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LWT - Food Science and Technology 60 (2015) 502e508

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LWT - Food Science and Technology

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

Encapsulation of Thyme essential oils in chitosan-benzoic acidnanogel with enhanced antimicrobial activity against Aspergillus flavus

Seyede Tahereh Khalili a, Afshin Mohsenifar b, c, *, Mina Beyki d, Sara Zhaveh d,Tavoos Rahmani-Cherati b, Alina Abdollahi b, Mansour Bayat e, Meisam Tabatabaei c, f, **

a Karaj Islamic Azad University, Karaj, Iranb Research and Development Department, Nanozino Co., Tehran, Iranc Nanosystems Research Team (NRTeam), Karaj, Irand Research and Science Branch, Islamic Azad University, Tehran, Irane Dept. of Pathobiology, Faculty of Veterinary Sciences, Islamic Azad University, Science and Research Branch, Tehran, Iranf Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran

a r t i c l e i n f o

Article history:Received 30 October 2013Received in revised form29 July 2014Accepted 30 July 2014Available online 2 September 2014

Keywords:Essential oilsMycotoxinsChitosan nanogelEncapsulationControlled release

* Corresponding author. Research and DevelopmenTehran, Iran. Tel.: þ98 2177061120; fax: þ98 2144580** Corresponding author. Tel.: þ989132865342; fax:

E-mail addresses: mohsenifar_a@yahoo.com (A.yahoo.com (M. Tabatabaei).

http://dx.doi.org/10.1016/j.lwt.2014.07.0540023-6438/© 2014 Published by Elsevier Ltd.

a b s t r a c t

This study was set to investigate the encapsulation of the Thyme essential oils using chitosan and benzoicacid-made nanogel in order to enhance its antifungal properties and half-life. To achieve this, the self-assembled polymer of chitosan and benzoic acid nanogel (CS-BA) was synthesized, its size and shapewere confirmed by spectrometric (FTIR) and microscopic methods (TEM and SEM) and was then used inencapsulating the essence. Under sealed condition, the minimum inhibitory concentration of the CS-BAencapsulated essential oils was recorded at 300 mg/l while the free Thyme extract could only completelyprevent the growth of Aspergillus flavus at an elevated concentration of 400 mg/l. Under non-sealedcondition, higher concentration of encapsulated Thyme essential oils (500 mg/l) was required to causecomplete fungi inhibition and free oils failed to lead to full inhibition even at concentrations as high as1000 mg/l. In vivo analysis also revealed significant anti-fugal properties of the encapsulated oils atconcentrations above 700 mg/l. Overall, due to the volatility and instability of free essential oils, CS-BAnanogel encapsulation was found to have significantly increased half-life and the anti-fungal propertiesof Thyme essential oils.

© 2014 Published by Elsevier Ltd.

1. Introduction

The fact that fungal attacks cause a huge damage in agriculturaloutturn and jeopardize billions of dollars in this industry every yearis undeniable. Mycotoxins can decrease the quality and value of theagricultural products and more importantly consumption ofmycotoxin-contaminated products could lead to injury or death inanimals and human (Yu Cleveland, Nierman,& Bennett, 2005; Zain,2011). Numerous studies have been conducted in order to restrainthe production of these compounds and especially aflatoxins(Groopman Kensler, & Wild, 2008; Jouany, 2007; Lowe & Arendt,2004). The results of such investigations have shown that someherbal extracts and essential oils are capable of preventing thegrowth of the aflatoxin-producing fungi and therefore, could besafely used in food and pharmaceutical industries (Baratta et al.,

t Department, Nanozino Co.,371.þ9832701067.Mohsenifar), meisam_tab@

1998; Beyki et al., 2014; Passone, Girardi, Ferrand, & Etcheverry,2012; Ribeiro et al., 2013). Among these beneficial extracts,Thyme essential oils due to its anti-fungal, anti-viral and anti-bacterial properties has attracted a great deal of attention duringthe last decades (Kumar, Shukla, Singh, Prasad, & Dubey, 2008;Lopez-Leon, Carvalho, Seijo, Ortega-Vinuesa, & Bastos-Gonz�alez,2005). Its properties have been previously investigated againstvarious pathogenic agents such as Botrytis cinerea, Salmonellaenteritidis, Salmonella typhimurium, Yersinia enterocolitica, Shigellaflexneri, Listeria monocytogenes, Shigella sonnei, Salmonella choler-easuis and Aspergillus niger (Rota, Carraminana, Burillo, & Herrera,2004; Soylu, Soylu, & Kurt, 2006).

On the other hand, essential oils are essentially volatile com-pounds and easily degradable at ambient temperature. As a result,increasing their activity and stability using different strategies suchas encapsulation has always been considered crucial (Herrero,Carmona, Jim�enez-Colmenero, & Ruiz-Capillas, 2014; Marques,2010; Parris, Cooke, & Hicks, 2005). Nanogels, networks of syn-thetic/bio polymers, are divided into two different types; nano-hydrogels and nano-organogels (micelle nanogels). The former

Table 1Composition of the thyme essential oils.

Component Composition (ml/l)

R-pinene 0.4R-terpinene 1.2p-cymene 20ç-terpinene 5.4a-caryophyllene 1.5Borneol 1.7Terpinen-4-ol 0.7Carvacrol 2.7Thymol 65.7

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are composed of hydrogels at nanoscale which could absorb asignificant amount of water easily (high degree of inflation in wa-ter) while the latter are hydrophobic nanogels with a tendency foroily substances. In fact, nano-organogels are micelle-like nano-particles which form aggregates in contact with water while se-questrating their hydrophobic regions at the center. Thehydrophobic region includes the oily substances attached to poly-meric backbones containing functional groups such as carboxyl,amine, aldehyde and so on.

Nanogels in general have high capacity and consistency.Encapsulation of antimicrobial essential oils within an nano-organogel has other benefits such as controlled and sustainedrelease of a certain amount of oils from the carrier e.g. nanogel aswell. This would allow the nutrients to be exposed to specific andadequate amounts of essential oils for a longer time (Chacko,Ventura, Zhuang, & Thayumanavan, 2012; Herrero et al., 2014;Raemdonck, Demeester, & De Smedt, 2009).

In the past decade, a variety of polysaccharide nanoparticleshave been investigated for encapsulation of compounds with bio-logical activity. Among those polysaccharide structures, chitosan(CS) which is produced by partial deacetylation of chitin, and is amajor compound of marines shells such as shrimp and crab hasbeen frequently used mainly due its biocompatibility (Borges,Cordeiro-da-Silva, Romeijn, Amidi, & Borchard, 2006; Yu et al,2005; Zain, 2011). Several studies have shown that CS-basednanogels could be used in tissue engineering, drug delivery andas carriers of various macromolecules (Dang & Leong, 2006;Raemdonck et al., 2009). In a study conducted by Zivanovic andChi (2005), CS films enriched with different essential oils werefound to possess more antimicrobial activity in comparisonwith CSfilms and free essential oils during a 5-day period. In fact, CS owesits unique features as a suitable carrier for pharmaceutical andother biological molecules to the existing amine groups on itspolymeric structure and the resulting positive charges (Agnihotri,Mallikarjuna, & Aminabhavi, 2004; Pedro, Cabral-Albuquerque,Ferreira, & Sarmento, 2009; Uzun, 2006).

The present study was set to produce nanogel through the for-mation of amid linkages between the existing amino groups of CS(a(1-4)-2-amino-2-deoxy b-D-glucan), and the carboxyl group ofBA (C6H5COOH). Moreover, the synthesized CS-benzoic acid (BA)nanogel was used to encapsulate Thyme essential oils in order toevaluate their potential synergistic effects in elimination of Asper-gillus flavus.

2. Materials and methods

2.1. Materials

CS and BA were purchased from Sigma (Germany). Ethylenedichloride (EDC) was obtained from Fluka. Acetic acid, tween 80and ethanol were purchased from Merck (Germany). The essentialoils of Thymus vulgaris (Thyme) was provided by Barij Essence Co.(Iran) (Table 1). A. flavus (ATCC5004) was provided by PastureInstitute (Iran). Potato Dextrose Agar (PDA) and Potato DextroseBroth (PDB) were purchased from Himedia (India).

2.2. Preparation and storage of A. flavus

The standard lineage of A. flavus (ATCC 5004) was obtained fromthe Pasture Institute of Iran and was cultured in falcon tubes con-taining PDA medium and stored in an incubator for 3e5 days todevelop spores. For long-term preservation of spores, glycerol wasadded to the tubes.

2.3. Preparation and numeration of spores

The PDA medium was incubated with spores for 3e5 days inorder to obtain fungalmasses. Some sporeswere then transferred toa medium containing physiologic serum and Tween 80 and vor-texed. The sporeswere counted using neobar lamella and a standardstock was prepared (150 spores/ml) and was stored at 4 �C.

2.4. Nanogel formulation and analysis

BAwas coupled to CS by the formation of amide linkages throughan EDC-mediated reaction following the method proposed by Chen,Lee, and Park (2003) and Yuta, Ikeda, Yamaguchi, Aoyama, andAkiyoshi (2003). Nanogels were prepared by adding 3.125 mol ofCS to 100 ml of acetic acid (10 ml/l). Then, a mixture of 1.562 mol ofparahydroxy-benzoic acid and 4.686 mol EDC in 10 ml ethanol wasslowly added to the CS solution drop-wised and stored in dark for24 h before use. The pH was adjusted to 8.5e9 by adding 0.1 NNaOH. The white nanogel sediments were washed by ethanol andcentrifuged three times (5 min, 9000 � g). Nanogel particles weredispersed in acetic acid (10 ml/l), followed by a filtration through a0.2 mm-pore size filter. To confirm the formation of nanogels, FourierTransformation Infrared (FTIR) spectrum at 20 �C and at the range of500e4000 cm�1was performed using a FTIR-430 (Jascow, Japan).

CS-BA nanogel morphology was analyzed using scanning elec-tron microscopy (SEM) on a Philips: XL30 (Netherlands, http://www.philips.com). One drop of freshly prepared nanogel suspen-sion was deposited on carbon stickers, dried with air and coatedwith gold. Morphological characteristics of the CS-BA nanogelswere also examined by high resolution Transmission Electron Mi-croscopy (TEM) on an H-600; Philips (Netherlands, http://www.philips.com). Specimens were prepared as described by Beikiet al. (2014). The size distribution of the nanoparticles waschecked by particle size analyzer.

2.5. Encapsulating of Thyme essential oils in CS-BA nanogel

The Thyme essential oils was dissolved in ethanol (1:1, v/v) andmixtures of nanogels (500 mg/l) and essential oils (5000 mg/l),were prepared by sonication (70 kHz) for 5 min. The encapsulationefficiency was measured by a Shimadzu spectrophotometer (Japan,http://www.shimadzu.com), based on the optical density spectra ofthe essential oils (400e650 nm). The spectroscopic readings wereperformed after precipitating the nanogel by centrifugation(10,000 � g, 15 min).

2.6. Determination of minimum inhibitory concentrations (MIC) ofCS-BA nanogel, free and CS-BA nanogel encapsulated Thymeessential oils

Sterile Erlenmeyer flasks containing 25 ml of PDB medium,150 spores and different concentrations of CS-BA nanogel, as well asfree and CS-BA nanogel-encapsulated Thyme essential oils were

S.T. Khalili et al. / LWT - Food Science and Technology 60 (2015) 502e508504

stored in an incubator for 1 month in order to determine the MIC.Moreover, the MIC of T. vulgaris essential oils and CS-BA nanogel-encapsulated T. vulgaris essential oils were studied under twodifferent conditions; the flasks were sealed during the experimentand otherwise.

Antifungal activity (AF) was measured based on the followingequation:

AF ¼ N � SCd (1)

Where N is the number of colonies and SCd is the score given basedon colonies diameter (Cd, mm) (SCd ¼ 1 if 0.5 < Cd � 1; SCd ¼ 2 if1 < Cd � 2; SCd ¼ 3 if 2 < Cd � 3; SCd ¼ 4 if 3 < Cd � 4; SCd ¼ 5 if4 < Cd � 5; SCd ¼ 6 if 5 < Cd � 6; SCd ¼ 7 if 6 < Cd � 7; SCd ¼ 8 if7 < Cd � 8 and, SCd ¼ 9 if 8 < Cd � 9).

Fig. 1. Schematic figure of the coupling reaction between ben

2.7. T. vulgaris essential oils in vivo analysis

In order to study the antifungal effects of the CS-BA nanogel-encapsulated T. vulgaris essential oils in vivo, the fruit were firstwashed with detergent, and were sprayed with 2 ml of spore-containing solution (150 spores/ml). Subsequently, the fruitswere sprayed with different concentrations of the CS-BA nano-gel-encapsulated T. vulgaris essential oils ranging between0 and 900 mg/l and the fruit were stored in dark for onemonth at ambient temperature. Then, the spores were collectedby rinsing the fruits with 25 ml sterilized distilled water fol-lowed by centrifugation at 3000 � g for 10 min. Finally, theculture media were inoculated by the collected spores. After 5days, the developed colonies were counted. The experimentwas conducted in triplicates with 6 fruits designated to eachreplicate.

zoic acid (BA) and chitosan (CS) with EDC as cross-linker.

Fig. 2. Fourier transform infrared spectroscopy (FTIR) analysis obtained for, A: Chitosan (CS); B: Benzoic acid (BA); and, C: CS-BA nanogel.

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Fig. 3. A: Scaning Electron Microscopy of the CS-BA nanogel, B: Transmission ElectronMicroscopy of the CS-BA nanogel and size distribution of the CS-BA nanogel.

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2.8. Statistical analysis

All the experiments were performed in triplicates. The meansand standard deviations (SD) were calculated using MicrosoftExcell software.

3. Results and discussion

As mentioned earlier, the pathogenic fungi are one of the mostimportant groups of agricultural pests and aflatoxin-producers areamong the most common. Aflatoxins are toxic secondary metabo-lites produced by some species of Aspergillus (Reddy et al., 2010). Inrecent years, considerable efforts have been made in takingadvantage of nanotechnology in developing newantifungal recipes.These recipes include the application of biodegradable polymericnanoparticles, micelles, solid lipid nanoparticles, nanoliposomes,inorganic nanoparticles, magnetic nanoparticles, dandrimetrs,frofluids and quantumdots (Kim et al., 2008, 2009). Application ofnanoparticles provides several advantages for extracts deliverysystems which all are based on size of these systems. These ad-vantages include providing a controlled delivery or release systemfor extracts, enhancement of extracts' effects, and improving thestability of extracts against evaporation (Oh, Siegwart, &Matyjaszewski, 2007; Ryu et al., 2010). Alginate and CS areamong most-widely used polysaccharides in synthesis of nano-particles. Moreover, as their production methods are based onaqueous solutions and thus the use of environmentally harmfulorganic solutions are avoided, therefore, these polymers are ofgreat interest (Pedro et al., 2009; Uzun, 2006; Zain, 2011).

It has been reported that CS not only decreases the growth ofpathogens, it also clearly causes morphologic and structuralchanges in bacteria's molecules. On the other hand, connection ofCS's amino groups to abutting groups such as fatty acids couldcreate derivatives which bend to dual bonds, and this in turn en-ables them to form nanomicelles in water and to encapsulate fatsand oils e.g. herbal essential oils. Among the other attractiveproperties of CS polymers are being chemically inert, easy and low-cost preparation from chitin (without using toxic compounds);being immunogenic and non-carcinogenic and their antibacterialand antifungal properties (Crini & Badot, 2008; Ghaouth, Arul,Grenier, & Asselin, 1992; Nishimura, 1997; Zivanovic & Chi, 2005).Zheng and coworkers in 2003 showed that CS had anti-bacterialeffects on bacteria such as Escherichia coli, Bacillus megaterium,Bacillus cereus and Enterobacter sakazakii and also anti-fungal ef-fects on various fungi through destruction of their cell membranes.

BAwas used in this study as linker for production of CS polymer(nanogel). This was due to the fact that BA is also considered safe incomparison with the other known linkers such as glutaraldehydeand glycol which are toxic and have lots of side effects such as eye,skin and respiratory tracts irritation, headache, vomiting, fatigueand asthma (Nair, 2001; Natella, Nardini, Felice,& Scaccini,1999). Ina study by Kim and colleagues conducted in 2010, antifungalproperties of BA against Aspergillus species were examined and itwas found that this compound had potent antifungal propertiesagainst these fungi (Kim et al., 2010).

Having considered the above-mentioned features of CS and BA,in the present study CS-BA nanogels were synthesized by self as-semblymethod that led to the formation of non-polar heads inside-ward and polar heads outside-ward (Fig 1B). Moreover, EDC wasused as the cross-linker, which formed an amide linkage betweenthe carboxyl group of BA and the amino group of CSwithout leavinga spacer molecule (Albrecht, Moeller, & Groll, 2011; Lee, Jo, Kwon,Kim, & Jeong, 1998). The BA and CS concentrations and ratioswere determined so that for each 100 amine groups of CS, 75carboxyl groups of BA were available. This ratio not only led to the

formation of homogeneous nanogels (monodisperse) with spher-ical morphology but also the extra available amine groups (not-bound) caused the produced nanogel to be hydrophilic (Fig 1A). Theobtained FTIR spectra of CS, BA and CS-BA nanogels confirmed thatsuccessful connections between the amine groups of the CS and thecarboxyl groups of the BA took place. The main area of CS-BAnanogel spectrum located in the range of 1400e1700 cm�1 re-veals the formation of the nanogels (Fig 2AeC). More specifically,the first peak which appeared at 1410.79 cm�1 is associated withthe symmetric stretch of the carboxyl or amino groups while thesecond peak at 1569 cm�1 represents the asymmetric stretch of thecarboxyl or amino groups. The stretch observed at about 1610 cm�1

confirms the formation of O]C-NHR linkage (Fig. 2AeC). The SEMand TEM micrographs obtained show the globular (spherical)structure of the self-assembled nanoparticles with the particle sizeof less than 100 nm (Fig. 3A and B) (Coates, 2000, pp.10,815e10,837). Microscopic images also revealed the mono-dispersity of the synthesized nanogels. Fig. 3B also reveals the sizedistribution of the synthesized nanogel particles. The size of thesynthesized nanoparticles achieved in this study were adequatelysmall that was ascribed to several factors; first, the initial soni-casion performed converted the long chains of CS to smaller piecesand prevented the formation of long-chained nanogels. Anotherfactor that could have played an important role in reducing parti-cles size was the re-sonicasion and the third factor was passing thenanogel through the filter.

Measuring the MIC of CS-BA nanogel alone against A. flavus wasperformed at the concentrations of 1000e2000 mg/l and the con-centration of 1500 mg/l was found to be capable of completelyinhibiting the fungal growth. On the other hand, Thyme essential

Fig. 4. (A,B,C,D): The Comparison of anti-fungal activity of different concentration of free and encapsulated Thyme essential in sealed and non-sealed condition during 4 weeks.(Sealed condition: ━, non-sealed condition: ┅, free oils: C, CS-BA encapsulated oils:⃞, CS-BA nanogel: :).

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oils were also tested during a 4-week trial under sealed conditionand its MIC was recorded at 400 mg/l (Table 1). To the contrary, theessential oils did not cause full fungal inhibition under non-sealedconditions even at concentrations as high as 1000mg/l (Fig. 4AeD).Previous studies also showed that only high concentrations ofThyme essential oils ranging from 1000 to 5000 mg/l led to com-plete inhibition against different funga species (Beygi, Barzegar,Hamidi, & Naghdibadi, 2007; Klaric, Kosalec, Mastelic, Pieckova,& Pepeljnak, 2007; Pina-Vaz et al., 2004).

In this study, sonication was applied for encapsulating theessential oils by the produced nanogel. In fact, sonication frag-mentized the structure of CS-BA nanogel, and based on the self-assembly method, and since the internal space of the nanogelwas hydrophobe, the oils was trapped in the CS-BA nanogel. Theencapsulation efficiency was measured at 87% based on the opticaldensity spectra of the essential oils. Based on the results obtained inthe present study, the CS-BA nanogel encapsulation significantlyimproved the overall anti-fungal properties of the Thyme essentialoils as the MIC values recorded were at 300 and 500 mg/l undersealed and non-sealed conditions, respectively (Fig. 4AeD)(Table 1). This findingwas in linewith those of several other studies

Fig. 5. In vivo investigation of CS-BA nanogel-encapsulated Thymus vulgaris essential oils acolonies was counted after 5 days of incubation under ambient conditions.

in which CS polymers were used for encapsulating essential oils inorder to enhance their antimicrobial properties (Gutierrez, Barry-Ryan, & Bourke, 2008; Zivanovic & Chi, 2005).

The results obtained under non-sealed conditions showed thatdifferent concentrations of free Thyme essential oils were able todelay the growth of the fungi within a short period of time but couldnot completely inhibit it. The volatility of oils and exposure to airand other environmental factors must have affected the biologicalactivity of the investigated essential oils. On the other hand, theapplication of encapsulating nanogels with the Thyme essential oilsled to a complete inhibition at 500 mg/l concentration. This provesthat the encapsulation had significant effects on the half-life of theessential oils and could protect it against the environmental con-ditions and mainly evaporation. Moreover, the concurrent use ofessential oils and nanogel could have resulted in controlled releaseof the investigated essential oils from nanogel during the experi-mental period which this in turn enhanced the antifungal effects ofthe oils. On the other hand, the results of the in vivo application ofthe CS-BA nanogel-encapsulated oils on tomato samples within onemonth were found promising as full preservation of the samples atconcentrations above 700 mg/l was achieved (Fig. 5).

t different concentrations (0e900 mg/l) using tomato fruit. The number of developed

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4. Conclusions

CS-BA was synthesized using self-assembly reaction of water-soluble CS and BA and was characterized by SEM, TEM and FT-IR.The superior performance of T. vulgaris essential oils when encap-sulated by CS-BA nanogel under both sealed and non-sealed con-ditions in comparison with free oils was revealed. The in vivoexperiment also showed that the encapsulated oils at 700 mg/lconcentrationwas capable of preserving the quality the fruit duringthe 1-month storage period. Having considered the remarkablesynergic effect achieved when the CS-BA nanogel was used incombination with the studied essential oils, further investigationson these CS-BA nanogels and their preserving impact on othercrops are also suggested.

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