Food Chemistry 138 (2013) 2356–2364

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Food Chemistry

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Inhibition of citral degradation in an acidic aqueous environmentby polyoxyethylene alkylether surfactants

Masrat Maswal, Aijaz Ahmad Dar ⇑Department of Chemistry, University of Kashmir, Hazratbal, Srinagar 190 006, J&K, India

a r t i c l e i n f o

Article history:Received 8 September 2012Received in revised form 27 November 2012Accepted 11 December 2012Available online 28 December 2012

Keywords:CitralMicelleNon-ionic surfactantsMicroemulsionNanoemulsion

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.12.031

⇑ Corresponding author. Tel.: +91 (194) 2424900; fE-mail address: aijaz_n5@yahoo.co.in (A.A. Dar).

a b s t r a c t

Citral is a flavour component widely used in food and cosmetic industries, but is chemically unstable anddegrades over time in aqueous solutions due to acid-catalysed and oxidative reactions leading to loss ofdesirable flavour. The present study reveals the effect of non-ionic micellar solutions of Brij30 and Brij35on the extent of solubilisation and stabilisation of citral. The rate of chemical degradation of citral inacidic aqueous solutions was found to be highest, which was subsequently reduced significantly withinthese studied surfactant systems, suggesting protection of citral from an acidic environment once it isincorporated into the micelles. The work concludes that polyoxyethylene alkylether surfactants withlower HLB value, less dense hydrophilic corona and more hydrophobic core volume are efficient in solu-bilising and stabilising citral against an acidic environment.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Citral (3,7-dimethyl-2,6-octadienal) an acyclic monoterpenealdehyde occurs naturally in herbs, plants and citrus fruits (Ortiz,Gonzalez-Garcia, Ponce-Monter, Castaneda-Hernandez, & Aguilar-Robles, 2010) and is composed of natural mixture of two geometricisomers geranial (trans-citral, citral A) and neral (cis-citral, citral B)in 3:2 ratio (Choi, Decker, Henson, Popplewell, & McClements,2009). Citral is one of the most important natural flavouring com-pounds, which has intense lemon aroma and flavour and is widelyused as an additive in food, beverages and cosmetics with highconsumer acceptance (Djordjevic, Cercaci, Alamed, McClements,& Decker, 2007). Consumer demand for natural flavour ingredientsand more complex and authentic aroma profiles have resulted inan increased demand for the incorporation of citral into differentfood and beverage products (Djordjevic, Cercaci, Alamed, McCle-ments, & Decker, 2008). Since flavours are often used in acidifiedbeverages, the problem with citral is that it is chemically unstableand degrades rapidly during storage at acidic pH and under oxida-tive stress leading to the formation of compounds that produceundesirable off-flavours, resulting in loss of product quality and adecrease in shelf life limiting its application in food and cosmeticindustries (Yang, Tian, Ho, & Huang, 2011). Strategies to inhibit cit-ral degradation, such as reduction of storage temperature, alter-ation of pH and the partial pressure of oxygen are not practicalfor many foods and beverages (Djordjevic et al., 2007). Also citral

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is not fully water soluble, so the formulations of citral which in-crease its concentration and its physical and chemical stabilitiesfor use in foods and beverages would be a major benefit for thefood industries. Since citral degradation occurs predominantly inacidic aqueous solutions, the rate of its chemical degradation canbe altered appreciably by incorporating it into a colloidal disper-sion such as emulsion, microemulsion or micellar solution. In ear-lier studies, citral has been stabilised by encapsulating it inemulsions (Djordjevic et al., 2008; Yang, Tian, Ho, & Huang,2012; Yang et al., 2011) or micelles (Choi, Decker, Henson, Popple-well, & McClements, 2010; Rao & McClements, 2012) as encapsula-tion isolates citral from the reactive molecules, such as protons andfree radicals present in the aqueous medium. Yang et al. (2011)studied the effects of oil-in-water nanoemulsions combined withantioxidants on the stability of citral and the results suggested thatencapsulation of citral in the emulsions and the addition of appro-priate antioxidants could enhance chemical stability of citral dur-ing storage. Choi et al. (2009) investigated the impact ofcomposition of triacetin and medium chain triglycerols (MCT) onthe oil–water partitioning and chemical degradation of citral andconcluded that both MCT as emulsion droplets and triacetin asmicroemulsion droplets were able to appreciably slow its degrada-tion rate. The problem associated with these studies is that they in-volve a lipid phase, which is itself prone to undergo oxidation anddegradation at low pHs into products like 2-heptanone, 1-octen-3-ol and butanoic acid (Yang et al., 2011). An inverse relationship be-tween pH and lipid oxidation has been reported in literaturewherein the pro-oxidant species, which bring about the oxidationof lipids, become much more active at low pH resulting in the

M. Maswal, A.A. Dar / Food Chemistry 138 (2013) 2356–2364 2357

development of rancid flavour (Schafer & Buettner, 2000; Tichiv-angana & Morrissey, 1985). Djordjevic et al. (2008) used whey pro-tein isolate (WPI) and gum arabic (GA) stabilised oil-in-wateremulsions to stabilise citral and showed that the rate of degrada-tion of citral is the same in GA and WPI stabilised emulsions atpH 3. WPI was found to be more effective than GA to inhibit theoxidation of citral. However, the problem with this method is theisoelectric point associated with the proteins used for stabilisation.Depending on the pH of medium, proteins attain negative chargewhich promotes the oxidative reactions. Some authors employedhydrocarbon oils like octadecane (Mei et al., 2010) and hexadecane(Djordjevic et al., 2008) to stabilise citral which are not biocompat-ible and hence cannot be used in food industry. Choi et al. (2010)reported degradation of citral in micellar medium and concludedthat citral degradation was decreased by increasing Tween 80 con-centration as a result of incorporation of citral into the innerhydrophobic core of the micelle. As non-ionic surfactants are re-ported to be non toxic, biocompatible (Chu & Zhou, 1996), stableto pH and ionic strength of the medium (Lawrence & Rees, 2000),they have been used in a number of commercially available edibleformulations approved by FDA e.g., Movicol, Miralax, etc. and forsolubilisation and delivery of pharmaceuticals (Ayorinde, Gelain,Johnson, & Wan, 2000) and neutraceuticals (Brush et al., 1957).In addition, Brij35 has even been used for stability enhancementof citral (Choi et al., 2009; Mei et al., 2010). So any serious healtheffect regarding the investigated surfactant system is not expectedat low concentrations.

In this study, we have investigated the extent of solubilisationof citral and the rate of its degradation in aqueous polyoxyethylenealkyl ether surfactant (Brij30 and Brij35) solutions. The influence ofpH, temperature, surfactant concentration and the polyoxyethyl-ene chain length in the surfactant has been studied with the aimof identifying suitable conditions for preparing a stable micro-het-erogeneous medium for citral. The experimental results of thisstudy may be useful for designing a suitable formulation of citralrequired during various food, cosmetic and beverage applications.

2. Materials and methods

2.1. Materials

Citral was a Merck (India) product. Brij30 and Brij35 amphi-philes were Aldrich products. All chemicals were used as received.The chemical structures of citral and surfactants are presented inScheme 1. The stock solutions of citral, Brij30, and Brij35 were pre-pared in 50 mM phosphate buffer of pH 7, 3 and 1 with triple dis-tilled water and utilised to prepare the samples of desiredconcentrations.

2.2. Solubility and stability experiments

Stock nanoemulsions of 5.6 M citral were prepared in bufferedsurfactant solutions of Brij30 and Brij35 surfactants at various sur-factant concentrations (1–5 mM) at pH 7, 3 and 1 according to

C12H25CH2CH2OC12H254OH

Brij 30

O

Scheme 1. The structures of citral an

method of Rao and McClements (2012). Aliquots of stock nano-emulsion were then added to the vials containing 3 ml of corre-sponding concentrations of Brij30 and Brij35 surfactants to give arange of final citral concentrations (4–30 mM). The 5 ml samplevials were sealed with screw caps and then were agitated for a per-iod of 1 h on a magnetic stirrer at a temperature of (25 ± 0.5) �Cusing magnetic Teflon pieces previously placed in the vials. Theresulting colloidal dispersions were withdrawn periodically forturbidity measurements with a Schimazdu Spectrophotometer(model UV-1650). The turbidity of citral was determined at its kmax

using the formula, s ¼ � 1L

� �lnðI1=I2Þ, where s is the turbidity, L is

the path length and I1 and I2 are the transmitted intensities withand without citral in the surfactant solutions, respectively (Staf-ford, Ploplis, & Jacobs, 1990). This method was used to monitorchanges in characteristics of citral oil droplets after addition tomicellar solution as well as over time.

2.3. Analysis of degradation of citral using high performance liquidchromatography (HPLC) at different pHs

The liquid chromatography system consisted of a SchimadzuLC-20A with a SPD-M20A variable-wavelength UV detector (setat 237 nm), a CBM-20A/20Alite system controller, LC-20AB pumpand an injection valve with a 25 ll loop (Shimadzu, Kyoto, Japan).Separation was achieved using an Enable C18G column(250 mm � 4.6 mm, 5 lm) and a CTO-10ASvp column oven. Themobile phase used consisted of water:methanol (40:60, v/v), flow-ing at a rate of 0.5 ml min�1. The instrument was operated at 40 �C.

3. Results and discussion

3.1. Solubilisation capacity

Solubilisation is an important process in many areas of food sci-ence, such as encapsulation of lipophilic components, flavourdelivery, emulsion stability, detergency, development of microre-actors and absorption of lipophilic components within the humangastrointestinal tract (Garti, Spernath, Aserin, & Lutz, 2005). Mi-celles are capable of solubilising non-polar molecules into theirhydrophobic interiors, thus enabling them to be dispersed intoan aqueous phase in which they are normally insoluble. The aque-ous solubility of citral calculated spectrophotometrically wasfound to be 571 mg/ml tallying well with literature value of590 mg/ml (UN Environmental Programs, 2001) usinge = 2.82 L mmol�1 cm�1 determined from the calibration plot ofcitral in water. The solubilisation characteristic of citral in micellarsolution was expressed in terms of solubilisation capacity which isa measure of maximum amount of citral that can be incorporatedinto a given amount of surfactant. The solubilisation capacity ofsurfactant solutions was determined by measuring the turbidityof system containing different citral concentrations. As shown inthe prototype plot Fig.1, for lower citral concentrations the solu-tions appeared transparent, the turbidity of which increasedslightly when citral concentration was increased. However at high-

23

C3CH3

CH3 O

HOH

Brij 35

Citral

CH2CH2

H

d surfactants used in this study.

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idity

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-1)

[Citral] (mM)

Water 1 mM Brij30

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idity

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. sol

ubili

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[Surfactant] (mM)

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idity

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[Citral] (mM)

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26

Max

. sol

ubili

zatio

n ca

paci

ty, C

max

(mM

)

[Surfactant] (mM)

(a)

(b) (c)

Fig. 1. Influence of citral concentration on the turbidity of (a) buffer and 1 mM Brij30 surfactant solution, (b) Brij35 surfactant solutions and (c) Brij30 surfactant solutions.Inset: (variation of maximum solubilisation capacity, Cmax with the concentration of (b) Brij30 and (c) Brij35).

2358 M. Maswal, A.A. Dar / Food Chemistry 138 (2013) 2356–2364

er citral concentrations, a large linear increase in turbidity was ob-served resulting in formation of opaque solutions. Therefore, theplot of turbidity with citral concentration, showed two straightlines at relatively low and high concentrations, the point of inter-section of which gives the maximum solubilisation capacity, Cmax

of a given surfactant solution towards citral. As detailed by Raoand McClements (2012), at lower concentrations of citral all thecitral molecules present initially within the nanoemulsion dropletsget incorporated into the micelles present in surfactant solutionresulting in a slight increase in turbidity up to Cmax. However,above Cmax the micelles that are already saturated by citral mole-cules do not take up the oil and hence further addition of citralkeeps them in the form of nanoemulsion droplets in solutionresulting in a linear increase in turbidity with increase in citralconcentration. Fig. 1 also shows the effect of addition of citralnanoemulsion stock to the aqueous buffer solution without surfac-tant. In this case, a linear increase in turbidity with citral concen-tration suggests the presence of nanoemulsion droplets in waterwithout any solubilisation effect. Therefore Cmax observed in sur-factant solutions, corresponds to an important solubilisationparameter below which micelles are capable of solubilising citral,but above which micelles are saturated with citral and hence inca-pable of further solubilisation.

3.2. Influence of surfactant type and concentration on citralsolubilisation

The influence of surfactant concentration on the solubilisationof citral in Brij30 and Brij35 micelles is shown in Fig. 1. The Cmax

of citral increased with increasing surfactant concentration, indi-cating the solubility enhancement of citral within the micelles ofboth surfactants (inset of Fig. 1). The solubility of citral increasedmany-fold from 3.75 mM in aqueous buffer solution to 14.19 mMin 1 mM Brij35 and17.84 mM in 1 mM Brij30 surfactant systems.The value further increased by increasing the surfactant concentra-tion, to about 20.67 mM in 5 mM Brij35 and 25.58 mM in 5 mMBrij30 surfactant system, respectively. The solubilisation capacityof Brij30 micelles is greater compared to Brij35 micelles, whileboth show solubility enhancement as compared to that in water.Citral has an appreciable availability at the interface and in thewater phase due to its amphiphilic and surface active nature(Djordjevic et al., 2007). Therefore, it is envisaged that in additionto the micellar core solubilisation, citral could also get solubilisedin the micellar palisade layer resulting in solubility enhancementas compared to that in water. Our finding of the locus of solubilisa-tion (Section 3.4.) reveals that citral is indeed solubilised in boththe regions of micelles being predominantly solubilised within

225 250 275 3000.0

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1.0

Diethyl Ether Pet Ether Methanol Water Brij35 Brij30

Abs

orba

nce

Wavelength (nm)

0 2 4 6 8 10230

235

240

245

λ max

Polarity index

Fig. 2. Absorption spectra of citral in various solvents. Inset shows variation of kmax

with polarity index of solvent.

M. Maswal, A.A. Dar / Food Chemistry 138 (2013) 2356–2364 2359

the core of micelles in the case of Brij30, but in the case of Brij35micelles it is predominantly solubilised in the palisade layer. Dueto the small aggregation number of Brij35 micelles (40) (Bhat,Dar, & Rather, 2008), it is expected to have large micelle concentra-tion relative to Brij30 at a given surfactant concentration resultingin greater water–micelle interfacial area leading to appreciablepalisade layer solubilisation of the citral. Moreover, the polar inter-actions between carbonyl group/p electrons of citral and polyoxy-ethylene groups of surfactants would predominate over thehydrophobic interactions between the terpenic core of citral andthe non-polar tail of the surfactant due to the large number of oxy-ethylene groups (OE) (23) present in the headgroup of Brij35 sur-factant leading to more palisade layer solubilisation. However, incase of Brij30 micelles the predominant solubilisation occurringwithin the micellar core is facilitated by (a) the larger volume ofthe micellar core (aggregation number of 101 (Bhat et al., 2008))and (b) predominant magnitude of hydrophobic interactions be-tween the terpenic core of citral and the non-polar tail of the sur-factant because of less number of OE groups within the Brij30surfactant head group. Since the non-polar citral (Choi et al.,2010) would have a natural tendency to get partitioned into thehydrophobic environment, therefore any micelle that facilitatesits core solubilisation should act as a better solubilisation medium.In light of this discussion, since Brij30 micelles offer predominantmicellar core solubilisation as compared to Brij35 micelles, the for-mer has greater solubilisation capacity than latter. The higher tur-bidity of citral encapsulated within Brij30 micelles is a testimonyof the larger size of these micelles encapsulating citral.

3.3. Molar solubilisation ratio (MSR) and micelle-phase/aqueous-phase partitioning of citral

The variation of maximum solubilisation capacity, Cmax with thesurfactant concentration (inset of Fig. 1) shows that Cmax increaseslinearly with increase in surfactant concentration. Molar solubili-sation ratio (MSR) (Attwood & Florence, 1983) is equivalent to in-crease in solubilisate concentration per unit increase in micellisedsurfactant concentration. It is measure of the effectiveness of a sur-factant in solubilising a given solubilisate and is obtained from theslope of curve between solubilisate concentration and surfactantconcentration. The MSR value of citral in Brij30 surfactant system(1.94) is more than that of in Brij35 surfactant system (1.57). Theeffectiveness of solubilisation can also be expressed in terms ofthe partition coefficient, Km, of the citral between the micelle andaqueous phases and is defined as the ratio of mole fraction of thecitral in the micellar phase, Xm, to that in the aqueous phase, Xa.

Km ¼ Xm=Xa ð1aÞ

The value of Xm in terms of MSR can be written as

Xm ¼MSR

1þMSRð1bÞ

Xa can be expressed as Xa = [Scmc]Vm�Vm is the molar volume ofwater equal to 0.01805 L/mol at 25 �C and Scmc is the solubility ofthe solubilisate just below cmc of the surfactant which was takenequal to it aqueous solubility because solubility did not change upto the cmc of surfactants. With these expressions, Km for solubili-sation becomes

Km ¼MSR

ðScmcÞVmð1þMSRÞ ð1cÞ

The Km values so obtained for Brij30 and Brij35 were 9.79 � 103

and 9.07 � 103 respectively. Since both MSR and Km values arehigher in Brij30 as compared to that of Brij35, it shows that the for-mer is better solubilising medium for citral due to the reasonsmentioned in the Section 3.2.

3.4. Locus of solubilisation

The information about the locus of solubilisation of citral withintwo different micelles can be obtained by comparing kmax of citralwith that in different solvents. The UV spectra of citral in a numberof solvents with a variety of polarity index viz petroleum ether,diethyl ether, methanol, and water having polarity indices of 0.1,2.8, 5.1 and 9, respectively, along with the spectra in aqueousBrij30 and Brij35 micellar solutions are given in Fig. 2. As evidentfrom the figure, the kmax of citral shows a regular bathochromicshift with increasing polarity of medium. kmax increases from232 nm in petroleum ether to 247 nm in water through 236 nmin diethyl ether and 239 nm in methanol. Plot of kmax versus polar-ity index of solvent yield a straight line (inset of Fig. 2) indicating alinear increase in kmax of citral with polarity. The kmax obtained inBrij30 micellar medium was 241 nm in contrast to 244 nm inBrij35 micellar medium corresponding to polarity index of 5.8and 7.6, respectively. This indicates that the polarity sensed bythe solubilised citral is intermediate between methanol and water.It is known that the oxyethylene head groups of Brij micelles canbind water molecules which are proportional to the number ofoxyethylene groups (Renwick, Hughes, Pitman, & Vité, 1976).Therefore, Brij35 micelles with 23 oxyethylene groups in the headgroup is comprised of a palisade layer, which would be more hy-drated than the palisade layer of Brij30 micelles, where only fouroxyethylene groups are present. The kmax value of citral solubilisedin Brij30 micelles indicates it to be deep into the palisade layer ortowards the micellar core. However kmax of citral in Brij35 micellesindicates its solubilisation in the more hydrated region, which cor-responds to its palisade layer. It is also expected that the absorp-tion band in the two surfactant systems is a superposition of twounresolved peaks, one peak corresponding to that fraction of citralsolubilised in the inner hydrophobic core of micelles and the otherone for the fraction of citral solubilised in the outer hydrophiliccorona region of the micelle, with both regions having differentpolarities. For Brij30 micelles, the peak occurs more towards thenon-polar region indicating more fraction of citral is solubilisedwithin the micellar core owing to its larger micellar core volumeand less dense outer hydrophilic corona, while in the case of Brij35more appreciable amount of citral is solubilised within the outerhydrophilic corona as the peak lies further towards the polarregion.

3.5. Effect of pH on stability of citral in micellar media

The absorption spectra of citral corresponding to their Cmax con-centrations in aqueous buffer, pre-micellar and post-micellar con-

2360 M. Maswal, A.A. Dar / Food Chemistry 138 (2013) 2356–2364

centrations of both surfactants at pH 7, 3 and 1 are shown in Fig. 3.At pH 7, citral shows its characteristic UV spectra in all the studiedsystems, which indicates that it is quite stable at this pH. From theHPLC data (Fig. 4a) taken after 7 days, it is clear that citral does notdegrade at this pH. In addition to stability, the encapsulation of cit-ral within the micelles shows remarkable solubilisation enhance-ment. This many-fold increase in the solubility of citral in theaqueous micellar solutions in addition to maintaining its stabilitymay prove beneficial for the food and cosmetic industries. At pH3, the spectra of citral in aqueous buffer and in the pre-micellarconcentrations of both the surfactants show some degree of distor-tion as evident from Fig. 3b, which may be attributed to the degra-dation of citral due to its exposure to the acidic medium in thesesystems. It is clearly evident from the HPLC chromatogram shownin Fig. 4d that a significant amount of citral has degraded to varietyof products at pH 3. In the post-micellar concentrations of surfac-tants, citral is solubilised within the micelles; besides the solubilityenhancement of citral, the degradation is insignificant as the absor-bance peak values differ feebly from those of the same solutions atpH 7. At pH 1, the UV absorption spectra of citral in aqueous bufferand in pre-micellar region of both surfactants is entirely differentfrom its characteristic spectra indicating the complete degradationof citral owing to the strong acidic conditions prevailing in thesesystems. As observed in HPLC chromatogram (Fig. 4e), almost thewhole of the citral degraded to different products as the peak cor-responding to citral is missing. One of the main degradation endproducts of citral, ‘p-cymene’ has a reported kmax of 211.5 nm(Hirayama, 1967), while others like p-menthadien-8-ol andp-menthadien-4-ol are reported to absorb strongly in the regionof 214–218 nm (Baser & Buchbauer, 2009; FAO/WHO Expert Com-mittee, 2008; Urushibara & Hirota, 1961). Furthermore, the stablearomatic degradation product of citral, ‘a-p-dimethylstyrene’, has

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Abs

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(a)

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Waveleng

Fig. 3. Absorption spectra of citral in buffer solution, in Brij30 and Bri

a peak with maximum extinction coefficient at 210 nm (Das,Duportail, Richert, Klymchenko, & Mély, 2012; Matyjaszewski &Sigwalt, 1987). The presence of all these degradation products atlower pH may correspond to the addition peak observed near215 nm in Fig. 3c for aqueous and premicellar solution phases ofboth surfactant systems. Since the characteristic UV spectrum ofcitral is obtained in the post-micellar concentrations of Brij surfac-tants, the micellar nanostructures are thus capable of preventingthe citral degradation even under strong acidic conditions of pH1. In conclusion, the Brij surfactant micelles are capable of prevent-ing degradation of citral at all levels of acidic pH invoking its po-tential prospective use in cosmetic, pharmaceutical and foodindustries for citral stabilisation.

3.6. Characterisation of a few citral degradation products

HPLC has been used in the development of quantitative assaysfor citral (Rauber, Guterres, & Schapoval, 2005) and its metabolitesin biological matrices and pharmaceutical products (Hudaib, Bel-lardi, Rubies-Autonell, Fiori, & Cavrini, 2001). HPLC was used inthe current study to identify the degradation products of citral atlower pH’s. The HPLC profile of citral at pH 7 shows a high resolu-tion peak at a wavelength of 237 nm, using water:methanol(40:60, v/v) as mobile phase, given in Fig. 4a along with the HPLCprofiles of two main degradation products of citral, p-methyl ace-tophenone (Fig. 4b) and p-cresol (Fig. 4c) used as markers in thepresent study. Citral at pH 7 is stable and shows an intense absorp-tion, good selectivity and a single well resolved peak at a retentiontime of 3.777 min, while citral at pH 3 (Fig. 4d) and citral at pH 1(Fig. 4e) undergoes degradation and shows a range of absorptionpeaks with varying retention times at the set wavelength. TheHPLC profile of samples of citral at pH 3 and 1 elucidates that a

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Base line Water Brij35(0.01mM) Brij30(0.01mM) Brij35(3mM) Brij30(3mM)

275 300

(c)

Base lineWater Brij35(0.01mM) Brij30(0.01mM Brij35(3mM) Brij30(3mM)

th (nm)

j35 both above and below cmc at (a) pH 7, (b) pH 3 and (c) pH 1.

Fig. 4. HPLC profile of (a) citral at pH 7, (b) p-methyl acetophenone, (c) p-cresol, (d) citral at pH 3 and (e) citral at pH 1.

M. Maswal, A.A. Dar / Food Chemistry 138 (2013) 2356–2364 2361

number of degradation products are formed along with p-cresoland p-methyl acetophenone, and the concentration of citral inthese samples decreases as is indicated by the intensity of peaks,which corresponds to citral at retention times of 3.721 and3.622, respectively. The presence of p-cresol and p-methyl aceto-phenone in these samples was identified by the comparison ofthe retention times with the authentic markers used, the peakswith retention times at 3.088 and 2.947 corresponds to the pres-

ence of p-methyl acetophenone while the peaks with retentiontime of 5.837 and 5.735 corresponds to the presence of p-cresolin the samples of citral at pH 3 and 1, respectively.

3.7. Kinetics of citral degradation

The effect of solution pH on the kinetics of chemical degrada-tion of citral was studied spectrophotometrically over a seven

2362 M. Maswal, A.A. Dar / Food Chemistry 138 (2013) 2356–2364

day period. The kinetics of chemical degradation of citral in aque-ous solutions was studied at a citral concentration of 380 mg/ml,which is below the saturation level. However, in micellar solutionsthe initial concentration was below Cmax corresponding to a givensurfactant concentration. The rate of citral degradation has beenmodelled by assuming a first-order reaction in accordance withearlier studies (Choi et al., 2009; Djordjevic et al., 2007; Yanget al., 2011):

CðtÞ ¼ C0 expð�ktÞ

where C0 is the initial citral concentration, C(t) is the citral concen-tration remaining at time t, and k is the degradation rate constant. Afew prototype plots showing variation of ln(C(t)/C0) versus time atdifferent pH values in aqueous and 1 mM Brij30 and 1 mM Brij35micellar solutions are shown in Fig. 5. The values of k in aqueousand micellar solutions of Brij30 and Brij35 at different pH valueswere determined by carrying out a linear regression on plots ofln(C(t)/C0) versus t and are plotted in Fig. 6a as a function of surfac-tant concentration at various pH values. The data in Fig. 5 showsthat there is little change in citral concentration over the periodof 7 days at pH 7, even in aqueous solution, which is in conformitywith the results of HPLC. In addition the degradation rate constantof citral is very low in aqueous Brij30 and Brij35 micellar media atpH 7, which does not vary much with concentration of surfactant(Fig. 6a). k showed values of 0.0138, 0.116 and 0.255 day�1 in aque-ous solutions at pH 7, 3 and 1 respectively indicating the rate ofdegradation to be highest at acidic pH values. In aqueous micellarmedia, k was very low at all pH values as compared to that in pureaqueous solutions with a maximum at pH 1. This shows that thesurfactant micelles were capable of decreasing k due to partitioning

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o)

Tim

Fig. 5. Variation of ln(Ct/C0) with time (t) at pH 7, 3 and 1 for (a) buffer solution, (b) 1 mMcoefficients of linear fits were greater than 0.98 for all studied systems.

of citral molecules into the micelles isolating them from the acidpresent in aqueous environment and hence retarding the degrada-tion rate. In addition, the increase in surfactant concentration leadsto further decrease in value of k due to increase in micellar concen-tration and hence the partitioning of citral within the micelles(Fig. 6a). Fig. 6a indicates that Brij30 micelles are more capable ofstabilising citral from chemical degradation than Brij35 micellesand relatively stable microemulsions of citral were formed withinBrij30 micelles. As described in earlier section due to the large corevolume and lower Laplace pressure inside the Brij30 micelles, citralis mainly solubilised within the micellar core and hence proves tobe a better solubilisation medium both in terms of solubilityenhancement and protection from chemical deterioration of citral.For Brij35, an appreciable amount of citral is solubilised in the pal-isade layer, where it is in direct contact with reactive pro-oxidativespecies and is hence prone to undergoing degradation, explainingthe relatively lower potency of Brij35 micelles to protect citral fromdegradation.

Though the formulations of citral with Brij surfactants showedexcellent physical stability in the temperature range of 25–40 �C,the rate of degradation, however, increased with increase in tem-perature. The variation of k with temperature in 3 mM Brij30,3 mM Brij35 and aqueous solution at pH 1 is given in Fig. 6b. Theincrease in rate of degradation with temperature is more in thecase of aqueous solution than in the micellar solutions, indicatingthat the temperature effect on the increase in degradation rate isconsiderably suppressed by the presence of micelles. The compar-ative account of variation of rate of degradation of citral with tem-peratures solubilised within different surfactant micelles showsthat in Brij35 micelles, where citral is preferentially solubilised in

1 2 3 4 5 6

-0.14

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

(b)

Ln (C

(t)/C

o)

Time (days)

pH 7 pH 3 pH 1

4 5 6e (days)

pH 7 pH 3 pH 1

and Brij30 surfactant solution and (c) 1 mM Brij35 surfactant solution. Regression

1 2 3 4 50.000

0.005

0.010

0.015

0.020 Brij35 at pH 7 Brij35 at pH 3 Brij35 at pH 1 Brijj30 at pH 7 Brij30 at pH 3 Brij30 at pH 1

Deg

rada

tion

cons

tant

(k)

Surfactant conc (mM)

(a)

24 26 28 30 32 34 36 38 40 420.00

0.02

0.2

0.3

0.4

0.5(b)

Deg

rada

tion

cons

tant

(k)

Temperature ( oC)

Brij30 Brij35 Water

Fig. 6. Variation of Degradation constant (k) with; (a) concentrations of Brij30 and Brij35 surfactant at pH 7, 3 and 1 at 25 �C and (b) temperature (in �C) for 3 mM Brij30,3 mM Brij35 and aqueous solution at pH 1.

M. Maswal, A.A. Dar / Food Chemistry 138 (2013) 2356–2364 2363

the palisade layer/interfacial layer, the effect is more pronouncedas compared to that in Brij30 micelles, wherein citral is preferen-tially solubilised in the micellar core. This points to the easy accessof pro-oxidant species to interfacially solubilised citral moleculesin Brij35 micelles leading to significant increase in the probabilityof encounters between two reactants with temperature. These re-sults suggest that a surfactant system with a lower HLB value, lessdense hydrophilic corona and more hydrophobic core volume isefficient in solubilising and stabilising citral.

4. Conclusions

In summary, this study reports the solubilisation and stabilisa-tion of citral by micelles of polyoxyethylene alkylether based non-ionic surfactants. The solubilisation capacity strongly depends onconcentration and number of oxyethylene groups present in thehead group of surfactant. The locus of solubilisation of citral ismainly towards the non-polar region of Brij30 while in case ofBrij35 more appreciable amount of citral is solubilised within theouter corona indicating the significant effect of number of oxyeth-ylene groups per head group of surfactant in citral encapsulation.The encapsulation of citral results in increased chemical stabilityof citral in acidic pH range where aqueous solutions without mi-celles are incapable of stabilising the citral. Brij30 proves to be abetter solubilising as well as stabilising medium for citral as com-pared to Brij35 micellar medium at all acidic pH’s. This study con-cludes that Brij surfactants with a lower HLB value, less densehydrophilic corona and more hydrophobic core volume are effi-cient in solubilising and stabilising citral against an acidicenvironment.

Acknowledgement

We are thankful to the Head of the Department of Chemistry,University of Kashmir, for his constant encouragement andinspiration.

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