oly

0900

tials deasestinglikesco

sulated formulation has potential to be applied to other volatile compounds.Crown Copyright 2013 Published by Elsevier Ltd. All rights reserved.

en coial proor humproducum Linf med

(Chanwitheesuk, Teerawutgulrag, & Rakariyatham, 2005;

ulata, Luqman, & Nikhat, 2010; Kumar, Shukla, Singh, & Dubey,

2001; Bihari, Shankar, Kumar, Keshari, & Ellaiah, 2010), anti-inammatory (Singh, Majumdar, & Rehan, 1996; Singh, Taneja, &Majumdar, 2007), antitussive (Nadig & Laxmi, 2005), and antican-cer (Shimizu et al., 2013) properties. Their health benets also in-clude the inhibition of cholesterol synthesis (Khanna et al., 2010;Pattanayak, Behera, Das, & Panda, 2010) and the improvement of

coacervate phase around an active compound, which is suspended2004). Coacerva-ple and cosingle hy

loid, while the latter requires two oppositely charged hydrocol-

complex coacervation with regard to cost saving and exible oper-ation. To induce the phase separation, simple coacervation usesinexpensive inorganic salts, whereas complex coacervation usesrelatively expensive hydrocolloids. Furthermore, complex coacer-vation is more sensitive to even a small pH change. Nevertheless,for both types of coacervation, gelatin is the most frequently ap-plied encapsulating material.

Gelatin is a mixture of peptides and proteins obtained by apartial hydrolysis of collagen found in skin, bones, and connective

Corresponding author. Tel.: +66 2 5625074; fax: +66 2 5794096.

Food Chemistry 150 (2014) 313320

Contents lists availab

he

lseE-mail address: pakamon.c@ku.ac.th (P. Chitprasert).2010), anthelmintic (Asha, Prashanth, Murali, Padmaja, & Amit, loids. Simple coacervation offers important advantages overJuntachote, Berghofer, Siebenhandl, & Bauer, 2007; Maisuthisakul,Pasuk, & Ritthiruangdej, 2008; Manosroi, Dhumtanom, & Manosroi,2006), antibacterial (Burt, 2004), antifungal (Amber, Aijaz, Immac-

or emulsied in the same reaction media (Gouin,tion is generally classied into two types: simcoacervation. The former is usually formed by a0308-8146/$ - see front matter Crown Copyright 2013 Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.10.159mplexdrocol-tial oils (Baliga, Shivashankara, Azmidah, Sunitha, & Palatty, 2013).The main constituents of the holy basil essential oil (HBEO) arephenolic and terpenoid derivatives including methyl eugenol(42.58%) followed by caryophyllene (26.88%) and eugenol(10.66%). There have been reports on their antioxidant

tive techniques. Microencapsulation also offers controlled-releasedelivery and improves the handling properties of the essential oils.One commonly used method for essential oil microencapsulation iscoacervation. It is basically phase separation of hydrocolloids fromthe initial solutions and subsequent deposition of a newly formedEssential oilGelatinResponse surface methodology

1. Introduction

In recent years, there has beresearching and utilising the benecto develop natural health products ffrom general fragrances and avourature have shown that Ocimum sanctholy basil, has emerged as a source onsiderable interest inperties of essential oilsans and animals, apartts. Reviews of the liter-n., commonly known asicinally valuable essen-

the digestive performance (Baliga, Shivashankara, Azmidah,Sunitha, & Palatty, 2013). Even though HBEO possesses all thesehealth-promoting properties, a disadvantage of HBEO is a shortshelf life due to its volatility nature and its ready chemical degra-dation in response to light, heat, moisture, and oxygen.

To protect the essential oils against the deteriorating effects ofprocessing and storage conditions, as well as retard evaporation,microencapsulation has been considered as one of the most effec-MicroencapsulationHoly basil

HBEO and gelatin. Under storage conditions at 60 C for 49 days equivalent to 25 C for 18 months, smalldecreases in the HBEO retention rate and the antioxidant activity were observed. Thus, the microencap-Analytical Methods

Optimisation of microencapsulation of hby response surface methodology

Polin Sutaphanit, Pakamon Chitprasert Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 1

a r t i c l e i n f o

Article history:Received 17 January 2013Received in revised form 1 October 2013Accepted 26 October 2013Available online 11 November 2013

Keywords:Optimisation

a b s t r a c t

To protect holy basil essencoacervation of gelatin waface methodology (RSM) wwhich provided the great95.41%, respectively. Scanncapsule was honeycomb-transform infrared spectro

Food C

journal homepage: www.ebasil essential oil in gelatin

, Thailand

oil (HBEO) from volatilisation and oxidation, microencapsulation by simpleveloped. An optimal encapsulating condition obtained from response sur-a gelatin concentration of 11.75% (w/v) and an HBEO amount of 31 ml,yield, oil content, and encapsulation efciency of 98.80%, 66.50%, andelectron microscope (SEM) revealed that the internal surface of the micro-networks containing nonhomogeneous distributions of HBEO. Fourier

py (FTIR) indicated that there was no signicant interaction between the

le at ScienceDirect

mistry

vier .com/locate / foodchem

lating condition was determined employing RSM with a central

(0.31%), respectively. Gelatin from porcine skin (300 Bloom, type

equations were plotted using the Statistica 6.0 computer software

were dissolved in dichloromethane to a nal volume of 1 ml. The

od CA), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchasedfrom Sigma Aldrich (St. Louis, MO, USA). Sodium dodecyl sulphate(SDS) and Tween 80 were purchased from Ajak Finechem (NSW,Australia). Glutaraldehyde was obtained from Merck (Darmstadt,Germany). All chemicals used in this experiment were analyticalgrade.

2.2. Preparation of HBEO-loaded gelatin microcapsules

The HBEO-loaded gelatin microcapsules were prepared by sim-ple coacervation via a glutaraldehyde crosslinking method. In areaction vessel, 416% (w/v) of a 30 ml aqueous solution of gelatincontaining 1 mM SDS/g of gelatin was mixed with differentamounts (7.533.1 ml) of HBEO under stirring at 450 rpm and40 C. Then, the pH was adjusted to 3.54.0 by adding 10% (v/v)acetic acid solution and the mixture was stirred at 450 rpm for25 min to form a stable emulsion. Sodium sulphate solution (20%w/v, 300 ml) was added to the emulsion and the mixing speedwas raised to 550 rpm. The system was cooled down slowly to5 C. The crosslinking of the gelatin microcapsules was achievedby the slow addition of 3.525 ml of glutaraldehyde solution con-sisting of 25% (v/v) glutaraldehyde and acetonewater. The tem-perature was subsequently raised to 40 C and the pH wasadjusted to 7 with continuous stirring for 60 min. The precipitatedcomposite design (CCD). The effects of the gelatin concentrationand HBEO amount on the yield, oil content, and encapsulation ef-ciency were assessed. SEM, particle size analyser, FTIR, colorimeter,and X-ray diffraction were used to characterise the physical andchemical properties. The shelf life of the HBEO-loaded gelatinmicrocapsules was also evaluated under accelerated conditions.

2. Materials and methods

2.1. Materials

The steam distilled HBEO was purchased from Thai-China Fla-vours and Fragrances Industry Co., Ltd., Thailand. It was analysedfor chemical constituents by gas chromatographymass spectrom-etry (GCMS). Its major components were methyl eugenol (42.58%)followed by b-caryophyllene (26.88%), eugenol (10.66%), b-elem-ene (5.99%), naphthalene (2.97%), germacrene (2.59%), a-humulene(1.73%), caryophyllene oxide (1.08%), and small amounts of cam-phor (0.64%), chavical (0.58%), linalool (0.20%), and a-selinenetissues of animals. Owing to its biodegradability, biocompatibility,nontoxicity, low cost, water-solubility, lm forming, and emulsify-ing ability, it is an ideal encapsulating material for essential oilsincluding clove oils (Thimma & Tammishetti, 2003), camphor oils(Chang, Leung, Lin, & Hsu, 2006), citronella oils (Hsieh, Chang, &Gao, 2006; Maji, Baruah, Dube, & Hussain, 2007; Solomon, Sahle,Gebre-Mariam, Asres, & Neubert, 2012), and peppermint oils (Donget al., 2008). However, to the best of our knowledge thus far, therehas been no report on the microencapsulation of HBEO using gela-tin via simple coacervation. Furthermore, most of the studies notedabove have essentially focused on the effects of the formulationparameters on release behaviour. However, the stability of theencapsulated essential oils during storage has not beeninvestigated.

Therefore, this study aimed to develop the microencapsulatedHBEO by simple coacervation. Gelatin was used as the wall mate-rial and crosslinked with glutaraldehyde. The optimised encapsu-

314 P. Sutaphanit, P. Chitprasert / Fomicrocapsules were washed twice with 0.3% tween 80 under stir-ring at room temperature and ltered under vacuum. They werecollected and dried at 60 C till constant dry weight was obtained.suspension was gently mixed, kept for 20 min and successivelysonicated for 10 min. The microcapsule residues were ltered(StatSoft Inc.).

2.4. Physical and chemical characterizations of HBEO-loaded gelatinmicrocapsules

2.4.1. Determination of yield, oil content, and encapsulation efciencyThe obtained microcapsules were characterised in terms of

yield, oil content, and encapsulation efciency for the evaluationof the response variables using the RSM. The calculation of yieldwas performed using Eq. (2) below:

Yield % WMWT

100 2

where WM is the weight of the microcapsules and WT is the totalweight of the microcapsules and inapplicable clusters adhering toturbines.

A standard curve of HBEO was created to determine the oilcontent and encapsulation efciency of the microcapsules. TheHBEO sample was diluted with dichloromethane to form a seriesof standard solutions at different known concentrations. Thesolutions were scanned over the range of 275400 nm using aUV visible spectrophotometer (Hekios c, Thermospectonic,England) and the absorption maximum was observed at 289 nm.The absorbance values at this wavelength for various concentra-tions of the HBEO standard solutions were recorded and plotted.The data were t to a linear regression line with the equation,y 1:8082x 0:0662R2 0:998.

To determine the HBEO content, 80 mg of the microcapsulesThe nished microcapsules were stored in a hermetically sealedglass bottle at room temperature until analysis.

2.3. Experimental design

RSM was used to determine the optimum encapsulating condi-tions. The effects of two independent variables: gelatin concentra-tions (X1: 416% w/v) and HBEO amounts (X2: 7.537.5 ml) onthree response variables: yield (Y1), oil content (Y2), and encapsu-lation efciency (Y3) were evaluated using CCD. The factors andtheir levels used in the design are shown in Table 1. The ve codedlevels (1.414, 1, 0, +1, and +1.414) of the two variables wereincorporated into the design leading to 13 experiments. All exper-iments were carried out in triplicate. The effects of the indepen-dent variables, X1 and X2, on the responses Y were evaluatedusing the second-order polynomial regression Eq. (1):

Yn b0 b1X1 b2X2 b12X1X2 b11X21 b22X22 1

where Yn represents the response variable, b0 is a constant, b1and b2 are the coefcients of the linear effects, b12 is the coef-cient of the interaction between the factors, and b11 and b22are the coefcients of the quadratic effect. These coefcients werecalculated using the SPSS 14.0 statistical computer software (SPSSInc.). The experimental data were analysed by multiple regres-sions to t the second-order polynomial equation. Analysis ofvariance (ANOVA) was performed to evaluate signicant differ-ences between the independent variables. To visualise the rela-tionships between the variables and the responses, the surfaceresponses and contour plots of the tted polynomial regression

hemistry 150 (2014) 313320through nylon syringe lters with a 0.22 lm pore size. The absor-bance of the HBEO solution was measured and its unknown con-centration was calculated based on the absorbance value and the

ATR-FTIR spectrometer (Bruker Optics, Germany).

od Cconstants from the linear regression curve. The oil content was cal-culated using Eq. (3):

Oil content % WOWM

100 3

where WO is the weight of the oil in the microcapsules. To evaluatethe encapsulation efciency (EE), Eq. (4) was used:

Encapsulation efficiency % WOWI

100 4

where WI is the weight of the oil initially added for themicroencapsulation.

2.4.2. Determination of oil loadingTo calculate the theoretical oil loading, Eq. (5) was used:

WI

Table 1Central composite design with independent and response variables for preparation ofHBEO-loaded gelatin microcapsules.

Runno.

Factors Response variables

X1 X2

Yield(%)

Oil Content(%)

EE (%) Oil loading(%)

1 14.24 33.10 98.08 68.47 95.99 387.412 14.24 11.90 62.65 60.47 100.09 139.283 5.67 11.90 96.67 64.38 95.24 344.334 5.67 33.10 95.94 66.48 51.88 957.755 16.00 22.50 74.48 63.14 87.82 234.386 10.00 37.50 97.22 68.28 68.21 6257 4.00 22.50 96.43 65.83 44.65 937.58 10.00 7.50 54.90 62.97 82.94 1259 10.00 22.50 99.35 67.45 95.63 375

10 10.00 22.50 96.84 67.29 95.22 37511 10.00 22.50 97.69 67.26 95.90 37512 10.00 22.50 98.14 66.94 97.08 37513 10.00 22.50 98.96 67.33 97.26 375

X1 = gelatin concentrations (%w/v).X2 = HBEO amounts (ml).

P. Sutaphanit, P. Chitprasert / FoOil loading % WC

100 5

where WC is the total weight of gelatin and glutaraldehyde.

2.4.3. Determination of average sizesThe size of the optimised HBEO-loaded gelatin microcapsules

was measured using a laser light scattering particle sizer (Master-sizer-2000, Malvern, UK) with a wet cell (Hydro 2000 MU (A)) and0.3% Tween 80 as a suspending solution. The area-volume meandiameter, D[4,3], was recorded.

2.4.4. Scanning electron microscope (SEM) studiesAn SEM (JSM-5600, JEOL, Japan) at an accelerating voltage of

15 kV was used to characterise the external and internal morphol-ogy of the optimised HBEO-loaded gelatin microcapsules. The sam-ples were incubated under 2% (v/v) osmium tetroxide vapour for1 h, washed twice in distilled water for 10 min each time, and con-secutively dehydrated in 30%, 50%, 70% and 95% (v/v) ethanol solu-tion for 10 min and in 100% (v/v) ethanol solution three times for10 min each time. Then, they were dried under vacuum using acritical point dryer. The microcapsules were xed on an aluminumstub with double-sided adhesive tape. To characterise the innersurfaces, the microcapsules were cut with a razor blade prior to x-ation. All samples were sputter-coated with gold using an ion sput-tering coater (IB2, Giko Engineering, Japan).2.5. Stability of HBEO-loaded gelatin microcapsules under acceleratedstorage conditions

2.5.1. Determination of HBEO retention rateThe optimised HBEO-loaded gelatin microcapsules were kept in

amber glass bottles with airtight lids and stored in the dark underaccelerated storage conditions at 60 C for 49 days. Samples weretaken at various times to determine the HBO retention rate andantioxidant activity using Eq. (6):

HBEO retention rate % CIC0

100 6

where C0 and CI are the HBEO amounts in the microcapsules beforeand after storage, respectively. The HBEO was extracted using thesame method as described in Section 2.4.1.

2.5.2. Determination of antioxidant activityThe antioxidant activity of the microcapsules was measured by

DPPH assay. Briey, 1 ml of the microcapsules dissolved in dichlo-romethane was mixed with 1 ml of 0.2 mMDPPH. The mixture wasthen homogenised and left for 20 min in the dark. The absorbancewas measured at 517 nm against methanol as a blank using aUVVIS spectrophotometer (Hekios c, Thermospectonic, England).All samples were analysed in triplicate. The DPPH radical scaveng-ing activity was calculated using Eq. (7):

DPPH radical scavenging activity %

A0 AM ASA0

100 7

where A0 is the absorbance of the control (DPPH only), AM is theabsorbance of DPPH mixed with the samples, and AS is the absor-bance of the samples without DPPH.

2.5.3. Determination of colourThe colour of the microencapsulated HBEO was measured be-

fore and after storage using a CIE colorimeter (Miniscan XE, HunterAssociates Laboratory, Inc., VA, USA). This system uses three valuesto describe the precise location of a colour inside a three-dimen-sional visible colour space. The measurements were displayed inL, a and b values [(L = 0 (black) to 100 (white), a = 60 (green)to +60 (red), and b=60 (blue) to +60 (yellow)]. The colorimeterwas calibrated against a standard white tile (L = 93.66,a = 0.75, and b = 1.07) before colour measurements. The totalcolour change (DE) was calculated using Eq. (8), where the sub-script 0 indicates the initial colour of the microcapsule beforestorage as a reference. Colour was measured three times intriplicate.

DE L0 L2 a0 a2 b0 b

2q

8

2.5.4. Determination of crystallinityThe crystal structures of the HBEO-loaded gelatin microcap-

sules before and after storage as well as the gelatin powder weredetermined using an XRD diffractometer (Philips model XPert2.4.5. Fourier transform infrared spectroscopy (FTIR) studiesThe FTIR spectra of the optimised HBEO-loaded gelatin micro-

capsules, gelatin solution, and HBEOwere characterised. They wererecorded in the range of 4000500 cm1 on a TENSOR series

hemistry 150 (2014) 313320 315MPD, Netherlands) with Cu-Ka (wavelength 1.5406 ) radiationat 40 kV and 30 mA. The samples were scanned between 2h = 5and 50 at a scan rate of 2.4/min.

ANOVA was performed to evaluate the signicance of the coef-

od Ccients of the quadratic polynomial models (Table 2). On the basisof the regression coefcients and the p-value, it was found that thelinear, quadratic, and interaction terms of both the gelatin concen-tration and HBEO amount had signicant effects on the oil content(P < 0.05). The variable with the greatest effect on the encapsula-2.6. Statistical analysis

The data were presented as mean value standard deviation(SD). The statistical signicance was set at P < 0.05. P values < 0.05were regarded as signicant.

3. Results and discussion

3.1. Optimisation of microencapsulation of HBEO by RSM

In the present study, the HBEO-loaded gelatin microcapsuleswere successfully prepared by a simple coacervation method viacovalent crosslinking with glutaraldehyde. To minimise the exper-imental runs and times for the optimisation of the microencapsu-lation parameters, the two-factor CCD was applied on the basis ofve coded levels of the two independent variables, resulting inthirteen experiments with ve replicates at the centre of theexperimental design (Table 2). The independent variables, (the gel-atin concentration and the HBEO amount) were set on the basis ofa preliminary screening, which took into account the physical andchemical properties of the microcapsules in terms of the yield, oilcontent, and encapsulation efciency. For example, the gelatinconcentrations of less than 4% (w/v) not only produced smallamounts of the microcapsules, but also had low encapsulation ef-ciency. Clearly, a large amount of HBEO was not enclosed in themicrocapsules and it was consequently lost during the separationprocess. On the other hand, the gelatin concentrations of greaterthan 16% (w/v) caused an excessively viscous solution leading toirregular clusters of the microcapsules. Meanwhile, the HBEOamounts of 7.537.5 ml were properly selected for the gelatin con-centrations within the used range of 416% (w/v). According to thepreliminary study, other important microencapsulation variablessuch as the mixing speed, gelatin volume, glutaraldehyde concen-tration, and hardening time were kept constant at 450 rpm, 30 ml,10 mmol/g of gelatin, and 60 min, respectively.

Thirteen formulations of the HBEO microcapsules with variousamounts of HBEO and gelatin were fabricated and their propertiesare displayed in Table 1. All the batches within the experimentaldesign showed a variation in the yield, oil content, and encapsula-tion efciency in the range 54.9098.96%, 62.9768.47%, and44.65100.09%, respectively. The results were analysed by SPSS14.0 and the regression equations describing the mathematicalrelationships between the independent and response variables ob-tained are shown in Eqs. (9)(11):

Yield % 68:521 1:6072X1 3:0182X2 0:2397X21 0:2019X1X2 0:0862X22 9

Oil content % 60:0272 0:6611X1 0:216X2 0:0784X21 0:0328X1X2 0:0075X22 10

Encapsulation efficiency % 36:4561 9:8412X1 0:2279X2 0:5775X21 0:2197X1X2 0:07X22: 11

316 P. Sutaphanit, P. Chitprasert / Fotion efciency was the quadratic term of the gelatin concentration,followed by the linear term of the gelatin concentration and thequadratic term of the HBEO amount (P < 0.05), while the interac-tion term did not exert a signicant effect (P > 0.05). The yieldwas signicantly inuenced by the linear and quadratic terms ofthe HBEO amount, and the interaction between the two variables(P < 0.05), but not by the linear and quadratic terms of the gelatinconcentration (P > 0.05).

To validate the models obtained from the RSM, additionalexperiments were needed. The effects of the gelatin concentrationand the HBEO amount on the yield, oil content, and encapsulationefciency are shown in the response surfaces and their respectivecontour plots in Fig. 1. The optimised encapsulation conditionswere determined by superimposing the contour plots of all threeresponses. The optimum zone in which every point represented acombination of the gelatin concentration and the HBEO amountgave the maximum values of all the responses. Then, veencapsulation conditions (11.5% (w/v), 29 ml; 12% (w/v), 30 ml;11.75% (w/v), 31 ml; 10.5% (w/v), 28 ml; and 11% (w/v), 28 ml)randomly selected from that zone were repeated in triplicate tocompare the model predictions with the experimental data. Thepredicted values of the yield, oil content, and encapsulationefciency were in good agreement with the observed ones (datanot shown) indicating that the models were applicable.

From the experimental results, it was observed that the opti-mised conditions corresponding to the centre of the experimentaldesign (runs no. 913) had an oil-to-wall volume ratio of 0.75:1.Chang et al. (2006) also successfully used a coacervation methodto fabricate gelatin-gum arabic microcapsules containing camphoroil and polystyrene with an oil-to-wall volume ratio of 0.75:1. Theother parameter indicating the success of microencapsulation wasoil loading which was the ratio of the HBEO amount to the totalamounts of gelatin and crosslinkers. It was calculated using Eq.(5) and the results are shown in Table 2. The optimised oil loadingwas found to be 375%. At higher oil loading conditions (runs no. 4,6, and 7), some oil droplets were not encapsulated in the gelatinmicrocapsules and consequently were lost during the separationprocess as reported by Maji et al. (2007). They suggested that theconcentration of gelatin should be sufcient to encapsulateZanthoxylum limonella oil via a coacervation technique. On theother hand, under lower oil loading conditions, especially withhigh gelatin concentrations (16% (w/v) in run no. 5), highly viscousgelatin droplets containing the HBEO were not properly cross-linked and they nally appeared as large clusters adhering to theturbine at the end of the crosslinking process. These clusters werenot applicable for use and this resulted in a low yield, low oilcontent, and low encapsulation efciency.

When considering the encapsulation efciency obtained fromthe optimised conditions, the simple coacervation process devel-oped in this study was an effective approach to microencapsulateHBEO. The encapsulation efciency was an indicator of not onlythe oil retention ability of gelatin, but also chemical stability ofHBEO. The nearly 100% encapsulation efciency suggested thatthere was only a small physical loss of HBEO during the polymericchain crosslinking and washing of the microcapsules to remove theexcess oil on the surface. In addition, the chemical degradation ofHBEO was negligible due to the mild encapsulation conditions.

3.2. Physical and chemical properties of HBEO-loaded gelatinmicrocapsules

3.2.1. Size and surface morphologyThe primary physical and chemical characteristics of the opti-

mised microcapsules that indicate their potential applications infood, feed, and pharmaceutical industries were analysed. The vol-ume moment mean diameter (D[4,3]) obtained from the particle

hemistry 150 (2014) 313320size analyser was 392.30 lm. The internal and external surfacemorphology was examined by SEM. The photographs of the pre-pared microcapsules revealed that the microcapsule was almost

ber of the much larger pores was expected to contain HBEO and

radical DPPH. Similar to the HBEO retention rate, a small decrease

EO

n co

od Cthese multi-core features of the microcapsule resulted in the highHBEO content of about 67%. The morphological defects visible inthe bottom right corner of the image were also observed, whichwas possibly due to the razor cuts.

3.2.2. FTIR spectraThe FTIR was used to conrm the presence of HBEO and gelatin,

and to investigate their possible interactions. The FTIR spectrum ofthe HBEO displayed the characteristic peaks of its main compo-nents, methyl eugenol, eugenol, and caryophyllene (Fig. 3a). Thespectrum at 3074 cm1 was assigned to the asymmetric CHstretching vibrations in @CH2. The bands observed at 2928 and2858 cm1 indicated the asymmetric and symmetric CH stretch-ing vibrations in CH2. The peaks at 1638, 1607, 1592, and1513 cm1 were assigned to the alkene/aromatic C@C stretchingvibrations (Dhoot, Auras, Rubino, Dolan, & Soto-Valdez, 2009). Thespherical in shape with folding and rough external surfaces(Fig. 2a). The high magnication image displayed a sponge-likestructure with a large number of micron-sized pores randomly dis-tributed on the external surface (Fig. 2b). The appearance of thesepores was attributed to the loss of water trapped by the gelatinnetwork during the sample preparation for SEM. The internal mor-phology of the surface was examined in cross-section view(Fig. 2c). The crosslinked gelatin network was honeycomb-like aspreviously reported (Benjakul, Oungbho, Visessanguan,Thiansilakul, & Roytrakul, 2009). The honeycomb walls, containingwater-lled pores had a thickness of about 0.4 lm. The large num-

Table 2Analysis of variance of regression coefcients calculated for microencapsulation of HB

Independent variable Oil content EE

Regression coefcient Signicance level (P) Regressio

Constant 60.027 0.000 38.638

LinearX1 0.661 0.022 10.862X2 0.216 0.042 0.345Quadratic

X21 0.0784 0.000 0.627X22 0.03282 0.002 0.0587

InteractionX1X2 0.00748 0.000 0.218

EE = encapsulation efciency.Signicance at P < 0.05.

P. Sutaphanit, P. Chitprasert / Foscissor CH bending vibrations in @CH2 appeared at 1463 cm1.The asymmetric and symmetric CH bending vibrations in CH3were observed at 1451 and 1368 cm1, respectively. The bandsbetween 994 and 544 cm1 represented the CH bending of thealkene/aromatic groups (Sajomsang et al., 2012).

The FTIR spectrum of gelatin is shown in Fig. 3b. The bandappearing at 3238 cm1 represented the NH stretching vibrationsof amide groups designated as amide A. The carbonyl C@O stretch-ing vibrations with contributions from in-phase bending of theNH bond and stretching of the CN bond occurred at 1637 cm1

and are referred as the amide I band. The amide II band appearedat 1568 and 1455 cm1 attributed to the deformation of the NHbond and the out-of-phase combination of CN stretch (Bandekar,1992; Hashim et al., 2010; Lavialle, Adams, & Levin, 1982). Thesmall peak representing the amide III band was also found at1244 cm1.

In case of the gelatin microcapsules containing HBEO, most ofthe characteristic peaks of HBEO remained unchanged, indicatingthe successful incorporation of HBEO into the microcapsules andthe chemical stability of the HBEO after encapsulation. In otherin the antioxidant activity was detected throughout the 49 days ofstorage (Fig. 4b). These results provided evidence showing the abil-ity of the gelatin microcapsules to protect the HBEO against phys-ical and chemical loss during storage. However, the gelatinmicrocapsule noticeably changed its colour with a decrease in L,but an increase in both a and b. Thus, it became darker with aDE value of 30.81. This was likely due to Maillard product forma-words, there was no signicant chemical interaction betweenHBEO and gelatin. In addition, it was observed that the broad peakat 3283 cm1 in the spectrum of the gelatin solution shifted to3351 cm1 in that of the HBEO-loaded gelatin microcapsule. Thisconrmed that in order to form gelatin gel, the amino group of gel-atin was crosslinked with glutaraldehyde.

3.3. Stability of HBEO-loaded gelatin microcapsules under acceleratedconditions

To assess the effect of microencapsulation on the HBEO shelflife, the stability testing was conducted under accelerated condi-tions at 60 C for 49 days equivalent to 25 C for 18 months. Themeasurements of chemical properties (the HBEO retention rateand antioxidant activity) as well as physical properties (colourand crystallinity) were performed during storage. The time proleof the HBEO retention rate is displayed in Fig. 4a. No signicantchange of the microencapsulated HBEO amount was observed.The antioxidant activity of HBEO was measured in terms of thehydrogen-donating or radical-scavenging ability, using the stable

in gelatin matrices.

Yield

efcient Signicance level (P) Regression coefcient Signicance level (P)

0.283 70.697 0.009

0.044 1.398 0.6130.845 2.776 0.029

0.012 0.201 0.0610.091 0.0816 0.003

0.063 0.201 0.011

hemistry 150 (2014) 313320 317tion from the reaction between the carbonyl groups of HBEO andthe amino groups of gelatin at high temperature.

Besides the colour change, the conformational rearrangement ofthe HBEO-loaded gelatin microcapsule occurred in Fig. 4ce illus-trate the XRD patterns of the gelatin powder, as well as theHBEO-loaded gelatin microcapsule before and after storage. TheXRD pattern of the gelatin powder showed a crystalline structurecorresponding to the characteristic peaks at 2h around 8 and 20(Fig. 4c). Similar results were reported by Ki et al. (2005), whofound that the gelatin powder gave a typical XRD pattern of a crys-talline material due to its a-helix and triple-helical structure,which was mainly by the formation of inter-chain hydrogen bondsbetween carbonyl and amine groups (Rivero, Garca, & Pinotti,2010). However, the XRD pattern of the HBEO-loaded gelatinmicrocapsules became amorphous after encapsulation due to theconformational change from helical to random coil and b-sheetconformations of the gelatin during gel formation (Fig. 4d). Theobservation of the amorphous pattern may also indicate that theamorphous HBEO is molecularly dispersed in the amorphous gela-tin matrix. However, the gelatin gel under accelerated storage

od C318 P. Sutaphanit, P. Chitprasert / Foconditions partially underwent conformational transition from therandom coil and b-sheet conformation to the helical form(Arvanitoyannis, Psomiadou, Nakayama, Aiba, & Yamamoto,

Fig. 1. Response surfaces (ac) and contour plots (df) showing inuence of gelatin concencapsulation efciency.

Fig. 2. SEM photographs of (a) whole, (b) external, and (c) internhemistry 150 (2014) 3133201997). The gelatin renaturation into a collagen-like structure in-creased the crystallinity as observed from the higher intensity ofthe peak at 2h around 20 in the XRD pattern (Fig. 4e). The higher

entrations and HBEO amounts on (a and d) yield, (b and e) oil content, and (c and f)

al surfaces of optimized HBEO-loaded gelatin microcapsules.

od CP. Sutaphanit, P. Chitprasert / Focrystallinity resulted in a harder microcapsule and partial expul-sion of HBEO to the microcapsule surface. Therefore, the HBEOretention rate and the antioxidant activity of the microcapsulewere reduced due to the possible oxidation and evaporation of

techniques. Polymer, 50(6), 14701482.Dong, Z.-J., Xia, S.-Q., Hua, S., Hayat, K., Zhang, X.-M., & Xu, S.-Y. (2008).

Fig. 3. FTIR spectra of (a) HBEO, (b) gelatin solution, and (c) optimized HBEO-loadedgelatin microcapsules.

Fig. 4. Stability of optimized HBEO-loaded gelatin microcapsules under acceleratedstorage at 60 C for 49 days in terms of (a) HBEO retention rate, (b) DPPH radicalscavenging activity, and XRD patterns of (c) gelatin powder, (d) optimized HBEO-loaded gelatin microcapsules before storage at 60 C for 49 days, and (e) optimizedHBEO-loaded gelatin microcapsules after storage at 60 C for 49 days.Optimization of cross-linking parameters during production oftransglutaminase-hardened spherical multinuclear microcapsules by complexcoacervation. Colloids and Surfaces B: Biointerfaces, 63(1), 4147.

Gouin, S. (2004). Microencapsulation: Industrial appraisal of existing technologiesand trends. Trends in Food Science & Technology, 15(78), 330347.

Hashim, D. M., Man, Y. B. C., Norakasha, R., Shuhaimi, M., Salmah, Y., & Syahariza,the surface oil. The renaturation of the gelatin lm matrix was alsoreported to cause the release of incorporated BHT and a-tocoph-erol during storage at 28 C for 6 weeks (Jongjareonrak, Benjakula,Visessanguan, & Tanaka, 2008).

4. Conclusion

HBEO, an effective natural antioxidant, was successfully micro-encapsulated into the gelatin matrix by the simple coacervationmethod via glutaraldehyde crosslinking. The optimal encapsulat-ing conditions providing high yield, encapsulation efciency, andoil content were attained using the RSM. Due to the suitable char-acteristics of the optimised HBEO-loaded gelatin microcapsules,the protection of HBEO against physical and chemical loss underaccelerated storage conditions at 60 C for 49 days was achievable.The microencapsulated formulation developed in this study is ex-pected to be able to be applied to other bioactive substances thatare sensitive to detrimental factors such as oxygen, light, and mois-ture. Nevertheless, to elucidate more benets and applications ofthe HBEO-loaded gelatin microcapsule, further studies on HBEO re-lease are needed.

Acknowledgments

This research was co-funded by the Thailand Research Fund-Master Research Grants (TRF-MAG), No. MRGWI535S019, and Bet-ter Pharma Co., Ltd., Thailand.

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320 P. Sutaphanit, P. Chitprasert / Food Chemistry 150 (2014) 313320

Optimisation of microencapsulation of holy basil essential oil in gelatin by response surface methodology1 Introduction2 Materials and methods2.1 Materials2.2 Preparation of HBEO-loaded gelatin microcapsules2.3 Experimental design2.4 Physical and chemical characterizations of HBEO-loaded gelatin microcapsules2.4.1 Determination of yield, oil content, and encapsulation efficiency2.4.2 Determination of oil loading2.4.3 Determination of average sizes2.4.4 Scanning electron microscope (SEM) studies2.4.5 Fourier transform infrared spectroscopy (FTIR) studies

2.5 Stability of HBEO-loaded gelatin microcapsules under accelerated storage conditions2.5.1 Determination of HBEO retention rate2.5.2 Determination of antioxidant activity2.5.3 Determination of colour2.5.4 Determination of crystallinity

2.6 Statistical analysis

3 Results and discussion3.1 Optimisation of microencapsulation of HBEO by RSM3.2 Physical and chemical properties of HBEO-loaded gelatin microcapsules3.2.1 Size and surface morphology3.2.2 FTIR spectra

3.3 Stability of HBEO-loaded gelatin microcapsules under accelerated conditions

4 ConclusionAcknowledgmentsReferences