- Email: [email protected]
Encapsulation of rosemary essential oil
Accepted Manuscript Encapsulation of rosemary essential oil Hazal Turasan, Serpil Sahin, Gulum Sumnu PII:
S0023-6438(15)00399-0
DOI:
10.1016/j.lwt.2015.05.036
Reference:
YFSTL 4692
To appear in:
LWT - Food Science and Technology
Received Date: 8 January 2015 Revised Date:
19 May 2015
Accepted Date: 24 May 2015
Please cite this article as: Turasan, H., Sahin, S., Sumnu, G., Encapsulation of rosemary essential oil, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.05.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Encapsulation of rosemary essential oil
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Hazal Turasan, Serpil Sahin*, Gulum Sumnu
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Department of Food Engineering, Middle East Technical University, 06800, Ankara,
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Turkey
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*Corresponding author
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Tel:+90 312 210 5627; E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Encapsulation protects sensitive food ingredients against oxygen, heat, moisture, pH
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and it masks the unwanted taste of nutrients. The objective of the study was to
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encapsulate the rosemary essential oil in micron size and to find the optimum coating
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material formulation by investigating the physicochemical properties and storage
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stability of microcapsules. In the capsule preparation two different ratios of whey
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protein concentrate (WP) and maltodextrin (MD) (1:3 and 3:1), three different core to
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coating ratios (1:40, 1:20 and 1:10) and two different dextrose equivalent (DE) MD
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(DE:13-17 and DE:4-7) were used. Emulsions were analyzed for their particle size
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distributions and freeze dried capsules were analyzed for their drying efficiencies,
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encapsulation efficiencies, surface morphologies, and concentrations of 1,8-cineole
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during storage. Increasing WP:MD ratio was found to increase both drying and
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encapsulation efficiencies. Also, capsules having core to coating ratio of 1:20 and MD
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with DE:13-17 gave the highest drying and encapsulation efficiency values. Changing
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DE value of MD did not have any significant effect on particle size distributions and
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surface morphologies of the capsules. Lastly, encapsulation was found to be an effective
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method for increasing the storage stability of 1,8-cineole.
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Keywords
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Microencapsulation, rosemary essential oil, whey protein concentrate, maltodextrin,
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dextrose equivalence
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Chemical compounds studied in this article: 1,8- cineole (another name: 1,3,3-
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Trimethyl-2-oxabicyclo [2.2.2]octane) (PubChem CID: 2758); maltodextrin (PubChem
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CID: 62698); Magnesium chloride (MgCl2) (PubChem CID: 5360315); dipotassium
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phosphate (K2HPO4) (PubChem CID: 24450); potassium dihydrogen phosphate
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(KH2PO4) (PubChem CID: 516951); n-hexane (PubChem CID: 8058).
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ACCEPTED MANUSCRIPT 1. Introduction
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During the last decades, the demands of the consumers from the food production
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industry have remarkably increased. People no longer see the food to appease the
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hunger but as a source to get the required nutrients which are supposed to help with the
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nutrition-related diseases and contribute to both physical and mental well-being of
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individuals (Bigliardi & Galati, 2013). This trend forced the food researchers and the
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producers to concentrate more on the production of foods that meets the requirements of
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humans for healthier lives (Bigliardi & Galati, 2013). Novel technologies are adopted
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especially to overcome the problem of active compound deterioration. These relatively
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more advanced techniques are based on the idea of coating the desired active compound
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(Bigliardi & Galati, 2013). Some of the entrapment techniques are microencapsulation,
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coating with edible films or vacuum impregnation.
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Rosemary (Rosmarinus officinalis L.) is a long-lasting evergreen aromatic herb
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(Bousbia, et al., 2009). The usage of rosemary oil dates back to 1500s (Guenther, 1948).
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Before refrigeration was invented rosemary oil was used for food preservation purposes
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as well as medical antiseptic, and astringent purposes (Bousbia, et al., 2009). Through
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the years, the utilization area of rosemary oil has not been changed much. Rosemary
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essential oil includes phenolic constituents in its composition (Başer & Buchbauer,
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2010). Due to this composition, which mainly involves monoterpenes like 1,8-cineole,
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α-pinene, camphor, camphene, rosemary essential oil has many therapeutical effects
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(Katerinopoulos et al., 2005; Başer & Buchbauer, 2010). Among these effects, the most
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well-known ones are its antioxidant effects (Valgimigli, 2012; Estévez et al., 2007),
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antimicrobial effects (Issabeagloo et al., 2012), pediculicidal, aromatherapeutical and
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anticarcinogenic activities (Başer & Buchbauer, 2010). However, due to its high
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volatility and its susceptibility against environmental effects, rosemary essential oil
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ACCEPTED MANUSCRIPT needs further protective actions, especially to increase its uptake and bioavailability.
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Encapsulation is one of the most efficient techniques to preserve it. It is a process in
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which, small solid particles, liquid components or gaseous materials are coated by or
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entrapped within another inert shell material, which isolates and protects the core
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material from environmental factors (Zhu et al., 2012; Kuang, Oliveira, & Crean, 2010;
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Ghosh, 2006; Desai & Park, 2005). Also this technique helps to mask the unwanted
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taste and the odor of the ingredient, to prevent the evaporation of the volatile
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components and to prevent the contact of the ingredient with oxygen to prevent
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oxidation. However, there are some important factors influencing microencapsulation.
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To achieve successful microencapsulation process, the choice of coating materials, the
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homogenization techniques and the drying techniques must be chosen carefully. In the
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literature, although there are many studies on the effects of rosemary essence on health
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problems and about the chemical composition of it, there is only a limited number of
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researches on microencapsulation of rosemary essential oil. In these studies, the main
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drying technique used is spray drying, the emulsions are generally prepared by using
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high-speed blenders and usually gum Arabic is used as the coating material (Fernandes,
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et al., 2013; Janiszewska & Witrowa-Rajchert, 2009; Teodoro et al., 2014). The main
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objective of this study was to develop a new and different technique for
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microencapsulation of rosemary essential oil. This is achieved by employing freeze
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drying as the encapsulation technique. Maltodextrin is chosen as the coating material
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since it has excellent oxygen-blocking property; however its lack of emulsification
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characteristic creates a need for an additional coating material (Runge, 2004; Sheu &
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Rosenberg, 1998). Preliminary studies showed that gums cannot be used as additional
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coating materials since their solutions reach high viscosities even in low concentrations.
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Whey protein concentrate is chosen as the second coating material since they show low
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ACCEPTED MANUSCRIPT viscosities even in high concentrations and they are excellent emulsifiers (Runge, 2004;
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Vardhanabhuti & Foegeding, 1999). Investigating the optimum coating material
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formulation for obtaining the highest drying and encapsulation efficiencies were other
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aims of this study. To even further increase the efficiency results, ultrasonication is
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added to the homogenization step since its effects on reducing particle sizes is proven
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by others (Jafari, He, & Bhandari, 2007a). The effects of different coating formulations
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on particle size and surface morphology of the capsules were analyzed as well as the
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storage stabilities of the encapsulated products.
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2. Experimental
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2.1 Materials
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Rosemary (Rosemarinus Officinalis L.) essential oil, maltodextrin (MD) in two different
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dextrose equivalences (DE) (DE: 4.0-7.0 and DE: 13.0-17.0), magnesium chloride
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(MgCl2),
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(KH2PO4), n-hexane and 1,8-cineole were purchased from Sigma Aldrich Chemical Co.
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(St. Louis, MO, USA). Whey protein concentrate (WP) containing 80 g protein/100 g
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solid, was supplied by Tunçkaya Kimyevi Maddeler (Tuzla, İstanbul). The Patcote
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502K anti-foaming agent, which was used in the drying efficiency analyses, was
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supplied from Hydrite Chemical Company (Brookfield, WI, U.S.A.).
phosphate (K2HPO4),
potassium
dihydrogen
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2.2
Preparation of Microcapsules
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2.2.1 Preparation of Coating Materials
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The maltodextrin (MD) solutions were prepared in distilled water and in two different
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concentrations (10g/100g solution and 30g/100g solution) for two different dextrose
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equivalences (DE: 4.0-7.0 and DE: 13.0-17.0). The solutions were pre-mixed for 10 min
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ACCEPTED MANUSCRIPT by a magnetic stirrer (Heidolph MR 3001 K, Heidolph Instruments GmbH & Co,
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Schwabach, Germany) and then left in a shaking water bath (GFL 1086, Burgwedel,
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Germany) at 25⁰C at 90 rpm for one night (18 h) to obtain full hydration.
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Whey protein concentrate (WP) solutions were prepared in phosphate buffer solution
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and in two different concentrations (10g/100g solution and 30g/100g solution). The
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preparation procedure of the phosphate buffer is adapted from Kuhlmann (2006). WP
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solutions were also prepared by using the magnetic stirrer and were left in the shaking
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water bath for one night (18 h) at 25⁰C to get full hydration.
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Prior to the addition of core material, the coating material solutions were weighed and
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mixed. From both whey protein solution and maltodextrin solution, 60 g were weighed,
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which makes a 120 g of total coating material mixture. The mixture of coating materials
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were prepared in the following ratios by choosing the suitable concentrations of WP and
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MD solutions: a) WP: MD (DE: 4.0-7.0) = 1:3 b) WP: MD (DE: 4.0-7.0) = 3:1 c) WP:
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MD (DE: 13.0-17.0) = 1:3 and d) WP: MD (DE: 13.0-17.0) = 3:1. All the mixtures were
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prepared in mass ratios. The total soluble solid contents of the mixtures were kept at
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20g/100g solution.
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Rosemary essential oil was added in three different core-to-coating ratios: 1:40, 1:20
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and 1:10. First, pre emulsions were homogenized in high-speed homogenizer (IKA T25
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digital Ultra-Turrax, Selangor, Malaysia) at 8000 rpm for 5 min and then were
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homogenized by using Ultrasonic Homogenizer (Sonic Ruptor 400, OMNI International
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the Homogenizer Company, Georgia, USA). Ultrasonic homogenizer was equipped
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with solid titanium 1" Solid and Tapped tip with a diameter of 25.4 mm and a length of
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12.70 cm. The ultrasonication process was performed for 15 min at 40% power of 20
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kHz using 50% pulse. To prevent excessive heating during homogenization, the beakers
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were placed in 4⁰C water baths. The emulsions were frozen at -18⁰C immediately after
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the homogenization procedure to prevent any coalescence or flocculation.
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Fully frozen emulsions were dried in a freeze drier (Christ, Alpha 2-4 LD plus,
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Germany) for 48 h at -50⁰C and at 1.9 Pa. After lyophilization, dried samples were
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grinded into powder form with a glass rod. For SEM analysis, the capsules were further
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grinded with coffee grinder for 15 s.
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Storage of the Microcapsules
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The storage stability of the microencapsulated rosemary oil was investigated at 35.3 %
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± 0.1 % relative humidity and at 15⁰C. To obtain this relative humidity, saturated
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aqueous solution of magnesium chloride (MgCl2) was prepared and placed into the
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desiccators. Before the placement of the samples, the salt solution was kept overnight in
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the desiccators to reach equilibrium. In order to see the difference, the encapsulated and
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non-encapsulated (fresh) rosemary oil samples were stored. Five grams of specimen
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from each sample were taken at certain time intervals during 40 days of storage. The
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samples were then analyzed for their 1,8-cineole concentrations by GC-MS. As the
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reference compound, 1,8-cineole was chosen since it is one of the major constituents of
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rosemary essential oil (Surburg & Panten, 2006).
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2.4
Analysis of Emulsions and Microcapsules
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2.4.1
Particle Size Analysis of Emulsions
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ACCEPTED MANUSCRIPT Particle size distributions of emulsions were analyzed with Mastersizer 2000 (Malvern
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Instruments Limited, Worcestershire, UK). The mean particle size of the emulsions was
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represented with Sauter mean diameter, D32 (µm), and was calculated with the
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following equation (McClements, 2005);
∑n d D32 = ∑n d
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i
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(1)
Span, was calculated with the following formula (Karimi & Mohammadifar, 2014);
Span =
[d (0.9) − d (0.1)]
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d (0.5)
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(2)
where, d(0.9), d(0.1), and d(0.5) are the diameters at 90%, 10%, and 50% of cumulative
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volume, respectively. During particle size analysis, the sonication was applied to the
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emulsions. However, during the analyses of emulsions, which were prepared in order to
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see the effect of ultrasonication only, the sonication of Mastersizer 2000 (Malvern
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Instruments Limited, Worcestershire, UK) was switched off to have an accurate result.
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2.4.2
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To determine the efficiency of the encapsulation process, two types of efficiency
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analyses were adopted: drying efficiency and encapsulation efficiency.
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Efficiency Analysis of Microcapsules
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2.4.2.1
Drying Efficiency
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For the drying efficiency analysis, a hydrodistillation technique with Clevenger
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apparatus was chosen to measure the oil retention. First, 10 g of microcapsules was
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dissolved in 250 ml of distilled water in a 500 ml flask. In order to prevent foaming, one
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droplet of Patcote 502K anti-foaming agent (Hydrite Chemical Company, WI, U.S.A.)
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was added by a syringe and mixed with the solution. After 3 h of distillation in
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Clevenger apparatus, the volume of the total oil was read from the volumetric arm and
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multiplied with the density of rosemary essential oil (0.908 g/ml) to estimate the actual
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oil content in the capsules. The drying efficiency was then calculated according to the
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following equation (Jafari, He, & Bhandari, 2007b):
Drying Efficiency (%) =
[Oil content of microcapsules] × 100 [Oil content of emulsions]
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2.4.2.2
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To measure the surface oil content, an analysis with Soxhlet apparatus was performed
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(Baranauskiené et al., 2007). Five grams of dried powder was weighed and trapped in
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filtration paper and washed for 3 h with 250 ml of n-hexane in the Soxhlet apparatus.
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The excess hexane was then evaporated in a vacuum rotary evaporator (Heidolph
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Laborota 4000 efficient; Heidolph Instruments GmbH & Co, Schwabach, Germany) at
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33.5 kPa and at 40⁰C. The surface oil was then concentrated under nitrogen dryer
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(Turbovap LV Concentration Evaporator Workstation; Biotage, Charlotte, NC, USA).
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Total oil content was determined by Clevenger apparatus. Then, the encapsulation
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efficiency was calculated using the following formula (Jafari, He, & Bhandari, 2007b);
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Encapsulation Efficiency (%) =
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Encapsulation Efficiency
(3)
[Total oil content − Surface oil content ] × 100 [Total oil content ]
(4)
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2.4.3
Surface Morphology Analysis of Microcapsules
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To analyze the structures and the surface morphologies of microcapsules of rosemary
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essential oil, scanning electron microscope was used. Samples were grinded for 15 s
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with coffee grinder (ARZUM AR151 Mulino Coffee Grinder, Turkey) to eliminate the
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structural differences. Then, they were coated with the mixture of gold/palladium by
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ACCEPTED MANUSCRIPT HUMMLE VII Sputter Coating Device (ANATECH, Union city, CA, USA). The
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scanning electron microscopy (SEM) (JSM-6400 Electron Microscope, Jeol Ltd.,
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Tokyo, Japan) was equipped with NORAN System 6 X-ray Microanalysis System and
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Semafore Digitizer. The images of the microcapsules were taken at two different
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magnifications; 500× and 5000×.
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GC-MS Analysis
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The stored microcapsules and fresh rosemary oil were analyzed for their 1, 8-cineole
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content. The oil was extracted from the samples were analyzed with Gas
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chromatography/Mass spectrometry (GC-MS) system (Agilent Technologies 6890N
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Network GC System coupled to Agilent Technologies 5973 Network Mass Selective
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Detector, USA). Quantitative analyses could be done using FID with a capillary column
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(Agilent 19091s-433 HP-5MS with a 5 g phenyl/95 g methylpolysiloxane stationary
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phase and with a size of 30m x 0.25mm x 0.25µm). An Agilent Tecnologies 7683B
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Series Injector (Thailand) was used to inject the sample. The data were analyzed by
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MSD ChemStation software program and helium was used as the carrier gas.
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The following GC-MS conditions were used during the analyses: split ratio 16.5:1;
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injection volume 1 µL; oven temperature program, holding at 40°C for 1 min, rising to
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180°C with 4°C/min with a total run time 39 min; MSD transfer line temperature,
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230°C; MSD quadrupole temperature, 150°C. Solvent delay was for 4.0 min. The GC
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analysis was performed with the following conditions: H2 flow rate, 35 mL/min; air
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flow rate, 400 mL/min; make-up flow rate of 48.8 mL/ min with a make-up gas type,
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He; FID temperature, 275°C.
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Prior to injection, the samples were diluted with n-hexane at a ratio of 1:100 (µl:µl).
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The calibration curve for 1,8-cineole was prepared with five different concentrations in
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ml/ml (1/50, 1/100, 1/200, 1/400, 1/800 ml/ml) with n-hexane. Correction of variation
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values (R2) of all calibration curves were obtained as greater than 0.99.
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Statistical Analysis
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To understand if there was a significant difference between the samples, the results were
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analyzed by analysis of variance (ANOVA) (p≤ 0.05). When a significant difference
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was found between the samples, Duncan's Multiple Comparison Test was applied (p≤
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0.05) by using SAS software version 9.1 (SAS Institute Inc., NC, USA).
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Results and discussion
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3.1
Drying Efficiency
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Fig. 1 represents the drying efficiency results of microcapsules. The results show that,
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there was no significant difference (p>0.05) between the drying efficiencies of
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microcapsules prepared with core to coating ratios of 1:40 and 1:20, for both MD with
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DE value of 13-17 and 4-7. However, the drying efficiency was significantly lower
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when core to coating ratio was 1:10 (p≤0.05). This difference could be explained by the
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oil load of the emulsions. As the amount of oil used in the preparation of the emulsions
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increased, the solid content in the mixture became insufficient to cover and entrap the
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excessive amount of oil. Therefore, more non-entrapped volatile oil was lost under the
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vacuum of freeze drying. According to the results, the formulation with the MD with
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DE:13-17 gave higher drying efficiency than MD with DE:4-7, which becomes more
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obvious as the core to coating ratios increase to 1:10. This indicated that as DE value
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increased, the retention of the volatiles in the wall matrix increased. These results show
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accordance with the findings of Shah et al., (2012) and Sheu & Rosenberg (1998).
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According to Sheu & Rosenberg (1998), as DE value increases, the proportion of low
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ACCEPTED MANUSCRIPT molecular weight carbohydrates increases and provides less disrupted capsules during
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drying which results in higher drying efficiencies. Also as particles get smaller, the
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drying rates increases, which enhances the solidification rate and also retention of
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volatiles.
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Fig. 1 also represents the effects of different WP:MD and core to coating ratios on
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drying efficiency values. For samples having MD with different DE vaues and for all
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core to coating ratios drying efficiency decreased noticeably as WP:MD ratio changed
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from 3:1 to 1:3. As Sheu & Rosenberg (1998) explained, the positive effect of
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increasing concentration of whey proteins on drying efficiency is due to their
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contribution to surface morphology of the capsules. Since whey proteins have positive
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effects on both drying rate and mechanical properties of the wall matrix, they decrease
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the number of surface cracks on the capsules, which results in increased drying
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efficiency.
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3.2
Encapsulation Efficiency
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The effect of different WP:MD ratios on the encapsulation efficiency values of the
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microcapsules are illustrated in Fig. 2. For MD with DE:13-17, the increase in WP
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concentration increased encapsulation efficiency values significantly for all core to
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coating ratios (p≤0.05). The capsules prepared by MD with DE:4-7 also showed similar
290
results (p≤0.05) (data not shown). One of the reasons of this trend is the good
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emulsification properties of whey proteins (Jafari, et al., 2008). In addition,
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maltodextrins lack surface-active properties and this makes them poor wall materials
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especially when they are used for encapsulation of volatile core materials (Sheu &
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Rosenberg, 1998). Also, whey proteins are effective on encapsulation efficiency since
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the unfolding and adsorption on the oil-water interfaces change protein structures and
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ACCEPTED MANUSCRIPT causes the formation of a resistant and stable layer over the oil droplets with decrease in
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WP (Jafari, et al., 2008). Thus, the increase in whey protein concentration results in
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higher encapsulation efficiencies. This can also be explained by the reduction in
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viscosity of the wall material solutions (Jafari, et al., 2008). Although the solid content
300
ratio remained the same when WP:MD ratio was changed from 3:1 to 1:3, the amount
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of larger particles (whey proteins) decreased which caused a decrease in the viscosity of
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the coating solution. Thus, as the viscosity of the wall material decreased, the
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encapsulation efficiency values also decreased.
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Fig. 3 represents the results of encapsulation efficiency values for two types of MD and
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three different core to coating ratios. When only the difference between MD types is
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considered, it is seen that the encapsulation efficiencies of the capsules prepared with
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MD having different DE values were significantly different from each other (p≤0.05). It
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was seen that for all core to coating ratios, the formulation with MD having DE:13-17
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gave higher encapsulation efficiency results than formulation with DE:4-7. The results
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indicated that MD with higher DE value helped to entrap more oil inside of the capsule
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and left less of the total oil on the surface. The positive effect of increasing DE value is
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due to the decreasing permeability of capsule to oxygen (Jafari et al., 2008).
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Another parameter that Fig. 3 represents about the encapsulation efficiencies of
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microcapsules is the core to coating ratios of the emulsions. For both DE values of MD,
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encapsulation efficiency values of capsules prepared with core to coating ratio of 1:40
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and 1:20 were not significantly different (p>0.05). In general, it can be seen that the
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encapsulation efficiencies decreased with the increasing oil content. The reason of this
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inverse relation between the oil amount and encapsulation efficiencies was the
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insufficiency of the solid materials to produce a strong structural layer around the oil
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droplets and cover them completely when the oil amount is increased. Consequently,
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ACCEPTED MANUSCRIPT this leads to lower encapsulation efficiency. It is seen that, microcapules having
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WP:MD ratio of 1:3 have much lower drying and encapsulation results in general.
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Therefore they are excluded from further analyses, since they are clearly not good
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candidates to have optimum formulation.
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Particle Size
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The particle size distributions of emulsions prepared with WP and MD at a ratio of 3:1
328
as coating material were analyzed only, since coating with WP and MD at a ratio of 1:3
329
resulted in lower drying and encapsulation efficiency values. Table 1 shows the particle
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size distributions, span values and the specific surface area (SSA) values of the
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emulsions prepared with MD having different DE values, different core to coating ratios
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(1:40, 1:20 and 1:10) with a WP:MD ratio of 3:1. As can be seen from Table 1, there are
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no significant difference between the D32(µm) values of emulsions prepared with
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DE:13-17 and DE:4-7 in all core to coating ratios (1:40, 1:20 and 1:10) (p>0.05). From
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this results, it can be inferred that the differences between the drying and encapsulation
336
efficiencies of capsules prepared with MD having different DE values were not the
337
consequences of different particle sizes of the emulsions. Another factor that Table 1
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shows was the effect of the ratio of core material to the wall materials on particle size of
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emulsions. The effect of different core to coating ratios can also be seen separately for
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DE values of 4-7 and 13-17 in Fig. 4 and Fig. 5, respectively. For both types of MD, the
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particle size distribution curve shifted slightly to larger particle size side of the graph as
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core to coating ratio changed from 1:40 to 1:20 and 1:10. This means that as the oil
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concentration increased in the emulsion, the particle size of the emulsion became larger.
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The Sauter mean diameter values gave the same outcome as well. Hogan et al., (2001)
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and Taneja et al., (2013) also reported similar results, in which they found that particle
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ACCEPTED MANUSCRIPT sizes increased with increasing core to wall ratios. This phenomenon could be related to
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coalescence. Because, as oil concentration increased, the protein amount became
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insufficient for the adsorption at the core/wall interfaces which led to coalescence and
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an increase in the droplet size in the emulsion. Additionally, the span values and the
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SSA values of the emulsions changed with the increasing oil ratio as expected (Table 1).
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In order to see the effect of the ultrasonication technique on the particle size distribution
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of the emulsion, sample with DE:13-17, WP:MD ratio of 3:1 and core to coating ratio of
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1:20 was analyzed before and after the ultrasonication step of homogenization. In Table
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2, the particle size values are given for sample with and without ultrasonication (just
355
high speed homogenization). Table 2 clearly showed that ultrasonication had a
356
significant effect on decreasing the particle size. Also it can be seen from Fig. 6 that
357
while the majority of the particles were in a range between 0.1-1 µm for ultrasonicated
358
sample, for the sample, which was homogenized only with high speed mixer, the range
359
was between 0.5-10 µm. The span of the particle size distribution curves and the SSA
360
values were also negatively correlated with particle size values (Table 2). Based on
361
these results, it can be said that ultrasonication has a positive impact on emulsification.
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3.4
Surface Morphology of Microcapsules
364
From different core to coating ratios and WP:MD ratios, samples having the highest
365
encapsulation efficiencies, that is the samples having core to coating ratio of 1:20 and
366
WP:MD ratio of 3:1, were analyzed. The SEM images of the samples with 500×
367
magnification are illustrated in Fig. 7. From the images it can be seen that both sample
368
with DE:13-17 (Fig. 7A) and sample with DE:4-7 (Fig. 7B) had almost evenly
369
distributed particles throughout the images. Fig. 7 also shows that, there was no
370
difference between the sizes of the particles of two samples prepared with MD having
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emulsions, in which there is no significant difference between D32 (µm) values of the
373
emulsions. From Fig. 7, it can also be seen that both samples had smooth surfaces free
374
of cracks and dents. This lack of surface deformations could be explained by the high
375
content of whey protein concentration of wall matrices. Also it can be concluded that a
376
change in DE value did not cause a significant difference in surface morphologies of the
377
capsules.
378
In the comparison of capsules with MD having DE:13-17 (Fig. 8A) and capsules with
379
MD having DE:4-7 (Fig. 8B) under 5000× magnifications, it can be seen that powders
380
with DE:13-17 had more porous structures with more holes within the capsules. The
381
pores are the residues of entrapped rosemary oil cavitations, resulting from the
382
volatilization of the oil during the exposure. The result is also in accordance with the
383
encapsulation efficiency results of the capsules with same formulations.
384
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3.5
Storage Stability of Microcapsules
386
Rosemary essential oil coated with WP and MD at a ratio of 3:1 and core to coating
387
ratio of 1:20 were chosen for stability analysis. Experiments were performed with MD
388
having DE values of both 4.0-7.0 and 13.0-17.0. Fig. 9 shows the 1,8-cineole
389
concentrations of three samples during 40 days of storage under 35.3% relative
390
humidity. As can be seen from the figure, for all the three samples, the 1,8-cineole
391
concentrations decreased during storage as expected. This decrease of 1,8-cineole was
392
highly due to its volatilization. In the encapsulated oils, the volatilization of 1,8-cineole
393
was lower than non-encapsulated rosemary oil. The reason of this was the barrier effect
394
of the wall materials. Also, the increasing permeability of the wall material allows the
395
oxidation of the encapsulated oil over time which also decreases the 1,8-cineole
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397
decrease of 1,8-cineole concentration of non-encapsulated oil shows the barrier effect of
398
the wall materials more clearly. For the first 30 days of storage, the concentrations of
399
1,8-cineole for the capsules encapsulated with MD with DE:4-7 were higher than the
400
concentrations of 1,8-cineole in capsules prepared using MD having DE:13-17. This
401
result indicated that MD with DE:4-7 achieved the retention of 1,8-cineole better than
402
DE:13-17. After 30 days of storage, the effect of different DE values on stability was
403
lost and the retention percentages of MD became almost equal. Higher retention
404
performance of MD having lower DE value has been investigated by many researchers.
405
Ersus & Yurdagel (2007) linked the worse storage stability of higher DE MD to their
406
lower molecular weight with shorter chains, which are more susceptible to structural
407
deformations than MD with low DE values. However, the main reason why lower DE
408
MD exhibited better storage stability functions was their higher glass transition
409
temperature. As DE value increased, the molecular weight of MD decreased which also
410
lowered the glass transition temperature of the MD. Because of this, during storage at
411
high relative humidity environments, MD with high DE had higher hygroscopicity
412
which led to caking and loss of volatile components (Desorby, Netto, & Labuza, 1997).
413
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4.
Conclusion
415
In order to obtain the best microencapsulation formulation for the encapsulation of
416
rosemary essential oil, drying efficiency and encapsulation efficiency analyses were
417
conducted in the capsules prepared with MD having two different DE values, different
418
core to coating ratios and WP:MD ratios. Among the coating formulations, WP:MD
419
ratio of 3:1 provided the highest drying and encapsulation efficiencies for both type of
420
MD and the best core to coating ratio giving higher drying and encapsulation
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422
found to yield better drying and encapsulation efficiency values as compared to MD
423
having DE:4-7. Analyses also showed that the DE value did not have any
424
morphological effect on the capsules or any particle size difference on the emulsions.
425
Lastly, GC-MS analyses showed that powders prepared with MD having DE:4-7 was
426
found to have the higher retention values of 1,8-cineole during the first 30 days of
427
storage. However, after 30 days of storage, the retention powers of MD became almost
428
equal. Thus at the end, the optimum coating material formulation was chosen to be core
429
to coating ratio of 1:20 and WP:MD ratio of 3:1 with DE value of 13-17.
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430 References
432
Baranauskiené, R., Bylaité, E., Zukauskaité, J., & Venskutonis, R. P. (2007). Flavor
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Başer, K. C., & Buchbauer, G. (2010). Handbook of Essential Oils: Science,
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Desai, K. G., & Park, H. J. (2005). Recent developments in microencapsulation of food ingredients. Drying Technology, 1361–1394.
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Desorby, S. A., Netto, F. M., & Labuza, T. P. (1997). Comparison of spray-drying,
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carrot (Daucuscarota L.) by spray drier. Journal of Food Engineering, 805–812.
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Estévez, M., Ramírez, R., Ventanas, S. & Cava, R. (2007). Sage and rosemary essential
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Fernandes, R. V., Borges, S. V., Botrel, D. A., Silva, E. K., Gomes da Costa, J. M., &
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Queiroz, F. (2013). Microencapsulation of rosemary essential oil: characterization of
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particles. Drying Technology, 1245–1254.
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Ghosh, S. K. (2006). Functional Coatings and Microencapsulation: A General
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Perspective. In S. K. Ghosh, Functional Coatings by Polymer Microencapsulation
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Hogan, S. A., McNamee, B. F., O’Riordan, E. D., & O’Sullivan, M. (2001).
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Issabeagloo, E., Kermanizadeh, P., Taghizadieh, M., & Forughi, R. (2012).
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Antimicrobial effects of rosemary (Rosmarinus officinalis L.) essential oils against
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Staphylococcus spp. African Journal of Microbiology Research, 5039-5042.
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Jafari, S. M., He, Y., & Bhandari, B. (2007a). Role of Powder Particle Size on the
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Encapsulation Efficiency of Oils during Spray Drying. Drying Technology: An
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International Journal, 1081-1089.
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Jafari, S. M., He, Y., & Bhandari, B. (2007b). Encapsulation of nanoparticles of d-
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limonene by spray drying: role of emulsifiers and emulsifying techniques. Drying
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Technology: An International Journal, 1069-1079.
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Jafari, S. M., Assadpoor, E., He, Y., & Bhandari, B. (2008). Encapsulation efficiency of
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food flavours and oils during spray drying. Drying Technology, 816–835.
Janiszewska, E. & Witrowa-Rajchert, D. (2009). The influence of powder morphology
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Karimi, N., & Mohammadifar, M. A. (2014). Role of water soluble and water swellable
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Katerinopoulos, H. E., Pagona, G., Afratis, A., Stratigakis, N., & Roditakis, N. (2005).
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Composition and insect attracting activity of the essential oil of Rosmarinus
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officinalis. Journal of Chemical Ecology, 111-122.
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Kuang, S. S., Oliveira, J. C., & Crean, A. M. (2010). Microencapsulation as a tool for
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incorporating bioactive ingredients into food. Critical Reviews in Food Science and
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Kuhlmann, W. D. (2006). Buffer solutions. 69120 Heidelberg, Germany: Division of
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Runge, F. E. (2004). Multiple-core Encapsulation. In P. Vilstrup, Microencapsulation of Food Ingredients (pp. 133-145). Surrey: Leatherhead Publishing.
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Shah, B., Ikeda, S., Davidson, P. M., & Zhong, Q. (2012). Nanodispersing thymol in
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Sheu, T. Y., & Rosenberg, M. (1998). Microstructure of microcapsules consisting of
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whey proteins and carbohydrates. Journal of Food Science, 491-494.
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Taneja, A., Ye, A., Jones, J. R., Archer, R., & Singh, H. (2013). Behaviour of oil
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droplets during spray drying of milk-protein-stabilised oil-in-water emulsions.
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International Dairy Journal, 15-23.
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Teodoro, R. A. R., Fernandes, R. V., Botrel, D. A., Borges, S. V. & de Souza, A. U.
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(2014). Characterization of microencapsulated rosemary essential oil and its
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antimicrobial effect on fresh dough. Food and Bioprocess Technology. me Vol 7,
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Valgimigli, L. (2012). Essential Oils as Natural Food Additives: Composition,
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Applcations, Antioxidant and Antimicrobial Properties. New York: Nova Science
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Vardhanabhuti, B., & Foegeding, A. E. (1999). Rheological Properties and
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Characterization of Polymerized Whey Protein Isolates. Journal of Agricultural and
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Food Chemistry, 3649−3655.
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Zhu, G., Xiao, Z., Zhou, R., & Yi, F. (2012). Fragrance and flavor microencapsulation
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technology. Advanced Materials Research, 440-445.
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ACCEPTED MANUSCRIPT Figure captions
517
Figure 1 Drying efficiencies of microcapsules encapsulated with different formulations;
518
( ): Capsules with MD:WP ratio of 3:1 and MD having DE:13-17, (■): Capsules with
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MD:WP ratio of 1:3 and MD having DE:13-17 ( ): Capsules with MD:WP ratio of 3:1
520
and MD having DE:4-7, (■): Capsules with MD:WP ratio of 1:3 and MD having DE:4-
521
7. Different letters represent significant difference (p ≤ 0.05).
522
Figure 2 Encapsulation efficiencies of microcapsules encapsulated by MD with DE:13-
523
17 at different core to coating ratios and different WP:MD ratios; (■): 3:1 and (■): 1:3.
524
Different letters represent significant difference (p≤0.05)
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Figure 3 Encapsulation efficiencies of microcapsules encapsulated with WP and MD at
526
a ratio of 3:1, different core to coating ratios and MDs having different DE values; (■):
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DE:13-17, (■): DE:4-7. Different letters represent significant difference (p ≤ 0.05)
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Figure 4 Particle size distributions of emulsions prepared with WP:MD ratio of 3:1,
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MD with DE:4-7 and different core to coating ratios; 1:40 (solid line), 1:20 (dashed
530
line) and 1:10 (dotted line).
531
Figure 5 Particle size distributions of emulsions prepared with WP:MD ratio of 3:1,
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MD with DE:13-17 and different core to coating ratios; 1:40 (solid line), 1:20 (dashed
533
line) and 1:10 (dotted line).
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Figure 6 Particle size distributions of emulsions prepared with ultrasonic
535
homogenization (solid line) and Ultra-turrax homogenization (dashed line) with
536
WP:MD ratio of 3:1, MD with DE:13-17 and core to coating ratio of 1:20.
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Figure 7 Scanning Electron Microscope images (500× magnification) of microcapsules
538
having WP:MD ratio of 3:1 and core to coating ratio of 1:20 prepared by MD having
539
different DE values; (A): DE:13-17, (B): DE:4-7
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Figure 81 Scanning Electron Microscope images (×5000 magnification) of
541
microcapsules prepared with WP:MD ratio of 3:1 and core to coating ratio of 1:20 and
542
MD having different DE values; (A): DE:13-17, (B): DE:4-7
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Figure 9 1,8-cineole concentration of non-encapsulated oil (♦), capsules prepared with
544
maltodextrin having DE:13-17 (■) and DE:4-7 (▲) for 40 days of storage at 33.3% RH.
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Table 1 Particle size analyses of emulsions prepared with MD having different DE
548
values and different core to coating ratios with WP: MD ratio of 3:1
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Table 2 The effect of homogenization technique on the particle size results of the
550
emulsion prepared by MD with DE:13-17 and core to coating ratio of 1:20.
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Table 1 Particle size analyses of emulsions prepared with MD having different DE values and
Core:Coating
D32(µm)
4-7
1:40
0.187c*
4-7
1:20
0.207b
4-7
1:10
13-17
1:40
13-17
1:20
AC C
SSA (m2/g)
32.1a
5.295ab
29.0b
0.255a
3.708b
23.5c
0.188c
20.813a
31.9a
0.205b
12.423ab
29.2b
4.238b
24.4c
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16.769a
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13-17
Span
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DE values
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different core to coating ratios with WP: MD ratio of 3:1
0.246a
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Table 2 The effect of homogenization technique on the particle size results of the emulsion
High-speed homogenizer
Span
1.428
13.039
High-speed+ Ultrasonic
0.254
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homogenizer
Area (m2/g)
SC
Technique
Specific Surface
D32(µm)
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prepared by MD with DE:13-17 and core to coating ratio of 1:20
26.948
4.2
23.6
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ACCEPTED MANUSCRIPT • MD having DE:13-17 yielded better drying and encapsulation efficiency values • Core to coating ratio of 1:20 gave higher drying and encapsulation efficiency • WP:MD ratio of 3:1 provided highest drying and encapsulation efficiency values.
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• DE value had no effect on particle size of the emulsions.
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• Retention powers of both type of MD became almost equal after 40 days of storage.
S0023-6438(15)00399-0
DOI:
10.1016/j.lwt.2015.05.036
Reference:
YFSTL 4692
To appear in:
LWT - Food Science and Technology
Received Date: 8 January 2015 Revised Date:
19 May 2015
Accepted Date: 24 May 2015
Please cite this article as: Turasan, H., Sahin, S., Sumnu, G., Encapsulation of rosemary essential oil, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.05.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Encapsulation of rosemary essential oil
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Hazal Turasan, Serpil Sahin*, Gulum Sumnu
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Department of Food Engineering, Middle East Technical University, 06800, Ankara,
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Turkey
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*Corresponding author
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Tel:+90 312 210 5627; E-mail: [email protected]
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1
ACCEPTED MANUSCRIPT Abstract
27
Encapsulation protects sensitive food ingredients against oxygen, heat, moisture, pH
28
and it masks the unwanted taste of nutrients. The objective of the study was to
29
encapsulate the rosemary essential oil in micron size and to find the optimum coating
30
material formulation by investigating the physicochemical properties and storage
31
stability of microcapsules. In the capsule preparation two different ratios of whey
32
protein concentrate (WP) and maltodextrin (MD) (1:3 and 3:1), three different core to
33
coating ratios (1:40, 1:20 and 1:10) and two different dextrose equivalent (DE) MD
34
(DE:13-17 and DE:4-7) were used. Emulsions were analyzed for their particle size
35
distributions and freeze dried capsules were analyzed for their drying efficiencies,
36
encapsulation efficiencies, surface morphologies, and concentrations of 1,8-cineole
37
during storage. Increasing WP:MD ratio was found to increase both drying and
38
encapsulation efficiencies. Also, capsules having core to coating ratio of 1:20 and MD
39
with DE:13-17 gave the highest drying and encapsulation efficiency values. Changing
40
DE value of MD did not have any significant effect on particle size distributions and
41
surface morphologies of the capsules. Lastly, encapsulation was found to be an effective
42
method for increasing the storage stability of 1,8-cineole.
43
Keywords
44
Microencapsulation, rosemary essential oil, whey protein concentrate, maltodextrin,
45
dextrose equivalence
46
Chemical compounds studied in this article: 1,8- cineole (another name: 1,3,3-
47
Trimethyl-2-oxabicyclo [2.2.2]octane) (PubChem CID: 2758); maltodextrin (PubChem
48
CID: 62698); Magnesium chloride (MgCl2) (PubChem CID: 5360315); dipotassium
49
phosphate (K2HPO4) (PubChem CID: 24450); potassium dihydrogen phosphate
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(KH2PO4) (PubChem CID: 516951); n-hexane (PubChem CID: 8058).
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ACCEPTED MANUSCRIPT 1. Introduction
52
During the last decades, the demands of the consumers from the food production
53
industry have remarkably increased. People no longer see the food to appease the
54
hunger but as a source to get the required nutrients which are supposed to help with the
55
nutrition-related diseases and contribute to both physical and mental well-being of
56
individuals (Bigliardi & Galati, 2013). This trend forced the food researchers and the
57
producers to concentrate more on the production of foods that meets the requirements of
58
humans for healthier lives (Bigliardi & Galati, 2013). Novel technologies are adopted
59
especially to overcome the problem of active compound deterioration. These relatively
60
more advanced techniques are based on the idea of coating the desired active compound
61
(Bigliardi & Galati, 2013). Some of the entrapment techniques are microencapsulation,
62
coating with edible films or vacuum impregnation.
63
Rosemary (Rosmarinus officinalis L.) is a long-lasting evergreen aromatic herb
64
(Bousbia, et al., 2009). The usage of rosemary oil dates back to 1500s (Guenther, 1948).
65
Before refrigeration was invented rosemary oil was used for food preservation purposes
66
as well as medical antiseptic, and astringent purposes (Bousbia, et al., 2009). Through
67
the years, the utilization area of rosemary oil has not been changed much. Rosemary
68
essential oil includes phenolic constituents in its composition (Başer & Buchbauer,
69
2010). Due to this composition, which mainly involves monoterpenes like 1,8-cineole,
70
α-pinene, camphor, camphene, rosemary essential oil has many therapeutical effects
71
(Katerinopoulos et al., 2005; Başer & Buchbauer, 2010). Among these effects, the most
72
well-known ones are its antioxidant effects (Valgimigli, 2012; Estévez et al., 2007),
73
antimicrobial effects (Issabeagloo et al., 2012), pediculicidal, aromatherapeutical and
74
anticarcinogenic activities (Başer & Buchbauer, 2010). However, due to its high
75
volatility and its susceptibility against environmental effects, rosemary essential oil
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ACCEPTED MANUSCRIPT needs further protective actions, especially to increase its uptake and bioavailability.
77
Encapsulation is one of the most efficient techniques to preserve it. It is a process in
78
which, small solid particles, liquid components or gaseous materials are coated by or
79
entrapped within another inert shell material, which isolates and protects the core
80
material from environmental factors (Zhu et al., 2012; Kuang, Oliveira, & Crean, 2010;
81
Ghosh, 2006; Desai & Park, 2005). Also this technique helps to mask the unwanted
82
taste and the odor of the ingredient, to prevent the evaporation of the volatile
83
components and to prevent the contact of the ingredient with oxygen to prevent
84
oxidation. However, there are some important factors influencing microencapsulation.
85
To achieve successful microencapsulation process, the choice of coating materials, the
86
homogenization techniques and the drying techniques must be chosen carefully. In the
87
literature, although there are many studies on the effects of rosemary essence on health
88
problems and about the chemical composition of it, there is only a limited number of
89
researches on microencapsulation of rosemary essential oil. In these studies, the main
90
drying technique used is spray drying, the emulsions are generally prepared by using
91
high-speed blenders and usually gum Arabic is used as the coating material (Fernandes,
92
et al., 2013; Janiszewska & Witrowa-Rajchert, 2009; Teodoro et al., 2014). The main
93
objective of this study was to develop a new and different technique for
94
microencapsulation of rosemary essential oil. This is achieved by employing freeze
95
drying as the encapsulation technique. Maltodextrin is chosen as the coating material
96
since it has excellent oxygen-blocking property; however its lack of emulsification
97
characteristic creates a need for an additional coating material (Runge, 2004; Sheu &
98
Rosenberg, 1998). Preliminary studies showed that gums cannot be used as additional
99
coating materials since their solutions reach high viscosities even in low concentrations.
100
Whey protein concentrate is chosen as the second coating material since they show low
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ACCEPTED MANUSCRIPT viscosities even in high concentrations and they are excellent emulsifiers (Runge, 2004;
102
Vardhanabhuti & Foegeding, 1999). Investigating the optimum coating material
103
formulation for obtaining the highest drying and encapsulation efficiencies were other
104
aims of this study. To even further increase the efficiency results, ultrasonication is
105
added to the homogenization step since its effects on reducing particle sizes is proven
106
by others (Jafari, He, & Bhandari, 2007a). The effects of different coating formulations
107
on particle size and surface morphology of the capsules were analyzed as well as the
108
storage stabilities of the encapsulated products.
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2. Experimental
111
2.1 Materials
112
Rosemary (Rosemarinus Officinalis L.) essential oil, maltodextrin (MD) in two different
113
dextrose equivalences (DE) (DE: 4.0-7.0 and DE: 13.0-17.0), magnesium chloride
114
(MgCl2),
115
(KH2PO4), n-hexane and 1,8-cineole were purchased from Sigma Aldrich Chemical Co.
116
(St. Louis, MO, USA). Whey protein concentrate (WP) containing 80 g protein/100 g
117
solid, was supplied by Tunçkaya Kimyevi Maddeler (Tuzla, İstanbul). The Patcote
118
502K anti-foaming agent, which was used in the drying efficiency analyses, was
119
supplied from Hydrite Chemical Company (Brookfield, WI, U.S.A.).
phosphate (K2HPO4),
potassium
dihydrogen
phosphate
120
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2.2
Preparation of Microcapsules
122
2.2.1 Preparation of Coating Materials
123
The maltodextrin (MD) solutions were prepared in distilled water and in two different
124
concentrations (10g/100g solution and 30g/100g solution) for two different dextrose
125
equivalences (DE: 4.0-7.0 and DE: 13.0-17.0). The solutions were pre-mixed for 10 min
5
ACCEPTED MANUSCRIPT by a magnetic stirrer (Heidolph MR 3001 K, Heidolph Instruments GmbH & Co,
127
Schwabach, Germany) and then left in a shaking water bath (GFL 1086, Burgwedel,
128
Germany) at 25⁰C at 90 rpm for one night (18 h) to obtain full hydration.
129
Whey protein concentrate (WP) solutions were prepared in phosphate buffer solution
130
and in two different concentrations (10g/100g solution and 30g/100g solution). The
131
preparation procedure of the phosphate buffer is adapted from Kuhlmann (2006). WP
132
solutions were also prepared by using the magnetic stirrer and were left in the shaking
133
water bath for one night (18 h) at 25⁰C to get full hydration.
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Prior to the addition of core material, the coating material solutions were weighed and
137
mixed. From both whey protein solution and maltodextrin solution, 60 g were weighed,
138
which makes a 120 g of total coating material mixture. The mixture of coating materials
139
were prepared in the following ratios by choosing the suitable concentrations of WP and
140
MD solutions: a) WP: MD (DE: 4.0-7.0) = 1:3 b) WP: MD (DE: 4.0-7.0) = 3:1 c) WP:
141
MD (DE: 13.0-17.0) = 1:3 and d) WP: MD (DE: 13.0-17.0) = 3:1. All the mixtures were
142
prepared in mass ratios. The total soluble solid contents of the mixtures were kept at
143
20g/100g solution.
144
Rosemary essential oil was added in three different core-to-coating ratios: 1:40, 1:20
145
and 1:10. First, pre emulsions were homogenized in high-speed homogenizer (IKA T25
146
digital Ultra-Turrax, Selangor, Malaysia) at 8000 rpm for 5 min and then were
147
homogenized by using Ultrasonic Homogenizer (Sonic Ruptor 400, OMNI International
148
the Homogenizer Company, Georgia, USA). Ultrasonic homogenizer was equipped
149
with solid titanium 1" Solid and Tapped tip with a diameter of 25.4 mm and a length of
150
12.70 cm. The ultrasonication process was performed for 15 min at 40% power of 20
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kHz using 50% pulse. To prevent excessive heating during homogenization, the beakers
152
were placed in 4⁰C water baths. The emulsions were frozen at -18⁰C immediately after
153
the homogenization procedure to prevent any coalescence or flocculation.
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156
Fully frozen emulsions were dried in a freeze drier (Christ, Alpha 2-4 LD plus,
157
Germany) for 48 h at -50⁰C and at 1.9 Pa. After lyophilization, dried samples were
158
grinded into powder form with a glass rod. For SEM analysis, the capsules were further
159
grinded with coffee grinder for 15 s.
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160 2.3
Storage of the Microcapsules
162
The storage stability of the microencapsulated rosemary oil was investigated at 35.3 %
163
± 0.1 % relative humidity and at 15⁰C. To obtain this relative humidity, saturated
164
aqueous solution of magnesium chloride (MgCl2) was prepared and placed into the
165
desiccators. Before the placement of the samples, the salt solution was kept overnight in
166
the desiccators to reach equilibrium. In order to see the difference, the encapsulated and
167
non-encapsulated (fresh) rosemary oil samples were stored. Five grams of specimen
168
from each sample were taken at certain time intervals during 40 days of storage. The
169
samples were then analyzed for their 1,8-cineole concentrations by GC-MS. As the
170
reference compound, 1,8-cineole was chosen since it is one of the major constituents of
171
rosemary essential oil (Surburg & Panten, 2006).
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172 173
2.4
Analysis of Emulsions and Microcapsules
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2.4.1
Particle Size Analysis of Emulsions
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ACCEPTED MANUSCRIPT Particle size distributions of emulsions were analyzed with Mastersizer 2000 (Malvern
176
Instruments Limited, Worcestershire, UK). The mean particle size of the emulsions was
177
represented with Sauter mean diameter, D32 (µm), and was calculated with the
178
following equation (McClements, 2005);
∑n d D32 = ∑n d
179
i
i
i
2
(1)
Span, was calculated with the following formula (Karimi & Mohammadifar, 2014);
Span =
[d (0.9) − d (0.1)]
181
d (0.5)
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3
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(2)
where, d(0.9), d(0.1), and d(0.5) are the diameters at 90%, 10%, and 50% of cumulative
183
volume, respectively. During particle size analysis, the sonication was applied to the
184
emulsions. However, during the analyses of emulsions, which were prepared in order to
185
see the effect of ultrasonication only, the sonication of Mastersizer 2000 (Malvern
186
Instruments Limited, Worcestershire, UK) was switched off to have an accurate result.
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2.4.2
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To determine the efficiency of the encapsulation process, two types of efficiency
190
analyses were adopted: drying efficiency and encapsulation efficiency.
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Efficiency Analysis of Microcapsules
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2.4.2.1
Drying Efficiency
193
For the drying efficiency analysis, a hydrodistillation technique with Clevenger
194
apparatus was chosen to measure the oil retention. First, 10 g of microcapsules was
195
dissolved in 250 ml of distilled water in a 500 ml flask. In order to prevent foaming, one
196
droplet of Patcote 502K anti-foaming agent (Hydrite Chemical Company, WI, U.S.A.)
197
was added by a syringe and mixed with the solution. After 3 h of distillation in
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ACCEPTED MANUSCRIPT 198
Clevenger apparatus, the volume of the total oil was read from the volumetric arm and
199
multiplied with the density of rosemary essential oil (0.908 g/ml) to estimate the actual
200
oil content in the capsules. The drying efficiency was then calculated according to the
201
following equation (Jafari, He, & Bhandari, 2007b):
Drying Efficiency (%) =
[Oil content of microcapsules] × 100 [Oil content of emulsions]
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2.4.2.2
205
To measure the surface oil content, an analysis with Soxhlet apparatus was performed
206
(Baranauskiené et al., 2007). Five grams of dried powder was weighed and trapped in
207
filtration paper and washed for 3 h with 250 ml of n-hexane in the Soxhlet apparatus.
208
The excess hexane was then evaporated in a vacuum rotary evaporator (Heidolph
209
Laborota 4000 efficient; Heidolph Instruments GmbH & Co, Schwabach, Germany) at
210
33.5 kPa and at 40⁰C. The surface oil was then concentrated under nitrogen dryer
211
(Turbovap LV Concentration Evaporator Workstation; Biotage, Charlotte, NC, USA).
212
Total oil content was determined by Clevenger apparatus. Then, the encapsulation
213
efficiency was calculated using the following formula (Jafari, He, & Bhandari, 2007b);
214
Encapsulation Efficiency (%) =
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Encapsulation Efficiency
(3)
[Total oil content − Surface oil content ] × 100 [Total oil content ]
(4)
216
2.4.3
Surface Morphology Analysis of Microcapsules
217
To analyze the structures and the surface morphologies of microcapsules of rosemary
218
essential oil, scanning electron microscope was used. Samples were grinded for 15 s
219
with coffee grinder (ARZUM AR151 Mulino Coffee Grinder, Turkey) to eliminate the
220
structural differences. Then, they were coated with the mixture of gold/palladium by
9
ACCEPTED MANUSCRIPT HUMMLE VII Sputter Coating Device (ANATECH, Union city, CA, USA). The
222
scanning electron microscopy (SEM) (JSM-6400 Electron Microscope, Jeol Ltd.,
223
Tokyo, Japan) was equipped with NORAN System 6 X-ray Microanalysis System and
224
Semafore Digitizer. The images of the microcapsules were taken at two different
225
magnifications; 500× and 5000×.
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226 2.4.4
GC-MS Analysis
228
The stored microcapsules and fresh rosemary oil were analyzed for their 1, 8-cineole
229
content. The oil was extracted from the samples were analyzed with Gas
230
chromatography/Mass spectrometry (GC-MS) system (Agilent Technologies 6890N
231
Network GC System coupled to Agilent Technologies 5973 Network Mass Selective
232
Detector, USA). Quantitative analyses could be done using FID with a capillary column
233
(Agilent 19091s-433 HP-5MS with a 5 g phenyl/95 g methylpolysiloxane stationary
234
phase and with a size of 30m x 0.25mm x 0.25µm). An Agilent Tecnologies 7683B
235
Series Injector (Thailand) was used to inject the sample. The data were analyzed by
236
MSD ChemStation software program and helium was used as the carrier gas.
237
The following GC-MS conditions were used during the analyses: split ratio 16.5:1;
238
injection volume 1 µL; oven temperature program, holding at 40°C for 1 min, rising to
239
180°C with 4°C/min with a total run time 39 min; MSD transfer line temperature,
240
230°C; MSD quadrupole temperature, 150°C. Solvent delay was for 4.0 min. The GC
241
analysis was performed with the following conditions: H2 flow rate, 35 mL/min; air
242
flow rate, 400 mL/min; make-up flow rate of 48.8 mL/ min with a make-up gas type,
243
He; FID temperature, 275°C.
244
Prior to injection, the samples were diluted with n-hexane at a ratio of 1:100 (µl:µl).
245
The calibration curve for 1,8-cineole was prepared with five different concentrations in
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ACCEPTED MANUSCRIPT 246
ml/ml (1/50, 1/100, 1/200, 1/400, 1/800 ml/ml) with n-hexane. Correction of variation
247
values (R2) of all calibration curves were obtained as greater than 0.99.
248 2.4.5
Statistical Analysis
250
To understand if there was a significant difference between the samples, the results were
251
analyzed by analysis of variance (ANOVA) (p≤ 0.05). When a significant difference
252
was found between the samples, Duncan's Multiple Comparison Test was applied (p≤
253
0.05) by using SAS software version 9.1 (SAS Institute Inc., NC, USA).
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Results and discussion
256
3.1
Drying Efficiency
257
Fig. 1 represents the drying efficiency results of microcapsules. The results show that,
258
there was no significant difference (p>0.05) between the drying efficiencies of
259
microcapsules prepared with core to coating ratios of 1:40 and 1:20, for both MD with
260
DE value of 13-17 and 4-7. However, the drying efficiency was significantly lower
261
when core to coating ratio was 1:10 (p≤0.05). This difference could be explained by the
262
oil load of the emulsions. As the amount of oil used in the preparation of the emulsions
263
increased, the solid content in the mixture became insufficient to cover and entrap the
264
excessive amount of oil. Therefore, more non-entrapped volatile oil was lost under the
265
vacuum of freeze drying. According to the results, the formulation with the MD with
266
DE:13-17 gave higher drying efficiency than MD with DE:4-7, which becomes more
267
obvious as the core to coating ratios increase to 1:10. This indicated that as DE value
268
increased, the retention of the volatiles in the wall matrix increased. These results show
269
accordance with the findings of Shah et al., (2012) and Sheu & Rosenberg (1998).
270
According to Sheu & Rosenberg (1998), as DE value increases, the proportion of low
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ACCEPTED MANUSCRIPT molecular weight carbohydrates increases and provides less disrupted capsules during
272
drying which results in higher drying efficiencies. Also as particles get smaller, the
273
drying rates increases, which enhances the solidification rate and also retention of
274
volatiles.
275
Fig. 1 also represents the effects of different WP:MD and core to coating ratios on
276
drying efficiency values. For samples having MD with different DE vaues and for all
277
core to coating ratios drying efficiency decreased noticeably as WP:MD ratio changed
278
from 3:1 to 1:3. As Sheu & Rosenberg (1998) explained, the positive effect of
279
increasing concentration of whey proteins on drying efficiency is due to their
280
contribution to surface morphology of the capsules. Since whey proteins have positive
281
effects on both drying rate and mechanical properties of the wall matrix, they decrease
282
the number of surface cracks on the capsules, which results in increased drying
283
efficiency.
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3.2
Encapsulation Efficiency
286
The effect of different WP:MD ratios on the encapsulation efficiency values of the
287
microcapsules are illustrated in Fig. 2. For MD with DE:13-17, the increase in WP
288
concentration increased encapsulation efficiency values significantly for all core to
289
coating ratios (p≤0.05). The capsules prepared by MD with DE:4-7 also showed similar
290
results (p≤0.05) (data not shown). One of the reasons of this trend is the good
291
emulsification properties of whey proteins (Jafari, et al., 2008). In addition,
292
maltodextrins lack surface-active properties and this makes them poor wall materials
293
especially when they are used for encapsulation of volatile core materials (Sheu &
294
Rosenberg, 1998). Also, whey proteins are effective on encapsulation efficiency since
295
the unfolding and adsorption on the oil-water interfaces change protein structures and
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297
WP (Jafari, et al., 2008). Thus, the increase in whey protein concentration results in
298
higher encapsulation efficiencies. This can also be explained by the reduction in
299
viscosity of the wall material solutions (Jafari, et al., 2008). Although the solid content
300
ratio remained the same when WP:MD ratio was changed from 3:1 to 1:3, the amount
301
of larger particles (whey proteins) decreased which caused a decrease in the viscosity of
302
the coating solution. Thus, as the viscosity of the wall material decreased, the
303
encapsulation efficiency values also decreased.
304
Fig. 3 represents the results of encapsulation efficiency values for two types of MD and
305
three different core to coating ratios. When only the difference between MD types is
306
considered, it is seen that the encapsulation efficiencies of the capsules prepared with
307
MD having different DE values were significantly different from each other (p≤0.05). It
308
was seen that for all core to coating ratios, the formulation with MD having DE:13-17
309
gave higher encapsulation efficiency results than formulation with DE:4-7. The results
310
indicated that MD with higher DE value helped to entrap more oil inside of the capsule
311
and left less of the total oil on the surface. The positive effect of increasing DE value is
312
due to the decreasing permeability of capsule to oxygen (Jafari et al., 2008).
313
Another parameter that Fig. 3 represents about the encapsulation efficiencies of
314
microcapsules is the core to coating ratios of the emulsions. For both DE values of MD,
315
encapsulation efficiency values of capsules prepared with core to coating ratio of 1:40
316
and 1:20 were not significantly different (p>0.05). In general, it can be seen that the
317
encapsulation efficiencies decreased with the increasing oil content. The reason of this
318
inverse relation between the oil amount and encapsulation efficiencies was the
319
insufficiency of the solid materials to produce a strong structural layer around the oil
320
droplets and cover them completely when the oil amount is increased. Consequently,
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ACCEPTED MANUSCRIPT this leads to lower encapsulation efficiency. It is seen that, microcapules having
322
WP:MD ratio of 1:3 have much lower drying and encapsulation results in general.
323
Therefore they are excluded from further analyses, since they are clearly not good
324
candidates to have optimum formulation.
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325 3.3
Particle Size
327
The particle size distributions of emulsions prepared with WP and MD at a ratio of 3:1
328
as coating material were analyzed only, since coating with WP and MD at a ratio of 1:3
329
resulted in lower drying and encapsulation efficiency values. Table 1 shows the particle
330
size distributions, span values and the specific surface area (SSA) values of the
331
emulsions prepared with MD having different DE values, different core to coating ratios
332
(1:40, 1:20 and 1:10) with a WP:MD ratio of 3:1. As can be seen from Table 1, there are
333
no significant difference between the D32(µm) values of emulsions prepared with
334
DE:13-17 and DE:4-7 in all core to coating ratios (1:40, 1:20 and 1:10) (p>0.05). From
335
this results, it can be inferred that the differences between the drying and encapsulation
336
efficiencies of capsules prepared with MD having different DE values were not the
337
consequences of different particle sizes of the emulsions. Another factor that Table 1
338
shows was the effect of the ratio of core material to the wall materials on particle size of
339
emulsions. The effect of different core to coating ratios can also be seen separately for
340
DE values of 4-7 and 13-17 in Fig. 4 and Fig. 5, respectively. For both types of MD, the
341
particle size distribution curve shifted slightly to larger particle size side of the graph as
342
core to coating ratio changed from 1:40 to 1:20 and 1:10. This means that as the oil
343
concentration increased in the emulsion, the particle size of the emulsion became larger.
344
The Sauter mean diameter values gave the same outcome as well. Hogan et al., (2001)
345
and Taneja et al., (2013) also reported similar results, in which they found that particle
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ACCEPTED MANUSCRIPT sizes increased with increasing core to wall ratios. This phenomenon could be related to
347
coalescence. Because, as oil concentration increased, the protein amount became
348
insufficient for the adsorption at the core/wall interfaces which led to coalescence and
349
an increase in the droplet size in the emulsion. Additionally, the span values and the
350
SSA values of the emulsions changed with the increasing oil ratio as expected (Table 1).
351
In order to see the effect of the ultrasonication technique on the particle size distribution
352
of the emulsion, sample with DE:13-17, WP:MD ratio of 3:1 and core to coating ratio of
353
1:20 was analyzed before and after the ultrasonication step of homogenization. In Table
354
2, the particle size values are given for sample with and without ultrasonication (just
355
high speed homogenization). Table 2 clearly showed that ultrasonication had a
356
significant effect on decreasing the particle size. Also it can be seen from Fig. 6 that
357
while the majority of the particles were in a range between 0.1-1 µm for ultrasonicated
358
sample, for the sample, which was homogenized only with high speed mixer, the range
359
was between 0.5-10 µm. The span of the particle size distribution curves and the SSA
360
values were also negatively correlated with particle size values (Table 2). Based on
361
these results, it can be said that ultrasonication has a positive impact on emulsification.
362
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3.4
Surface Morphology of Microcapsules
364
From different core to coating ratios and WP:MD ratios, samples having the highest
365
encapsulation efficiencies, that is the samples having core to coating ratio of 1:20 and
366
WP:MD ratio of 3:1, were analyzed. The SEM images of the samples with 500×
367
magnification are illustrated in Fig. 7. From the images it can be seen that both sample
368
with DE:13-17 (Fig. 7A) and sample with DE:4-7 (Fig. 7B) had almost evenly
369
distributed particles throughout the images. Fig. 7 also shows that, there was no
370
difference between the sizes of the particles of two samples prepared with MD having
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ACCEPTED MANUSCRIPT different DE values. This result was also in accordance with the particle size analysis of
372
emulsions, in which there is no significant difference between D32 (µm) values of the
373
emulsions. From Fig. 7, it can also be seen that both samples had smooth surfaces free
374
of cracks and dents. This lack of surface deformations could be explained by the high
375
content of whey protein concentration of wall matrices. Also it can be concluded that a
376
change in DE value did not cause a significant difference in surface morphologies of the
377
capsules.
378
In the comparison of capsules with MD having DE:13-17 (Fig. 8A) and capsules with
379
MD having DE:4-7 (Fig. 8B) under 5000× magnifications, it can be seen that powders
380
with DE:13-17 had more porous structures with more holes within the capsules. The
381
pores are the residues of entrapped rosemary oil cavitations, resulting from the
382
volatilization of the oil during the exposure. The result is also in accordance with the
383
encapsulation efficiency results of the capsules with same formulations.
384
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3.5
Storage Stability of Microcapsules
386
Rosemary essential oil coated with WP and MD at a ratio of 3:1 and core to coating
387
ratio of 1:20 were chosen for stability analysis. Experiments were performed with MD
388
having DE values of both 4.0-7.0 and 13.0-17.0. Fig. 9 shows the 1,8-cineole
389
concentrations of three samples during 40 days of storage under 35.3% relative
390
humidity. As can be seen from the figure, for all the three samples, the 1,8-cineole
391
concentrations decreased during storage as expected. This decrease of 1,8-cineole was
392
highly due to its volatilization. In the encapsulated oils, the volatilization of 1,8-cineole
393
was lower than non-encapsulated rosemary oil. The reason of this was the barrier effect
394
of the wall materials. Also, the increasing permeability of the wall material allows the
395
oxidation of the encapsulated oil over time which also decreases the 1,8-cineole
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ACCEPTED MANUSCRIPT concentration of encapsulated oil. Especially for the first 10 days of storage, the sharp
397
decrease of 1,8-cineole concentration of non-encapsulated oil shows the barrier effect of
398
the wall materials more clearly. For the first 30 days of storage, the concentrations of
399
1,8-cineole for the capsules encapsulated with MD with DE:4-7 were higher than the
400
concentrations of 1,8-cineole in capsules prepared using MD having DE:13-17. This
401
result indicated that MD with DE:4-7 achieved the retention of 1,8-cineole better than
402
DE:13-17. After 30 days of storage, the effect of different DE values on stability was
403
lost and the retention percentages of MD became almost equal. Higher retention
404
performance of MD having lower DE value has been investigated by many researchers.
405
Ersus & Yurdagel (2007) linked the worse storage stability of higher DE MD to their
406
lower molecular weight with shorter chains, which are more susceptible to structural
407
deformations than MD with low DE values. However, the main reason why lower DE
408
MD exhibited better storage stability functions was their higher glass transition
409
temperature. As DE value increased, the molecular weight of MD decreased which also
410
lowered the glass transition temperature of the MD. Because of this, during storage at
411
high relative humidity environments, MD with high DE had higher hygroscopicity
412
which led to caking and loss of volatile components (Desorby, Netto, & Labuza, 1997).
413
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414
4.
Conclusion
415
In order to obtain the best microencapsulation formulation for the encapsulation of
416
rosemary essential oil, drying efficiency and encapsulation efficiency analyses were
417
conducted in the capsules prepared with MD having two different DE values, different
418
core to coating ratios and WP:MD ratios. Among the coating formulations, WP:MD
419
ratio of 3:1 provided the highest drying and encapsulation efficiencies for both type of
420
MD and the best core to coating ratio giving higher drying and encapsulation
17
ACCEPTED MANUSCRIPT efficiencies was found to be 1:20 for both DE values. Also, MD having DE:13-17 was
422
found to yield better drying and encapsulation efficiency values as compared to MD
423
having DE:4-7. Analyses also showed that the DE value did not have any
424
morphological effect on the capsules or any particle size difference on the emulsions.
425
Lastly, GC-MS analyses showed that powders prepared with MD having DE:4-7 was
426
found to have the higher retention values of 1,8-cineole during the first 30 days of
427
storage. However, after 30 days of storage, the retention powers of MD became almost
428
equal. Thus at the end, the optimum coating material formulation was chosen to be core
429
to coating ratio of 1:20 and WP:MD ratio of 3:1 with DE value of 13-17.
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430 References
432
Baranauskiené, R., Bylaité, E., Zukauskaité, J., & Venskutonis, R. P. (2007). Flavor
433
Retention of Peppermint (Mentha piperita L.) Essential oil spray-dried in modified
434
starches during encapsulation and storage. Journal of Agricultural and Food
435
Chemistry, 3027−3036.
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Başer, K. C., & Buchbauer, G. (2010). Handbook of Essential Oils: Science,
437
Technology, and Applications. New York: CRC Press, Taylor & Francis Group.
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Bigliardi, B., & Galati, F. (2013). Innovation trends in the food industry: The case of
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Bousbia, N., Vian, M. A., Ferhat, M. A., Petitcolas, E., Meklati, B. Y., & Chemat, F.
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(2009). Comparison of two isolation methods for essential oil from rosemary leaves:
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Hydrodistillation and microwave hydrodiffusion and gravity. Food Chemistry, 355–
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362.
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functional foods. Trends in Food Science & Technology, 118-129.
Desai, K. G., & Park, H. J. (2005). Recent developments in microencapsulation of food ingredients. Drying Technology, 1361–1394.
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Desorby, S. A., Netto, F. M., & Labuza, T. P. (1997). Comparison of spray-drying,
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drum-drying and freeze-drying for b-carotene encapsulation and preservation.
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Journal of Food Science, 1158-1162. Ersus, S., & Yurdagel, U. (2007). Microencapsulation of anthocyanin pigments of black
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carrot (Daucuscarota L.) by spray drier. Journal of Food Engineering, 805–812.
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Estévez, M., Ramírez, R., Ventanas, S. & Cava, R. (2007). Sage and rosemary essential
452
oils versus BHT for the inhibition of lipid oxidative reactions in liver pâté. LWT
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Food Science and Technology. Vol 40, Issue 1, 58–65.
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Fernandes, R. V., Borges, S. V., Botrel, D. A., Silva, E. K., Gomes da Costa, J. M., &
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Figure 1 Drying efficiencies of microcapsules encapsulated with different formulations;
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( ): Capsules with MD:WP ratio of 3:1 and MD having DE:13-17, (■): Capsules with
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MD:WP ratio of 1:3 and MD having DE:13-17 ( ): Capsules with MD:WP ratio of 3:1
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and MD having DE:4-7, (■): Capsules with MD:WP ratio of 1:3 and MD having DE:4-
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7. Different letters represent significant difference (p ≤ 0.05).
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Figure 2 Encapsulation efficiencies of microcapsules encapsulated by MD with DE:13-
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17 at different core to coating ratios and different WP:MD ratios; (■): 3:1 and (■): 1:3.
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Different letters represent significant difference (p≤0.05)
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Figure 3 Encapsulation efficiencies of microcapsules encapsulated with WP and MD at
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a ratio of 3:1, different core to coating ratios and MDs having different DE values; (■):
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DE:13-17, (■): DE:4-7. Different letters represent significant difference (p ≤ 0.05)
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Figure 4 Particle size distributions of emulsions prepared with WP:MD ratio of 3:1,
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MD with DE:4-7 and different core to coating ratios; 1:40 (solid line), 1:20 (dashed
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line) and 1:10 (dotted line).
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Figure 5 Particle size distributions of emulsions prepared with WP:MD ratio of 3:1,
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MD with DE:13-17 and different core to coating ratios; 1:40 (solid line), 1:20 (dashed
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line) and 1:10 (dotted line).
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Figure 6 Particle size distributions of emulsions prepared with ultrasonic
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homogenization (solid line) and Ultra-turrax homogenization (dashed line) with
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WP:MD ratio of 3:1, MD with DE:13-17 and core to coating ratio of 1:20.
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Figure 7 Scanning Electron Microscope images (500× magnification) of microcapsules
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having WP:MD ratio of 3:1 and core to coating ratio of 1:20 prepared by MD having
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different DE values; (A): DE:13-17, (B): DE:4-7
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Figure 81 Scanning Electron Microscope images (×5000 magnification) of
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microcapsules prepared with WP:MD ratio of 3:1 and core to coating ratio of 1:20 and
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MD having different DE values; (A): DE:13-17, (B): DE:4-7
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Figure 9 1,8-cineole concentration of non-encapsulated oil (♦), capsules prepared with
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maltodextrin having DE:13-17 (■) and DE:4-7 (▲) for 40 days of storage at 33.3% RH.
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Table 1 Particle size analyses of emulsions prepared with MD having different DE
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values and different core to coating ratios with WP: MD ratio of 3:1
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Table 2 The effect of homogenization technique on the particle size results of the
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emulsion prepared by MD with DE:13-17 and core to coating ratio of 1:20.
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Table 1 Particle size analyses of emulsions prepared with MD having different DE values and
Core:Coating
D32(µm)
4-7
1:40
0.187c*
4-7
1:20
0.207b
4-7
1:10
13-17
1:40
13-17
1:20
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32.1a
5.295ab
29.0b
0.255a
3.708b
23.5c
0.188c
20.813a
31.9a
0.205b
12.423ab
29.2b
4.238b
24.4c
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Span
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Table 2 The effect of homogenization technique on the particle size results of the emulsion
High-speed homogenizer
Span
1.428
13.039
High-speed+ Ultrasonic
0.254
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Area (m2/g)
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Technique
Specific Surface
D32(µm)
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26.948
4.2
23.6
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ACCEPTED MANUSCRIPT • MD having DE:13-17 yielded better drying and encapsulation efficiency values • Core to coating ratio of 1:20 gave higher drying and encapsulation efficiency • WP:MD ratio of 3:1 provided highest drying and encapsulation efficiency values.
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• DE value had no effect on particle size of the emulsions.
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• Retention powers of both type of MD became almost equal after 40 days of storage.