- Email: [email protected]
Preparation and properties of cinnamon-thyme-ginger composite essential oil nanocapsules
Industrial Crops & Products 122 (2018) 85–92
Contents lists available at ScienceDirect
Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Preparation and properties of cinnamon-thyme-ginger composite essential oil nanocapsules
T
⁎
Jing Hu , Yudi Zhang, Zuobing Xiao, Xuge Wang School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, 201418, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Composite essential oils Nanocapsules Chitosan Ionic gelification Antibacterial activity
Essential oils (EO) as one of natural antimicrobials possess excellent antibacterial, antifungal and antioxidant properties. However, their main components are easy to be oxidated and deteriorated when EO was exposed to the oxygen, light and heat. In this study, cinnamon-thyme-ginger composite essential oil nanocapsules (CEONPs) were prepared with chitosan as the wall via ionic gelification reaction. The effect of the mass ratio of chitosan (CS) to tripolyphosphate (TPP), composite essential oils (CEO) and surfactant concentration and pH on the properties of the CEO-NPs were investigated in detail. The morphology and structures of CEO-NPs were measured by dynamic light scattering (DLS), fourier transformation infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), gas chromatography–mass spectrometry (GC–MS) and ultraviolet spectrum (UV). Meanwhile, the antibacterial property of CEO-NPs was determined by inhibition zone method. CEO-NPs with the size of 215 nm and zeta potential of 25.12 mv were obtained when the mass ratio of CS/TPP, CEO and fatty alcohol polyoxyethylene ether-9 (AEO-9) concentration and pH were 8:1, 1.8 g/L, 0.7 g/L and 5.26. FTIR demonstrated that CEO has been encapsulated into NPs. TGA showed that the thermal stability of CEO-NPs was improved compared with CEO and the loading capacity of CEO was 13.4%. GC–MS displayed that more than 90% ingredients of CEO had been encapsulated into NPs. UV indicated that CEO-NPs had the excellent sustainedrelease property. Furthermore, CEO-NPs showed a long-lasting antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus. In all, CEO-NPs can be used as a potential long-term natural preservative.
1. Introduction Recently, natural antimicrobials have attracted more attention due to the increased consumer awareness on the safety (Benjemaa et al., 2018). Essential oils (EO) are aromatic natural oily liquids obtained from flowers, leaves, fruits, stems and other part of plants, which can be prepared by steam distillation, fermentation, or by expression (Baran et al., 2007; Benelli and Pavela, 2018; Burt, 2004; Tiziana Baratta et al., 1998) and exhibit excellent antibacterial activities, antifungal and antioxidant properties. For example, cinnamon oil obtained from foliages and barks showed the antioxidant and antimicrobial activities because of the high eugenol and cinnamic aldehyde content (Yildirim et al., 2017). Thyme oil isolated from herb thyme had the strong antimicrobial activity, which could replace synthetic fungicides (Vilaplana et al., 2018; Ryu et al., 2018). Ginger oil produced by fresh ginger exhibited antibacterial and antioxidant properties (Nile and Park, 2015). However, EO is the mixture of volatile compounds including terpenes, aromatic hydrocarbons, esters, phenols and other natural substances. When EO is exposed to the environment of oxygen, light ⁎
Corresponding author. E-mail addresses: [email protected], [email protected] (J. Hu).
https://doi.org/10.1016/j.indcrop.2018.05.058 Received 11 October 2017; Received in revised form 22 May 2018; Accepted 22 May 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
and heat, most ingredients can be easily oxidated and deteriorated (Perdones et al., 2012; Carvalho et al., 2016). Encapsulation technology provided an effective approach to keep the stabilization of EO and prevent the loss of volatile ingredients (Campelo et al., 2017; Wang et al., 2018; Calvo et al., 2012). Natural polysaccharides including chitosan (Tan et al., 2018), maltodextrin (Medina-Torres et al., 2016)， cyclodextrin (da Rosa et al., 2013) and gum arabic (Binsi et al., 2017) have been widely used to encapsulate EO due to their non-toxic, biodegradability and biocompatibility (Xiao and Grinstaff, 2017; Olejnik et al., 2016; Tabart et al., 2012; Sotelo-Boyás et al., 2017; Cota-Arriola et al., 2013). Cinnamon EO was encapsulated with β-cyclodextrin by inclusion complexation method and showed the high antifungal activity against Botrytis sp (Munhuweyi et al., 2018). Simon-Brown et al. (2016) reported that ginger EO microcapsule (the particle size: 8.215.3 μm) was prepared with maltodextrin (MD) and/or gum arabic (GA) as the walls by spray drying and exhibited effective antioxidant activity to prolong food shelf-life. Especially, chitosan (CS) obtained from deacetylation of chitin, as the second most abundant polysaccharide showed the excellent
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
2.3.2. Chemical structure analysis The chemical structures of ginger EO, cinnamon EO, thyme EO, CEO-NPs after freeze-drying and CS were determined with a VERTEX 70 FTIR spectrophotometer with ATR accessory (Bruker, Ettlingen, Germany) in the range from 4000 to 600 cm−1.
synergism antimicrobial effect in combination with EOs (Gharsallaoui et al., 2007; Ali and Ahmed, 2018; Xu et al., 2017). Patchouli oil CS microcapsules with diameter in the range of 1–20 μm were prepared via complex coacervation method and displayed high antibacterial activity against Staphylococcus aureus and Escherichia coli in cotton fabrics (Liu et al., 2013). In addition, Velmurugan et al. (2017) prepared orange and lavender EO CS nanoparticles with the size of 213.6 and 273.8 nm and then applied them in leather finishing against B. cereus, B. subtilis, Aspergillus fumigatus, Macrophomina phaseolina. In our previous study, CS nanoparticles loaded with cinnamon EO (CE-NPs) with three sizes of 112 nm, 215 nm and 527 nm were prepared by ionic gelification reaction. Then these CE-NPs were applied in the package preservation of the chilled pork. CE-NPs with 527 nm led to a significant decrease of microbial growth, pH, peroxide value, 2-thiobarbituric acid and sensory scores of the pork than the other treatments at 4 °C during 15 day (Hu et al., 2015). Up to now, few studies about the encapsulation of composite essential oil (CEO) with CS as wall material have been reported. In this study, cinnamon-thyme-ginger composite essential oil nanocapsules (CEO-NPs) with CS as the wall were prepared successfully via ionic gelification method. The influence of the mass ratio of CS to TPP, CEO and surfactant content and pH on the particle size and zeta potential of CEO-NPs were investigated in detail. Then the morphology and structure of CEO-NPs were characterized by DLS, FT-IR, TGA, GC–MS and UV. Finally, the antibacterial activity of CEO-NPs against Escherichia coli, Bacillus subtilis and Staphylococcus aureus were studied.
2.3.3. Determination of CEO loading capacity and thermal stability In order to obtain the thermal stability and CEO loading capacity of CEO-NPs, CEO, CS-NPs and CEO-NPs after freeze-drying were performed on a Q5000 thermal analyzer (TA Instruments, USA) from 25 °C to 600 °C at 10 °C/ min heating rate under nitrogen atmosphere with a flow of 20 mL/min. 2.3.4. Determination of the encapsulated aromatic compounds in CEO-NPs via GC–MS In order to identify the main ingredients of CEO encapsulated in CEO-NPs, the analysis of cinnamon EO, thyme EO, ginger EO and CEONPs after-drying was performed with GC–MS (Agilent Technologies Inc., New York, USA). CEO-NPs were firstly pretreated as the followings. 0.1 g CEO-NPs after freeze-drying was diluted in 5 mL ethanol. The suspension was treated with the ultrasonication at 800 W for 30 min until the shell of CEO-NPs was completely destroyed and CEO was dissolved in the ethanol. Then, the suspension was centrifuged at 13000 rpm for 20 min and the supernatant was collected. After that, 1, 2-Dichlorobenzene of 100ug/mL as an internal standard (ISTD) was added in the solution to determine the content (%) of CEO ingredients. Meanwhile, each pure aromatic compound in the three EOs was also been determined as its standard. An Agilent 6890N gas chromatograph with a 5873N mass detection in the range 30–450 mass/charge was used with a HP-INNOWAX polar column (60 m × 0.25 mm i.d. × 0.25 μm film, Agilient). The carrier gas was ultrapurified helium at a flow rate of 1.0 mL/min. The injection volume was 0.2 μL with a spilt ratio of 20:1. The initial column temperature was held at 40 °C for 6 min, programmed to ramp to 100 °C at a rate of 3 °C/min for 2 min, then to 230 °C at a rate of 5 °C/min and held at this temperature for 20 min. The detector ion source temperature was set at 230 °C. Electron impact ionization was performed at electron energy of 70 eV. Identification of the main components of CEO was achieved by comparing mass spectra with those in the NIST08 library.
2. Materials and methods 2.1. Materials Chitosan (CS, average molecular weight = 150000) was obtained from Sigma-Aldrich, USA. Sodium tripolyphosphate (TPP), fatty alcohol polyoxyethylene ether-9 (AEO-9), glacial acetic acid (HAC) and sodium hydroxide (NaOH) were supplied by Shanghai National Chemical Reagent Co., Ltd., China. Cinnamon, thyme and ginger essential oil were purchased from ZhengzhouXomolon Food Flavor Co., Ltd., China. All chemicals were used as received without any further purification. Deionized water was used for all experiments.
2.3.5. Sustained-release property of CEO-NPs In order to study the sustained-release property of CEO-NPs, 1 g CEO-NPs after freeze-drying were placed at 40 °C for 1, 2, 3, 5, 7, 9, 12, 15, 18 days, separately. Then, these CEO-NPs were added into 20 mL dichloromethane. The other extract processes were the same as above. The volatile components in the CEO released from CEO-NPs after different placing times were determined by UV–vis spectrum (UV2100, Unico, shanghai). (E)-cinnamaldehyde and thymol were selected as the model compounds and determined at 286 nm and 275 nm, respectively.
2.2. Preparation of CEO-NPs CEO-NPs were similarly obtained according to the previously described method (Xiao et al., 2014a,b; Hu et al., 2015). Typically, CEO was obtained by blending three EOs (the mass ratio of cinnamon EO, thyme EO and ginger EO: 4:3:3). CEO (0.09 g) was mixed with AEO-9 (0.035 g) under 1000 rpm stirring for 5 min at 25 °C. Then the mixture was added into 15 mL TPP aqueous solution to form O/W emulsion under the homogenization for 10 min. After that, 35 mL CS aqueous solution was added into the above emulsion to keep the weight ration of CS/TPP 8/1 with a stirring speed of 400 rpm at 30 °C. Meanwhile, the pH of the emulsion was controlled to 5.26 with 1 mol/L sodium hydroxide aqueous solution and the reaction was kept for 2 h. Finally, CEO-NPs were purified by the centrifugation at 15 000 rpm for 30 min at 5 °C. CS-NPs were also prepared as the above process without CEO.
2.4. Antibacterial experiment Antibacterial activities of CEO, CS-NPs, CEO-NPs against Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 25923) were carried out by the zone of inhibition method. Bacterial strains were cultured overnight at 37 °C in the nutrient agar plates. The nutrient agar medium in a Petri dish was inoculated with 0.1 mL 107–108 cfu/mL bacteria. The sterile filter paper discs (6 mm diameter) were impregnated with 1 mL CEO and aqueous dispersions of CEO-NPs and CS-NPs (0.2 g/mL) and dried for 5 min by an ultraviolet lamp. Meanwhile, gentamicin sulfate and sterile saline were tested as the positive and negative controls, respectively. The dishes were incubated at 37 °C for 1, 5, 7 and 9 days. The diameter of the inhibition zones around each of the discs was taken as measure of the antimicrobial activity. Each experiment was carried out 6 times and the mean diameter of the inhibition zone was measured.
2.3. CEO-NPs characterization 2.3.1. Particle size and zeta potential The particle size, polydispersity index (PDI) and zeta potential of CEO-NPs in three replicates were measured by the Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Each sample was determined using a solid state He-Ne laser of 633.0 nm at 25 °C with an angle detection of 90 °C. 86
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
Fig. 2. Effect of CEO concentration on the particle size and zeta potential of CEO-NPs.
Fig. 1. Effect of the mass ratio of CS to TPP on the particle size and zeta potential of CEO-NPs.
2.5. Statistical analysis All tests were performed in triplicate, and the experiments data were averaged. Variances of each value in each group (ANOVA) were analyzed with the Duncan test of the SPSS (Version 17.0 for Windows) (Hu et al., 2015). Correlations between variables (P < 0.05) were determined by correlation analysis using Pearson's linear correlation coefficient.
3. Results and discussion 3.1. The effect of the mass ratio of CS to TPP Fig. 1 demonstrates that the influence of the mass ratio of CS to TPP on the particle size and zeta potential of CEO-NPs. With the increase of CS/TPP mass ratio, the mean diameter of CEO-NPs increased firstly and then decreased. When the mass ratio of CS to TPP was 8:1, the size and particle size distribution (PDI) of CEO-NPs were minimum (215 nm and 0.26) as shown in Fig. S1. This phenomenon was different from the previous study (Xiao et al., 2014a,b). Generally the higher the CS content, the larger the size of CS-NPs. However, during this experiment the intermolecular hydrogen bond formed among CS molecules was easy to keep the ionic gelation reaction between CS and TPP (Fan et al., 2012). Meanwhile, the improved electrostatic repulsions between CEONPs effectively maintained their stability due to the enhancement of the positively charged amino groups of CS (Rodrigues et al., 2012; Janes et al., 2001). Furthermore, with CS/TPP mass ratio rising, the zeta potential of CEO-NPs increased. This is attributed to that the more positive charge CS molecules can produce many free amino groups on the surface of CEO-NPs.
Fig. 3. Effect of AEO-9 concentration on the particle size and zeta potential of CEO-NPs.
3.3. The effect of AEO-9 concentration Fig. 3 displays the effect of different AEO-9 concentrations on the size and zeta potential of CEO-NPs. Both the mean size and zeta potential of CEO-NPs mainly decreased with AEO-9 concentration increased. A stable emulsion can be obtained with AEO-9 as emulsifier dispersed between the interface of water and oil droplets (Hu et al., 2015; Sadeghi and Fayazi, 2012; Zhang et al., 2017). Many emulsifiers are useful for the dissolution of CEO into the micelles to form small micelles. Simultaneously, CEO-NPs with the small size can be prepared by the complex coacervation interactions between the positively charged protonated amino groups on CS and the negatively charged phosphate groups on TPP.
3.2. The effect of CEO concentration 3.4. The effect of pH The effect of different CEO concentrations on the particle size and zeta potential of CEO-NPs is indicated in Fig. 2. When CEO concentration increased from 0.8 to 1.8 g/L, the size and zeta potential of CEO-NPs had no significant change. Especially the average size of CEONPs was the smallest with 1.8 g/L CEO. For the reason is that CS can completely encapsulate the dispersed CEO droplets. Then with CEO concentration increasing, the average size of CEO-NPs enhanced, but their zeta potentials decreased. This is attributed to that more CEO dissolved into the micelles led to the size enhancement of CEO-NPs. As well, the excessive CEO could adhere on the surface of CEO-NPs, which could reduce the surface charges of NPs and cause their aggregation.
The influence of pH on the particle size and zeta potential of CEONPs is shown in Fig. 4. CS is an alkaline polysaccharide, insoluble in neutral or alkaline condition (Benavides et al., 2016). The protonation degree of CS was significantly affected by pH of the solution (Shu and Zhu, 2002). Thus, pH value has significant effect on both zeta-potential and particle size of CEO-NPs. With pH increased, the zeta potential of CEO-NPs was decreased due to the reduced protonation degree of CS molecules. When the pH value ranged from 3.5 to 5.26, the particle size of CEO-NPs decreased gradually. After that, their sizes increased with pH rising. pH could affect the ionic cross-linking process of CS NPs, which belongs to the pH sensitive characters of the whole 87
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
Fig. 7. TG and DTG curves of CS-NPs (a, d), CEO-NPs (b, e), CEO (c, f). Fig. 4. Effect of pH on the particle size and zeta potential of CEO-NPs.
3.5. Particle size and particle size distribution Fig. 5 showed the particle size and PDI of the CEO-NPs. The average particle size and PDI of CEO-NPs were 215 nm and 0.26, respectively. CEO can be emulsified in the micelles formed by AEO-9. The positively charged CS can be interacted with the negatively charged TPP via the complex coacervation to encapsulate CEO. 3.6. Chemical structure analysis Fig. 6 displays the differences among the structures of CS(a), ginger EO(b), thyme EO(c), cinnamon EO(d) and CEO-NPs(e). In the FTIR spectrum of CS, the absorption peaks at 2824 cm−1 and 1593 cm−1 were attributed to the stretching vibration of CeH groups and the deformation vibration absorption peak of −NH2, respectively. The absorption peak at 1079 cm−1 corresponded to the stretching vibration of the CeO group of CS. Generally, cinnamon, thyme and ginger EO contain aldehydes, phenols, and ketone monomers (Yu et al., 2017; Blaise et al., 1997). The obvious adsorption peaks of cinnamon EO appeared at 1678 cm−1, 1626 cm−1, 1450 cm−1, 1289 cm−1, which corresponded to the stretching vibration of C]O, C]C, eOH of aromatic compounds. The strong peaks at 748 cm−1 and 689 cm−1 were attributed to benzene rings CeH vibration absorption and the vibration absorption of alkenes, respectively. In the spectrum of thyme EO, the peak at 3001 cm−1 was associated with the stretching vibration of CeH in benzene ring. The absorption peaks at 1619 cm−1, 1583 cm−1,1515 cm−1,1458 cm−1 were four unequal intensity ones, which corresponded to the characteristic peaks of thymol. The peaks at 1429 cm−1and 1226 cm−1 were attributed to the bending vibration of −OH and CeO. The main components of ginger EO were terpene compounds. The absorption peaks at 1708 cm−1, 1608 cm−1 were associated to C]C and C]CeC]C stretching vibration. The peaks at 2927 cm−1 and 2858 cm−1 were affected by the bending vibrations of −CH2 and −CH3 groups, respectively. In the FTIR spectrum of CEONPs, the characteristic absorption peaks of cinnamon, thyme and ginger EO all appeared. The vibration absorption peak of amino group was shifted to1675 cm−1, and the new absorption peaks at 1533 cm−1 appeared, related to the cross-linking between the phosphate groups of TPP and amino groups of CS(Yang et al., 2009). Meanwhile, the absorption peaks at 1137 cm−1 and 880 cm−1 corresponded to the vibration and flexural vibration of P = O and P-O. In all, the above results indicated that the CEO can be successfully encapsulated into CEO-NPs.
Fig. 5. Particle size distribution of CEO-NPs.
Fig. 6. FTIR of CS(a), ginger EO(b), thyme EO(c), cinnamon EO(d) and CEONPs(e).
process (Perdones et al., 2016). When pH was low, the complex coacervation interactions between the positively charged CS and the negatively charged TPP are easy to be processed. Otherwise, the reduced protonation degrees of CS molecules weaken the complex coacervation reaction, which could lead to the aggregation of CEO-NPs.
3.7. CEO loading capacity and thermal stability The TGA results of CS-NPs (a), CEO-NPs (b) and CEO(c) are shown in Fig. 7. There were two thermal decomposition stages for CEO. The first weight loss (74.23%) from 106 °C to 155 °C corresponded to the decomposition of many aromatic compounds with the low boiling point 88
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
Table 1 Composition of three kinds of EOs and CEO-NPs identified by GC–MS. Compound
α-Pinene Camphene Hexanal Phellandrene β-Myrcene (+)-4-Carene D-Limonene Eucalyptol γ-Terpinene o-Cymene α-Terpinolene Diacetone alcohol 2-Furanmethanol α-ylangene α-Copaene Decanal Benzaldehyde 2-Nonanol Linalyl acetate β-Linalool Isobornyl acetate (R)-Terpinen-4-ol Patchoulene Aromandendrene β-Terpineol Alloaromadendrene Cis-β-Farnesene Isoborneol Humulene Cis-α-Bisabolene β-Curcumene L-α-Terpineol Endo-Borneol γ-Muurolene Zingiberene β-Bisabolene α-Muurolene Citral β-Panasinsene α-Farnesene Geranyl acetate Copaene β- Sesquiphellandrene 2-phenylethyl ester Anethole Geraniol Trans-Calamenene γ-Elemene Benzenemethanol Phenylethyl Alcohol Caryophyllene oxide Nerolidol (E)-Cinnamaldehyde γ-eudesmol (−)-Spathulenol Cinnamyl acetate Eugenol Thymol Methyleugenol γ-Muurolene Carvacrol α-Bisabolol α-Cadinol Trans-Isoeugenol Isoaromadendrene epoxide Coumarin Benzyl Benzoate Total Monoterpene hydrocarbon Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes
Relative content (%) RIa
RIb
Cinnamon
Thyme
Ginger
CEO-NPs
1019 1062 1085 1166 1170 1181 1199 1210 1249 1275 1285 1368 1448 1491 1500 1505 1539 1518 1548 1573 1598 1612 1619 1624 1637 1664 1670 1680 1688 1689 1702 1705 1713 1730 1736 1738 1740 1746 1751 1755 1764 1767 1786 1833 1847 1849 1855 1857 1890 1927 2019 2043 2072 2135 2148 2175 2189 2192 2199 2202 2225 2233 2256 2276 2281 2429 2485
1012 1075 1084 1160 1173 1149 1196 1210 1243 1268 1288 1352 1428 1489 1500 1502 1530 1510 1555 1581 1582 1616 1639 1637 1624 1657 1663 1672 1682 1701 1712 1690 1715 1725 1726 1729 1738 1733 1746 1752 1759 1519 1783 1806 1847 1847 1850 1651 1875 1931 2008 2042 2084 2130 2152 2174 2186 2198 2030 1701 2225 2232 2255 2309 2291 2437 2655
0.13 0.02 — 0.02 — — 0.88 0.52 0.02 0.95 0.02 0.03 0.02 0.04 0.38 — 1.60 — 2.39 2.19 — 1.13 — 0.13 — — — 0.18 0.05 — 0.28 0.34 — — — 0.43 — — — 0.18 — — 0.51 1.35 — — — — 0.04 0.76 — 0.47 64.94 — 0.16 10.78 4.30 1.66 — — — — — 0.22 — 0.52 0.22 97.86 1.09 4.36 2.00 0.63
0.79 0.07 — 0.11 0.70 1.24 2.99 14.96 1.09 24.60 0.88 — — — 0.14 — — — 3.50 1.09 0.35 13.76 0.16 0.53 0.09 0.21 — 0.40 — — — 1.96 0.75 — — — 0.14 — — — — — — — 0.09 — 0.21 — 0.39 — 0.47 — 0.33 — 0.18 — — 23.15 — — 4.28 — — — — — — 99.61 7.87 33.01 1.39 0.65
0.66 1.77 1.34 4.25 — 0.14 0.82 1.45 — 1.24 0.10 — — — 0.92 1.65 — 0.10 — 0.08 — 0.83 — — — 0.41 0.70 0.22 — 0.14 0.49 0.94 1.14 1.39 37.72 11.17 — 0.92 1.68 6.31 0.17 0.54 14.96 — — 0.20 — 0.43 — — — 0.66 0.87 0.14 — — — — 0.09 0.09 1.17 0.10 0.14 0.15 0.65 — — 98.94 7.74 5.03 76.95 1.69
— 0.08 0.21 1.12 — 0.18 1.92 3.53 1.10 6.86 0.43 0.31 — — 0.14 — — — 2.12 1.23 — 2.01 — 0.21 0.09 0.18 — 0.63 — 0.43 0.53 1.54 0.72 — 10.91 1.53 — — 0.10 1.47 — — 3.87 — — — — — — — — 0.12 30.79 — — 2.44 1.65 15.20 — — 0.94 — — — — — — 94.59 4.83 9.75 19.37 0.12
(continued on next page) 89
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
Table 1 (continued) Compound
Relative content (%) RIa
RIb
Aromatic compounds
Cinnamon
Thyme
Ginger
CEO-NPs
89.78
56.69
7.53
60.52
Ra: based on a homologous series of normal alkanes. Rb: Literature retention indices.
LC: CEO loading capacity of CEO-NPs Fig. 7
3.8. CEO encapsulation and sustained-release property Table 1 shows the chemical compositions of cinnamon EO, thyme EO, ginger EO and CEO encapsulated in CEO-NPs. 36 components were identified from cinnamon EO by GC–MS representing 97.86% of the total EO. Cinnamon EO consisted of aromatic compounds (89.78%), monoterpoids (4.36%), sesquiterpene hydrocarbons (2.0%), monoterpene hydrocarbons (1.09%) and sesquiterpenoids (0.63%). The major components of cinnamon EO were (E)-Cinnamaldehyde (64.94%), cinnamyl acetate (10.782%) and eugenol (4.30%). In thyme EO there were identified 31 compounds, which represents 99.61% of the total EO. The main compounds were o-cymene (24.60%) followed by thymol (23.15%), eucalyptol (14.96%) and (R)-terpinen-4-ol (13.76%). Besides, 33 compounds were identified from ginger EO by GC–MS representing 98.94% of the total oil. Sesquiterpene hydrocarbons (76.95%) occupied the highest proportion among all the components. Especially, zingiberene (37.72%), β-sesquiphellandrene (14.96%), and β-Bisabolene (11.17%) were the major components of ginger EO. The chemical components of three kinds of EO were similar with those of the previous studies (Li et al., 2013; Lu et al., 2011; Noori et al., 2018). CEO was obtained by blending cinnamon EO, thyme EO and ginger EO according to the mass ratio of 4/3/3. When CEO was encapsulated into CEO-NPs, there were 33 compounds identified from CEO by GC–MS representing 94.59% of the total CEO. The encapsulated aromatic ingredients included aromatic compounds (60.52%), monoterpoids (9.75%), sesquiterpene hydrocarbons (19.37%), monoterpene hydrocarbons(4.83%) and sesquiterpenoids (0.12%). The main compounds were (E)-cinnamaldehyde (30.794%), thymol(15.20%), zingiberene(10.91%), o-Cymene(6.858%) and β-Sesquiphellandrene (3.87%). Compared with the pure cinnamon EO, thyme EO and ginger EO, more than 90% aromatic ingredients in CEO have been encapsulated into NPs. Table 1 In order to reflect the sustained-release property of aromatic components encapsulated in CEO-NPs, (E)-cinnamaldehyde and thymol were selected as the model fragrances to study their suatined-release form CEO-NPs during 18 days. Fig. 8 displays the release comparison of
Fig. 8. Release profiles of (E)-cinnamaldehyde and thymol from CEO-NPs.
in CEO. The second weight loss occurred from 155 to 269 °C, which was due to those with the high boiling point such as zingiberene, cinnamaldehyde, thymol and so on. The weight losses of CS-NPs and CEONPs below 100 °C were 3.2% and 4.1% respectively, due to the vaporization of residual moisture in NPs. For CS-NPs, the main weight loss (46.7%) occurred at 160–360 °C due to the depolymerization and decomposition of glucosamine of CS. The main weight loss of CEO-NPs was observed at 160–460 °C. The first weight loss of 160–270 °C was ascribed to the thermal decomposition of CS structure and some aromatic components encapsulated in CEO-NPs. The second weight loss occurred from 270 to 460 °C, which was due to the thermal decomposition of other aromatic components with the high boiling point encapsulated in CEO-NPs. The thermal stability of CEO encapsulated in NPs was enhanced. The total weight loss of CEO-NPs was 53.2%. Based on the same quality of CS in CEO-NPs and CS-NPs, the CEO LC was calculated as 13.4% according to Eq. (1) (Xiao et al., 2014a,b).
We − LC Wc = 1 − W1 − LC 1 − W2
(1)
We: CEO-NPs weight loss (%); Wc：CS-NPs weight loss (%); W1:and W2: CS-NPs and CEO-NPs weight loss (%), respectively; Table 2 Antimicrobial activity of CEO-NPs. Attribute
Escherichia coli
Bacillus subtilis
Staphylococcus aureus
Treatment
CEO CS-NPs CEO-NPs CEO CS-NPs CEO-NPs CEO CS-NPs CEO-NPs
Different days (mm) 1
5
7
9
16.87 ± 0.42dz 5.77 ± 0.35cdx 11.56 ± 0.71ay 14.32 ± 1.00dz 7.35 ± 1.32dx 11.66 ± 0.40by 17.02 ± 0.59dz 8.21 ± 1.11dx 12.26 ± 1.00by
7.22 ± 1.11cy 4.65 ± 0.45cx 11.89 ± 0.11bz 6.58 ± 1.00cy 5.68 ± 0.58cx 12.98 ± 0.71cz 5.69 ± 0.55cx 6.21 ± 0.44cy 13.56 ± 0.45dz
1.23 ± 0.36bx 3.11 ± 0.22by 12.02 ± 0.41cz 0.65 ± 0.11bx 2.98 ± 0.31by 12.51 ± 0.49cz 0.23 ± 0.05bx 2.12 ± 0.57by 12.99 ± 0.62cz
0.00 ± 0.11ax 0.98 ± 0.78ay 11.02 ± 0.76az 0.00 ± 0.14ax 0.25 ± 0.05ay 10.77 ± 0.98az 0.00 ± 0.08ax 0.18 ± 0.11ay 11.21 ± 0.68az
Different letters (a, b, c, and d) in the same row indicate differences among mean values, P < 0.05. Different letters(x, y, and z)in the same column indicate differences among mean values, P < 0.05. 90
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
(E)-cinnamaldehyde and thymol from CEO-NPs. (E)-cinnamaldehyde and thymol released 38.05% and 26.01% during the first 7 day quickly, and then they reached equilibrium in 9 day. Finally, the cumulative released amount of (E)-cinnamaldehyde and thymol were 38.77% and 27.67% at 18 day. This demonstrated that CEO-NPs had the excellent sustained-release property.
and National key R & D projects (2016YFA0200300) were appreciated.
3.9. Antibacterial activity
References
The antibacterial activity of CEO, CS-NPs and CEO-NPs against Escherichia coli, Bacillus subtilis and Staphylococcus aureus was assessed by inhibition zones as shown in Table 2. Initially, all of CEO, CS-NPs and CEO-NPs produced obvious inhibition zones against the tested bacteria for 1 day incubation. CEO showed the highest inhibition zone following by CEO-NPs and CEO in this order. To specific, the inhibition zone diameters of CEO against Escherichia coli, Bacillus subtilis and Staphylococcus aureus were 16.87 ± 0.42 mm, 14.32 ± 1.00 mm and 17.02 ± 0.59 mm for 1 day incubation, respectively. Then the diameters of the inhibition zone of CEO and CS-NPs were decreased with the incubation time delayed. Especially, CEO produced no observable inhibition zones against three tested bacteria at 9 day. However, the inhibition zone sizes of CEO-NPs against all the bacteria were no any observable decrease after 9 day incubation. The inhibition zone diameters of CEO-NPs against Escherichia coli were 11.56 ± 0.71 mm for 1 day, 11.89 ± 0.11 mm for 5 day, 12.02 ± 0.41 mm for 7 day and 11.02 ± 0.76 mm for 10 day, respectively. And the inhibition zone diameters of CEO-NPs against Bacillus subtilis were 11.66 ± 0.40 mm for 1 day, 12.98 ± 0.71 mm for 5 day, 12.51 ± 0.49 mm for 7 day and 10.77 ± 0.98 mm for 9 day, respectively. Meanwhile, the inhibition zone diameters of CEO-NPs against Staphylococcus aureus were 12.26 ± 1.00 mm for 1 day, 13.56 ± 0.45 mm for 5 day, 12.99 ± 0.62 mm for 7 day and 11.21 ± 0.68 mm for 9 day, respectively. This demonstrated that CEO-NPs possessed the long-term antimicrobial effect. CEO was obtained by mixing cinnamon EO, thyme EO and ginger EO. According to the above GC–MS results, many aromatic ingredients in CEO such as (E)-cinnamaldehyde and thymol displayed the excellent antimicrobial activity against a variety of bacteria (Guarda et al., 2011; Ultee et al., 2002; Wang et al., 2018). CS has a certain antibacterial activities (Yu et al., 2017). Thus, the synergistic effect of both of the encapsulated aromatic ingredients in CEO-NPs and CS enhanced the antimicrobial activity of CEO-NPs. It can be seen that CEO-NPs have a long lasting antibacterial activity on Escherichia coli, Bacillus subtilis and Staphylococcus aureus due to CEO-NPs can gradually release the antimicrobial active substances in CEO (Giner et al., 2012).
Ali, A., Ahmed, S., 2018. A Review on chitosan and its nanocomposites in drug delivery. Int. J. Biol. Macromol. 109, 273–286. Baran, S., Reuss, W.A., von. Könige, S.H., Kalemba, D., 2007. Composition of the essential oil of Abies koreana Wils. Flavour Fragr. J. 22, 78–83. Benavides, S., Cortés, P., Parada, J., Wendy, F., 2016. Development of alginate microspheres containing thyme essential oil using ionic gelation. Food Chem. 204, 77–83. Benelli, G., Pavela, R., 2018. Beyond mosquitoes—essential oil toxicity and repellency against bloodsucking insects. Ind. Crops Prod. 117, 382–392. Benjemaa, M., Neves, M.A., Falleh, H., Isoda, H., Ksour, R., Nakajima, M., 2018. Nanoencapsulation of thymus capitatus essential oil: formulation process, physical stability characterization and antibacterial efficiency monitoring. Ind. Crops Prod. 113, 414–421. Binsi, P.K., Nayak, N., Sarkar, P.C., Jeyakumari, A., Ashraf, P.M., Ninan, G., Ravishankar, C.N., 2017. Structural and oxidative stabilization of spray dried fish oil microencapsulates with gum arabic and sage polyphenols: characterization and release kinetics. Food Chem. 219, 158–168. Blaise, O., Simardb, R.E., Holley, R.A., Piette, G.J.P., Bégina, A., 1997. Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms. Int. J. Food Microbiol. 37, 155–162. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in. foods—a review. Int. J. Food Microbiol. 94, 223–253. Calvo, P., Castaño, Á.L., Lozano, M., González-Gómez, D., 2012. Influence of the microencapsulation on the quality parameters and shelf-life of extra-virgin olive oil encapsulated in the presence of BHT and different capsule wall components. Food Res. Int. 45, 256–261. Campelo, P.H., do Carmo, E.L., Zacarias, R.D., Yoshida, M.I., Ferraz, V.P., Fernandes, R.V.d.B., Botrel, D.A., Borges, S.V., 2017. Effect of dextrose equivalent on physical and chemical properties of lime essential oil microparticles. Ind. Crops Prod. 102, 105–114. Carvalho, I.T., Estevinho, B.N., Santos, L., 2016. Application of microencapsulated essential oils in cosmetic and personal healthcare products-a review. Int. J. Cosmetic Sci. 38, 109–119. Cota-Arriola, O., Cortez-Rocha, M.O., Burgos-Hernandez, A., Ezquerra-Brauer, G.M., Plascencia-Jatomea, M., 2013. Controlled release matrices and micro/nanoparticles of chitosan with antimicrobial potential: development of new strategies for microbial control in agriculture. J. Sci. Food. Agric. 93, 1525–1536. Fan, W., Yan, W., Xu, Z.S., Ni, H., 2012. Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique. Colloids Sur. B: Biointerfaces 90, 21–27. Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A., Saurel, R., 2007. Applications of spray-drying in microencapsulation of food ingredients: an overview. Food Res. Int. 40, 1107–1121. Giner, M.J., Vegara, S., Funes, L., Martí, N., Saura, D., Micol, V., Valero, M., 2012. Antimicrobial activity of food-compatible plant extracts and chitosan against naturally occurring micro-organisms in tomato juice. J. Sci. Food. Agric. 92, 1917–1923. Guarda, A., Rubilar, J.F., Miltz, F., Galotto, M.J., 2011. The antimicrobial activity of microencapsulated thymol and carvacrol. Int. J. Food Micro. 146, 144–150. Hu, J., Wang, X.G., Xiao, Z.B., Bi, W.C., 2015. Effect of chitosan nanoparticles loaded with cinnamon essential oil on the quality of chilled pork. LWT-Food Sci. Technol. 63, 519–526. Janes, K.A., Calvo, P., Alonso, M.J., 2001. Polysaccharide colloidal particles as delivery systems for macromolecules. Adv. Drug Deliv. Rev. 47, 83–97. Li, Y.Q., Kong, D.X., Wu, H., 2013. Analysis and evaluation of essential oil components of cinnamon barks using GC?MS and FTIR spectroscopy. Ind. Crops Prod. 41, 269–278. Liu, J., Liu, C., Liu, Y., Chen, M., Hu, Y., Yang, Z., 2013. Study on the grafting of chitosan–gelatin microcapsules onto cotton fabrics and its antibacterial effect. Colloids Sur. B: Biointerfaces. 109, 103–108. Lu, F., Ding, Y.C., Ye, X.Q., Ding, Y.T., 2011. Antibacterial effect of cinnamon oil combined with thyme or clove oil. Agr. Sci. in China. 10, 1482–1487. Medina-Torres, L., Santiago-Adame, R., Calderas, F., Gallegos-Infante, J.A., GonzálezLaredo, R.F., Rocha-Guzmán, N.E., Núñez-Ramírez, D.M., Bernad-Bernad, M.J., Manero, O., 2016. Microencapsulation by spray drying of laurel infusions (Litsea glaucescens) with maltodextrin. Ind. Crops Prod. 90, 1–8. Munhuweyi, K., Caleb, O.J., Van Reenen, A.J., Opara, U.L., 2018. Physical and antifungal properties of b-cyclodextrin microcapsules and nanofibre films containing cinnamon and oregano essential oils. LWT-Food Sci. Technol. 87, 413–422. Nile, S.H., Park, S.W., 2015. Chromatographic analysis, antioxidant, anti-inflammatory, and xanthine oxidase inhibitory activities of ginger extracts and its reference compounds. Ind. Crops Pro. 70, 238–244. Noori, S., Zeynali, F., Almasi, H., 2018. Antimicrobial and antioxidant efficiency of nanoemulsion-based edible coating containing ginger (zingiber officinale) essential oil and its effect on safety and quality attributes of chicken breast fillets. Food Control. 84, 312–320. Olejnik, A., Kowalska, K., Olkowicz, M., Juzwa, W., Dembczynski, R., Schmidt, M., 2016.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.05.058.
4. Conclusion CEO-NPs with the size of 215 nm and zeta potential of 25.12 mv were prepared by ionic gelification reaction. The sizes and zeta potentials of CEO-NPs were strongly dependent on the mass ratio of CS to TPP, pH, as well as AEO-9 and EO concentration. TGA illustrated that CEO loading capacity of CEO-NPs was 13.4% and their thermal stability was significantly improved in contrast to that of pure essential oil. GC–MS displayed that 33 identified compounds in CEO had been encapsulated into NPs. UV showed that the cumulative released amount of (E)-cinnamaldehyde and thymol in CEO were 38.77% and 27.67% at 18 day, which demonstrated that CEO-NPs had the excellent sustainedrelease property. Moreover, CEO-NPs showed a long-lasting antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus. Acknowledgements The financial supports from National Nature Science Foundation of China (51773116), Shanghai Natural Science Foundation (17ZR1429800), Shanghai Capacity-building Project (18090503500) 91
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
essential for action against the food-borne pathogen bacillus cereus. Appl. Environ. Microbiol. 68, 1561–1568. Velmurugan, P., Ganeshan, V., Nishter, N.F., Jonnalagadda, R.R., 2017. Encapsulation of orange and lavender essential oils in chitosan nanospherical particles and its application in leather for aroma enrichment. Sur. Interfaces. 9, 124–132. Vilaplana, R., Pazmiño, L., Valencia-Chamorro, S., 2018. Control of anthracnose, caused by colletotrichum musae, on postharvest organic banana by thyme oil. Postharvest Biol. Technol. 138, 56–63. Wang, J.G., Li, Y.Z., Gao, Y.F., Xie, Z.F., Zhou, M.H., He, Y.Y., Wu, H., Zhou, W.Y., Dong, X.M., Yang, Z.H., Yang, H., 2018. Cinnamon oil-loaded composite emulsion hydrogels with Lantibacterial activity prepared using concentrated emulsion templates. Ind. Crops Prod. 112, 281–289. Xiao, R., Grinstaff, M.W., 2017. Chemical synthesis of polysaccharides and polysaccharide mimetics. Prog. Polym. Sci. 74, 78–116. Xiao, Z.B., Tian, T., Hu, J., Wang, M.X., Zhou, R.J., 2014a. Preparation and characterization of chitosan nanoparticles as the delivery system for tuberose fragrance. Flavour Fragr. J. 29, 22–34. Xiao, Z.B., Liu, W.L., Zhu, G.Y., Zhou, R.J., Niu, Y.W., 2014b. Production and characterization of multinuclear microcapsules encapsulating lavender oil by complex coacervation. Flavour Fragr. J. 29, 166–172. Xu, W.R., Xie, W.J., Huang, X.Q., Chen, X., Huang, N., Wang, X., Liu, J., 2017. The graphene oxide and chitosan biopolymer loads TiO2 for antibacterial and preservative research. Food Chem. 221, 267–277. Yang, W.J., Fu, J., Wang, T., He, N.Y., 2009. Chitosan/sodium tripolyphosphate nanoparticles: preparation, characterization and application as drug carrier. J. Biomed. Nanotechnol. 5, 591–595. Yildirim, S.T., Oztop, M.H., Soyer, Y., 2017. Cinnamon oil nanoemulsions by spontaneous emulsification: formulation, characterization and antimicrobial activity. LWT-Food Sci. Technol. 84, 122–128. Yu, D.W., Xu, Y.S., Jiang, Q.X., Xia, W.S., 2017. Effects of chitosan coating combined with essential oils on quality and antioxidant enzyme activities of grass carp (ctenopharyngodon idellus) fillets stored at 4 °C. Int. J. Food Sci.&Technol. 52, 404–412. Zhang, H.C., Jun, J.Y., Zhao, Y.Y., 2017. Preparation and characterization of cellulose nanocrystals films incorporated with essential oil loaded β-chitosan beads. Food Hydrocol. 69, 164–172. da Rosa, C.G., Borges, C.D., Zambiazi, R.C., Nunes, M.R., Benvenutti, E.V., da Luz, S.R., D’Avila, R.F., Rutz, J.K., 2013. Microencapsulation of gallic acid in chitosan, β-cyclodextrin and xanthan. Ind. Crops Prod. 46, 138–146.
A gastrointestinally digested ribes nigrum L. fruit extract inhibits inflammatory response in a co-culture model of intestinal caco-2cells and RAW264.7 macrophages. J. Agric. Food Chem. 64, 7710–7721. Perdones, A., Sánchez-González, L., Chiralt, A., Vargas, M., 2012. Effect of chitosan–lemon essential oil coatings on storage-keeping quality of strawberry. Postharvest Biol. Technol. 70, 32–41. Perdones, Á., Chiralt, A., Vargas, M., 2016. Properties of film-forming dispersions and films based on chitosan containing basil or thyme essential oil. Food Hydrocol. 57, 271–279. Rodrigues, S., da Costa, A.M., Grenha, A., 2012. Chitosan/carrageenan nanoparticles: effect of cross-linking with tripolyphosphate and charge ratios. Carbohydr. Poly. 89, 282–289. Ryu, V., McClements, D.J., Corradini, M.G., McLandsborough, L., 2018. Effect of ripening inhibitor type on formation, stability, and antimicrobial activity of thyme oil nanoemulsion. Food Chem. 245, 104–111. Sadeghi, F., Fayazi, A., 2012. Analysis of crystalline structure of sodium tripolyphos phate: effect of pH of solution and calcination conditions. Ind. Eng. Chem. Res. 51, 1093–1098. Shu, X.Z., Zhu, K.J., 2002. The influence of multivalent phosphate structure on the properties of ionically cross-linked chitosan films for controlled drug release. Eur. J. Pharm. Biopharm. 54, 235–243. Simon-Brown, K., Solval, K.M., Chotiko, A., Alfaro, L., Reyes, V., Liu, C., Dzandu, B., Kyereh, E., Goldson Barnaby, A., Thompson, I., Xu, Z., Sathivel, S., 2016. Microencapsulation of ginger (zingiber officinale) extract by spray drying technology. LWT − Food Sci. Technol. 70, 119–125. Sotelo-Boyás, M., Correa-Pacheco, Z., Bautista-Baños, S., Corona-Rangel, M.L., 2017. Physicochemical characterization of chitosan nanoparticles and nanocapsules incorporated with lime essential oil and their antibacterial activity against food-borne pathogens. LWT − Food Sci. Technol. 77, 15–20. Tabart, J., Franck, T., Kevers, C., Pincemail, J., Serteyn, D., Defraigne, J.O., Dommes, J., 2012. Antioxidant and anti-inflammatory activities of Ribes nigrum extracts. Food Chem. 131, 1116–1122. Tan, P.Y., Tan, T.B., Chang, H.W., Tey, B.T., Chan, E.S., Lai, O.M., Baharin, B.S., Nehdi, I.A., Tan, C.P., 2018. Effects of storage and yogurt matrix on the stability of tocotrienols encapsulated in chitosan-alginate microcapsules. Food Chem. 241, 79–85. Tiziana Baratta, M., Damien Dorman, H.J., Deans Stanley, G., Cristina Figueiredo, G., Barroso Jose, G., Ruberto, G., 1998. Antimicrobial and antioxidant properties of some commercial essential oils. Flavour Fragr. J. 13, 235–244. Ultee, A., Bennik, M.H.J., Moezelar, R., 2002. The phenolic hydroxyl group of carvacrol is
92
Contents lists available at ScienceDirect
Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Preparation and properties of cinnamon-thyme-ginger composite essential oil nanocapsules
T
⁎
Jing Hu , Yudi Zhang, Zuobing Xiao, Xuge Wang School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, 201418, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Composite essential oils Nanocapsules Chitosan Ionic gelification Antibacterial activity
Essential oils (EO) as one of natural antimicrobials possess excellent antibacterial, antifungal and antioxidant properties. However, their main components are easy to be oxidated and deteriorated when EO was exposed to the oxygen, light and heat. In this study, cinnamon-thyme-ginger composite essential oil nanocapsules (CEONPs) were prepared with chitosan as the wall via ionic gelification reaction. The effect of the mass ratio of chitosan (CS) to tripolyphosphate (TPP), composite essential oils (CEO) and surfactant concentration and pH on the properties of the CEO-NPs were investigated in detail. The morphology and structures of CEO-NPs were measured by dynamic light scattering (DLS), fourier transformation infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), gas chromatography–mass spectrometry (GC–MS) and ultraviolet spectrum (UV). Meanwhile, the antibacterial property of CEO-NPs was determined by inhibition zone method. CEO-NPs with the size of 215 nm and zeta potential of 25.12 mv were obtained when the mass ratio of CS/TPP, CEO and fatty alcohol polyoxyethylene ether-9 (AEO-9) concentration and pH were 8:1, 1.8 g/L, 0.7 g/L and 5.26. FTIR demonstrated that CEO has been encapsulated into NPs. TGA showed that the thermal stability of CEO-NPs was improved compared with CEO and the loading capacity of CEO was 13.4%. GC–MS displayed that more than 90% ingredients of CEO had been encapsulated into NPs. UV indicated that CEO-NPs had the excellent sustainedrelease property. Furthermore, CEO-NPs showed a long-lasting antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus. In all, CEO-NPs can be used as a potential long-term natural preservative.
1. Introduction Recently, natural antimicrobials have attracted more attention due to the increased consumer awareness on the safety (Benjemaa et al., 2018). Essential oils (EO) are aromatic natural oily liquids obtained from flowers, leaves, fruits, stems and other part of plants, which can be prepared by steam distillation, fermentation, or by expression (Baran et al., 2007; Benelli and Pavela, 2018; Burt, 2004; Tiziana Baratta et al., 1998) and exhibit excellent antibacterial activities, antifungal and antioxidant properties. For example, cinnamon oil obtained from foliages and barks showed the antioxidant and antimicrobial activities because of the high eugenol and cinnamic aldehyde content (Yildirim et al., 2017). Thyme oil isolated from herb thyme had the strong antimicrobial activity, which could replace synthetic fungicides (Vilaplana et al., 2018; Ryu et al., 2018). Ginger oil produced by fresh ginger exhibited antibacterial and antioxidant properties (Nile and Park, 2015). However, EO is the mixture of volatile compounds including terpenes, aromatic hydrocarbons, esters, phenols and other natural substances. When EO is exposed to the environment of oxygen, light ⁎
Corresponding author. E-mail addresses: [email protected], [email protected] (J. Hu).
https://doi.org/10.1016/j.indcrop.2018.05.058 Received 11 October 2017; Received in revised form 22 May 2018; Accepted 22 May 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
and heat, most ingredients can be easily oxidated and deteriorated (Perdones et al., 2012; Carvalho et al., 2016). Encapsulation technology provided an effective approach to keep the stabilization of EO and prevent the loss of volatile ingredients (Campelo et al., 2017; Wang et al., 2018; Calvo et al., 2012). Natural polysaccharides including chitosan (Tan et al., 2018), maltodextrin (Medina-Torres et al., 2016)， cyclodextrin (da Rosa et al., 2013) and gum arabic (Binsi et al., 2017) have been widely used to encapsulate EO due to their non-toxic, biodegradability and biocompatibility (Xiao and Grinstaff, 2017; Olejnik et al., 2016; Tabart et al., 2012; Sotelo-Boyás et al., 2017; Cota-Arriola et al., 2013). Cinnamon EO was encapsulated with β-cyclodextrin by inclusion complexation method and showed the high antifungal activity against Botrytis sp (Munhuweyi et al., 2018). Simon-Brown et al. (2016) reported that ginger EO microcapsule (the particle size: 8.215.3 μm) was prepared with maltodextrin (MD) and/or gum arabic (GA) as the walls by spray drying and exhibited effective antioxidant activity to prolong food shelf-life. Especially, chitosan (CS) obtained from deacetylation of chitin, as the second most abundant polysaccharide showed the excellent
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
2.3.2. Chemical structure analysis The chemical structures of ginger EO, cinnamon EO, thyme EO, CEO-NPs after freeze-drying and CS were determined with a VERTEX 70 FTIR spectrophotometer with ATR accessory (Bruker, Ettlingen, Germany) in the range from 4000 to 600 cm−1.
synergism antimicrobial effect in combination with EOs (Gharsallaoui et al., 2007; Ali and Ahmed, 2018; Xu et al., 2017). Patchouli oil CS microcapsules with diameter in the range of 1–20 μm were prepared via complex coacervation method and displayed high antibacterial activity against Staphylococcus aureus and Escherichia coli in cotton fabrics (Liu et al., 2013). In addition, Velmurugan et al. (2017) prepared orange and lavender EO CS nanoparticles with the size of 213.6 and 273.8 nm and then applied them in leather finishing against B. cereus, B. subtilis, Aspergillus fumigatus, Macrophomina phaseolina. In our previous study, CS nanoparticles loaded with cinnamon EO (CE-NPs) with three sizes of 112 nm, 215 nm and 527 nm were prepared by ionic gelification reaction. Then these CE-NPs were applied in the package preservation of the chilled pork. CE-NPs with 527 nm led to a significant decrease of microbial growth, pH, peroxide value, 2-thiobarbituric acid and sensory scores of the pork than the other treatments at 4 °C during 15 day (Hu et al., 2015). Up to now, few studies about the encapsulation of composite essential oil (CEO) with CS as wall material have been reported. In this study, cinnamon-thyme-ginger composite essential oil nanocapsules (CEO-NPs) with CS as the wall were prepared successfully via ionic gelification method. The influence of the mass ratio of CS to TPP, CEO and surfactant content and pH on the particle size and zeta potential of CEO-NPs were investigated in detail. Then the morphology and structure of CEO-NPs were characterized by DLS, FT-IR, TGA, GC–MS and UV. Finally, the antibacterial activity of CEO-NPs against Escherichia coli, Bacillus subtilis and Staphylococcus aureus were studied.
2.3.3. Determination of CEO loading capacity and thermal stability In order to obtain the thermal stability and CEO loading capacity of CEO-NPs, CEO, CS-NPs and CEO-NPs after freeze-drying were performed on a Q5000 thermal analyzer (TA Instruments, USA) from 25 °C to 600 °C at 10 °C/ min heating rate under nitrogen atmosphere with a flow of 20 mL/min. 2.3.4. Determination of the encapsulated aromatic compounds in CEO-NPs via GC–MS In order to identify the main ingredients of CEO encapsulated in CEO-NPs, the analysis of cinnamon EO, thyme EO, ginger EO and CEONPs after-drying was performed with GC–MS (Agilent Technologies Inc., New York, USA). CEO-NPs were firstly pretreated as the followings. 0.1 g CEO-NPs after freeze-drying was diluted in 5 mL ethanol. The suspension was treated with the ultrasonication at 800 W for 30 min until the shell of CEO-NPs was completely destroyed and CEO was dissolved in the ethanol. Then, the suspension was centrifuged at 13000 rpm for 20 min and the supernatant was collected. After that, 1, 2-Dichlorobenzene of 100ug/mL as an internal standard (ISTD) was added in the solution to determine the content (%) of CEO ingredients. Meanwhile, each pure aromatic compound in the three EOs was also been determined as its standard. An Agilent 6890N gas chromatograph with a 5873N mass detection in the range 30–450 mass/charge was used with a HP-INNOWAX polar column (60 m × 0.25 mm i.d. × 0.25 μm film, Agilient). The carrier gas was ultrapurified helium at a flow rate of 1.0 mL/min. The injection volume was 0.2 μL with a spilt ratio of 20:1. The initial column temperature was held at 40 °C for 6 min, programmed to ramp to 100 °C at a rate of 3 °C/min for 2 min, then to 230 °C at a rate of 5 °C/min and held at this temperature for 20 min. The detector ion source temperature was set at 230 °C. Electron impact ionization was performed at electron energy of 70 eV. Identification of the main components of CEO was achieved by comparing mass spectra with those in the NIST08 library.
2. Materials and methods 2.1. Materials Chitosan (CS, average molecular weight = 150000) was obtained from Sigma-Aldrich, USA. Sodium tripolyphosphate (TPP), fatty alcohol polyoxyethylene ether-9 (AEO-9), glacial acetic acid (HAC) and sodium hydroxide (NaOH) were supplied by Shanghai National Chemical Reagent Co., Ltd., China. Cinnamon, thyme and ginger essential oil were purchased from ZhengzhouXomolon Food Flavor Co., Ltd., China. All chemicals were used as received without any further purification. Deionized water was used for all experiments.
2.3.5. Sustained-release property of CEO-NPs In order to study the sustained-release property of CEO-NPs, 1 g CEO-NPs after freeze-drying were placed at 40 °C for 1, 2, 3, 5, 7, 9, 12, 15, 18 days, separately. Then, these CEO-NPs were added into 20 mL dichloromethane. The other extract processes were the same as above. The volatile components in the CEO released from CEO-NPs after different placing times were determined by UV–vis spectrum (UV2100, Unico, shanghai). (E)-cinnamaldehyde and thymol were selected as the model compounds and determined at 286 nm and 275 nm, respectively.
2.2. Preparation of CEO-NPs CEO-NPs were similarly obtained according to the previously described method (Xiao et al., 2014a,b; Hu et al., 2015). Typically, CEO was obtained by blending three EOs (the mass ratio of cinnamon EO, thyme EO and ginger EO: 4:3:3). CEO (0.09 g) was mixed with AEO-9 (0.035 g) under 1000 rpm stirring for 5 min at 25 °C. Then the mixture was added into 15 mL TPP aqueous solution to form O/W emulsion under the homogenization for 10 min. After that, 35 mL CS aqueous solution was added into the above emulsion to keep the weight ration of CS/TPP 8/1 with a stirring speed of 400 rpm at 30 °C. Meanwhile, the pH of the emulsion was controlled to 5.26 with 1 mol/L sodium hydroxide aqueous solution and the reaction was kept for 2 h. Finally, CEO-NPs were purified by the centrifugation at 15 000 rpm for 30 min at 5 °C. CS-NPs were also prepared as the above process without CEO.
2.4. Antibacterial experiment Antibacterial activities of CEO, CS-NPs, CEO-NPs against Escherichia coli (ATCC 25922), Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 25923) were carried out by the zone of inhibition method. Bacterial strains were cultured overnight at 37 °C in the nutrient agar plates. The nutrient agar medium in a Petri dish was inoculated with 0.1 mL 107–108 cfu/mL bacteria. The sterile filter paper discs (6 mm diameter) were impregnated with 1 mL CEO and aqueous dispersions of CEO-NPs and CS-NPs (0.2 g/mL) and dried for 5 min by an ultraviolet lamp. Meanwhile, gentamicin sulfate and sterile saline were tested as the positive and negative controls, respectively. The dishes were incubated at 37 °C for 1, 5, 7 and 9 days. The diameter of the inhibition zones around each of the discs was taken as measure of the antimicrobial activity. Each experiment was carried out 6 times and the mean diameter of the inhibition zone was measured.
2.3. CEO-NPs characterization 2.3.1. Particle size and zeta potential The particle size, polydispersity index (PDI) and zeta potential of CEO-NPs in three replicates were measured by the Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Each sample was determined using a solid state He-Ne laser of 633.0 nm at 25 °C with an angle detection of 90 °C. 86
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
Fig. 2. Effect of CEO concentration on the particle size and zeta potential of CEO-NPs.
Fig. 1. Effect of the mass ratio of CS to TPP on the particle size and zeta potential of CEO-NPs.
2.5. Statistical analysis All tests were performed in triplicate, and the experiments data were averaged. Variances of each value in each group (ANOVA) were analyzed with the Duncan test of the SPSS (Version 17.0 for Windows) (Hu et al., 2015). Correlations between variables (P < 0.05) were determined by correlation analysis using Pearson's linear correlation coefficient.
3. Results and discussion 3.1. The effect of the mass ratio of CS to TPP Fig. 1 demonstrates that the influence of the mass ratio of CS to TPP on the particle size and zeta potential of CEO-NPs. With the increase of CS/TPP mass ratio, the mean diameter of CEO-NPs increased firstly and then decreased. When the mass ratio of CS to TPP was 8:1, the size and particle size distribution (PDI) of CEO-NPs were minimum (215 nm and 0.26) as shown in Fig. S1. This phenomenon was different from the previous study (Xiao et al., 2014a,b). Generally the higher the CS content, the larger the size of CS-NPs. However, during this experiment the intermolecular hydrogen bond formed among CS molecules was easy to keep the ionic gelation reaction between CS and TPP (Fan et al., 2012). Meanwhile, the improved electrostatic repulsions between CEONPs effectively maintained their stability due to the enhancement of the positively charged amino groups of CS (Rodrigues et al., 2012; Janes et al., 2001). Furthermore, with CS/TPP mass ratio rising, the zeta potential of CEO-NPs increased. This is attributed to that the more positive charge CS molecules can produce many free amino groups on the surface of CEO-NPs.
Fig. 3. Effect of AEO-9 concentration on the particle size and zeta potential of CEO-NPs.
3.3. The effect of AEO-9 concentration Fig. 3 displays the effect of different AEO-9 concentrations on the size and zeta potential of CEO-NPs. Both the mean size and zeta potential of CEO-NPs mainly decreased with AEO-9 concentration increased. A stable emulsion can be obtained with AEO-9 as emulsifier dispersed between the interface of water and oil droplets (Hu et al., 2015; Sadeghi and Fayazi, 2012; Zhang et al., 2017). Many emulsifiers are useful for the dissolution of CEO into the micelles to form small micelles. Simultaneously, CEO-NPs with the small size can be prepared by the complex coacervation interactions between the positively charged protonated amino groups on CS and the negatively charged phosphate groups on TPP.
3.2. The effect of CEO concentration 3.4. The effect of pH The effect of different CEO concentrations on the particle size and zeta potential of CEO-NPs is indicated in Fig. 2. When CEO concentration increased from 0.8 to 1.8 g/L, the size and zeta potential of CEO-NPs had no significant change. Especially the average size of CEONPs was the smallest with 1.8 g/L CEO. For the reason is that CS can completely encapsulate the dispersed CEO droplets. Then with CEO concentration increasing, the average size of CEO-NPs enhanced, but their zeta potentials decreased. This is attributed to that more CEO dissolved into the micelles led to the size enhancement of CEO-NPs. As well, the excessive CEO could adhere on the surface of CEO-NPs, which could reduce the surface charges of NPs and cause their aggregation.
The influence of pH on the particle size and zeta potential of CEONPs is shown in Fig. 4. CS is an alkaline polysaccharide, insoluble in neutral or alkaline condition (Benavides et al., 2016). The protonation degree of CS was significantly affected by pH of the solution (Shu and Zhu, 2002). Thus, pH value has significant effect on both zeta-potential and particle size of CEO-NPs. With pH increased, the zeta potential of CEO-NPs was decreased due to the reduced protonation degree of CS molecules. When the pH value ranged from 3.5 to 5.26, the particle size of CEO-NPs decreased gradually. After that, their sizes increased with pH rising. pH could affect the ionic cross-linking process of CS NPs, which belongs to the pH sensitive characters of the whole 87
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
Fig. 7. TG and DTG curves of CS-NPs (a, d), CEO-NPs (b, e), CEO (c, f). Fig. 4. Effect of pH on the particle size and zeta potential of CEO-NPs.
3.5. Particle size and particle size distribution Fig. 5 showed the particle size and PDI of the CEO-NPs. The average particle size and PDI of CEO-NPs were 215 nm and 0.26, respectively. CEO can be emulsified in the micelles formed by AEO-9. The positively charged CS can be interacted with the negatively charged TPP via the complex coacervation to encapsulate CEO. 3.6. Chemical structure analysis Fig. 6 displays the differences among the structures of CS(a), ginger EO(b), thyme EO(c), cinnamon EO(d) and CEO-NPs(e). In the FTIR spectrum of CS, the absorption peaks at 2824 cm−1 and 1593 cm−1 were attributed to the stretching vibration of CeH groups and the deformation vibration absorption peak of −NH2, respectively. The absorption peak at 1079 cm−1 corresponded to the stretching vibration of the CeO group of CS. Generally, cinnamon, thyme and ginger EO contain aldehydes, phenols, and ketone monomers (Yu et al., 2017; Blaise et al., 1997). The obvious adsorption peaks of cinnamon EO appeared at 1678 cm−1, 1626 cm−1, 1450 cm−1, 1289 cm−1, which corresponded to the stretching vibration of C]O, C]C, eOH of aromatic compounds. The strong peaks at 748 cm−1 and 689 cm−1 were attributed to benzene rings CeH vibration absorption and the vibration absorption of alkenes, respectively. In the spectrum of thyme EO, the peak at 3001 cm−1 was associated with the stretching vibration of CeH in benzene ring. The absorption peaks at 1619 cm−1, 1583 cm−1,1515 cm−1,1458 cm−1 were four unequal intensity ones, which corresponded to the characteristic peaks of thymol. The peaks at 1429 cm−1and 1226 cm−1 were attributed to the bending vibration of −OH and CeO. The main components of ginger EO were terpene compounds. The absorption peaks at 1708 cm−1, 1608 cm−1 were associated to C]C and C]CeC]C stretching vibration. The peaks at 2927 cm−1 and 2858 cm−1 were affected by the bending vibrations of −CH2 and −CH3 groups, respectively. In the FTIR spectrum of CEONPs, the characteristic absorption peaks of cinnamon, thyme and ginger EO all appeared. The vibration absorption peak of amino group was shifted to1675 cm−1, and the new absorption peaks at 1533 cm−1 appeared, related to the cross-linking between the phosphate groups of TPP and amino groups of CS(Yang et al., 2009). Meanwhile, the absorption peaks at 1137 cm−1 and 880 cm−1 corresponded to the vibration and flexural vibration of P = O and P-O. In all, the above results indicated that the CEO can be successfully encapsulated into CEO-NPs.
Fig. 5. Particle size distribution of CEO-NPs.
Fig. 6. FTIR of CS(a), ginger EO(b), thyme EO(c), cinnamon EO(d) and CEONPs(e).
process (Perdones et al., 2016). When pH was low, the complex coacervation interactions between the positively charged CS and the negatively charged TPP are easy to be processed. Otherwise, the reduced protonation degrees of CS molecules weaken the complex coacervation reaction, which could lead to the aggregation of CEO-NPs.
3.7. CEO loading capacity and thermal stability The TGA results of CS-NPs (a), CEO-NPs (b) and CEO(c) are shown in Fig. 7. There were two thermal decomposition stages for CEO. The first weight loss (74.23%) from 106 °C to 155 °C corresponded to the decomposition of many aromatic compounds with the low boiling point 88
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
Table 1 Composition of three kinds of EOs and CEO-NPs identified by GC–MS. Compound
α-Pinene Camphene Hexanal Phellandrene β-Myrcene (+)-4-Carene D-Limonene Eucalyptol γ-Terpinene o-Cymene α-Terpinolene Diacetone alcohol 2-Furanmethanol α-ylangene α-Copaene Decanal Benzaldehyde 2-Nonanol Linalyl acetate β-Linalool Isobornyl acetate (R)-Terpinen-4-ol Patchoulene Aromandendrene β-Terpineol Alloaromadendrene Cis-β-Farnesene Isoborneol Humulene Cis-α-Bisabolene β-Curcumene L-α-Terpineol Endo-Borneol γ-Muurolene Zingiberene β-Bisabolene α-Muurolene Citral β-Panasinsene α-Farnesene Geranyl acetate Copaene β- Sesquiphellandrene 2-phenylethyl ester Anethole Geraniol Trans-Calamenene γ-Elemene Benzenemethanol Phenylethyl Alcohol Caryophyllene oxide Nerolidol (E)-Cinnamaldehyde γ-eudesmol (−)-Spathulenol Cinnamyl acetate Eugenol Thymol Methyleugenol γ-Muurolene Carvacrol α-Bisabolol α-Cadinol Trans-Isoeugenol Isoaromadendrene epoxide Coumarin Benzyl Benzoate Total Monoterpene hydrocarbon Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes
Relative content (%) RIa
RIb
Cinnamon
Thyme
Ginger
CEO-NPs
1019 1062 1085 1166 1170 1181 1199 1210 1249 1275 1285 1368 1448 1491 1500 1505 1539 1518 1548 1573 1598 1612 1619 1624 1637 1664 1670 1680 1688 1689 1702 1705 1713 1730 1736 1738 1740 1746 1751 1755 1764 1767 1786 1833 1847 1849 1855 1857 1890 1927 2019 2043 2072 2135 2148 2175 2189 2192 2199 2202 2225 2233 2256 2276 2281 2429 2485
1012 1075 1084 1160 1173 1149 1196 1210 1243 1268 1288 1352 1428 1489 1500 1502 1530 1510 1555 1581 1582 1616 1639 1637 1624 1657 1663 1672 1682 1701 1712 1690 1715 1725 1726 1729 1738 1733 1746 1752 1759 1519 1783 1806 1847 1847 1850 1651 1875 1931 2008 2042 2084 2130 2152 2174 2186 2198 2030 1701 2225 2232 2255 2309 2291 2437 2655
0.13 0.02 — 0.02 — — 0.88 0.52 0.02 0.95 0.02 0.03 0.02 0.04 0.38 — 1.60 — 2.39 2.19 — 1.13 — 0.13 — — — 0.18 0.05 — 0.28 0.34 — — — 0.43 — — — 0.18 — — 0.51 1.35 — — — — 0.04 0.76 — 0.47 64.94 — 0.16 10.78 4.30 1.66 — — — — — 0.22 — 0.52 0.22 97.86 1.09 4.36 2.00 0.63
0.79 0.07 — 0.11 0.70 1.24 2.99 14.96 1.09 24.60 0.88 — — — 0.14 — — — 3.50 1.09 0.35 13.76 0.16 0.53 0.09 0.21 — 0.40 — — — 1.96 0.75 — — — 0.14 — — — — — — — 0.09 — 0.21 — 0.39 — 0.47 — 0.33 — 0.18 — — 23.15 — — 4.28 — — — — — — 99.61 7.87 33.01 1.39 0.65
0.66 1.77 1.34 4.25 — 0.14 0.82 1.45 — 1.24 0.10 — — — 0.92 1.65 — 0.10 — 0.08 — 0.83 — — — 0.41 0.70 0.22 — 0.14 0.49 0.94 1.14 1.39 37.72 11.17 — 0.92 1.68 6.31 0.17 0.54 14.96 — — 0.20 — 0.43 — — — 0.66 0.87 0.14 — — — — 0.09 0.09 1.17 0.10 0.14 0.15 0.65 — — 98.94 7.74 5.03 76.95 1.69
— 0.08 0.21 1.12 — 0.18 1.92 3.53 1.10 6.86 0.43 0.31 — — 0.14 — — — 2.12 1.23 — 2.01 — 0.21 0.09 0.18 — 0.63 — 0.43 0.53 1.54 0.72 — 10.91 1.53 — — 0.10 1.47 — — 3.87 — — — — — — — — 0.12 30.79 — — 2.44 1.65 15.20 — — 0.94 — — — — — — 94.59 4.83 9.75 19.37 0.12
(continued on next page) 89
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
Table 1 (continued) Compound
Relative content (%) RIa
RIb
Aromatic compounds
Cinnamon
Thyme
Ginger
CEO-NPs
89.78
56.69
7.53
60.52
Ra: based on a homologous series of normal alkanes. Rb: Literature retention indices.
LC: CEO loading capacity of CEO-NPs Fig. 7
3.8. CEO encapsulation and sustained-release property Table 1 shows the chemical compositions of cinnamon EO, thyme EO, ginger EO and CEO encapsulated in CEO-NPs. 36 components were identified from cinnamon EO by GC–MS representing 97.86% of the total EO. Cinnamon EO consisted of aromatic compounds (89.78%), monoterpoids (4.36%), sesquiterpene hydrocarbons (2.0%), monoterpene hydrocarbons (1.09%) and sesquiterpenoids (0.63%). The major components of cinnamon EO were (E)-Cinnamaldehyde (64.94%), cinnamyl acetate (10.782%) and eugenol (4.30%). In thyme EO there were identified 31 compounds, which represents 99.61% of the total EO. The main compounds were o-cymene (24.60%) followed by thymol (23.15%), eucalyptol (14.96%) and (R)-terpinen-4-ol (13.76%). Besides, 33 compounds were identified from ginger EO by GC–MS representing 98.94% of the total oil. Sesquiterpene hydrocarbons (76.95%) occupied the highest proportion among all the components. Especially, zingiberene (37.72%), β-sesquiphellandrene (14.96%), and β-Bisabolene (11.17%) were the major components of ginger EO. The chemical components of three kinds of EO were similar with those of the previous studies (Li et al., 2013; Lu et al., 2011; Noori et al., 2018). CEO was obtained by blending cinnamon EO, thyme EO and ginger EO according to the mass ratio of 4/3/3. When CEO was encapsulated into CEO-NPs, there were 33 compounds identified from CEO by GC–MS representing 94.59% of the total CEO. The encapsulated aromatic ingredients included aromatic compounds (60.52%), monoterpoids (9.75%), sesquiterpene hydrocarbons (19.37%), monoterpene hydrocarbons(4.83%) and sesquiterpenoids (0.12%). The main compounds were (E)-cinnamaldehyde (30.794%), thymol(15.20%), zingiberene(10.91%), o-Cymene(6.858%) and β-Sesquiphellandrene (3.87%). Compared with the pure cinnamon EO, thyme EO and ginger EO, more than 90% aromatic ingredients in CEO have been encapsulated into NPs. Table 1 In order to reflect the sustained-release property of aromatic components encapsulated in CEO-NPs, (E)-cinnamaldehyde and thymol were selected as the model fragrances to study their suatined-release form CEO-NPs during 18 days. Fig. 8 displays the release comparison of
Fig. 8. Release profiles of (E)-cinnamaldehyde and thymol from CEO-NPs.
in CEO. The second weight loss occurred from 155 to 269 °C, which was due to those with the high boiling point such as zingiberene, cinnamaldehyde, thymol and so on. The weight losses of CS-NPs and CEONPs below 100 °C were 3.2% and 4.1% respectively, due to the vaporization of residual moisture in NPs. For CS-NPs, the main weight loss (46.7%) occurred at 160–360 °C due to the depolymerization and decomposition of glucosamine of CS. The main weight loss of CEO-NPs was observed at 160–460 °C. The first weight loss of 160–270 °C was ascribed to the thermal decomposition of CS structure and some aromatic components encapsulated in CEO-NPs. The second weight loss occurred from 270 to 460 °C, which was due to the thermal decomposition of other aromatic components with the high boiling point encapsulated in CEO-NPs. The thermal stability of CEO encapsulated in NPs was enhanced. The total weight loss of CEO-NPs was 53.2%. Based on the same quality of CS in CEO-NPs and CS-NPs, the CEO LC was calculated as 13.4% according to Eq. (1) (Xiao et al., 2014a,b).
We − LC Wc = 1 − W1 − LC 1 − W2
(1)
We: CEO-NPs weight loss (%); Wc：CS-NPs weight loss (%); W1:and W2: CS-NPs and CEO-NPs weight loss (%), respectively; Table 2 Antimicrobial activity of CEO-NPs. Attribute
Escherichia coli
Bacillus subtilis
Staphylococcus aureus
Treatment
CEO CS-NPs CEO-NPs CEO CS-NPs CEO-NPs CEO CS-NPs CEO-NPs
Different days (mm) 1
5
7
9
16.87 ± 0.42dz 5.77 ± 0.35cdx 11.56 ± 0.71ay 14.32 ± 1.00dz 7.35 ± 1.32dx 11.66 ± 0.40by 17.02 ± 0.59dz 8.21 ± 1.11dx 12.26 ± 1.00by
7.22 ± 1.11cy 4.65 ± 0.45cx 11.89 ± 0.11bz 6.58 ± 1.00cy 5.68 ± 0.58cx 12.98 ± 0.71cz 5.69 ± 0.55cx 6.21 ± 0.44cy 13.56 ± 0.45dz
1.23 ± 0.36bx 3.11 ± 0.22by 12.02 ± 0.41cz 0.65 ± 0.11bx 2.98 ± 0.31by 12.51 ± 0.49cz 0.23 ± 0.05bx 2.12 ± 0.57by 12.99 ± 0.62cz
0.00 ± 0.11ax 0.98 ± 0.78ay 11.02 ± 0.76az 0.00 ± 0.14ax 0.25 ± 0.05ay 10.77 ± 0.98az 0.00 ± 0.08ax 0.18 ± 0.11ay 11.21 ± 0.68az
Different letters (a, b, c, and d) in the same row indicate differences among mean values, P < 0.05. Different letters(x, y, and z)in the same column indicate differences among mean values, P < 0.05. 90
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
(E)-cinnamaldehyde and thymol from CEO-NPs. (E)-cinnamaldehyde and thymol released 38.05% and 26.01% during the first 7 day quickly, and then they reached equilibrium in 9 day. Finally, the cumulative released amount of (E)-cinnamaldehyde and thymol were 38.77% and 27.67% at 18 day. This demonstrated that CEO-NPs had the excellent sustained-release property.
and National key R & D projects (2016YFA0200300) were appreciated.
3.9. Antibacterial activity
References
The antibacterial activity of CEO, CS-NPs and CEO-NPs against Escherichia coli, Bacillus subtilis and Staphylococcus aureus was assessed by inhibition zones as shown in Table 2. Initially, all of CEO, CS-NPs and CEO-NPs produced obvious inhibition zones against the tested bacteria for 1 day incubation. CEO showed the highest inhibition zone following by CEO-NPs and CEO in this order. To specific, the inhibition zone diameters of CEO against Escherichia coli, Bacillus subtilis and Staphylococcus aureus were 16.87 ± 0.42 mm, 14.32 ± 1.00 mm and 17.02 ± 0.59 mm for 1 day incubation, respectively. Then the diameters of the inhibition zone of CEO and CS-NPs were decreased with the incubation time delayed. Especially, CEO produced no observable inhibition zones against three tested bacteria at 9 day. However, the inhibition zone sizes of CEO-NPs against all the bacteria were no any observable decrease after 9 day incubation. The inhibition zone diameters of CEO-NPs against Escherichia coli were 11.56 ± 0.71 mm for 1 day, 11.89 ± 0.11 mm for 5 day, 12.02 ± 0.41 mm for 7 day and 11.02 ± 0.76 mm for 10 day, respectively. And the inhibition zone diameters of CEO-NPs against Bacillus subtilis were 11.66 ± 0.40 mm for 1 day, 12.98 ± 0.71 mm for 5 day, 12.51 ± 0.49 mm for 7 day and 10.77 ± 0.98 mm for 9 day, respectively. Meanwhile, the inhibition zone diameters of CEO-NPs against Staphylococcus aureus were 12.26 ± 1.00 mm for 1 day, 13.56 ± 0.45 mm for 5 day, 12.99 ± 0.62 mm for 7 day and 11.21 ± 0.68 mm for 9 day, respectively. This demonstrated that CEO-NPs possessed the long-term antimicrobial effect. CEO was obtained by mixing cinnamon EO, thyme EO and ginger EO. According to the above GC–MS results, many aromatic ingredients in CEO such as (E)-cinnamaldehyde and thymol displayed the excellent antimicrobial activity against a variety of bacteria (Guarda et al., 2011; Ultee et al., 2002; Wang et al., 2018). CS has a certain antibacterial activities (Yu et al., 2017). Thus, the synergistic effect of both of the encapsulated aromatic ingredients in CEO-NPs and CS enhanced the antimicrobial activity of CEO-NPs. It can be seen that CEO-NPs have a long lasting antibacterial activity on Escherichia coli, Bacillus subtilis and Staphylococcus aureus due to CEO-NPs can gradually release the antimicrobial active substances in CEO (Giner et al., 2012).
Ali, A., Ahmed, S., 2018. A Review on chitosan and its nanocomposites in drug delivery. Int. J. Biol. Macromol. 109, 273–286. Baran, S., Reuss, W.A., von. Könige, S.H., Kalemba, D., 2007. Composition of the essential oil of Abies koreana Wils. Flavour Fragr. J. 22, 78–83. Benavides, S., Cortés, P., Parada, J., Wendy, F., 2016. Development of alginate microspheres containing thyme essential oil using ionic gelation. Food Chem. 204, 77–83. Benelli, G., Pavela, R., 2018. Beyond mosquitoes—essential oil toxicity and repellency against bloodsucking insects. Ind. Crops Prod. 117, 382–392. Benjemaa, M., Neves, M.A., Falleh, H., Isoda, H., Ksour, R., Nakajima, M., 2018. Nanoencapsulation of thymus capitatus essential oil: formulation process, physical stability characterization and antibacterial efficiency monitoring. Ind. Crops Prod. 113, 414–421. Binsi, P.K., Nayak, N., Sarkar, P.C., Jeyakumari, A., Ashraf, P.M., Ninan, G., Ravishankar, C.N., 2017. Structural and oxidative stabilization of spray dried fish oil microencapsulates with gum arabic and sage polyphenols: characterization and release kinetics. Food Chem. 219, 158–168. Blaise, O., Simardb, R.E., Holley, R.A., Piette, G.J.P., Bégina, A., 1997. Antibacterial activity of selected fatty acids and essential oils against six meat spoilage organisms. Int. J. Food Microbiol. 37, 155–162. Burt, S., 2004. Essential oils: their antibacterial properties and potential applications in. foods—a review. Int. J. Food Microbiol. 94, 223–253. Calvo, P., Castaño, Á.L., Lozano, M., González-Gómez, D., 2012. Influence of the microencapsulation on the quality parameters and shelf-life of extra-virgin olive oil encapsulated in the presence of BHT and different capsule wall components. Food Res. Int. 45, 256–261. Campelo, P.H., do Carmo, E.L., Zacarias, R.D., Yoshida, M.I., Ferraz, V.P., Fernandes, R.V.d.B., Botrel, D.A., Borges, S.V., 2017. Effect of dextrose equivalent on physical and chemical properties of lime essential oil microparticles. Ind. Crops Prod. 102, 105–114. Carvalho, I.T., Estevinho, B.N., Santos, L., 2016. Application of microencapsulated essential oils in cosmetic and personal healthcare products-a review. Int. J. Cosmetic Sci. 38, 109–119. Cota-Arriola, O., Cortez-Rocha, M.O., Burgos-Hernandez, A., Ezquerra-Brauer, G.M., Plascencia-Jatomea, M., 2013. Controlled release matrices and micro/nanoparticles of chitosan with antimicrobial potential: development of new strategies for microbial control in agriculture. J. Sci. Food. Agric. 93, 1525–1536. Fan, W., Yan, W., Xu, Z.S., Ni, H., 2012. Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique. Colloids Sur. B: Biointerfaces 90, 21–27. Gharsallaoui, A., Roudaut, G., Chambin, O., Voilley, A., Saurel, R., 2007. Applications of spray-drying in microencapsulation of food ingredients: an overview. Food Res. Int. 40, 1107–1121. Giner, M.J., Vegara, S., Funes, L., Martí, N., Saura, D., Micol, V., Valero, M., 2012. Antimicrobial activity of food-compatible plant extracts and chitosan against naturally occurring micro-organisms in tomato juice. J. Sci. Food. Agric. 92, 1917–1923. Guarda, A., Rubilar, J.F., Miltz, F., Galotto, M.J., 2011. The antimicrobial activity of microencapsulated thymol and carvacrol. Int. J. Food Micro. 146, 144–150. Hu, J., Wang, X.G., Xiao, Z.B., Bi, W.C., 2015. Effect of chitosan nanoparticles loaded with cinnamon essential oil on the quality of chilled pork. LWT-Food Sci. Technol. 63, 519–526. Janes, K.A., Calvo, P., Alonso, M.J., 2001. Polysaccharide colloidal particles as delivery systems for macromolecules. Adv. Drug Deliv. Rev. 47, 83–97. Li, Y.Q., Kong, D.X., Wu, H., 2013. Analysis and evaluation of essential oil components of cinnamon barks using GC?MS and FTIR spectroscopy. Ind. Crops Prod. 41, 269–278. Liu, J., Liu, C., Liu, Y., Chen, M., Hu, Y., Yang, Z., 2013. Study on the grafting of chitosan–gelatin microcapsules onto cotton fabrics and its antibacterial effect. Colloids Sur. B: Biointerfaces. 109, 103–108. Lu, F., Ding, Y.C., Ye, X.Q., Ding, Y.T., 2011. Antibacterial effect of cinnamon oil combined with thyme or clove oil. Agr. Sci. in China. 10, 1482–1487. Medina-Torres, L., Santiago-Adame, R., Calderas, F., Gallegos-Infante, J.A., GonzálezLaredo, R.F., Rocha-Guzmán, N.E., Núñez-Ramírez, D.M., Bernad-Bernad, M.J., Manero, O., 2016. Microencapsulation by spray drying of laurel infusions (Litsea glaucescens) with maltodextrin. Ind. Crops Prod. 90, 1–8. Munhuweyi, K., Caleb, O.J., Van Reenen, A.J., Opara, U.L., 2018. Physical and antifungal properties of b-cyclodextrin microcapsules and nanofibre films containing cinnamon and oregano essential oils. LWT-Food Sci. Technol. 87, 413–422. Nile, S.H., Park, S.W., 2015. Chromatographic analysis, antioxidant, anti-inflammatory, and xanthine oxidase inhibitory activities of ginger extracts and its reference compounds. Ind. Crops Pro. 70, 238–244. Noori, S., Zeynali, F., Almasi, H., 2018. Antimicrobial and antioxidant efficiency of nanoemulsion-based edible coating containing ginger (zingiber officinale) essential oil and its effect on safety and quality attributes of chicken breast fillets. Food Control. 84, 312–320. Olejnik, A., Kowalska, K., Olkowicz, M., Juzwa, W., Dembczynski, R., Schmidt, M., 2016.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.indcrop.2018.05.058.
4. Conclusion CEO-NPs with the size of 215 nm and zeta potential of 25.12 mv were prepared by ionic gelification reaction. The sizes and zeta potentials of CEO-NPs were strongly dependent on the mass ratio of CS to TPP, pH, as well as AEO-9 and EO concentration. TGA illustrated that CEO loading capacity of CEO-NPs was 13.4% and their thermal stability was significantly improved in contrast to that of pure essential oil. GC–MS displayed that 33 identified compounds in CEO had been encapsulated into NPs. UV showed that the cumulative released amount of (E)-cinnamaldehyde and thymol in CEO were 38.77% and 27.67% at 18 day, which demonstrated that CEO-NPs had the excellent sustainedrelease property. Moreover, CEO-NPs showed a long-lasting antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus. Acknowledgements The financial supports from National Nature Science Foundation of China (51773116), Shanghai Natural Science Foundation (17ZR1429800), Shanghai Capacity-building Project (18090503500) 91
Industrial Crops & Products 122 (2018) 85–92
J. Hu et al.
essential for action against the food-borne pathogen bacillus cereus. Appl. Environ. Microbiol. 68, 1561–1568. Velmurugan, P., Ganeshan, V., Nishter, N.F., Jonnalagadda, R.R., 2017. Encapsulation of orange and lavender essential oils in chitosan nanospherical particles and its application in leather for aroma enrichment. Sur. Interfaces. 9, 124–132. Vilaplana, R., Pazmiño, L., Valencia-Chamorro, S., 2018. Control of anthracnose, caused by colletotrichum musae, on postharvest organic banana by thyme oil. Postharvest Biol. Technol. 138, 56–63. Wang, J.G., Li, Y.Z., Gao, Y.F., Xie, Z.F., Zhou, M.H., He, Y.Y., Wu, H., Zhou, W.Y., Dong, X.M., Yang, Z.H., Yang, H., 2018. Cinnamon oil-loaded composite emulsion hydrogels with Lantibacterial activity prepared using concentrated emulsion templates. Ind. Crops Prod. 112, 281–289. Xiao, R., Grinstaff, M.W., 2017. Chemical synthesis of polysaccharides and polysaccharide mimetics. Prog. Polym. Sci. 74, 78–116. Xiao, Z.B., Tian, T., Hu, J., Wang, M.X., Zhou, R.J., 2014a. Preparation and characterization of chitosan nanoparticles as the delivery system for tuberose fragrance. Flavour Fragr. J. 29, 22–34. Xiao, Z.B., Liu, W.L., Zhu, G.Y., Zhou, R.J., Niu, Y.W., 2014b. Production and characterization of multinuclear microcapsules encapsulating lavender oil by complex coacervation. Flavour Fragr. J. 29, 166–172. Xu, W.R., Xie, W.J., Huang, X.Q., Chen, X., Huang, N., Wang, X., Liu, J., 2017. The graphene oxide and chitosan biopolymer loads TiO2 for antibacterial and preservative research. Food Chem. 221, 267–277. Yang, W.J., Fu, J., Wang, T., He, N.Y., 2009. Chitosan/sodium tripolyphosphate nanoparticles: preparation, characterization and application as drug carrier. J. Biomed. Nanotechnol. 5, 591–595. Yildirim, S.T., Oztop, M.H., Soyer, Y., 2017. Cinnamon oil nanoemulsions by spontaneous emulsification: formulation, characterization and antimicrobial activity. LWT-Food Sci. Technol. 84, 122–128. Yu, D.W., Xu, Y.S., Jiang, Q.X., Xia, W.S., 2017. Effects of chitosan coating combined with essential oils on quality and antioxidant enzyme activities of grass carp (ctenopharyngodon idellus) fillets stored at 4 °C. Int. J. Food Sci.&Technol. 52, 404–412. Zhang, H.C., Jun, J.Y., Zhao, Y.Y., 2017. Preparation and characterization of cellulose nanocrystals films incorporated with essential oil loaded β-chitosan beads. Food Hydrocol. 69, 164–172. da Rosa, C.G., Borges, C.D., Zambiazi, R.C., Nunes, M.R., Benvenutti, E.V., da Luz, S.R., D’Avila, R.F., Rutz, J.K., 2013. Microencapsulation of gallic acid in chitosan, β-cyclodextrin and xanthan. Ind. Crops Prod. 46, 138–146.
A gastrointestinally digested ribes nigrum L. fruit extract inhibits inflammatory response in a co-culture model of intestinal caco-2cells and RAW264.7 macrophages. J. Agric. Food Chem. 64, 7710–7721. Perdones, A., Sánchez-González, L., Chiralt, A., Vargas, M., 2012. Effect of chitosan–lemon essential oil coatings on storage-keeping quality of strawberry. Postharvest Biol. Technol. 70, 32–41. Perdones, Á., Chiralt, A., Vargas, M., 2016. Properties of film-forming dispersions and films based on chitosan containing basil or thyme essential oil. Food Hydrocol. 57, 271–279. Rodrigues, S., da Costa, A.M., Grenha, A., 2012. Chitosan/carrageenan nanoparticles: effect of cross-linking with tripolyphosphate and charge ratios. Carbohydr. Poly. 89, 282–289. Ryu, V., McClements, D.J., Corradini, M.G., McLandsborough, L., 2018. Effect of ripening inhibitor type on formation, stability, and antimicrobial activity of thyme oil nanoemulsion. Food Chem. 245, 104–111. Sadeghi, F., Fayazi, A., 2012. Analysis of crystalline structure of sodium tripolyphos phate: effect of pH of solution and calcination conditions. Ind. Eng. Chem. Res. 51, 1093–1098. Shu, X.Z., Zhu, K.J., 2002. The influence of multivalent phosphate structure on the properties of ionically cross-linked chitosan films for controlled drug release. Eur. J. Pharm. Biopharm. 54, 235–243. Simon-Brown, K., Solval, K.M., Chotiko, A., Alfaro, L., Reyes, V., Liu, C., Dzandu, B., Kyereh, E., Goldson Barnaby, A., Thompson, I., Xu, Z., Sathivel, S., 2016. Microencapsulation of ginger (zingiber officinale) extract by spray drying technology. LWT − Food Sci. Technol. 70, 119–125. Sotelo-Boyás, M., Correa-Pacheco, Z., Bautista-Baños, S., Corona-Rangel, M.L., 2017. Physicochemical characterization of chitosan nanoparticles and nanocapsules incorporated with lime essential oil and their antibacterial activity against food-borne pathogens. LWT − Food Sci. Technol. 77, 15–20. Tabart, J., Franck, T., Kevers, C., Pincemail, J., Serteyn, D., Defraigne, J.O., Dommes, J., 2012. Antioxidant and anti-inflammatory activities of Ribes nigrum extracts. Food Chem. 131, 1116–1122. Tan, P.Y., Tan, T.B., Chang, H.W., Tey, B.T., Chan, E.S., Lai, O.M., Baharin, B.S., Nehdi, I.A., Tan, C.P., 2018. Effects of storage and yogurt matrix on the stability of tocotrienols encapsulated in chitosan-alginate microcapsules. Food Chem. 241, 79–85. Tiziana Baratta, M., Damien Dorman, H.J., Deans Stanley, G., Cristina Figueiredo, G., Barroso Jose, G., Ruberto, G., 1998. Antimicrobial and antioxidant properties of some commercial essential oils. Flavour Fragr. J. 13, 235–244. Ultee, A., Bennik, M.H.J., Moezelar, R., 2002. The phenolic hydroxyl group of carvacrol is
92