided by the encapsulating layer to the oil inthe core of the microcapsules. It is evident from the results discussedabove that the PV of free as well as encapsulated oil remainedbelow the limit of up to 5 meq peroxides/kg of oil under the CodexAlimentarius Commission standards [51]. High oxidative stability of en-capsulatedflaxseed oil as compared to free oil could also be attributed tothe antioxidative properties of casein and whey proteins due to theirfree-radical scavenging and metal ion chelation properties [52]. Thespecific antioxidative feature of caseins has been attributed to theirhigh content of phosphoseryl groups which have metal binding ability[53]. In general, milk proteins show free-radical scavenging propertiesdue to various amino acids such as cysteine, tyrosine, tryptophan, phe-nylalanine and histidine [31] and free sulphydryl groups. Present resultsare in good agreementwith thefindings of Tonon et al. [26],who report-ed lowest PV (b1.5 meq peroxides/kg) in microencapsulated flaxseedoil prepared by using modified starch, WPC and gum Arabic. Similarly,Goyal et al. [54] developed flaxseed oil emulsions and reported~20.98% increase in PV after four weeks of low (4–7 °C) temperaturestorage.

4.10. In-vitro release behavior of flaxseed oil from microcapsules undersimulated gastric fluid (SGF) conditions

Percent release of flaxseed oil from the microcapsules of differentMFOP formulations exposed to simulated gastric fluid (SGF, pH 1.2) isshown in Table 6. It is evident from the data that the percent oil released(4.39 to 4.45%) from FO/WPC and FO-AO/WPC microcapsules was notsignificantly different (p b 0.05). This low percent release of flaxseedoil frommicrocapsules could be attributed to thehighly globular confor-mation of whey proteins, due to which whey proteins are highly resis-tant to peptic hydrolysis in their native state [55]. Another interestingfinding of the studywas that the percent oil releasedwas comparativelyhigh in case of MFOP formulation wherein sodium caseinate was the

months storage at room temperature (30–35 °C) of differentMFOP preparations. FO/WPC:AO/WPC: antioxidant added flaxseed oil microencapsulated with WPC-80; FO/NaCas:ifferent superscript letterswithin amonth indicate significant difference among the values

Table 6In-vitro percent release of flaxseed oil from microcapsules under SGF and SGF + SIFconditions.

MFOP preparation Percent release of flaxseed oil frommicrocapsules

SGF (2 h)⁎ SGF + SIF (2 + 3 h)

FO/WPC 4.39 ± 0.53a 23.12 ± 7.06a

FO-AO/WPC 4.45 ± 0.44a 20.00 ± 3.66a

FO/NaCas 10.09 ± 1.17b 40.54 ± 1.75b

SGF: simulated gastric fluid; SIF: simulated intestinal fluid; MFOP: microencapsulatedflaxseed oil powder; FO/WPC: flaxseed oil powder encapsulated with WPC; FO-AO/WPC: antioxidant added flaxseed oil powder encapsulatedwithWPC; FO/NaCas: flaxseedoil powder with sodium caseinate. Values are mean ± S.D. (n = 3).⁎ Value in bracket indicates the time of incubation in hours.

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encapsulating agent. This can be explained by the fact that openand flexible conformation of caseins makes them highly susceptible topeptic enzymes [56,57]. Our results are in agreement with the resultsof Shen et al. [58], who reported 4.4–6.1% EPA and ≤1.5% DHA releaseafter digestion of tuna oil powder under the exposure of SGF.

4.11. In vitro release behavior of flaxseed oil from microcapsules undersequential exposure of SGF + SIF conditions

During the gastric digestion, the food is first digested by gastricpepsin at low pH ~2.0, and then reaches the intestine having pH ~ 6.8.Higher pH and presence of food stimulates the pancreatic enzymes forfurther digestion and absorption of food components. Therefore, tomimic the gastric digestion conditions, the effect of sequential exposureof SGF and SIF was evaluated on the basis of % oil release from variousMFOP formulations. It is clear from the data (Table 6) that in FO/WPCand FO-AO/WPC formulations, oil release was 20.00 to 23.12%, and thedifference between them was statistically non-significant (p b 0.05).However, a higher % of oil release (40.54%) was observed in FO/NaCasformulation. The higher oil percent release observed in sequential expo-sure (SGF + SIF) of reconstituted MFOP formulations, as compared toSGF conditions alone could be due to higher degradation of the micro-capsules in SGF + SIF containing pancreatin (amylase and trypsin),which hydrolyzes both proteins and carbohydrate resulting in thechange in capsule structure (such as large pore formation) with subse-quent oil release. Higher oil release in FO/NaCas formulation could alsobe attributed to the simpler/primary structure of caseins over thecomplex (secondary & tertiary) conformations of whey proteins [59].Present results are in agreement with findings of Shen et al. [57], whoreported that free fatty acids release increased from 5.2 ± 4.1 (SGF) to78.3 ± 1.7% (SGF+ SIF) during in-vitro digestion of microencapsulatedtuna oil powder. However, Karaca et al. [10] reported a very high %release of oil under SGF (36.6–43.4%) and SGF + SIF (84.5–92.6%)conditions in flaxseed oil powder. This large difference in % oil releasemay be due to the difference in the technique used in the powderpreparation.

Table 7Effect of MFOP fortification on sensory evaluation of market milk.

Omega-3 fortified milk Color and appearance(Scores out of 10)

Mouthfeel (score out of 3

Days of storage

0 5 0 5

Control 8.83 ± 0.24a 8.67 ± 0.26a 27.67 ± 0.82a 27.67FO/WPC 8.33 ± 0.23a 8.67 ± 0.24a 28.33 ± 0.43a 27.67FO-AO/WPC 8.33 ± 0.47a 8.77 ± 0.25a 27.33 ± 0.94a 28.33FO/NaCas 9.00 ± 0.36a 8.70 ± 0.12a 26.83 ± 0.82a 26.67

MFOP: microencapsulated flaxseed oil powder; FO/WPC: Milk fortified with flaxseed oil powdepowder encapsulated with WPC; FO/NaCas: Milk fortified with flaxseed oil powder with sodiusignificantly different within the column at p b 0.05.

4.12. Effect of MFOP fortification on sensory characteristics of market milk

Liquidmilk, themost consumed formof dairy products [60]was sen-sorial analyzed after omega-3 fortification during storage of five days forcolor and appearance, taste, odor and mouthfeel. It is evident from thedata (Table 7) that there was no significant difference in color and ap-pearance, mouthfeel and odor scores among the samples and controlthroughout the storage period (p b 0.05). However, scores for taste de-creased significantly (p b 0.05) from the initial score 36 on zero day to31.6 on 5th day of storage for control samples. Similarly, as a result ofstorage the scores for fortified milk samples were reduced to 31.9from its initial score of 34.8 at day zero. There was no significant differ-ence (p b 0.05) in taste scores among the fortified samples and controlon the same day of evaluation. Thus, the fortifiedmilk remained accept-able till 5 days of storage. However, a rancid odor was observed in forti-fied milk sample by sensory panel on sixth day onwards and was notevaluated for other sensory attributes. The results also indicated thattype of fortificant, i.e., formulation of flaxseed oil powder, did not signif-icantly affect the overall acceptability of fortified milk. Let et al. [61]studied themilk enrichedwith fish oil and observed thatfish andmetal-lic odor increased significantly during 8 days of storage. According toICMR [62], 1.6 g of ALA is recommended per day for adults. Assumingthe standard serving size of fluid milk as 250 mL, approximate ~34.0%of the RDA can be met by per serving of developed fortified milk evenafter 5 days of storage (calculated by fatty acid profile data, which isnot described here).

5. Conclusions

In the present study, polyunsaturated flaxseed oil was stabilizedthrough microencapsulation technique using milk proteins. Developedpowder showed good oxidative aswell as storage stability and sphericalshaped microcapsules without any cracks or fissures suggesting betterencapsulation and protection of highly susceptible flaxseed oil. Dissolu-tion and reconstitution behavior showed improved solubility andnarrower particle size distribution ofWPC-encapsulatedflaxseed oilmi-crocapsules. As compared to sodium caseinate, WPC showed better mi-croencapsulation efficiency and lower surface fats in the formulations.Peroxide value of encapsulated flaxseed oil, which has been a majorconcern in previous studies conducted by other researchers, showed~80% lesser value than that of permissible limit given by CodexAlimentarius Commission. However, no significant effect of antioxidantwas observed on peroxide value of flaxseed oil in FO-AO/WPC formula-tion. In-vitro release behavior showed that percent release of flaxseedoil was higher under sequential exposure (simulated SGF + SIF) thanthat of only SGF conditions. Between the employed encapsulatingagents, sodium caseinate, which showed almost twice percent releasethan that of WPC, was found to be better releasing medium undersimulated conditions. MFOP fortified market milk (at 1% level) wasobserved to be comparable with control in term of sensory characteris-tics up to 5 days of storage. The prepared flaxseed oil powder showed

0) Odor (score out of 20) Taste (score out of 40)

0 5 0 5

± 1.25a 17.67 ± 0.40a 17.33 ± 0.50a 36.10 ± 1.04a 31.67 ± 0.42a

± 1.25a 16.67 ± 0.26a 16.57 ± 0.31a 35.57 ± 1.17a 32.37 ± 0.45a

± 0.47a 16.67 ± 0.94a 16.60 ± 1.23a 34.67 ± 1.25a 31.33 ± 1.89a

± 0.47a 18.00 ± 1.27a 15.67 ± 0.47a 34.43 ± 1.74a 32.00 ± 1.63a

r encapsulated withWPC; FO-AO/WPC: Milk fortified with antioxidant added flaxseed oilm caseinate. Values are mean ± S.D. (n = 9). Values with different superscript letters are

536 A. Goyal et al. / Powder Technology 286 (2015) 527–537

excellent storage stability (in terms of peroxide value) for a period of atleast six month at room temperature (35 ± 1 °C). Thus, flaxseed oilpowder formulated in present study could be used as a fortifyingagent in commercial food and supplementary products.

Conflicts of interest

No conflicts of interest to declare.

Acknowledgments

The first author is thankful to Dr. Rohit Sharma (Assistant Professor)and Dr. Rasane Prasad Jayprakash (Assistant Professor) for assisting inediting of themanuscript. The authors are also thankful to Director, Na-tional Dairy Research Institute, for providing financial assistance for car-rying out the researchwork. The authors thankfully acknowledge KamaniOil Industries Pvt. Ltd., Khopoli, Maharashtra, India for supplying refinedflaxseed oil for the present study.

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Ankit Goyal studied Biochemistry from Kurukshetra Univer-sity and Food Science & Technology from Haryana Agricul-tural University, India. He obtained his Doctorate in DairyChemistry from National Dairy Research Institute, India;and worked in the area of development of functional dairyfoods. Currently, he is working as an Assistant Professor inMansinhbhai Institute of Dairy & Food Technology, Gujarat,India. He has published several research, review and techni-cal articles in national and international journals.

Vivek Sharma studied Chemistry and Dairy Chemistry fromNational Dairy Research Institute, Karnal, India and obtainedhis Doctorate degree from the same institute. He haseighteen years' research experience in the field of physico-chemical analysis of milk and dairy products. He has alsoworked in the field of artificial sweeteners, milk fat analysisand development of value-added food and dairy products.Recently, his group was granted a patent for developing thetechnology of preparation 85% ‘less cholesterol ghee (milkfat)’. He is working as a Principal Scientist in department ofDairy Chemistry and has published several research andreview articles.

Manvesh K. Sihag obtained his Doctorate degree in DairyChemistry from National Dairy Research Institute, Karnal,India. During PhD, he worked on the development of ironand vitamin-A enriched pearl millet basedweaning food for-mula. Currently, he is working as an Assistant Professor inMansinhbhai Institute of Dairy & Food Technology, Gujarat,India. He has published several research, review and techni-cal articles in national and international journals.

Dr. Tomar obtained his degree in Dairy Microbiology fromNational Dairy Research Institute, Karnal, India and currentlyworking as a Principal Scientist in the same department. Hiscurrent research area is the production of ‘FunctionalBiomolecules’ by scanning electron microscopy. He has wideexperience in physico-chemical and microbiological analysisof biomolecules as well as food and dairy products. Havingvarious awards such as Young Scientist award, he has severalresearch & review articles and book chapters published inNational and International journals/books.

537logy 286 (2015) 527–537

Dr. Arora obtained his Masters' and Doctorate degree inDairy Chemistry and currently working as Principle Scientistin the same department of Dairy Chemistry, National DairyResearch Institute, India. His area of expertise is the stabilitydetermination and applications of artificial sweeteners &functional ingredients in dairy products. Having severalresearch and review articles published in national and inter-national journals, various patents have also been granted tohim for his contribution in research.

Dr. Latha Sabikhi is working as a Principle Scientist in dept.of Dairy Technology, National Dairy Research Institute, India.Her current area of research is functional and probiotic foods.She is the Fellow of the National Academy of Dairy Science(India) and conferred International Professional WomenOpportunity Award by Consorzio Ricerca Filiera Lattiero-Casearia (CoRFiLaC), Italy — 2006. Currently working on theherbal bioactive components, she has published severalresearch and review articles in national and internationaljournals.

Dr. Ashish Kumar Singh obtained his Masters' and Doctor-ate degree in Food Technology and currently workingas Principle Scientist in Department of Dairy Technology,National Dairy Research Institute, India. His area ofspecialization is composite dairy foods, functional foods &nutraceuticals. Having several research and review articlespublished in national and international journals, variouspatents have also been granted to him for his contributionin research.