Crayfish chitosan for microencapsulation of coriander (Coriandrum sativum L.) essential oil

International Journal of Biological Macromolecules 92 (2016) 125–133

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Crayfish chitosan for microencapsulation of coriander (Coriandrum sativum L.) essential oil Fatih Duman a,∗ , Murat Kaya b a b

Department of Biology, Faculty of Science, Erciyes University, 38039, Kayseri, Turkey Department of Biotechnology and Molecular Biology, Faculty of Science and Letters Aksaray University, 68100, Aksaray, Turkey

a r t i c l e

i n f o

Article history: Received 30 March 2016 Received in revised form 16 June 2016 Accepted 20 June 2016 Available online 21 June 2016 Keywords: Microcapsules Antioxidant Antimicrobial

a b s t r a c t In this study, chitosan, which was obtained from the waste shells of crayfish (Astacus leptodactylus), was used for the encapsulation of the essential oil isolated from coriander (Coriandrum sativum L.) via the spray drying method. The obtained capsules were characterized using SEM, FT-IR, TGA and XRD. The size of the microcapsules was between 400 nm − 7 ␮m. It was determined that the swelling characteristic of the capsules was pH sensitive. The release showed bi-phasic characteristics and the maximum degree was reached after 72 h. Antimicrobial activity studies showed that pure chitosan more effective than the capsule. The antioxidant activity was recorded concentration-dependent. In contrast the antimicrobial activity, antioxidant activity of the capsule was found much higher than the oil and the pure chitosan. Consequently, it was determined that this product could be used in the food and pharmaceutical industries as a natural antioxidant and antimicrobial agent. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Encapsulation is a packaging technology in which gas, liquid or solid materials are enclosed in miniature capsules, generally in the food industry. In this way, the release of the matter is under control. This technology is used for the controlled release of active ingredients to protect them from the surroundings during storage to maintain the flavor, to prevent the flavor being tasted in the mouth and to increase the biological availability of the ingredients [1]. Thanks to encapsulation, a greater effect can be obtained with a much lower amount of active ingredient [2]. Many natural or synthetic polymers have been used for the purpose of controlled release [3–5]. Chitosan as the derivative obtained from aminopolysaccharide chitin [6] has become a preferred product recently in biomedicine, pharmacy and cosmetics [7–9] because of some remarkable characters, such as nontoxicity, mucous adhesiveness, biodegradability and biocompatibility [10–12]. Coriander (Coriandrum sativum L.) is a cosmopolite widely cultured medicinal plant. Its seeds contain up to 1% essential oil [13]. It has been assessed to be one of the most important essential oil plants in the world [14]. In folk medicine it is used for the elimination of stomach problems, fevers and rheumatism [14]. The

∗ Corresponding author. E-mail address: [email protected] (F. Duman). http://dx.doi.org/10.1016/j.ijbiomac.2016.06.068 0141-8130/© 2016 Elsevier B.V. All rights reserved.

extracted oil of this plant is also used as a sweetener in liqueur, cocoa and chocolate industries. When the plant is green, its fresh parts are used as flavor for soups and foods, and it is widely used in the making of souse. In addition, the concentrated oil is a valuable ingredient that is used in the perfume industry. The oxidation of herbal oil acids in long term preservation and storage can cause the breakdown of the taste and the formation of toxins. Consequently, the protection of essential oils against harsh environmental circumstances is important in health and economic terms. In the literature, there are different methods that have been used for the encapsulation of essential oils. For example, Hosseini, Zandi, Rezaei and Farahmandghavi [15] encapsulated oregano (Origanum vulgare L.) with chitosan using a two-step emulsion-gelation method, and determined the physico-chemical characterization and in vitro release features. In a study conducted by Ribeiro, Ribeiro, Camurc¸a-Vasconcelos, Macedo, Santos, Paula, Araujo Filho, Magalhães and Bevilaqua [16] the essential oil of Eucalyptus staigeriana was emulsified with chitosan and the anthelminthic effects of the obtained product were determined. Recently, spray drying has been a widely used method for the encapsulation of herbal extracts [17], essential oils [11,18], phytochemicals [19] and phenolic compounds [2,20]. The features that make the spray drying technique superior to the others are a) simplicity, b) low price and c) adjustable microcapsule size [21]. Some factors such as deacetylation degree, surface morphology and molecular weight of the chitosan used for encapsulation affect

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the encapsulation efficiency and release features [22,23]. prasanth Koppolu, Smith, Ravindranathan, Jayanthi, Kumar and Zaharoff [23] used chitosan for the microencapsulation of protein and a vaccine; they determined that when the molecular weight increases, the release ratio decreases in an inversely proportional manner. In addition, the physicochemical features of chitosan obtained from different organisms are different. For example, Hajji, Younes, Ghorbel-Bellaaj, Hajji, Rinaudo, Nasri and Jellouli [24] examined three different marine species (␣-chitin from waste shells of crab and shrimp and ␤-chitin from cuttlefish bones) and determined that the physico-chemical characteristics of the three species were different from each other. Generally, commercial chitosan is preferred for encapsulation studies. However, in this study, chemically isolated pure chitosan obtained from Astacus leptodactylus was used for encapsulation. A. leptodactylus is distributed in eastern Europe and western Asia. This species lives in rivers, ponds and lakes [25]. As the essential oil of coriander has a broad usage area, it was selected for encapsulation in this study. According to the literature survey, we could not find any studies focusing on the encapsulation and release characteristics of coriander essential oil. This study was carried with three main aims: 1) to obtain chitosan from A. leptodactylus and use it to encapsulate the essential oil of coriander via spray drying, 2) determine the physico-chemical characteristics of the pure chitosan and encapsulated product and 3) determine the encapsulation efficiency, controlled release, antioxidant and antimicrobial features.

2. Materials and methods 2.1. Sample collection The crayfish, belonging to the species A. leptodactylus, that were used in this study were dead individuals obtained from purification grills at a water supply reservoir (Aksaray, Turkey), on 30 November 2013. Dead crayfish are collected daily to maintain the quality of the water in the reservoir. In this study, a natural waste product is converted into a useful product. 2.2. Chitin isolation and chitosan production The shells of the crayfish were dried at room temperature, and then ground in a mortar to a fine powder. Then 20 g of the powder were refluxed with 2 M HCl at 100 ◦ C for 3 h; and, the samples were washed with double distilled water up to neutral pH and minerals were removed from the shells by filtering. Later, the demineralized samples were refluxed with 2 M NaOH at 100 ◦ C for 18 h to remove the protein from the shells. Deproteinized samples were exposed to a mixture containing a 4:2:1 ratio of distilled water, methanol and chloroform at room temperature for 2 h. After that, samples were washed with distilled water to obtain a neutral pH. By this means, the oil and pigments were removed. Eventually, samples were dried in a drying oven at 50 ◦ C for 48 h and chitin was produced. Then 1.5 g dried chitin were refluxed with 60% NaOH at 100 ◦ C for 4 h, and then the samples were washed with distilled water to a neutral pH. Using this process, the deacetylation of the chitin was conducted and pure chitosan was obtained. Finally, wet chitosan samples were dried in a drying oven at 40 ◦ C for 48 h. 2.3. Extraction of essential oils from coriander The upper parts of coriander plants were sampled from a greenhouse that belongs to the Agriculture Faculty, Erciyes University in 2013. Collected samples were dried on a laboratory bench at room temperature (22–24 ◦ C) and then blended. 100 g of powdered plant

material were hydrodistilled within 1 l water in a Clevenger device for 4 h and essential oil was obtained. 2.4. Encapsulation process In our study, the microencapsulation of essential oil was conducted using the method and mix ratios of Sansone at al. (2014) [13]. 0.5% of acetic acid was used for dissolving chitosan. Chitosan and the essential oil were sprayed in the ratio 1:1 in a spray dryer (Büchi Mini Spray Dryer B-290). The spray drying conditions were air inlet temperature 120 ◦ C, outlet temperature 68–71 ◦ C, rate of feed 5 ml/min and air flow 600 l/h. Spray dried samples were gathered and stored under a vacuum at ambient temperature for 48 h. 2.5. Characterization of chitin, chitosan and encapsulated samples The chitin and chitosan samples obtained from crayfish and the prepared microcapsule samples were photographed using a scanning electron microscopy (QUANTA FEG 250). The samples were coated with gold using a Sputter Coater (Cressingto Auto 108). The ATR FT-IR analyses of chitin, chitosan and microcapsule samples were performed with a Perkin Elmer FT-IR spectrometer over the frequency range of 4000–625 cm−1 . The TG/DTG curves at the thermal degradation of chitin, chitosan and the encapsulated samples were analyzed at a heating rate of 10 ◦ C min−1 via an EXSTAR S11 7300. X-ray diffraction analyses of the chitin, chitosan and encapsulated samples were made using a Rigaku D max 2000 system at 40 kV, 30 mA and 2␪ with a scan angle from 5◦ to 45◦ at the heating rate of 10 ◦ C min−1 . Molecular weight of the crayfish chitosan was measured using an Ubbelohde Dilution Viscometer. Five different concentrations of the chitosan were prepared using the solvent system (0.1 M acetic acid + 0.2 M NaCl (1:1, v/v)) for determining the viscosity-average molecular weight. All the treatments were conducted at 25 ◦ C in triplicate and the mean value was determined. Mark-Houwink equation was followed for determination of molecular weight as given below; [] : kMv˛ (1) [␩]: intrinsic viscosity. Mv: viscosity average molecular weight. k and ␣: Mark-Houwink-Sakurada constants. The constants used were k = 1.81 × 10−3 and ␣ = 0.93. Flash 2000 Elemental analyzer was used for determining the percentage of C, N and H in the chitosan sample. Deacetylation degree (DA) of the chitosan was calculated using the formula: DA = 100−[(C/N − 5.14)/1.72] × 100(2) 2.6. Swelling capacity 0.2 g of microcapsules was weighted. To simulate gastric liquid, a pH 2.2 solution was prepared (HCl/KCl). To simulate the intestinal liquid, a pH 6.8 phosphate-buffered saline (PBS) solution was prepared. Microcapsules were added into the solutions, and were left for 48 h in the pH 6.8 solution and 2 h in the pH 2.2 solution. Swollen samples were put into a centrifuge tube together with some filter paper at the bottom centrifuged at 500 rpm for 3 min and weighed. To determine the swelling percentage (SP), the formula given below was used. SP(wt%) = [(w − w0 )/w0 ] × 100(1) In this formula, w: weight after immersion and w0 : beginning weight of microcapsule (dry weight).

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Fig. 1. Scanning electron microscopy pictures of a) chitin, b) chitosan and c, d, e, f) capsules (coriander essential oil encapsulated by chitosan).

2.7. Encapsulation efficiency

2.8. In vitro release studies

10 mg of dried microcapsules was weighted. Then, the microcapsules were digested in 5 ml 2 M HCl, and 2 ml methanol was added. They were left in boiling water for 30 min, and then centrifuged at 3000 rpm for 10 min. The supernatant’s absorption was read at 538 nm in a UV spectrophotometer. The main component of coriander essential oil (linalool, about 83%) taken into consideration to calculations. The released linalool concentration was calculated using the following equation, which was optimized for linalool by Indumathi, Durgadevi, Nithyavani and Gayathri [26]:

In vitro release studies were conducted at pH 6.8 (PBS). For this, 0.1 g dried microcapsules were weighted and put into a dialysis bag (cutoff molecular weight: 14 kDa). Then 10 ml PBS (pH 6.8) solution was added. The dialysis bag was immersed in a 200 ml medium solution in a beaker. The beaker was mixed consistently at 100 rpm at room temperature, and 5 ml of the medium was taken from the beaker at time intervals, with the removed volume being replaced with new medium. The concentration of the coriander oil released to the medium was calculated according to the formula given above at 538 nm in a UV–vis spectrophotometer. To calculate the cumulative release percentage (CRP), the following formula was used:

Y = 0.012x + 0.011(R 2 = 0.982)(2) In this formula, Y is the absorbance and x is the concentration in ppm. The encapsulation efficiency (EE) was calculated according to the formula below.

CRP =

t

Mt

t=0 M0

× 100

(4)

EE% = (Amount of essential oil in capsule/Initial amount of essential oil)× 100(3)

Where, M0 represents the essential oil amount at the beginning and Mt represents the essential oil amount at the current time. The analyses were repeated three times.

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Fig. 2. FT-IR spectrum of a) chitin, b) chitosan and c) capsule (coriander essential oil encapsulated by chitosan).

2.9. Antioxidant and antimicrobial activity To determine the antioxidant activity, the 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging method was used. The same procedure was used for pure chitosan, microcapsules and coriander oil. Briefly, different amounts (1, 2, 3, 4 and 5 mg/ml) of samples were suspended in 1 ml ethanol. Briefly, different amounts (1, 2, 3, 4 and 5 mg/ml) of microcapsules were suspended in 1 ml ethanol. After, centrifugation at 900 rpm for 10 min, 100 ␮l samples were taken from the supernatant and 3.9 ml DPPH was added. It was left in the dark for 24 h and absorbance was determined at 517 nm. In our study we used the microorganism strains Aeromonas hydrophila ATCC 7965 (Gram-negative), Escherichia coli ATCC 25922 (Gram-negative), Escherichia coli O157 (Gram-negative), Klebsiella pneumoniae FMC 5 (Gram-negative), Pseudomonas aeruginosa ATCC 27853 (Gram-negative), Salmonella typhimurium NRRLE 4463 (Gram-negative), Yersinia enterocolitica ATCC 1501 (Gramnegative), Bacillus cereus RSKK 863 (Gram-positive), Listeria monocytogenes 1/2 B (Gram-positive) and Candida albicans ATCC 1223 (yeast). The agar diffusion method was used for the determination of antimicrobial activities. Firstly, pure chitosan,

microcapsules and coriander oil were dissolved in Dimethyl sulfoxide (DMSO) (10 mg/ml) and diluted with distilled water to the intended concentration. Then the agar diffusion method was used to determine the antimicrobial activity of the chitosan encapsulated microcapsules for each microorganism given above. Each microorganism was suspended in sterile nutrient broth. Yeast (C. albicans) was suspended in malt extract broth and each microorganism was diluted to ca. 106 –107 colony forming units (cfu)/mL. Then 250 ␮l of each microorganism was added into a flask containing 25 ml sterile Mueller-Hinton agar or malt extract agar at 45 ◦ C and poured into Petri dishes (9 cm in diameter). The petri dishes containing agar were left at 4 ◦ C for 1 h. In the agar, four equidistant holes were made using sterile cork borers and loaded with 10 mg/ml (50 ␮l) of the dissolved microcapsule solution. Yeast (C. albicans) was incubated at 25 ◦ C for 24–48 h in the inverted position. The other microorganisms were incubated at 37 ◦ C for 18–24 h. At the end of the period, all plates were examined for zones of growth inhibition, and the diameters of these zones were measured in millimeters. Tetracycline (10 mg ml−1 ) and Natamycin (30 mg ml−1 ), standard antibiotics, were used as positive controls.

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Fig. 3. Thermogravimetric analysis (TGA) and derivative thermogravimetric (DTG) analysis of the capsule (coriander essential oil encapsulated by chitosan).

3. Results and discussion 3.1. Characterization SEM micrographs of the oil-encapsulated chitosan microspheres were taken at 5000 X, 10000 X, 20000 X and 40000 X magnifications, and are shown in Fig. 1. The diameters of the oil-encapsulated chitosan microspheres were in the range 400 nm to 7 ␮m. The big spheres were pumpkin shaped, whereas the small ones were oval shaped. There were pores with varying sizes between 20 and 70 nm on the surface of the microspheres. The pores were bigger on the surface of the big spheres than on the surface of the small spheres. As seen from Fig. 1, the capsules were separated from each other. The surface morphology of the chitosan consists of nanofibers and nanopores [27]. We identified fibers and pores on the surface of the chitin and chitosan obtained from crayfish in the present study (Fig. 1). After encapsulation, the nanofibers were lost and only pores were still present, but the size of the pores was observed to be smaller. These small pores could be very important for releasing the oils from the capsule. The FTIR bands of chitin, chitosan, the capsule and coriander essential oil are shown in Fig. 2. The recorded FT-IR bands at 1654 cm−1 , 1619 cm−1 and 1550 cm−1 show that the chitin extracted from the crayfish was in the ␣ form [28]. The observed bands appearing at 1659 cm−1 ␯ ( C O) in NHCOCH3 group (Amide I band) and 1590 cm−1 ␯ (NH2 ) in NHCOCH3 group (Amide II band) show that the chitosan was obtained after deacetylation of chitin in an alkali solution [29]. The OH and NH stretching bands at 3356 cm−1 in the chitosan were shifted to lower wavenumbers, which indicated changes in the chitosan structure following the encapsulation procedure. These changes can be attributed to the interactions of the OH and NH groups of chitosan with the C O of the essential oils [11]. Furthermore, symmetric and asymmet-

ric stretching of the amine group (1540 cm−1 ) was observed at a lower wavenumber (1403 cm−1 ), which confirmed the presence of hydrogen bonds. Some characteristics bands (–C H stretching: 2921 cm−1 , CH3 in NHCOCH3 : 1378 cm−1 , Amide III band: 1259 cm−1 , C O stretching: 1062 cm−1 , pyranose ring skeletal vibrations: 891 cm−1 ) for chitosan were also observed in the encapsulated formation. Also, chemical structure of coriander essential oil was analyzed via FT-IR. The observed major bands are as follows: 2920 and 2854 cm−1 (asymmetrical and symmetrical stretching vibration of methylene group), 1744 cm−1 (ester carbonyl functional group of the triglycerides), 1460 cm−1 (bending vibrations of the CH2 and CH3 aliphatic groups), 1377 cm−1 (bending vibrations of CH2 groups) and 1144 cm−1 (C O stretching). Upon the encapsulation, the peak (1744 cm−1 ) assigned to the ester carbonyl functional group of the triglycerides was observed as a shoulder in Fig. 2c. The bands corresponded to the asymmetrical and symmetrical stretching vibration of methylene group was disappeared after the encapsulation procedure (Fig. 2c). TGA analysis of the microcapsules showed four main decomposition steps from 30 ◦ C to 650 ◦ C (Fig. 3). The first mass loss (8.9%) occurred between 0 and 100 ◦ C and was due to water evaporation. The second mass loss (15.3%) recorded between 100 and 200 ◦ C was due to the decomposition of oil. The third mass loss (36.7%) observed between 200 and 350 ◦ C was due to the decomposition of chitosan molecules in the structure [29]. The last mass loss (13.8%) between 350 and 650 ◦ C (max = 364 ◦ C) was due to the chitin molecules that could not be deacetylated [30]. In total, 75.6% of the capsule was degraded and 24.4% was not degraded, and this un-degraded part could be the source of the ash content. The maximum degradation temperatures (DTGmax) for the steps 1, 2, 3 and 4 were 55.5 ◦ C, 115.9 ◦ C, 256.7 ◦ C and 364.3 ◦ C respectively (Fig. 3).

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Fig. 4. X-ray diffraction (XRD) of a) chitin, b) chitosan and c) capsule (coriander essential oil encapsulated by chitosan).

The XRD peaks of the chitin extracted from crayfish were 9.42, 12.98, 19.44, 20.4, 23.5 and 26.86◦ . In addition, peaks at 10.44 and 19.42◦ were observed for crayfish chitosan (Fig. 4). The peaks observed in the present study were the characteristic peaks for chitin and chitosan [24,30]. The crystalline index values of this crayfish chitin and chitosan were calculated as 87% and 69% respectively [31]. After XRD analysis of the encapsulated sample, a large peak from 20 to 22◦ was observed. However, a weak peak at 9.16◦ was newly appeared due to the crystalline structure (Fig. 4). The CrI value of the encapsulated sample was recorded as 49%. As can be seen, the CrI values decreased and the peaks were altered after the encapsulation of the oils. These changes indicated that the oil was successfully capsulated by the chitosan. Molecular weight of the chitosan was recorded as 3.119 kDa. The percentage of C, N and H in the chitosan was measured as 41.29, 7.31 and 6.75 respectively. DA value was calculated as 70.44 by using the elemental analysis results. 3.2. Swelling studies In this study, the swelling properties of the microcapsules that encapsulated the chitosan were determined for both simulated gastric liquid (pH 2.2) and intestinal liquid (pH 6.8). It was seen that the swelling properties of the microcapsules that encapsulated the chitosan were pH- sensitive. While the swelling degree was 560% in the gastric liquid, this ratio was 260% for the intestinal ´ Milaˇsinovic, ´ Djordjevic, ´ Kruˇsic, ´ Kneˇzevic-Jugovi ´ ´ liquid. Trifkovic, c, Nedovic´ and Bugarski [20] used chitosan to encapsulate the polyphenols of Thymus serphllum and they determined that their obtained microcapsules underwent greater swelling under acidic ´ Milaˇsinovic, ´ than basic conditions. Our results agree with Trifkovic,

´ Kruˇsic, ´ Kneˇzevic-Jugovi ´ ´ Nedovic´ and Bugarski [20]. Djordjevic, c, The increased swelling ratio under acidic conditions may be due to the protonation of the amino groups that exist on the chitosan. Polymer chains will have a positive change, so a pushing effect will occur and consequently swelling will be realized. It was seen that the microcapsules dropped to the bottom when they were exposed to pH 6.8, and they were stable at the end of the experiments. This can be explained because the protonated carboxyl groups were dominant at the higher pH. In this situation, an osmotic pressure increase together with ion mobility and swelling occurred. This characteristic shows that the obtained microcapsule can be used as a drug carrier. 3.3. Encapsulation efficiency and in vitro release In this study, the encapsulation efficiency of coriander with chitosan was determined as 28.4%. In a similar study conducted by Hosseini, Zandi, Rezaei and Farahmandghavi [15], oregano essential oil was encapsulated with chitosan, and it was determined that the efficiency of encapsulation changed according to the essential oil-chitosan ratio. Further, the authors found the encapsulation efficiency was between 5.45% and 24.72%. In our study, we obtained a similar rate. Another factor affecting the encapsulation efficiency is the molecular weight of the chitosan used. The use of chitosan with a lower molecular weight increases the fecundity. Li, Zhuang, Wang, Sun, Nie and Pan [32] determined that using low molecular weight chitosan for encapsulation of liposome increased the coating efficiency and product stability. There are a lot of mechanisms that involve the release of essential oil after encapsulation with different kinds of materials, such as surface erosion, diffusion and desorption [33]. It can be seen form

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131

Fig. 5. In vitro release profile of coriander essential oil from capsule at room temperature in PBS at pH 6.8. The data are expressed as the mean ± SD (n = 3).

Fig. 5 that the essential oil release from the microcapsule reached the maximum after 72 h. It can be determined that the essential oil release from chitosan is bi-phasic. While the release rate was high for the first 24 h, the subsequent release was slowed, and 56.7% of the total release occurred in the first phase (Fig. 5). The main reason for the rapid release in the first stage can be explained by the essential oil that was attached to the surface of the microcapsule dispersing into the environment. In the second phase, the essential oil that was in the chitosan matrix was dispersed into the environment. However, this stage was fairly slow and consequently it reaches a plateau. As a result, the chitosan from crayfish can be used for the controlled release of essential oil. When the results obtained from this study were compared with earlier studies [15,32], it could be seen that there were differences in terms of release rate. The reasons for these differences could be a) physicochemical dissimilarity in terms of chitosan used, b) surface morphology discrepancy and c) differences in terms of nanoporous structure. Consequently, not only the molecular weight of chitosan used but also specific characters, such as surface morphology and structure (being fibrillar or porous, etc.) should be taken into account in release studies.

3.4. Antioxidant and antimicrobial activity The antioxidant properties of herbal essential oils may be associated with the presence of OH groups in their structures [34]. These OH groups take in charge as a hydrogen donor, and they offer protection from the free radical that occurs. Deepa and Anuradha [35] revealed the antioxidant and free radical scavenging activity of the ˇ coriander plant. Samojlik, Lakic, Mimica-Dukic´ı, Ðakovic´ı-Svajcer and Boˇzin [36] determined that the main component of coriander essential oil was linalool at 82.9%, which is a very high ratio. In this study, we found 54.7% antioxidant activity for 5 mg/l coriander essential oil [13]. Similarly, in a related study conducted by, it was determined coriander essential oils were able to reduce the stable DPPH in a dose-dependent manner reaching 50% neutralization for 4.05 ␮l/ml for C. sativum. However, antioxidant activity of crayfish chitosan was found as 13.5% for 5 mg/ml. Kaya, Baran, Erdo˘gan, Mentes¸, Özüsa˘glam and C¸akmak [37] acquired chitosan from the Colorado potato beetle (Leptinotarsa decemlineata) and determined

the antioxidant activity of this newly synthetized chitosan as “moderate” (33.05% for 5 mg/ml). We found lower antioxidant activity for crayfish chitosan than Colorado potato beetle’s. It can be concluded that antioxidant scavenging activity of chitosan samples obtained from different species may be different each other. Difference may be originated from different factors such as deacetylation degree, molecular weight and so on [11]. As a result, antioxidant activity of the microcapsules was observed much higher than the oil and the pure chitosan. Encapsulation improved the antioxidant activity. As stated in the in vitro release study, in the first stage the linalool molecules located on the microcapsules should be released to the medium and consequently the antioxidant effect revealed. It is known that chitosan has an antioxidant activity on its own, and it has a high hydrogen delivery ability [38]. [37] acquired chitosan from the Colorado potato beetle and determined the antioxidant activity of this newly synthetized chitosan as “moderate”. In this study, we found that a microcapsule concentration of 5 mg/ml constitutes 49.8% antioxidant activity. In addition, the antioxidant effect of the microcapsule was concentration-dependent. For the same concentration, the activity of BHT (Buthylated hydroxytoluene), which is a synthetic antioxidant, was found to be 81.7% (Fig. 6). Having lower activity of microcapsules than BHT shows that there is a considerable competitive effect. Reducing of antimicrobial activity may be related to solubility or dispersion ability of microcapsules. Synthetic antioxidants such as BHT or BHA (Buthylated hydroxyanisole) are used in food production processes. However, due to negative effects on health, the use of this kind of synthetic antioxidant has been restricted [39]. To prevent this situation, the development of natural antioxidants is inevitable. It can be seen that the obtained product in this work could be used in the food industry as a natural antioxidant. The antimicrobial activity of coriander oil, crayfish chitosan and obtained microcapsule were tested on the organisms shown in Table 1. For the antimicrobial studies we deliberately selected food pathogens as the target microorganisms. When the antimicrobial activity results were compared with standard antibiotics, it was seen that the extract of our microcapsules was effective on both Gram (+) and Gram (−) bacteria. Although, earlier studies conducted by different researchers showed that coriander has

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Fig. 6. Antioxidant activity of capsules with different concentrations (mean ± SD; n = 3). Table 1 Antimicrobial activities of microcapsule, coriandrum oil and crayfish chitosan (inhibition zones, mm). Test microorganism

Standart antibiotics Coriandrum oil

Crayfish chitosan

Microcapsule

Tetracycline (10 mg ml−1 )

Gram (−) A. hydrophila E. coli E. coli O157 K. pneumoniae P. aeruginosa S. typhimurium Y. enterocolitica

– – – – – – –

34.0 32.0 42.0 36.0 41.0 22.0 20.0

9.0 9.0 9.0 – – 9.0 8.0

19.0 21.0 21.0 21.0 18.0 16.0 20.0

Gram (+) B. cereus L. monocytogenes

– –

38.0 38.0

8.0 –

26.0 20.0

Yeast C. albicans

–

–

–

Natamycin (30 mg ml−1 )

24.0

Inhibition zones include diameter of hole (6 mm), – ineffective.

important anti-bacterial effects [28,40,41], we could not observe an antibacterial effect for coriander oil. However, we observed important antibacterial activity on all studied bacteria strains for crayfish chitosan. Antimicrobial activity of chitosan encapsulated coriander oil was lower than pure crayfish chitosan. This result can be explained that surface of microcapsules are covered by oil which has no antibacterial effect and this situation surpass the effect of chitosan. From this result, it can be concluded that using of pure cray fish chitosan for antimicrobial purposes is more proper. Chitosan is a very important biopolymer for encapsulation of plant extracts. In recent years chitosan was used in many studies for encapsulation of materials such as polyphenols from strawberry extract [42], plasmid DNA and oil from Nigella sativa [43], Vitamin E and C [44] and ferulic acid [45]. Also in another study [46], C. sativum essential oil was encapsulated in chitosan/alginate/inulin microcapsules by using spray drying method and the size of microcapsules was found not uniform like the present study.

characterized by FTIR, XRD, TGA and SEM analyses. pH affected the swelling properties and the best swelling was observed at pH 2.2. The pores on the surface of the capsules provided an effective coating with suitable release properties. In this study, a natural waste product was converted into a useful product. When the antioxidant and antimicrobial activities of the capsules are considered, these newly produced capsules could find wider applications in some areas, such as the food, drug and medical industries. Conflict of interest statement The authors declare ‘no conflict of interest’ present in this work. Acknowledgments The authors are grateful to Dr. Mustafa C¸am for his encouragement for the encapsulation and Dr. Sevil Albayrak for providing microorganism strains.

4. Conclusion Chitosan obtained from the waste shells of crayfish was used for the encapsulation of essential oils isolated from coriander using a spray drying method. Obtaining chitosan from waste would reduce the encapsulation cost substantially. The produced capsules were

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