Research Article

Development of a soup powder enriched withmicroencapsulated linseed oil as a source ofomega-3 fatty acids

Monica Rubilar1,2, Eduardo Morales1, Karla Contreras1, Carolina Ceballos1, Francisca Acevedo1,

Mario Villarroel1,2 and Carolina Shene1,2

1 Center of Food Biotechnology and Bioseparations, BIOREN, Universidad de La Frontera, Temuco, Chile2 Agriaquaculture Nutritional Genomic Center, CGNA (R10C1001), Technology and Processes Unit,

Universidad de La Frontera, Temuco, Chile

The main objective of this study was to develop an optimized formulation of soup powder enriched with

omega-3 fatty acids. For this purpose, the process conditions for optimizing the microencapsulation

efficiency (ME) of linseed oil by spray drying were determined using the Taguchi methodology with

an orthogonal array L4(23). The effect of the variables on the ME, such as wall material concentration

(25 and 30%), linseed oil concentration (14 and 20%), and wall material type (gum arabic GA; and a

mixture maltodextrin/GA), was evaluated. The oxidative stability of the microcapsules obtained were

determined by the Rancimat method, and a morphological and size characterization of microcapsules

was performed by scanning electronic microscopy, confocal microscopy, and laser diffraction. The

optimization of the soup formulation was reached by means of RSM, using the central composite design,

two control factors (salty taste and consistency), and 11 design points. The hedonic test was applied to

measure the acceptability of the optimized formulation. Chemical characterization of optimized soup and

its oxidative stability were also evaluated. This study resulted in a healthy soup enriched with omega-3

which was highly acceptable to consumers.

Practical applications: Linseed oil is a healthy and nutritive oil, very rich in unsaturated fatty acids

such as omega-3. In addition to protecting the oil against oxidative damage, the microencapsulation of

oils in a polymeric matrix (mainly polysaccharides and proteins) also offers the possibility of controlled

release of the lipophilic functional ingredient and can be useful for the supplementation of foods with

PUFA. The soup powder enriched with microencapsulated linseed oil as a source of omega-3 formulated

in this study will contribute to the development of foods according to the functionality requirements of

current consumers and markets.

Keywords: Functional ingredient / Linseed oil / Microencapsulation / Spray dryer

Received: November 4, 2011 / Revised: February 8, 2012 / Accepted: March 13, 2012

DOI: 10.1002/ejlt.201100378

1 Introduction

In recent decades consumer demands in the field of func-

tional food production have increased considerably.

Functional foods play an outstanding role in promoting

health, increasing longevity, and preventing the onset of

chronic diseases.

Public health organizations such as the USDA in its

Dietary Reference Intakes of macronutrients, and the

WHO/FAO [1] in a report about diet, nutrition, and the

prevention of chronic diseases, have recommended daily

amounts for each type of fatty acid, saturated, monounsatu-

rated, polyunsaturated, and long-chain PUFA. Among the

Correspondence: Dr. Monica Rubilar, Center of Food Biotechnology and

Bioseparations, BIOREN, Universidad de La Frontera, Casilla 54-D,

Temuco, Chile

E-mail: mrubilar@ufro.cl

Fax: þ56 45 325053

Abbreviations: GA, gum arabic; IT, induction time; MD, maltodextrin; ME,

microencapsulation efficiency; MLO, microencapsulated linseed oil; OSI,

oxidative stability index; OTE, optimized theoretical equation; SEM,

scanning electronic microscopy; SQ, sensory quality; SQE, experimental

sensorial quality; SQT, theoretical sensorial quality

Eur. J. Lipid Sci. Technol. 2012, 114, 423–433 423

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PUFA, the most important families are the well-known n-3

and n-6 fatty acids. These two families are similar in that they

both comprise a precursor, namely ALA for the n-3 and

linoleic acid for the n-6 families and terminal products

obtained by a succession of elongations and desaturations

during metabolism; the two groups of fatty acids share

the same long-chain converting enzymes. These precursor

compounds are essential because the human body is unable

to synthesize them, although, it can metabolize them to

longer-chain derivatives. Furthermore, the diet must cover

the body’s needs for these fatty acids [2]. The absence

of EFA in a normal diet has been described as being

responsible for the development of a wide variety of diseases,

such as cardiovascular disorders, inflammatory processes,

viral infections, certain types of cancer, and autoimmune

disorders [3]. The administration of oils rich in EFA

has proved effective in the prevention and treatment of

these health issues [4]. Potential mechanisms for the cardio-

protective effect of n-3 PUFAs include anti-arrhythmic, anti-

inflammatory, hypotriglyceridemic effects, lowered blood

pressure, and improved endothelial function [5].These

potential health benefits have gained the interest of the

food industry, the medical community and consumers,

and have promoted increased numbers of products contain-

ing n-3 fatty acids [6].

Linseed oil is named as functional ingredient due to its

being an excellent dietary source of the ALA (C18:3n-3).

Linseed oil is highly unsaturated (90% of total fatty acids),

and particularly rich in ALA (55.0%), oleic acid (21.2%), and

linoleic acid (13.8%) [7].

One technological process aimed at protecting polyunsa-

turated oils against oxidation is microencapsulation. This

process is useful for masking or preserving flavors and aro-

mas, enhancing stability, and transferring bioactive liquid

lipids into easily handled powdery solids for food fortification

purposes [8]. Microencapsulated oils are essentially powdery

food products or ingredients comprising oil globules dis-

persed in a continuous matrix of saccharides and/or proteins.

The process of oil microencapsulation consists basically in

the preparation of an oil-in-water emulsion containing the

matrix components in the aqueous phase, which is then dried

[9]. Microencapsulation by spray drying has been found

effective for retarding or suppressing the oxidation of un-

saturated fatty acids [8, 10–14].

In this study, the process conditions for optimizing the

microencapsulation efficiency (ME) of linseed oil by spray

drying were determined in order to obtain an edible oil as a

powder ingredient, with an enhanced stability for oxidation

useful to food formulation. A soup powder enriched with

microencapsulated linseed oil (MLO) as a source of omega-3

was formulated to contribute to the development of foods

according to the functionality requirements of current con-

sumers and markets.

2 Materials and methods

2.1 Materials

Gum arabic (GA) and maltodextrin (MD; Sigma–Aldrich),

and solvents (chloroform, petroleum ether, hexane, and

methanol) were purchased from Merck. Corn, wheat flour,

basil, onion, pepper powder, reduced-sodium salt, skimmilk,

monosodium glutamate, corn starch, sodium caseinate, and

tricalcium phosphate were used for the soup formulation.

Linseed oil was obtained from the company Terrasol located

in Pucon, Chile, showing an initial peroxide value of

3.45 meq/Kg determined according to AOAC methods.

The frozen corn was dried for 9 h at 458C, then ground

and sieved to a fine powder with a particle size between

0.0117 and 0.0165 in. (mesh Nos. 40 and 50). The yield

of the sieved corn was 98%.

2.2 Linseed oil microencapsulation process

2.2.1 Experimental design for linseed oilmicroencapsulation

To optimize the linseed oil microencapsulation an exper-

imental Taguchi design was applied using the criterion bigger

is better and utilizing a matrix L4(23) with three-independent

variables: concentration of wall material (Factor A, 25 and

30%, i.e., g of polysaccharides in 100 g emulsion), oil con-

centration (Factor B, 14 and 20%), and type of wall material

(Factor C, GA and GA/MD; Table 1). The ratio of GA/MD

Table 1. Efficiency of linseed oil microencapsulation using the orthogonal array L4(23)

Design point

Orthogonal matrix L4(23) Microencapsulation efficiency (%)

A B C A B C Average

1 1 1 1 25 14 GA 66.0 � 9.21

2 1 2 2 25 20 MD/GA 54.6 � 6.46

3 2 1 2 30 14 MD/GA 90.7 � 1.19

4 2 2 1 30 20 GA 69.1 � 5.20

Coded variables and non-coded variables A, wall concentration (%); B, oil concentration (%); C, wall type (GA: gum arabic; MD/GA:

maltodextrin/gum arabic).

424 M. Rubilar et al. Eur. J. Lipid Sci. Technol. 2012, 114, 423–433

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used was 3:2 [15, 16]. The average for the level of each

factor and analyses of variance were calculated using the

software Qualitek-4. The optimized theoretical equation

(OTE) was determined considering the averages of

response with the greatest impact, identifying the most

important factors, and levels of work. Table 1 shows the

orthogonal matrix and the orthogonal array used with the

design factors.

2.2.2 Microencapsulation by spray dryer

In keeping with the experimental design, the linseed oil was

added to the encapsulating solution and then the mixture was

emulsified using a model 400DS Benchtop homogenizer at

18 000 rpm � g for 2 min.

The spray drying process was carried out in a LabPlant

SD-05 dryer (Huddersfield, England) on a laboratory scale,

with a 1.5 mm nozzle diameter and a spray chamber of

500 mm � 215 mm. Emulsions were fed into the main

chamber through a peristaltic pump. An air input tempera-

ture of 1408C and an outlet temperature of 958C were used,

with a drying air flow of 73 m3/h and a feed rate of 5.3 g/min

following the microencapsulation process conditions set out

by preliminary assays for spray drying.

Prior to its characterization, the microencapsulated oil

(powder product) was stored at �208C in sealed plastic bags

under vacuum according to Imagi et al. [17].

2.2.3 Determination of microencapsulation efficiency

For the determination of ME, it was necessary to quantify

the total oil in the microcapsule and the free oil (non-

encapsulated oil fraction).

The total oil of microcapsules was determined according

to the Association of Official Analytical Chemists [18]. The

Soxhlet extraction was accomplished in cellulose thimbles

containing 1 g of dried sample. Extractions were carried out

using 150 mL of petroleum ether for 5 h. The oil was con-

centrated using vacuum rotary evaporation of the solvent;

further lipid content was dried at 1058C until constant

weight.

According to Velasco et al. [9], the fraction of free oil was

determined by adding 100 mL of light petroleum ether (60–

808C) to 1 g ofmicroencapsulated powder, and stirring at RT

for 15 min. After filtering and evaporating the solvent in a

rotary evaporator, the extracted oil was dried until constant

weight.

TheMEwas calculated as reported by Velasco et al. [9] as

follows:

MEð%Þ ¼ microencapsulated oilðgÞtotal oilðgÞ � 100

The microencapsulated oil amount was calculated from the

difference between total oil and free oil amounts.

2.2.4 Characterization of microparticles

2.2.4.1 Determination of the oxidative stability of oil frommicrocapsules

For these analyses, the extraction of oil from the optimized

product (microcapsule obtained from the design point with

the highest ME) was carried out as described by Bligh and

Dyer [19]. Oil from 15 g of microcapsules was extracted with

15 mL of chloroform and 30 mL of methanol stirring for

2 min and 30 s. A second extraction was performed where

10 mL of chloroform were added to the mixture and hom-

ogenized for 30 s. Then the solution was diluted with water

and homogenized for 30 s. The mixture was centrifuged at

8872 � g for 10 min at 48C to separate the phases. The

solvent was evaporated in a rotary evaporator at 408C and

afterward with aN2 flow. The remaining residue was weighed

and the oil content was determined. The Rancimat (model

743, Metrohm) was used to determine the oxidative stability

of oil extracted from microcapsules. Consistent with Kobus

et al. [20], 3.5 g of oil sample previously extracted from the

microcapsule was used, and the temperature was fixed at

1008C in order to obtain the induction time (IT) and oxi-

dative stability index (OSI) at an air flow of 20 L/h.

2.2.4.2 Microcapsule morphology

The microcapsule morphology of design points 1 and 3 was

observed by means of scanning electronic microscopy (SEM)

and confocal microscopy. For SEM, the dried particles were

coated with carbon and then pure gold by an Edwars S150

Sputter Coater, and observed in a JEOL JSM – 6380 LV

scanning electronic microscope. For confocal microscopy,

the linseed oil was stained with Nile red prior to micro-

encapsulation by spray dryer. The microcapsules were

examined in a confocal microscope with a 488-nm argon

laser, 40 and 100� (oil immersion) objectives and the soft-

ware FV-ASW 1.7. All the settings for the confocal micro-

scope and the imaging of capsules were computer controlled.

Particle size distributions for microcapsules obtained

from design points 1 and 3 were measured by laser diffraction

(Malvern Mastersizer X, Malvern, UK). Samples were pre-

pared by suspending the particles in cyclohexane. On the

basis of the volume share distribution, a granulometric com-

position was determined using the characteristic parameters

D10 and D90, which are the equivalent volume diameters at

10 and 90% cumulative volume, respectively. The absorption

coefficient used was 0.1.

2.2.5 Fatty acid composition

Fatty acid composition of microcapsules obtained from the

design point with the highest ME, was obtained by GC

analyses. The fatty acid composition were determined with

a Clarus 500 chromatograph (Perkin Elmer) equipped with a

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FID and Fused Silica Capillary Column TVSS 2380

(60 m � 0.25 mm � 0.2 mm film thickness, Supelco). The

FAMEwere identified with a standard 37-component FAME

Mix (Supelco) and using methyl nonadecanoate (Sigma) as

the internal standard. Nitrogenwas used as the carrier gas. An

aliquot of the sample was injected under the following GC

conditions: the oven temperature was programmed at 1508Cfor 1 min, increasing to 1688C at a rate of 18C/min, holding

for 11 min, then increasing to 2308C at 68C/min, which was

maintained for 8 min. The temperature of the injector and

detector were 2508C.

2.3 Development of a corn soup powder enrichedwith microencapsulated linseed oil

2.3.1 Soup formulation

The soup was prepared containing 20% MLO as follows:

wheat flour (3.0 g), skim milk 0% fat (3.0 g), dehydrated

basil powder (0.1 g), dehydrated onion powder (0.3 g),

dehydrated pepper powder (0.2 g), corn (14 g: 8.0 g powder

of 0.00165 in. and 6.0 g of 0.0117 in.), monosodium gluta-

mate (0.2 g), sodium caseinate (0.2 g), tricalcium phosphate

(0.4 g), 50% reduced-sodium salt (2.0 g), corn starch

(4.0 g), and MLO (4.5 g).

The ingredients were homogenized with 200 mL of

water. Themixture was boiled for 15 min, stirring constantly.

The consistency and saltiness of the preparation were eval-

uated by a sensory panel in order to establish the levels of

incorporation and to obtain the optimal mixture.

2.3.2 Optimization of the soup formulation

To optimize the soup formulation, the RSM was used with

a factorial central composite design [21], considering two

control factors (X1: starch and X2: salt), three working levels

each, and two axial points (�a and a). Coded working levels

were: minimum level (�1), maximum level (þ1), and central

level (0), where the central point was repeated three

times, obtaining 11 experimental runs. The working levels

were determined through preliminary assays. The sensory

quality (SQ) response was submitted to regression and

variance analysis, the first to generate a fitted polynomial

equation (Eq. 1), and the second to determine the degree

of influence of the independent variables using the software

Design 6.0.

The polynomial equation (Eq. 1) for the RSM analysis

was as follows:

Y ¼ b0 þ b1X1 þ b2X2 þ b21X

21 þ b2

2X22 þ b12X1X2 (1)

where b0, b1, b2, and b12 represent the regression coefficients

for the constant, linear, quadratic, and interaction terms

respectively, and X1 and X2 represent the independent

variables.

2.3.3 Sensorial analysis of soup

The SQ of the optimized soup was defined as the sum of the

characteristics consistency and saltiness. In open panel ses-

sions, the percentages of the relative influence of features

were determined to obtain SQ using the following equation

(Eq. 2):

SQ|{z}

Y

¼ 0:41� consistency|fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl}

Y1

þ0:59� saltiness|fflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflffl}

Y2

(2)

where Y is the sensorial quality, Y1 the sensorial quality of

consistency, and Y2 is the sensorial quality of saltiness.

For determining the SQ of the optimized product, a panel

of 8 trained judges was composed using the sensory test of the

composite score [22] and a continuous descriptive analytical

scale with five ranges of responses and extremes of SQdefined

as very bad and very good. Eleven samples of soup (coded

A–K) and two scorecards (one for evaluating the consistency

and a second to evaluate the saltiness) were provided to

panelists. The SQ obtained by means of this evaluation

was called experimental sensorial quality (SQE) and the

theoretical sensorial quality (SQT) was obtained by

Software Design Expert 6.0.

2.3.4 Acceptability test

To determine the acceptability of the optimized product, the

hedonic test was applied [22] using a sensorial scale with

five sensorial points. For this purpose, 71 people ranging in

age from 10 to 50þ were selected. Basic instructions were

provided to consumers for a clear response.

2.3.5 Chemical characterization of corn soup

2.3.5.1 Proximal analyses

The chemical characterization (moisture, ash, protein, ether

extract, crude fiber, and nitrogen-free extract) was performed

on the optimized product and analyzed according to AOAC

standard procedures [18]. In order to determine the caloric

intake, the percentage of proteins, carbohydrates, and ether

extract of the soup, Atwater coefficients of 4.0 kcal/g for

proteins, 4.0 kcal/g for carbohydrates, and 9.0 kcal for lipids

were used [23].

2.3.5.2 Determination of oxidative stability of extracted oilfrom optimized soup

For these analyses, the extraction of oil from the optimized

soup was carried out as described by Bligh and Dyer [19],

with some modifications. Ten grams of homogeneous paste

was extracted with 10 mL of chloroform and 20 mL of

methanol, stirring for 2 min and 30 s, respectively. A second

extraction was performed where 20 mL of chloroform were

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added to the mixture and homogenized for 30 s. Then, the

solution was diluted with water and homogenized during

30 s. The mixture was centrifuged at 8872 � g for 10 min

at 48C to separate the phases. The solvent was evaporated in a

rotary evaporator at 408C and afterward with a N2 flow. The

remaining residue was weighed and the oil content was

determined.

The Rancimat was used to estimate the stability time at

208C (OSI20) of optimized soup. The stability time of

samples at five temperatures (80, 90, 100, 110, and

1208C) were determined and plotted to the log OSI [24].

The estimation OSI20 (Eq. 3) was expressed in hours.

LogðOSI20Þ ¼ aðTÞ þ b (3)

3 Results and discussion

3.1 Microencapsulation efficiency of linseed oil

The ME of linseed oil for the points of the experimental

design is shown in Table 1. The ME values of linseed oil

were found to be between 54.6% (value for design point 2)

and 90.7% (the value for design point 3). The results showed

that higher ME values were obtained with a high concen-

tration of encapsulating wall (30% wall material concen-

tration, 14% oil concentration, and MD/GA wall type).

According to Young et al. [25], ME can rise by increasing

the concentration of solids in the wall solution. According to

Jafari et al. [26], the most important factor determining the

retention of food oils during spray drying is the dissolved

solids content in the feed. A high solid content of the prepared

emulsion increases retention principally by reducing the time

required to form a semi-permeable membrane at the surface

of the drying particle. Moreover, higher total solids lead to

the increase of emulsion viscosity, preventing circulation

inside the droplets, and, thereby, resulting in a rapid skin

formation. With respect to the oil content, several reports

have observed that higher oil loads generally result in poorer

retention or lower encapsulation efficiency and higher surface

oil content of the powder [15, 27–30].

Different biomolecules have been microencapsulated by

spray drying using GA as a microencapsulation agent due to

the efficient protection offered by this wall material [31].

Researchers have tried to use a blend of GA with other wall

materials and/or to replace GA completely [32]. They have

shown that MD can successfully replace a part of GA as the

wall material, and they have determined the best ratios

between them. Turchiuli et al. [16] studied the microencap-

sulation of vegetable oil in a solution of MD/GA (2:3) by

spray drying and observed that the amount of free or non-

encapsulated oil on the surface of solidmicrocapsules was low

(<2% of total oil). Krishnan et al. [33] reported the micro-

encapsulation of cardamom oleoresin by dryer spraying using

GA, MD, and a modified starch as wall materials. The

microcapsules were evaluated by organic solvent extraction

of the non-volatile for 6 wk and the stability of volatile and

non-volatile compounds was determined. The results showed

that GA presented higher protection for the oleoresin than

MD and modified starch. Krishnan et al. [34] reported the

microencapsulation of cardamom oleoresin by spray drying

using binary and ternary mixtures of GA, MD, and modified

starch as the wall materials. The results obtained indicated

that the mixture 4:1:1 of GA/MD/modified starch showed a

higher protection from oxidation than the GA used alone.

The selection of the proper carrier is one of the most

important factors in the microencapsulation of oils, since it

can affect the viscosity of in-feed emulsion [35, 36].

Although, MD shows low viscosity, is a poor emulsifier,

and does not promote good retention of compounds during

the spray drying process, it does protect encapsulated ingre-

dients from oxidation [31, 37]. On the other hand, GA is

an excellent carrier for microencapsulation providing good

retention of oils upon drying [38]. The use of GA and MD

mixtures for the microencapsulation of oils is a good com-

promise between cost and effectiveness as wall materials.

Table 2 shows results concerning the magnitude of aver-

age difference by control factors and working levels. The

greater the difference, the higher the influence of the control

factors on the ME, and it is possible to identify the working

level that produces the best result. The concentration of

encapsulating wall (Factor A) showed a higher influence

on the ME of linseed oil with a difference of 19.6 units

between the response of working level 2 and working level

1, followed by the oil concentration (Factor B) with a differ-

ence of 16.49 units between the two working levels. Factor C

(type of wall material) did not generate an important impact

on the response, with a difference of 5.09 units.

Figure 1 shows the degree of incline of the slope as

response. The greater the difference between level 1 and level

2 for a variable, the higher the change magnitude of the

response. Therefore, the concentration of wall material (var-

iable A) presented the highest slope compared to the oil

concentration (variable B). Consequently, the higher the wall

material concentration and the lower the oil concentration,

the higher the ME.

Table 2. Levels selected as greater is better in the results obtained

for the microencapsulation efficiency response of linseed oil by

spray drying

Level

Control factors

A B C

N1 60.26 78.3 67.51

N2 79.86 61.81 72.6

Difference DN(2 � 1) 19.6 16.49 5.09

Factor A, encapsulating wall; B, oil concentration; C, type of

encapsulating wall.

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It should be taken into account that increasing the solids

concentration in the emulsion is favorable for an optimal

viscosity, which leads to a suppression of the internal circu-

lations and oscillations of droplets, thereby, improving reten-

tion [26]. However, should the optimal limit of viscosity be

increased, the retention will decrease due to a greater

exposure during atomization, the slow formation of discrete

droplets during atomization, and difficulties in droplet for-

mation [26]. As mentioned before, lower oil loads result in

higher retention [15, 27–30], as shown in this study.

Regarding the type of wall material, this variable did not

produce any significant change in the response since the

values were close to the average, restricting the effect of

control factors on the response. Furthermore, the combi-

nation (30% Factor A, 14% Factor B, and MD/GA

Factor C) ensures optimal conditions for obtaining a higher

ME of linseed oil. In addition, the Taguchi method made

it possible to determine which factors of the process

optimization most affect product quality with the minimum

amount of assays.

The results of theMEwere subject to an ANOVAwith the

aim of determining the degree of influence of the control

factors on the response. The Fexp values were 19.83, 14.03,

and 1.34 for Factors A, B, and C, respectively. The ANOVA

demonstrated that the determination coefficients (R2) for the

independent variables concentration of wall material (A), oil

concentration (B), and type of wall material (C) were 51.0,

36.0, and 3.4% respectively, to which A and B contributed

with 87% of the response. The analysis showed that the most

significant factor (p � 0.05) was the concentration of wall

material with a contribution (R2) of 51% to the ME.

However, the type of wall material did not significantly affect

(p � 0.05) the ME.

The determination coefficient for these three variables

was significant (p � 0.05), with R2 ¼ 90.4%, indicating a

high affinity or association of independent variables with the

ME of linseed oil.

The OTE for ME was determined by means of Eq. (4),

considering factors A and B together, which contributed 87%

of the response.

OTE ¼ T þ ðA2 � TÞ þ ðB1 � TÞ (4)

where T ¼ 70.1 is the total average of responses of exper-

imental runs, A and B are the control factors, and sub-indices

1 and 2 corresponding to working levels 1 and 2.

The OTE obtained (88.2%) represents the contribution

per working level of the two control factors.

As reported by Imagi et al. [17], a lower percentage of free

or non-encapsulated oil would indicate an efficient micro-

encapsulation process, thereby, improving the quality of the

microencapsulated product.

3.2 Determination of the oxidative stability of oil frommicrocapsules

The IT and the stability time (OSI) of the MLO by spray

drying was evaluated on total oil extracted frommicrocapsule

as an initial control of the optimized product (design point 3).

The IT represents the time elapsed since the start of measure-

ment until the beginning of the production of compounds

due to oxidation [39]. Therefore, a high IT value indicates

that the oil has greater oxidation stability. The results of the

Rancimat test for samples of MLO under optimal conditions

showed the value for IT of 2.83 � 0.62 h, and for OSI of

3.78 � 0.01 h.

Drusch [12] reported that lipid oxidation during the spray

drying depends on the drying temperature and on non-micro-

encapsulated oil, since this fraction is mainly what deterior-

ates during drying. Velasco et al. [40] reported that detection

of lipid oxidation in microencapsulated oils is critical because

this oxidation produces a loss of nutritional value and adverse

reactions in a wide range of foods on the market.

3.3 Microcapsule morphology

The samples of design points 1 and 3 obtained from spray

drying were analyzed by SEM.

Figure 2a shows the microcapsules obtained by design

point 3, which reached the highestME. The particle surface is

smooth and free of pores, which is essential for the stability of

the microcapsule as the pores facilitate the entry of oxygen

and the exit of the encapsulated material and, therefore, a

decrease in the oxidation of compounds such as fatty acids. In

recent studies, Fuchs et al. [41] encapsulated a vegetable oil

by spray drying using MD and GA as the carriers, leading

to the formation of small particles (<50 mm). The results

showed that the encapsulated oil was protected against oxi-

dation, contrary to the case of non-encapsulated oil.

Microcapsules with lower ME (design point 1) produced

with GA showed morphological differences. Figure 2b shows

collapsed or shrunken particles with rough surfaces.

According to Sheu and Rosenberg [42], microcapsules

obtained by spray drying using polysaccharides as the wall

0102030405060708090

A1 A2 B1 B2 C1 C2

Response

Working levels

Figure 1. Analysis of main effects of factors on the ME of linseed

oil.

428 M. Rubilar et al. Eur. J. Lipid Sci. Technol. 2012, 114, 423–433

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material show remarkable surface cracks and the formation of

dents or collapsed particles, which have been attributed to the

effects of the composition of encapsulating material, atom-

ization, and the drying parameters, among others. It has also

been reported that microcapsules with GA mixed with MD

and modified starch as wall materials showed improved

morphology, obtaining a more spherical shape [34].

Figure 3 clearly shows the presence of oil globules in the

microcapsules detected by the emitted fluorescence using

confocal microscopy.

Particle size distributions for microcapsules obtained

from design points 1 and 3 were measured by laser diffrac-

tion. For design point 1, the 10% of volume of particles were

distributed less to 5.8 mm, whereas, 90% of volume of

particles were distributed less to 46.6 mm. The distribution

mean was 17.6 mm. For design point 3, the 10% of volume of

particles were distributed less to 7.3 mm, whereas, 90% of

volume of particles were distributed less to 67.4 mm. The

distribution mean was 23.1 mm showing a homogeneous

distribution of microcapsules.

3.4 Fatty acid composition

To determine the fatty acid profile in this study (Table 3), the

sample with the highest ME yield of linseed oil by spray

drying (point design 3) was analyzed. Pure linseed oil was

used as control in order to evaluate the effect of the micro-

encapsulation treatment by spray drying on the fatty acid

profile of linseed oil.

Fifteen fatty acids in extracted linseed oil were identified

and quantified (Table 3). ALA was found in non-

encapsulated linseed oil (40.35%) and in MLO (34.97%).

Linoleic and oleic acids were also identified in non-

encapsulated oil (14.95 and 10.55%, respectively), and

MLO (8.45 and 5.46%, respectively).

The results in Table 3 showed that the homogenization of

the emulsion and/or the microencapsulation process by spray

drying affected the fatty acid composition of linseed oil.When

comparing the percentage of microencapsulated oil with the

untreated oil (control), a decrease in the percentage of fatty

acids was observed, probably due to the physical process of

encapsulation and chemical reactions that might change the

Figure 3. Confocal micrographies of MLO staining with nile red.

The presence of oil globules was detected in the microcapsules by

the fluorescence emitted using confocal microscopy.

Figure 2. Morphology of micropasules by SEM obtained for (a)

design point 3 and (b) design point 1. The particle surface of the

microcapsules obtained by design point 3 is smooth and free of

pores. The microcapsules of design point 1 produced with GA

showed collapsed or shrunken particles with rough surfaces.

Table 3. Fatty acid profile of linseed oil and microencapsulated

linseed oil

Fatty acid

Linseed oil

Microencapsulated

linseed oil

Fatty acid (%)

C14:0 Miristic 0.10 0.09

C15:0 Pentadecanoic 0.05 0.04

C16:0 Palmitic 1.89 1.22

C16:1 Palmitoleic 0.11 0.07

C17:0 Heptadecanoic 0.04 0.01

C17:1 Heptadecenoic 0.00 0.00

C18:0 Estearic 3.19 1.57

C18:1n9c Oleic 10.55 5.46

C18:2n6c Linoleic 14.95 8.45

C20:0 Eicosanoic 0.23 0.13

C18:3n3 a-Linolenic 40.35 34.97

C20:3n3 Eicosatrienoic 0.18 0.09

C20:4n6 Eicosatetraenoic 0.03 0.04

C22:2 Docosadienoic 0.11 0.06

C22:6n3 Docosahexaneoic 0.42 0.25

Eur. J. Lipid Sci. Technol. 2012, 114, 423–433 Development of soup enriched with omega-3 429

� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

nature of the original linseed oil. The amount of linolenic acid

decreased slightly; however, the amounts of oleic and linoleic

acid decreased by almost 50%.

3.5 Development of a soup powder enriched withmicroencapsulated linseed oil

3.5.1 Soup formulation optimization

The soup was prepared containing a fixed quantity of 14% of

MLO that provides approximately 40–80% of the RDI.

The European Academy of Nutritional Sciences EANS

and theHealth Council of theNetherlands suggest an average

intake of 2 g of ALA per day, and the International Society for

the Study of Fatty Acids and Lipids ISSFAL recommend

1.0 g of ALA per day.

The sensorial quality, expressed as the consistency and

saltiness of soup, was evaluated by panelists for establishing

the limits of the incorporation before optimization.

Consistency is a parameter related to texture, and saltiness

is one of the basic tastes and plays an important role for

humans. Saltiness, which is caused mainly by ionic materials,

is a good indicator of electrolyte balance in foods [43].

The optimization of the soup formulation was reached by

the RSM, using the central composite design, two control

factors (salty taste and consistence), and 11 design points.

The central points of the response of control factors showed

the best results with a description ranging between regular and

very good according to the sensorial scale used, qualifying

consistency as acceptable to suitable, and the salty taste as

identifiable to suitable. Regarding the SQE, the central points

presented the best and highest score, placing the response on

the sensory scale between fair to good which according to the

sensory scale used corresponds to still fair to good. In addition,

no difference was found between the SQE versus SQT values.

In order to determine the dependence degree of the

response with the independent variables, the experimental

data were submitted to regression analysis. A second-order

polynomial equation (Eq. 5) was obtained for the SQ (SQE)

of corn and basil soup.

YðSQÞ ¼ 3:75� 0:08X1 þ 0:13X2�

¼ :59X21 � 0:49X2

2 þ 0:03X1X2 (5)

The Fexp value ¼ 6.20 indicates that the model is signifi-

cant, with a 3.34% probability that this F value is due to

noise. The control factors, consistency and saltiness, in their

non-linear form showed a significant contribution of approxi-

mately 84% of the response variation. These control factors

generate a greater impact on the SQ compared with linear

forms that only contribute about 5%.

It was also demonstrated that the model has tight cause/

effect dependence between control factors and QS consider-

ing parameters such as R2 (88.61%), variation coefficient

(10.10%), and the signal/noise ratio 5.97 determining that

the reponse is robust and consistent. In addition, considering

that Fexp was significant (p < 0.05), it is possible to infer that

the equation has predictive capacity.

By analyzing the response surface for the soup, the best

combination obtained was corn starch 4.1 g and salt 2.3 g

(Fig. 4). Then, the soup was formulated based on the best

combination of corn starch and sodium-reduced salt to

obtain the best SQ (Table 4).

3.5.2 Sensorial analysis of optimized product

For the purposes of product validation, the optimized for-

mulation was evaluated by a new panel session. The QS

responses of panelists qualified the product according to

the sensorial scale between 3.41 and 5.00 with an average

of 4.40, indicating an acceptable, suitable, and good

2.43

2.76

3.10

3.43

3.76

Se

nsor

y Q

ualit

y

2.00

3.38

4.75

6.13

7.50

1.00

1.50

2.00

2.50

3.00

A: Corn starch (%) B: Salt (%)

Figure 4. Response surface of sensorial quality for the soup.

Table 4. Ingredients used in formulating soup for a portion of 32.3 g

powdered soup re-constituted in 200 mL water

Ingredient Amount (g) per size

Microencapsulated linseed oil 4.50

Dehydrated corn 14.00

Wheat flour 3.00

Dehydrated basil powder 0.08

Dehydrated onion powder 0.30

Dehydrated pepper powder 0.20

Skim milk 0% fat 3.00

50% Reduced-sodium salt 2.30

Corn starch 4.10

Monosodium glutamate 0.20

Sodium caseinate 0.20

Tricalcium phosphate 0.40

430 M. Rubilar et al. Eur. J. Lipid Sci. Technol. 2012, 114, 423–433

� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

consistency and an accepatble and good salty taste.

Therefore, the optimized product was validated by the pan-

elist session.

3.5.3 Acceptability test

According to the age range, the consumers qualified the

product with an average value of 4.6 from 10 to 19 years

old, 4.7 from 20 to 29 years old, 4.7 from 30 to 39 years old,

4.5 from 40 to 49 years old, and 4.7 for panelists over 50.

Furthermore, the product was qualified as a product that the

consumers like a lot with an average value of 4.6. Regarding

the product qualification, 66% of consumers like the soup a

lot and 34% of them like it. According to the salty taste, 55%

of consumers like the soup a lot, 37% of them like it, and 8%

of consumers showed a neutral response I do not like it and I

do not dislike it. With respect to the consistency, 54% of

consumers like the soup a lot, 42% of them like it, and 4% of

consumers showed a neutral response.

3.5.4 Soup characterization

A proximal analysis was performed to determine the chemical

composition of the soup (Table 5a) and to elaborate a nutri-

tion label [44] (Table 5b). These results were compared with

the composition of soups currently on the market (Table 5c).

Compared with soups currently on the market, the soup

formulated in this study presented similar energy and carbo-

hydrate values, an increased protein content (between 24 and

36% more) and 25% greater total fat content. However, it is

noted that the oil in the soup is unsaturated with a high

omega-3 content. The oxidative stability and the shelf-life

of oil extracted from the optimized soup were determined by

Rancimat test. The average IT was 14.85 h and the stability

time was 15.61 h. The determined shelf-life was 8.78months

(Table 6), which is less than the soups on the market

(12 months). The MLO and soup formulated in this study

contained no antioxidants so as to prevent the lipid per-

oxidation. Then, with the addition of antioxidants, the soup

formulated in this study could present a longer shelf-life.

In conclusion, by means of Taguchi methodology, the

combination of variables that yielded the highest ME of oil

(90.7%) was obtained from design point 3 using a wall

material concentration of 30%, oil concentration of 14%,

and a mixture of MD/GA as a type of wall material. The

results of this study demonstrated that both the wall material

concentration and the oil concentration significantly affect

(p < 0.05) the efficiency of microencapsulation of linseed oil

by spray drying. These variables together explained 87% of

the variation of ME. Using SEM and laser diffraction, the

microcapsules obtained under these conditions presented a

spherical shape, with a smooth surface, and homogenous

distribution. All characteristics provided stability of the

product.

The incorporation of MLO in an optimized formulation

of soup made it possible to provide a source of omega-3

for a high-consumption food (soup) with health benefits,

obtaining a product with added value highly acceptable to

consumers.

Table 5. (a) Chemical composition of the soup per 100 g of product,

(b) nutritional information of soup and (c) Nutritional composition of

commercial soups per 100 g of product

Parameter Optimized formulation

(a)

Moisture (%) 7.66

Proteins (%) 9.99

Total fat (%) 11.51

Ash (%) 10.94

Crude fiber (%) 1.5

Carbohydrate (%) 58.4

Sodium (mg) 1536

Calories (kcal) 377.15

Nutritional information

Portion: 32.3 g of powdered soup and 200 mL water

Servings per container: 5

100 g 1 portion

(b)

Energy (kcal) 377.15 127.5

Protein (g) 9.99 3.38

Fat (g) 11.51 3.9

Omega-3 fatty acid (mg) 979 316

Carbohydrates (g) 58.4 19.7

Crude fiber (g) 1.5 0.51

Sodium (mg) 1536 519.17

Parameter Range

(c)

Energy (kcal) 296–388

Protein (g) 7.3–8.0

Total fat (g) 2.7–9.2

Carbohydrate (g) 58.0–69.3

Sodium (mg) 2700–5800

Table 6. Determination of shelf-life of soup using lineal regression

of Log10(OSI) versus temperature (8C)

Temperature (8C) OSI Log OSI

80 74.49 1.87

90 36.82 1.57

100 15.61 1.19

110 8.34 0.92

120 3.86 0.59

OSI20 ¼ 10Log(�0.0322 � 20 þ 4.4449).

OSI20 ¼ 6322.66 h.

Eur. J. Lipid Sci. Technol. 2012, 114, 423–433 Development of soup enriched with omega-3 431

� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

This research was supported by funding from Conicyt through

Fondecyt project 1090516 and partially by the Research Office at

the Universidad de La Frontera through GAP technical support.

The authors have declared no conflict of interest.

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