Journal of Food Engineering 105 (2011) 585591

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Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Development of functional bread containing nanoencapsulated omega-3 fatty acids

Vural Gkmen a,b,, Bure Ata Mogol b, Roberta Barone Lumaga c, Vincenzo Fogliano c, Zoya Kaplun d, Eyal Shimoni d
a

Food Research Center, Hacettepe University, Beytepe Campus, 06800 Ankara, Turkey Department of Food Engineering, Hacettepe University, Beytepe Campus, 06800 Ankara, Turkey c Department of Food Science, University of Napoli Federico II, Napoli, Italy d Faculty of Biotechnology and Food Engineering, Technion Israel Institute of Technology, Haifa 32000, Israel
b

a r t i c l e

i n f o

a b s t r a c t
The aim of this study was to develop functional bread enriched with omega-3 fatty acids. High amylose corn starch was used to form nanosized complexes with ax seed oil that was converted to powder of microparticles by spray drying. The particles were then incorporated into bread formulation at different amounts to investigate their effects on bread quality characteristics. The effects of encapsulation on the formation of lipid oxidation products and thermal process contaminants including acrylamide and hydroxymethyl furfural (HMF) were determined. Encapsulation signicantly decreased lipid oxidation as measured by the formation of hexanal and nonanal in breads during baking. Increasing the amount of particles in dough signicantly decreased the formation of acrylamide and HMF in breads. Scanning electron microscopic analysis of bread demonstrated that particles added to dough remained intact in the crumb, but partially destroyed in the crust. Comparing to its free form, addition of nanoencapsulated ax seed oil increased nal product quality and safety by lowering lipid oxidation and formation of harmful compounds in breads during baking. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 6 January 2011 Received in revised form 15 March 2011 Accepted 17 March 2011 Available online 21 March 2011 Keywords: Nanoencapsulation Omega-3 fatty acids Functional bread Lipid oxidation Acrylamide

1. Introduction Encapsulation of active food ingredients is an important application that can be attained by nanotechnological approaches. Nanoencapsulation is useful for developing functional food products via delivery of bioactive compounds, such as vitamins, antioxidants, and enzymes (Baeumner, 2004). It provides enhanced product functionality and stability including controlled release of the core during storage (McClements et al., 2009). Long chain omega-3 polyunsaturated fatty acids (PUFAs) have a well-established role in lowering blood serum triacylglycerol and cholesterol concentration (Harris et al., 2008). On the other hand, PUFAs, such as sh oil and ax seed oil, are very susceptible to oxidation during processing and storage resulting in decreased nutritional value and sensory quality. One possible way to protect lipids against oxidation is encapsulation. Zimet and Livney (2009) showed that fatty acid molecules entrapped in electrostatic nanocomplexes consisting of b-lactoglobulin and pectin could be protected against oxidation during an accelerated shelf-life test. Borneo et al. (2007) have used encapsulated omega-3 fatty acids in cream-lled sandwich cookies. They have shown that it was
Corresponding author at: Department of Food Engineering, Hacettepe University, Beytepe Campus, 06800 Ankara, Turkey. Fax: +90 356 2521488. E-mail address: vgokmen@hacettepe.edu.tr (V. Gkmen).
0260-8774/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.03.021

possible to make shelf-stable fortied foods with high levels of long-chain omega-3 fatty acids without any adverse effect on sensory properties. This study aimed to develop functional breads enriched with the particles of nanoencapsulated omega-3 fatty acids. The effects of the amount of particles on the quality characteristics of bread were determined. The role of encapsulated omega-3 PUFA on lipid oxidation, and on the formation of thermal process contaminants were also investigated on both loaf bread model and bread crisp model where the thermal stress is amplied. 2. Material and methods 2.1. Materials High amylose corn starch (HACS) was used for the formation of nanocomplexes with ax seed oil. HACS is an unmodied corn starch, which contains approximately 70% amylose and approximately 28% amylopectin (fat, protein, calcium, and iron approximately 2%) (HYLON VII, National Starch, USA). HACS was chosen since it possesses the largest amylose portion ($70%), leading to obvious commercial advantage in the continuous process, since more amylose molecules will be available for the complexation with the guest. Amylose shows a strong ability to form complexes with a variety of molecules, compared to amylopectin, which

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exhibits a weak ability towards formation of inclusion complexes. Therefore, the amylose to amylopectin ratio is an important factor that produces variability in the ability of natural starches to bind lipids. The ax seed oil used contained 56% a-linolenic acid (Jerusalem LeSomsom, Israel). Wheat our, sugar, salt, shortening, ascorbic acid and yeast for preparing breads were supplied by MERK (Ankara, Turkey). Acrylamide (99+%) and HMF were purchased from Sigma (Diesenhofen, Germany). Potassium hexacyanoferrate, zinc sulfate, formic acid, potassium hydroxide, and phosphoric acid were of analytical grade and obtained from Merck (Darmstadt, Germany). HPLC gradient grade acetonitrile was purchased from J.T. Baker (Deventer, Holland). Oasis MCX solid phase extraction cartridges, Atlantis T3 column (150 4.6 mm, 3 lm), Atlantis dC18 column (4.6 300 mm, 5 lm), 0.45 lm syringe lters, and 2 ml autosampler vials were supplied by Waters Corp (Berkshire, England). Carboxenpolydimethylsiloxane solid phase micro-extraction bers and 40 ml headspace vials were purchased from Supelco (Bellefonte, PA, USA). HP-INNOWAX capillary column (30 m 250 lm i.d., 0.25 lm lm thickness) was supplied by Agilent Technologies (Wilmington, DE, USA). Ultra pure water was used throughout the experiments (MilliQ system, Millipore, Bedford, MA, USA). 2.2. Preparation of the powder of nano-encapsulated ax seed oil V-type starchoil complexes were produced via acidication of an alkali solution mixture of starch and ax seed oil based on a method previously described by others (Lesmes et al., 2008). Basically, 10% of HACS solution in 0.1 M KOH (w/v) was preheated to 80 C 1 C for 30 min and then cooled to 30 C 1 C. Flax seed oil was added to solution during constant stirring at ratio of 10% (10 g oil per 100 g HACS). This alkali mixture was then pressurized through a high-pressure homogenizer (Micro DeBee, BEE International, MA, USA) simultaneously with 0.1 M H3PO4 to yield a cloudy dispersion of nanoparticles. The pH of the mixture after homogenization was 5.35.8. The suspension of nanoparticles was converted into the powder of microparticles by spray drying. Spray drying operation was performed with a spray dryer type Production Minor (GEA Niro, Denmark), equipped with a rotary atomizer (GEA Niro, Denmark), adjusted to a wheel speed of 18,000 rpm. The air inlet temperature was 200 3 C, and outlet 80 3 C. Suspension ow was 7.71 l/h. During spray drying, nano-sized complexes occurred in solution phase formed micronsized aggregates (solids). 2.3. Characterization of the particles of nano-encapsulated ax seed oil 2.3.1. Lipid load Total lipid content of the powder was determined by Mojonnier method (AOAC, 2005). Lipid content was calculated using the following formula;

structures (Biais et al., 2006; Godet et al., 1995; Jouquand et al., 2006; Lalush et al., 2005; Le Bail et al., 2005).

2.3.3. Particle size distribution Mean particle size and particle size distribution were measured by Coulter counter (LS 320, Coulter Corporation, FL, USA). This was achieved by injecting samples into a water lled chamber of a LS230 coulter counter equipped with a Polarized Intensity Differential Scattering module (PIDS module) using three wavelengths and two polarization directions. The laser scattering data of the suspensions was collected over periods of 90 s and then processed using LS230 software, based on the general Fraunhofer optical model, with water dened as solvent.

2.4. Preparation of functional breads and bread crisps 2.4.1. Breads Bread was prepared using the AACC Method 1010B (AACC, 2000). The AACC formula consisted of wheat our (100 g), sugar (6 g), salt (1.5 g), shortening (3 g), ascorbic acid (50 mg/kg) and yeast suspension (5.3 ml). Flour and other ingredients were mixed until dough development. The particles of nano-encapsulated ax seed oil were directly added to dough at the levels of 0.0 (control), 1.0%, 2.5%, 5.0% and 10.0% (w/w) together with the other ingredients. Following rst fermentation (52 min), rst punch, second fermentation (25 min), second punch (13 min), panning, and proong (40 min) steps, the breads were baked at 220 C for 24 min in pans (7.3 cm height; 9.5 15.2 cm top; 7.5 13.2 cm bottom). The nal dough weight was approximately 150 g prior to baking. Baking experiments were run in two replicates for each formulation. Texture analyses were performed approximately after 2 h of baking.

2.4.2. Bread crisps Model bread crisps were prepared as they could represent a bread crust model. Briey, a dough prepared with a commercial our and water was used to prepare crisps added with two different amounts of free ax seed oil (FO) or encapsulated ax seed oil (EO). The recipes used to prepared dough are given in Table 1 Prepared dough was used to prepare crisps with 6 cm of diameter and weight of 3.0 0.1 g. They were baked in an oven (Rex, Italy) at 180 C for 10, 20, and 30 min. Ground bread and bread crisp samples were stored at 20 C until analyses of acrylamide, HMF and lipid oxidation products. Analyses were run in two replicates and mean values were reported.

2.5. Measurement of quality characteristics of functional breads 2.5.1. Loaf volume The volume of bread was determined by rapeseed displacement method using a loaf volumeter after the loaves were cooled to room temperature.

Total lipids % Weight of lipids=Total weight of sample

2.3.2. X-ray diffraction The formation of V-type complexes was veried by measuring the X-ray diffraction of powders using a Philips PW 3020 powder diffractometer equipped with a graphite crystal monochromator (Philips, The Netherlands). The operating conditions were Cu Ka1 radiation (0.154 nm), voltage 40 kV, and current 40 mA. Approximately 200 mg of sample powders were loaded onto an aluminum plate and scanned over the range 530 Bragg angles in steps of 0.02/s. Diffraction patterns were compared to what has already been extensively described in literature to be indicative of V-type

Table 1 The recipes used to prepare bread crisps with free and encapsulated ax seed oil. Ingredient Amount (%) Free Water Flour Flax seed oil HACS-ax seed oil complex
a

Encapsulateda 34.09 56.82 9.90

37.13 61.88 0.99

HACS-ax seed oil complex contained approximately 10% of oil.

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2.5.2. Texture Texture analyzer equipped with a 50 N load cell (TA Plus, Lloyd Instruments, UK) was used to determine texture prole of bread loaves. The force required to compress two slices (each 10.0 mm thickness) by 25% was measured with a TA4 probe (1.5 in. diameter) at a speed of 1 mm/s. Texture measurements were performed ve times for each sample and mean values were reported. 2.5.3. Color Color analyses of bread samples were performed using a computer vision based image analysis. The bread pictures were acquired using a digital camera and transferred to a personal computer. Color space parameters were determined in Lab by means of MATLAB code (Color_Lab.m) described earlier (Gkmen and Sugut, 2007). Color values of the crust of control bread were taken as the reference to calculate color differences (DE) of breads incorporated with the particles of ax seed oil. The DE was calculated by the following formula;

2.6. Measurement of neo-formed compounds 2.6.1. Acrylamide Bread samples were prepared for acrylamide analysis using a procedure described by us elsewhere (Gkmen et al., 2009). Briey, ground samples (1 g) were triple extracted with 10 mM formic acid (10, 5, and 5 ml). The combined extract was claried by Carrez clarication. The extract was further cleaned up using Oasis MCX SPE cartridge. The nal extract was ltered through 0.45 lm nylon lter and analyzed by LC-APCI-MS. An Agilent 1200 HPLC system (Waldbronn, Germany) consisting of a binary pump, an autosampler and a temperature controlled column oven, coupled to an Agilent 6130 MS detector equipped with multimode interface was used. The following interface parameters were applied: drying gas (N2, 20 psig) ow of 5 L/min, nebulizer pressure of 20 psig, drying gas temperature of 350 C, capillary voltage of 2000 V, and corona current of 5 lA. The analytical separation was performed on a Atlantis T3 column using the isocratic mixture of 10 mM formic acid at a ow rate of 0.3 ml/min at 25 C. The LC eluent was directed to the MS system from 10 to 16 min using MSD software. Ions monitored were m/z 72 and 55 for the quantication of acrylamide in the samples. The quantitation was based on a calibration curve in a concentration range between 0.01 and 0.10 lg/ml. The limit of quantitation was 5 ng/g. 2.6.2. HMF Bread samples were prepared for HMF analysis as described for acrylamide sample preparation procedure. HMF was analyzed by high-performance liquid chromatography (HPLC) (Gkmen and Senyuva, 2006). Agilent 1200 HPLC system (Waldbronn, Germany) consisting of a quaternary pump, an autosampler, a diode array detector and a temperature-controlled column oven was used. The chromatographic separations were performed on an Atlantis dC18 column, using the isocratic mixture of 10 mM formic acid solution and acetonitrile (90:10, v/v) at a ow rate of 1.0 ml/min at 25 C. Data acquisition was performed, acquiring chromatograms at the detection wavelength of 285 nm. HMF concentrations were calculated from the calibration curve that built with standard solutions of HMF dissolved in Milli-Q water (0.1 to 1.0 lg/ml). 2.6.3. Lipid oxidation products The lipid oxidation products in breads were analyzed by headspace GCMS just after baking and after 7 days storage at room temperature. One gram of bread slice was placed in a headspace vial (40 ml). The vial was tightly closed by means of a capper. A carboxenpolydimethylsiloxane solid phase micro-extraction ber was used to adsorb the volatile lipid oxidation compounds released from the sample. The ber was inserted into the vial and equilibrated at 40 C for 30 min prior to GCMS analysis. An Agilent 6890 gas chromatograph coupled to an Agilent 5973 single quadrupole mass spectrometer was used for the determination of hexanal and nonanal. The volatiles adsorbed onto the ber were desorbed in the injection port maintained at 250C for 5 min. The chromatographic separation was performed on a HP-INNOWAX column (30 m 250 lm i.d., 0.25 lm lm thickness). The temperature program for the GC was as follows: isothermal for 5 min at 50 C, increased at a rate of 5 C/min from 50 to 150 C, increased at a rate of 10 C/min from 150 to 220 C, and isothermal for 5 min. The ow of the carrier gas helium was 1.2 ml/min. The analysis was performed using electron ionization (70 eV) in scan mode. The peaks of aroma compounds appeared in GCMS chromatograms were identied by using NIST and Wiley libraries.

DE

q 2 2 2 L 0 L a0 a b0 b

where L 0 , a0 , and b0 correspond to the CIE color parameters of the crust of control, whereas L , a , and b correspond to the CIE color parameters of bread incorporated with the particles of nano-encapsulated ax seed oil.

2.5.4. Porosity The crumb porosity was analyzed by a computer vision based image analysis. High-resolution scanned images of bread slices were processed by means of the MATLAB code (Porosity_Index.m) to segment the images into two classes representing porous and nonporous regions of the crumb. The code used to segment bread crumb images is given below: % File PorosityIndex.m function [seg_im] = PorosityIndex(im_seg,u); u=im2double(u); [r c h]=size(im_seg); im=reshape(im2double(im_seg),rc,h); for i=1:4 dist(i,:)=sum((im-repmat(u(:,i),[1 rc])).^2); end [y loc]=min(dist); seg_im=zeros(rc,h); for i=1:4 pos=find(loc==i); seg_im(pos,:)=repmat(u(:,i),[length(pos) 1]); end seg_im=reshape(seg_im,[r c h]); figure(2); imshow(seg_im,[]); ratio1=length(find(loc==2))/ length(find(loc$=4)); ratio2=length(find(loc==3))/ length(find(loc$=4)); PorosityIndex=ratio1+ratio2 ; disp(PorosityIndex); The segmented images were used to calculate porosity index by the following formula:

Porosity index

Number pf pixels representing holes Total number of pixels

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V. Gkmen et al. / Journal of Food Engineering 105 (2011) 585591 Table 2 Some physical properties and texture prole parameters of breads incorporated with the particles of nano-capsulated ax seed oil (different letters in the rows indicate statistically signicant differences). Amount of particles* Control Weight (g) Specic volume (cm3/g) Porosity index Hardness (N) Cohesiveness Springiness index Gumminess (N) Chewiness (N mm)
*

2.7. Scanning electron microscope (SEM) analysis of functional breads Bread and bread crisp samples were lyophilized prior to SEM analysis. Lyophilized sample placed on a stub was coated with a thin layer (10 nm) of Au/Pd. A FEI Quanta 200 F model SEM (Oregon, USA) instrument equipped with a Schottky eld emission gun for optimal spatial resolution was used to image the samples. 2.8. Statistical analyses The data were subjected to analysis of variance (ANOVA) and least signicance difference test to determine difference between means at a signicance level of p < 0.05. 3. Results and discussion 3.1. Characteristics of nano-encapsulated ingredients Off-white colored powder of nano-encapsulated ax seed oil that is able to ow freely was obtained by spray drying. The oil content of the powder was 11.6 0.6 g ax seed oil per 100 g powder. The formation of V type complexes was veried by measuring their X-ray diffraction. The diffraction revealed Bragg angles (2h) of $17 and 20, which suggests mix type of B and V polymorphs. These Bragg angle values are typical for both crystalline types. Mean particle size and particle size distribution are important for the functionality of the inclusion complexes as a delivery system. Mean size based on laser diffraction of the particles was calculated as volume weighted mean average or surface weighted mean average. The analysis of particles dispersed in water showed a volume weighted mean average of 20.05 2.40 lm, and surface weighted mean average of 8.68 0.68 lm (Fig. 1). This veried the formation of aggregated nanoparticle complexes. 3.2. Bread quality characteristics The selected quality characteristics of breads are given in Table 2. Increasing the amount of particles also increased bread weight

1.0% 145.5a 3.6a,b 0.5a 5.3c,d 0.6a 0.9a 2.9c,d 16.8 c

2.5% 146.6a 3.2b,c 0.4b 6.6b,c 0.6a 0.9a 3.6b,c 21.6b

5.0% 148.4a 2.7c,d 0.4b 8.0b 0.5a 0.9a 4.3b 25.7b

10.0% 150.1a 2.3d 0.4b 14.4a 0.5a 0.8a 7.0a 37.8a

144.0a 3.9a 0.5a 4.2d 0.5a 0.9a 2.3d 13.5 c

Particles of spray dried HACS-ax seed oil complex containing approximately 10% oil.

Fig. 1. Particle size distribution curves calculated from laser diffraction of particles. Flax seed oil dispersed in water at pH 5.7. All scattering curves were processed using Mie scattering model.

and decreased bread volume, which led to increased loaf density. Since the pH of the particles of nano-encapsulated ax seed oil was 5.35.8, its inuence on the pH value of dough was limited. However, it was a fact that increasing the amount of particles decreased the relative concentration of gluten in the formulation adversely affecting its functionality. For example, addition of 10.0% of particles caused approximately 1.0% of dilution in gluten content of bread dough. This was the main reason for the loss of loaf expansion when higher amount of particles were used in the formulation. It should be stated here that high amylose corn starch was used as the coating material for the encapsulation of ax seed oil. The loaf expansion problem could be resolved by increasing gluten content approximately by 1.0% in the formulation. The addition of particles had signicant effects on the measured texture prole of breads. Of the texture parameters measured, hardness, gumminess and chewiness of breads linearly increased as the amount of particles increased (Table 2). Meanwhile cohesiveness and springiness parameters remained relatively stable. Hardness was one of the simply perceived feature of breads with added particles of nanoencapsulated ax seed oil. When the amount of particles exceeded 5.0%, the crumb structure became harder as easily noticed by manual examination of slices. Texture prole analyses of breads gave comparable results to those of sensory analysis. Adding the particles of nano-encapsulated ax seed oil at amounts exceeding 5.0% decreased the rate of drying during baking of bread loaves. This adversely affected the crumb porosity making the loaves harder. Crust color, measured by digital image analysis, was evaluated with calculated DE values. When 1.0% of particles were incorporated into bread formulation, DE value was found to be 4.92 1.60. DE values were found as 6.36 0.81, 11.00 0.68 and 11.50 1.13 for 2.5%, 5.0%, and 10.0% of particles added to breads, respectively. The difference in the crust colors of breads was obvious for breads incorporated with 5.0% and 10.0% of particles. In terms of surface browning of loaves, breads formulated with 5.0% and 10.0% of particles had lighter crust color while other breads having 0.0%, 1.0%, and 2.5% of particles had comparable browning in their crusts. During baking, the development of browning over the surface of bread is considered as an indication of the progress of the Maillard reaction. The rate of browning increases as the baking temperature increases. Initial moisture content of dough determines time/temperature prole of bread, thus, browning during baking. Since the addition of particles increased water absorption, the initial moisture contents were slightly higher for the dough incorporated with 5.0% and 10.0% of particles. Water absorption capacity for our contained with 0.0%, 1.0%, and 2.5% of particles was 62%. It increased to 65% and 70% for our contained with 5.0% and 10.0% of particles. These samples most probably had slightly lower tem-

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perature proles comparing to the control sample due to evaporative cooling. It was obvious from the visual examination of breads that the addition of particles inuenced the crumb porosity in terms of both number of holes and their size. A computer vision based image analysis algorithm was used to calculate the porosity index, which is dened as the total area of holes divided by the total area of slice. It was interesting to note that porosity index of bread crumb was inversely correlated with the amount of particles in dough formulation. 3.3. Neo-formed compounds 3.3.1. Lipid oxidation products Effect of encapsulation on lipid oxidation was determined by measuring hexanal and nonanal which are two marker compounds indicating the degree of lipid oxidation. The concentration of hexanal and nonanal in breads increased as the amount of particles increased from none to 10.0%. However, these markers signicantly decreased in breads after one week of storage at room temperature as shown in Fig. 2a. The formation of nonanal and hexanal was more pronounced in bread containing ax seed oil in the free form than in the encapsulated form (Fig. 2b). The data clearly demonstrated that use of nanoencapsulated ax seed oil reduces the formation of lipid oxidation products in breads. 3.3.2. Acrylamide The crust and the crumb parts of the bread have different risks in terms of the formation of thermal process contaminants. The

crumb temperature never exceeds 100 C during the baking process. The chemical reactions requiring higher temperatures do not occur or proceed at lower rates in the crumb. However, the temperature of crust approaches to oven temperature as soon as the moisture content decreases to a critical level. This makes the crust susceptible to form acrylamide during baking. Results showed that the crumb was free of acrylamide, while it is concentrated in the crust. Acrylamide concentration of was around 20 ng/g for bread without addition of nano particles and there was a gradual decrease in acrylamide concentration of breads as the amount of particles increased from none to 10.0% (Fig. 3a). This result revealed that using nanoencapsulated ax seed oil it is possible to reduce the risk associated to acrylamide formation in bread. However, recent studies have conrmed that lipid oxidation can contribute to the formation of acrylamide to a certain extend (Zamora and Hidalgo, 2008). It is therefore critically important to validate the safety of the incorporation of nanoencapsulated omega-3 fatty acids into bread. It is well known that polyunsaturated fatty acids are easily oxidized at extreme temperatures. Thermoxidation of polyunsaturated fatty acids can generate reactive carbonyls, which may further react with asparagine leading to acrylamide (Capuano and Fogliano, 2011). In the crisp model, water loss and temperature increase are faster than in the loaf, and consequently, browning reactions is much more diffused. For this reason, this model represents a suitable approach to investigate the role of added ingredients on acrylamide formation (Capuano et al., 2010). For both recipes, the results of crisp model study revealed that acrylamide formation become signicant after 20 min of baking at 180oC. There was further increase

Fig. 2. (a) Change of hexanal concentration in breads incorporated with different amounts of nanoencapsulated ax seed oil (different letters indicate statistically signicant differences), and (b) comparison of lipid oxidation risk in breads added with 0.5% of free and nanoencapsulated ax seed oil (p < 0.05).

Fig. 3. (a) Effect of particles of nanoencapsulated ax seed oil on acrylamide formation in breads (different letters indicate statistically signicant differences) and (b) comparison of acrylamide formation in bread crisps added with 1.0% of free and nanoencapsulated ax seed oil (p < 0.05).

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in acrylamide concentration of crisps after 30 min of baking under the same conditions (Fig. 3b). The results indicated that the crisps added with free ax seed oil as a source of omega fatty acids contained signicantly higher amounts of acrylamide than the crisps added with comparable amount of nanoencapsulated ax seed oil (p < 0.05). This can be explained considering that carbonyls arising from the thermoxidation of free ax seed oil during baking can promote the conversion of asparagine into acrylamide (Zamora and Hidalgo, 2008; Capuano et al., 2010). When the oil is encapsulated, reactive carbonyls are not available for this reaction due to prevention of its thermoxidation during baking. 3.3.3. HMF Fig. 4a showed how the amount of particles added modied the HMF formation on bread. Similar to acrylamide, HMF formation is concentrated on the crust layer while the crumb was free of HMF. The crust layer of control sample had approximately 60 mg/kg of HMF. It slightly increased when 1.0% and 2.5% of particles were added to bread. But, HMF concentration decreased when 5.0% or more particles were added to bread. Contrary to the results obtained for acrylamide in bread loaves, HMF formation was strongly increased by using the particles of nanoencapsulated ax seed oil, in a concentration-dependent way (Fig. 4b). This suggests that, in this experimental model, the starch-based coating material used to encapsulate ax seed oil give an important contribution to HMF formation. Its role could be related to:  amylose content, it can be supposed that the high amylose corn starch used as coating agent is more reactive than the starch naturally present in ours

 chemical state deriving by nano-encapsulation process (physico-chemical treatments, complexation with lipids)  physico-chemical changes derived from interaction between particle material and food matrix and/or thermal processing This particular behavior for HMF production in crisps model seems to conict with bread results, but it should be taken into account that very different dimensions and baking condition characterize each experiment. As consequence, completely different temperature and water activity curves over the time can be observed. In bread production, bigger dimension determine a lower temperature increase associated also with a smaller water activity loss. Therefore, the contribution of added particles to the water holding capacity results in an higher impact on HMF formation process, respect to the possible chemical role of starchy material. In a thinner food model, in which water loss occurs more rapidly and can be controlled only up to a certain extent, the others chemical aspects (as carbohydrates structure/composition) may play the major role.

Fig. 4. (a) Effect of particles of nanoencapsulated ax seed oil on acrylamide formation in breads (different letters indicate statistically signicant differences), and (b) comparison of HMF formation in bread crisps added with 1.0% of free and nanoencapsulated ax seed oil (p < 0.05).

Fig. 5. SEM images of bread incorporated with 10% nano-encapsulated HACSomega-3 complex, (a) crumb, and (b) crust.

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3.4. Processing stability of the particles The main potential benet offered by nano-encapsulation is the protection of bioactive ingredients. Foods form very complicated matrices with varying chemical composition and physical structure. Both chemical composition and physical structure are subject to change upon processing and during storage. The integrity of the particles of nano-encapsulated ingredients were analyzed by imaging them by scanning electron microscope (SEM). The crust and crumb layers were analyzed separately. The particles in the crust region were largely damaged due to more severe thermal conditions at crust regions during baking. However, the particles remained intact in the crumb regions as illustrated in Fig. 5. A temperature gradient occurs in crust and crumb parts during baking due to evaporative cooling phenomenon. As a result, crust layer temperature gradually increases to oven temperature as the baking proceeds (>200 C) while crumb temperature remains approximately at 100 C throughout the baking process. The particles of nano-encapsulated ingredients could be easily distinguished from the native starch globules. The size of the particles was approximately $5 lm, and non-homogenously distributed in the crumb.

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4. Conclusion In conclusion, the data presented in this paper demonstrated that it is possible to include in bread signicant amount of nanoencapsulated omega-3 fatty acids without affecting its sensorial properties. Moreover, the use of encapsulation allows reducing off avor and improving sensorial quality respect to the same amount of omega-3 as free oil. Beside these sensorial advantages, encapsulation also reduces thermoxidation of fatty acids during baking. This is not only an advantage through itself, but it can contribute to the reaction of these oxidation products with amino acids thus contributing to the formation of potentially harmful Maillard reaction products.

Acknowledgment The data presented in this manuscript was derived from NANOFOODS project funded by the European Commission (Project no: 222006).