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Beta-Carotene Stability in Extruded Snacks ProducedUsing Interface Engineered EmulsionsGulsah Caliskanab, Aaron S.L. Lima & Yrjӧ H. Roosa

a Food Technology, School of Food and Nutritional Sciences, University College Cork, Irelandb Food Engineering Department, Ege University, Izmir, TurkeyAccepted author version posted online: 10 Feb 2015.

To cite this article: Gulsah Caliskan, Aaron S.L. Lim & Yrjӧ H. Roos (2015): Beta-Carotene Stability in ExtrudedSnacks Produced Using Interface Engineered Emulsions, International Journal of Food Properties, DOI:10.1080/10942912.2014.973963

To link to this article: http://dx.doi.org/10.1080/10942912.2014.973963

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BETA-CAROTENE STABILITY IN EXTRUDED SNACKS PRODUCED USING INTERFACE ENGINEERED EMULSIONS Gulsah Caliskana,b, Aaron S.L. Lima, *Yrjӧ H. Roosa

aFood Technology, School of Food and Nutritional Sciences, University College Cork, Ireland

bFood Engineering Department, Ege University, Izmir, Turkey

ABSTRACT

The objectives of the present study were to produce snack-type extrudates and to investigate their

ability to encapsulate and protect β-carotene (0.05% w/w in sunflower oil) using single layer

(SL) and layer by layer (LBL) emulsions as an ingredient. The dry feed composed of wheat flour

(60% w/w dry solids), maltodextrin (DE 23-27, 20% w/w dry solids), and lactose (20% w/w dry

solids). The extrudates (0.6 aw) were ground and sealed in vials under vacuum, placed in vacuum

sealed plastic pouches and stored at 20, 40 and 60°C. Analysis of the beta-carotene content

during storage was carried out using HPLC with a C30 column and diode array detector. The

results showed rapid loss of β-carotene during the first 6 days at all temperatures. Further losses

of β-carotene at 20 and 40°C occurred gradually levelling off at 27 days. It was noted that the %

retention of β-carotene was generally higher in LBL extrudates with LBL upon storage for 27

days. It can be concluded that the LBL emulsion may enhance protection of bio sensitive

compounds in glassy membranes.

Keywords: extrusion; β-carotene; HPLC; layer by layer emulsion; microencapsulation

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INTRODUCTION

Extrusion is a significant food processing technology used to produce breakfast cereals, ready to

eat snack foods as well as other textured foods since mid-1930s. By using extrusion, raw

materials can be converted to various intermediate and finished food products.[1] Increased

production and consumption of snack foods has led to expanding choices of products being made

available to consumers.[2] ‘Snack foods’ are light meal products that are easy to handle, ready to

eat and small in size.[3] Extrusion processes enable continuous operation in cooking and shaping.

Extrusion is often a high temperature, short time process (HTST) that involves molecular

transformations and chemical reactions.[4] The high mechanical shear in the twin screw extrusion

process causes breaking of covalent bonds in biopolymers. The structural disruption and mixing

promotes changes in functional properties of food ingredients as well as providing texture.[5]

Starch as an ingredient plays a very important role in extrusion since changes in starch structure

such as gelatinization, dextrinization, fragmentation and fusion will affect the texture and

expansion of the final product.[3] Extrusion may denature proteins and produce complexes

between lipids and starch as well as between lipids and proteins.[6] The advantages of an

extrusion process includes the continuous production of high quality products, the capability to

produce products with textural advantages such as crispiness and mouthfeel, low operating cost,

high productivity and reduced cooking time.[7,8] The gelatinization of starch increases the

digestibility of the products. [9]

There has been increasing knowledge on the composition of food products and its influence on

foods nutritional quality. The presence of bioactive compounds in food products plays a

significant role in preventing chronic and degenerative diseases in humans.[10] Carotenoids

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provide 70% of the vitamin A in human diet. Due to its high antioxidant capacity as well as

provitamin A activity, β-carotene has received more attention compared to other carotenoids.[11]

β-carotene occurs naturally in plants either in crystalline form (carrots) or non-crystalline form

(mangoes).[12] Nonetheless, β-carotene is only partially soluble at room temperature in oil and

insoluble in water, β-carotene in crystalline form has poor bioavailability.[13] The beneficial

biological activity of carotenoids can be lost when they are exposed to low pH, high temperature,

light and oxygen. Beta-carotene degradation is usually caused by isomerisation[14] and

oxidation.[15]

The bioavailability and solubility of β-carotene can be improved by incorporating β-carotene in

the lipid phase of oil-in-water (O/W) emulsion. The lipophilic β-carotene can be dissolved into

the oil before homogenization to form an O/W emulsion.[16] The stability of an emulsion may be

increased with the application of layer by layer (LBL) technology on protein coated oil particles.

LBL emulsion has better stability towards changes in pH, ionic strength and heat in thermal

processing and drying, lipid oxidation, freeze thaw cycles and high salt concentrations. [17,18]

The thicker interfacial layer provides the particles with higher resistance towards disruptions.[19]

There are interests in the use of O/W emulsions as delivery system for lipophilic bioactive

compounds as they can be used in a wide range of food applications. Microencapsulation,

protection and delivery of bioactive compounds in food materials can be enhanced with the

application of layered interfaces of emulsion systems.[20] Oil droplets in such emulsions form

particles covered by alternating protein and polyelectrolyte layers and the particles become

entrapped within a continuous glass-forming wall matrix during extrusion. The objectives of the

present study were to produce snack-type extrudates and to investigate their solids ability to

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encapsulate and protect beta carotene prepared to an emulsion using single layer (SL) and layer

by layer (LBL) interface structures.

MATERIAL and METHOD

Material

Wheat flour (Musgrave Retail Partners, Cork, Ireland; 14.31% H2O), α-lactose monohydrate

(Sigma-Aldrich, St. Louis, Mo., U.S.A.; 2.39% H2O) and maltodextrin (MD250, GPC, U.S.A.,

6.26% H2O) were used as solid feed. Whey protein isolate (WPI, Isolac, Carbery Food

Ingredients, Balineen, Ireland) was used as emulsifier (the primary layer). Gum Arabic (GA)

(Sigma Aldrich G9752 Stenheim, Germany) was used as a polyelectrolyte (the secondary layer).

Sunflower oil (Musgrave Excellence™, Spain) was used as the lipid phase and the solvent for β-

carotene (crystalline Type I, synthetic, > 93% (UV), powders, Sigma-Aldrich, U.S.A.). All other

chemicals were purchased from Sigma-Aldrich.

Emulsions Preparation

Preparation of primary emulsions: WPI was dispersed in deionized water (12 %, w/w in oil) at

room temperature and stirred for 1 hour to enhance the hydration of the proteins. pH was

adjusted to 3.5 by citric acid (10% w/w). The oil phase was prepared by dispersing β-carotene

(0.05%, w/w) in sunflower oil at 50°C by mixing with magnetic stirrer in a beaker until a

homogeneous dispersion was obtained. Light exposure of the oil was avoided during the process

by covering the beaker with aluminium foil. The oil phase (400 g) and water phase (400 g) with

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oil:protein ratio of 60:1 were mixed, pre-homogenised using an Ultra-Turrax (T25 Digital, IKA-

Werke GmbH & Co. KG, Staufen, Germany) at 10 000 rpm for 60s. The pre-emulsions were

subsequently homogenized at room temperature using a two-stage valve homogenizer (APV-

1000, APV Homogenizer Group, Wilmington, MA, U.S.A.) with 3 cycles at 250 bar

(approximately 20% of the total pressure was applied for the second stage). The protein-

stabilised primary emulsion was used as a wet feed in the extruder.

Preparation of secondary emulsions by layer-by-layer electrodeposition technique: Layer-

by-layer (LBL) emulsions were prepared by firstly dispersing GA (0.15% w/w,) in deionized

water at room temperature and stirred for 1 hour. The GA solution was then adjusted to pH 3.5

with citric acid solution (10% (w/w)). The primary emulsion obtained earlier was mixed with

GA solution at room temperature and stirred for 30 minutes to form LBL . The protein-GA

stabilised LBL emulsion was used as a wet feed in the extruder.

Extrusion

The dry ingredients feed contained wheat flour (60%) 20% (w/w) for maltodextrin (DE 23-27)

and lactose, respectively. A homogenous mixture of these dry ingredients was prepared by using

a mixer (Kenwood KM330, Kenwood Limited, Hampshire, UK). The mixing process was

performed at 60 rpm for 5 min. The dry fed into a twin-screw pilot extruder (MPF model, APV

Baker, Peterborough, UK) was 73.4 g/min. The barrel had four heating zones and hosted twin

screws with screw diameter of 19 mm and length to diameter ratio (L/D) of 25:1. The screw

speed was adjusted as 300 rpm. The emulsions were diluted with water at a ratio of 4:1 (4 parts

emulsion w/w: 1 part water w/w) enabling them to be fed into the peristaltic pump. The diluted

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emulsion (wet feed) was supplied using a peristaltic pump (504U MK, Watson Marlow Ltd) at a

rate of 12.153 g/min. The temperatures in the four zones were adjusted to 105, 120, 145 and

155°C, respectively and these temperatures were kept constant during processing. To obtain

standard curve, the solid mixture was fed into the solid mixture feeder at the rates of 0-3 to give

values of 0-101.5 g/min (y=33.919x R2=0.9961) On the other hand, the diluted emulsion were

fed into peristaltic pump at rate of 0-25 giving values of 0-20.9 g/min (y=0.8629x R2=0.9967).

The extrudates were cooled to room temperature and ground with a mixer (KM330, Kenwood

Limited, Hampshire, UK, 30 s at minimum speed). Aliquots (2 g) of the powdered extrudates

were transferred to 10 mL clear glass vials (Schott, Müllheim, Germany).The vials were sealed

and closed with septa under vacuum in a freeze dryer (Lyovac, GT 2, Steris, Hurth Germany).

Closed vials were subsequently sealed in plastic packages (PA/PE 90, Fispak Ltd., Dublin,

Ireland) under vacuum (99%) using a vacuum packaging machine (Polar 80 KL, Henkelman B.

V., Den Bosch, The Netherlands). Samples were stored in temperature controlled incubators at

20 (cooling incubator, KBP 6151 series 6000, Termarks, Bergen, Norway), 40 (TS 8136,

Termarks) and 60°C (TS 8136, Termarks) and protected from light, water loss and uptake from

the environment. The packages with vials retained vacuum during the storage indicating a closed

system. Samples were analyzed at intervals during storage for up to 27 days.

Physical and Chemical Analysis

Determination of Moisture Content and Water Activity: The moisture content of the

extrudates was determined by difference in weight before and after drying in a vacuum oven at

65 °C for 24 h. The mean water content ± standard deviation (SD) of triplicate samples for each

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material was measured. The water activity of the extrudates was measured by water activity

meter (Aqua Lab 4TE, Decagon Devices, Inc., Pullman, WA). The mean water activity ±

standard deviation (SD) of duplicate samples for each material was measured after extrusion and

at every interval point before HPLC analysis.

Color Measurements: The color (L*, a*, and b* values) of the extrudates were measured by

using a Colorimeter (Model CR-300, Konica Minolta, Japan) and the results were expressed in

accordance with the CIE Lab System. The standard white tile was used as the reference.

Differential Scanning Calorimetry (DSC): Glass transition temperatures, Tg, of the extrudates

were measured using DSC (Mettler Toledo 821e with liquid N2 cooling). The extrudates were

milled and transferred into pre-weighted DSC aluminium pans (40 μL, Mettler Toledo

Schwerzenbach, Switzerland). The pans were hermetically sealed and reweighted. An empty pan

was used as a reference. The samples were scanned at 5°C/min from -60 to 50°C, cooled at

10°C/min to -60°C, and a second heating scan at 5°C/min was run from -60 to 100°C. The Tg

values were recorded using STARe software, version 8.10 (Mettler Toledo Schwerzenbach,

Switzerland) as onset temperatures of the glass transition.

Dynamic Mechanical Analyses (DMA): A dynamic mechanical analyser (Tritec 2000 DMA,

Triton Technology Ltd., UK) was used to determine the dynamic mechanical properties of the

extrudates.. Samples of the milled extrudates were prepared in metal pocket-forming sheets

(Triton Technology Ltd., UK). A thin sandwich pocket was formed by crimpling the sheet along

a pre-scored line which was then attached directly between the clamps of the DMA sample

assembly. (The length, width and thickness of the sample pocket between the clamps were

measured.) The samples were scanned between 0.5 to 20 Hz using the single cantilever bending

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mode from -50°C to 140°C with a cooling rate of 5°C/min and a heating rate of 3°C/min. Liquid

nitrogen was used for cooling. The α-relaxation temperature was determined from the peak of

loss modulus (E’’) above glass transition.

Extraction and HPLC Analysis: Two gram samples of the extrudates at various intervals of

storage were hydrated and suspended in 15mL of deionized water by vortexing (Scientific

Industries Inc., G-560E, NY, U.S.A.) at room temperature for 5 min to release suspended oil

particles. In order to destabilize emulsified droplets and extract beta-carotene, 4mL of

methanol:ethlyacetate (1:1 v/v) solution containing 0.25% butylated hydroxyl toluene (BHT)

was added and vortexed for 30 s. Oil was saponified by adding 1 mL of saturated (20%)

potassium hydroxide in methanol (2M) and vortexed for 30 s to separate the lipid carrier

(saponized fraction) from the β-carotene (unsaponised). Finally remaining β-carotene was

extracted using 1mL of dichlorometane and the sample was vortexed for 30s. The organic phase

was separated by adding 4mL of n-hexane and the sample was further vortexed for 30s. The

extracts were left to stand for 30min. The top layer was separated using a pipette and centrifuged

(Sigma 1-15, Model 78307, D-37520, Ostenode am Harz, Germany) at 10000rpm for 5 min. The

supernatant was filtered through a filter (Minisart RC 15, Sartorius Stedim Biotech GmbH,

Goettingen, Germany), upon transfer to 1 ml HPLC vials. Injections of 200 μL were used into

the HPLC system. The β-carotene contents of the extrudates were quantitated using an HPLC

(Dionex ICS3000, Sunnyvale, CA, U.S.A.), autosampler (AS-1, Dionex, Sunnyvale, CA,

U.S.A.), and photodiode-array detector (PDA ICS Series, Dionex, Sunnyvale, CA, U.S.A.). The

HPLC column was a 250 mm × 4.6 mm i.d., 5 μm, reversed-phase Acclaim C30 analytical

column with a 4 mm × 4 mm i.d. guard column of the same material (Dionex, Sunnyvale, CA,

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U.S.A.). An eluent gradient composed of acetonitrile at 85 to 65%, methanol:ethyl acetate (1:1)

at 15 to 35% and 0.5% acetic acid in water was used for separation of carotenoids that were

analysed at 450 nm. The amounts of β-carotene were calculated from the standard curve of all-

trans β-carotene. Standard curve was prepared using freshly prepared SL emulsion containing

all-trans β-carotene. The amount of emulsion present in the final extrudates was known and was

calculated to represent the emulsion present in the extrudates. Based on calculation, 0.07g of

emulsion was present in 1g sample. Standard curve was prepared using 0.025, 0.05, 0.1, 0.2, 0.3

and 0.4g of emulsions giving an equation of y=13.7246x (R2=0.98). β-carotene degradation data

were fitted to first-order kinetics: −kt = ln A/A0, and the rate constants (k) were derived from the

slopes of linear regression lines. Activation energy was obtained using the Arrhenius relationship

k=Ae-Ea/(RT).

Statistical Analysis

The data were analyzed using statistical software SPSS 16.0 (SPSS Inc., U.S.A.). The data were

also subjected to an analysis of variance (ANOVA) and Duncan’s multiple range test (α=0.05)

was used to determine the differences between means.

RESULTS AND DISCUSSION

Physical Properties of Extrudates and Colour

The moisture content of extrudates with SL and LBL emulsion was found to be as 7.47±0.05 g

H2O/100g of solids and 8.71±0.02 g H2O/100g of solids, respectively (P<0.05). The constant

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weights of samples during storage showed no loss of water. Changes in the aw of extrudates

during storage is shown in Figure 1. The initial water activity values were 0.5330±0.004 aw, and

0.5760±0.004 aw for SL and LBL extrudates, respectively (P<0.05). The aw remained constant

throughout storage of 27 days at the 3 different storage temperatures. A slight gradual decrease

during the first 3 days of storage for both extrudates was noted. SL and LBL extrudates showed a

similar Tg (onset) as they have the same wall materials with almost identical water content and

water activity. The extrudates has Tg of approximately 3±0.44°C (onset). Structural relaxation of

multicomponent food systems may provide more information on the changes in material

characteristics around and above the glass transition. DMA measurements were used for the

determination of the α-relaxation temperatures (Tα). The Tα of the extrudates were found to be as

55°C at 0.5 Hz.

Colour is an important quality factor which reflects the quality and sensory attractiveness of the

food materials.[21] The influence of storage temperature and emulsion type on the colour values

(L*, a*, and b*) of the extrudates are shown in Figure 2A, 2B and 2C respectively. The results

showed that the colour values (L*, a*, and b*) of the extruded snack were greatly influenced by

emulsion type (SL, and LBL), and storage temperature (P<0.05). The brightness (L*), and

yellowness (b*) values of extrudate with SL emulsion (71.83±0.23, and 35.49±0.38) were lower

than extrudate with LBL (73.06±0.57, and 41.34±0.34). However, redness value (a*) of

extrudates that contained SL emulsion (2.46±0.14) was found to be higher than for LBL

(1.05±0.20). As beta carotene was encapsulated in the oil phase, the thicker interfacial layer of

LBL emulsion compared to SL emulsion due to the presence of a layer of WPI and GA may have

reduced the a* value of the LBL sample. Harnsilawat et al.,[21] and Gu et al.,[23] reported that the

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thicker interfacial layer of LBL emulsion particles compared to a single emulsifier increased the

steric repulsion between particles. In addition, the particles have resistance towards disruptions

due to the thick interfacial layer.[19] The differences between the color values of extrudate snacks

stored at different temperatures were found to be statistically significant (P<0.05).

Generally, the L* values decreased and a* values increased with storage time especially at 60°C.

The same trends for carrot extrudates, and carrot pomace extrudates during storage were

observed by Kumar et al.[24] and Dar et al.[25]. The colour changes were rapid at the beginning of

storage but reduce with time. During storage, sugar crystallization of the wall material of

extrudates may occur resulting in the release of the oil containing β-carotene which has orange-

red color causing an increase in redness during storage. The brightness loss of the extrudates may

also be caused by non-enzymatic browning during the storage. Figure 2A and 2B showed that

when the storage temperature was increased from 20 to 60 °C rapid brightness loss and

increasing redness were observed (P<0.05).

Desorby et al.,[26] reported that while brightness and redness values are good indicators for β-

carotene degradation, yellowness value is not a sufficient indicator as they were not dominant.

The rapid colour changes in the samples at 60°C could be related with β-carotene loss in the

sample. Study by Zielinska and Markowski[27] found that high temperature leads to colour

degradation as a result of loss of carotenoids and β-carotene in carrots. Similar trend was

observed between β-carotene loss and the changes of L* and a* values where losses were higher

at 40°C compared to 20°C. Only small changes were observed in b* values and no trend could be

observed between β-carotene losses, and changes in b* values. However, Gaspar et al.,[28] stated

that L*, a*, b* values cannot be correlated with β-carotene loss.

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HPLC Analysis

The retention of noncrystalline β-carotene in extrudates containing SL and LBL emulsions was

monitored during storage at 20, 40 and 60°C for 27 days using HPLC. The mobile phase and

stationary phase of the C30 column used in our study were reported to effectively separate the

isomers of β-carotene. Three significant peaks of β-carotene were found in the HPLC

chromatograms (Figure 1B). The three β-carotene isomers were identified as 15-cis-β–carotene

(19.19±0.81 min), 13-cis-β–carotene (23.51±0.65 min), and all-trans-β-carotene (27.31 ±0.87

min). Total carotene content and the quantity of individual isomers were calculated from peak

area measurements and calibration data. The mechanism of β-carotene degradation has been

extensively reviewed by previous researchers. [16, 26, 29, 30] Generally, various factors during food

processing and storage, e.g., heat, acid, light, oxygen, metal ions, accelerate oxidation, and

isomerization of carotenoids, lead to the degradation and loss of bioavailability. [30, 31]

Autoxidation is known as the major cause of carotenoid loss in dehydrated foods.

The β-carotene losses after the extrusion process were 50.25% and 37.70% from the theoretical

amount for extrudates with SL and LBL emulsions, respectively. These losses were found to be

lower than the results of Guzman-Tello and Cheftel[29] and Emin et al.,[9] who reported 70-73%

reduction in β-carotene during the extrusion process, respectively. The results may show that the

encapsulation of beta carotene in the oil droplets increased the retention. It was also noted the

use of LBL emulsion reduced the loss of β-carotene suggesting higher ability of LBL emulsions

in protecting bioactive compounds. A study by Rao et al.,[32] on the stability of astaxanthin in oils

at different temperatures found significant losses of carotenoids during heating at 120, and

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150°C without changes in the fatty acid profiles of edible oils. The barrel temperature of the 4th

zone in the extruder in the study was 155°C. This could lead to high losses of β-carotene in the

material. In addition to the process conditions, food formulation, and moisture content of feed

which defines the food matrix are the major factors influencing carotenoids retention.[33] High

moisture content of feed can offer limited protection towards losses in protein water solubility,

quality, and molecular structure extrudates containing WPI at constant extrusion temperature.[34]

The wet feed of extrudates in this study were fed with diluted emulsion containing high water

content and this may provide the encapsulated β-carotene within the extrudates some protection

against losses. On the other hand, Pérez-Navarrete et al.,[3] reported that due to lipid degradation

from the high processing temperatures, the screw speed used for extrusion, the fatty acids in the

raw material form complexes with amylose making extraction more difficult.

β-carotene retention values were plotted versus storage times and the data were fitted using a

first order kinetics equation (Figure 3A). However, the reaction kinetic did not properly represent

the results obtained. It was observed that the β–carotene retention showed two different trends

giving two first order degradation of β–carotene slopes. During the first 6 days of storage, the

degradation rate was faster and the slope was found to be steeper. However, the loss of β–

carotene was slower upon storage from day 6 to 27. The higher initial loss in the first 6 days

could be due to the residual oxygen present within the matrix of the extrudates. The initial plot of

the degradation kinetics showed that the degradation kinetics was best fitted using two different

kinetic. The degradation kinetics of β-carotene followed a first order kinetics followed by a

second first order kinetics according to the first order kinetic plots obtained by using eqution –kt

= ln A/A0. Similar observation was also observed by Desorby et al.,[26] Carotenoids degradation

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is a typical first order reaction. [25, 26, 30,34-39] This same trend of carotenoids loss was also

observed in the other studies. [15, 25, 36] Even though oxidation is the major cause of carotenoid

degradation, isomerisation may play a major role in carotenoid degradation during processing.[41]

Chandler and Schwartz[42] found that heating causes increased in isomerization and at the same

time decreased all-trans-β-carotene. Significant amount of isomerisation was observed for both

extrudates which includes SL and LBL emulsions. The isomerisation amount of β-carotene was

found to be as 45.06% for extrudates with SL emulsion and 46.46% for extrudates with LBL

emulsion. At high temperatures (>120°C) where significant disruption of the food matrix

occurred, extensive β-carotene isomerization was observed. [43, 44]

The loss of β-carotene in the extrudates increased with increasing storage temperature. For

storage between 0-6 days, reaction rates were generally higher for samples with LBL emulsion.

Reaction rates of β-carotene losses were higher in LBL samples between 6 to 27 days as well.

The effect of storage temperatures on the β-carotene degradation followed the Arrhenius type of

equation both in 0-6 days; with activation energy of 25.40 kJ/mol in SL and 39.39 kJ/mol in LBL

(Table 1). Nevertheless, the activation energy of extrudate with LBL emulsion for 6-27 days

(11.01 kJ/mol) was found to be lower than extrudate with SL emulsion (17.27 kJ/mol). The fast

degradation in the LBL system at the beginning may result in it reaching constant value and stay

almost stable during storage at 6-27 days. The differences between activation energies of

extudates showed that the different behaviour of β-carotene degradation in the two emulsions.

The degradation kinetic of β-carotene was found to be significant. The activation energy reflects

the temperature dependent of any compound. The higher activation energy reflects that the β-

carotene degradation was more temperature dependent. The loss of β-carotene in LBL system

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was more temperature dependent at day 0-6. However, at day 6-27, loss of β-carotene was more

temperature dependent in SL system. It was noted that the beta-carotene amount was slightly

higher in extrudates with LBL at all temperatures. The % retention of β-carotene in LBL system

was higher upon storage for 27 days at 40°C and 60°C (Figure 3B). The higher stability of LBL

emulsion.[22, 45] may enhance protection of bioactive compounds in glassy membranes. The

retention percentage of beta-carotene was calculated as the ratio of the beta-carotene amount of

extrudates after extrusion to the beta-carotene amount of extrudates after storage period. The %

retention of β-carotene was 28.59% in LBL compared to 28.34% in SL upon storage at 40°C,

17.75% in LBL and 13.12% in SL upon storage at 60°C. However, lower % retention of β-

carotene was observed in LBL at 20°C. Despite the higher β-carotene retention in the LBL

during extrusion process, Serfert et al.,[46] reported that the oxidative stability of single layer

microcapsules with lecithin was higher than in the bilayer microcapsules which included lecithin

and chitosan.

Degradation of dispersed lipophilic compounds in hydrophilic solids depends upon matrix

stability and lipid physicochemical properties.[12] During storage, sugar crystallization of the wall

material of extrudates may occur resulting in the release of dispersed compounds with

subsequent exposure to oxygen and heat, and degradation of bioactive components. Further

losses of beta-carotene at 20, and 40°C occurred gradually levelling off at 27 days. However,

there was a rapid loss of beta-carotene content at 60°C during the storage period. In the glassy

state, most structural changes occur very slowly. [47] However, storing the samples at temperature

above the Tg caused rapid structural changes and sugar crystallization of the wall material of

extrudates and rapid release of the dispersed β-carotene. Overall, it can be seen that LBL system

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gave a better stability compared to SL system samples based in the % retention and rate constant

from day 6-27. The higher stability of LBL emulsion could play a role in protecting bioactive

compounds during extrusion giving a higher initial amount.

CONCLUSION

Extrudates were successfully produced using SL and LBL emulsions as wet feed. The results

showed that loss of β-carotene was higher in extrudates with LBL emulsion than in extrudates

with SL emulsion. Loss of β-carotene in LBL was more temperature dependence from day 0-6

and in SL from day 6-27. The % retention of β-carotene over storage was generally higher in

LBL. Modification to the LBL emulsion can be done to increase its stability towards heat as well

as mechanical stress in extrusion. The L* and b* values decreased while a* value of extrudates

increased with storage time. The formation of more resistant interfacial film by LBL however

does not provide protection against the loss of beta-carotene in extrudates after storage at room

temperature; conversely, the protection was effective at relatively high temperatures.

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Figure 1. A: Changes in aw values of SL, and LBL samples stored at 20, 40, and 60°C for 27 days, B: Peaks identified in chromatogram from SL and LBL extrudates stored at 20, 40, and 60°C

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Figure 2: Changes in A: L* values, B: a* values, C: b* values of SL, and LBL samples

stored at 20, 40, and 60°C for 27 days

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Figure 3. A: Beta-carotene retention (%) during storage at various temperatures for 27 days, B: Final retention percentages of beta-carotene for extrudates formulated with SL and LBL emulsions after storage at various temperatures for 27 days

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Table 1. Kinetic loss parameters of beta-carotene for extrudates prepared with single layer (SL) and layer-by-layer (LBL) emulsions after storage at 20, 40 and 60°C

Storage

Time (Days)

Sample Reaction

Rate, k (day-1)

R2 of

Reaction

Rate

Activation

Energy

(kJ/mol)

R2 of

Arrhenius

Plot

0-6

SL 20°C 0.0454 0.9951

SL 40°C 0.0553 0.9810 25.396 0.8367

SL 60°C 0.1621 0.8412

LBL 20°C 0.0304 0.7798

LBL 40°C 0.0577 0.9357 39.39 0.9462

LBL 60°C 0.2157 0.9898

6-27

SL 20°C 0.0207 0.7961

SL 40°C 0.0434 0.9624 17.268 0. 8031

SL 60°C 0.0479 0.9152

LBL 20°C 0.0489 0.9941

LBL 40°C 0.0402 0.9647 11.009 0.9609

LBL 60°C 0.0283 0.7275

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