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The innovative fabrication and applications of carvacrol nanoemulsions, carboxymethyl chitosan microgels and their composite films
Colloids and Surfaces B: Biointerfaces 175 (2019) 688–696
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
The innovative fabrication and applications of carvacrol nanoemulsions, carboxymethyl chitosan microgels and their composite films
T
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Kai Lei, Xinran Wang, Xiaozhou Li , Lin Wang College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanoemulsions Carvacrol Polymeric Microgels Films Active packaging
Carvacrol (CA) was firstly made into physically stable nanoemulsions using an innovative spontaneous emulsification method with carvacrol itself as oil phase without introducing other carrier oils. Self-crosslinking carboxymethyl chitosan (CMC) microgels via intermolecular hydrogen bonds were then fabricated using a simple solvent exchange method with ethanol as poor solvent. The CMC microgels obtained were rapidly deposited on the surface of tin foil using an electrospray technique to produce the CMC films. The stability of the films in water was further intensified by using Ca2+ as cross-linking agent. Also, the carvacrol nanoemulsions (CA-NEs) were loaded into the CMC films by utilizing swelling property of CMC microgels in aqueous solution. The results showed that the CA-NEs@CMC composite films possess satisfactory antioxidant activity, good antibacterial activity against S. aureus and E. coli, and excellent ability to extend the shelf life of wheat bread. We believe that the present strategies to prepare CA-NEs, CMC microgels and their composite films also apply to other types of essential oils and polymers, and the CA-NEs@CMC composite film is an excellent candidate for the active packaging.
1. Introduction Natural antimicrobial agents, essential oils have low extract cost, wide-spectrum antimicrobial activity and outstanding antioxidant performance [1,2] and therefore have attracted considerable scientific interests as functional ingredients in pharmaceutical, cosmetic, and food applications [3]. Carvacrol (CA) is a major component of oregano and thyme oil, which has been proven to be a very active antimicrobial agent against fungi, food-borne pathogens, yeast and molds, etc., besides, it is also an excellent antioxidant [4–6]. More importantly, carvacrol is classified as Generally Recognized as Safe (GRAS) substance by the Food and Drug Administration [7] and is registered by the European Commission as healthy with no risk to the consumers [8]. However, carvacrol is highly lipophilic with poor solubility and stability in aqueous media that limits its application in many industries [9,10]. Nanoemulsion can be generally classified into oil-in-water and water-in-oil types, is a colloidal nanodispersion of oil and water being stabilized by interfacial layer of surfactant. The size of the droplets in nanoemulsions is often much smaller, which means that they have much better stability to gravitational separation, flocculation, and coalescence than macroemulsions [11,12]. The major components of nanoemulsions include oil phase, emulsifying agent and aqueous phase. Among them, carrier oil within the oil phase is employed to dissolve active component, ⁎
however, the inappropriate select of carrier oil would seriously impact the formation, stability and performance of a nanoemulsion [12]. For that reason, we design that using carvacrol itself as the oil phase without introducing other carrier oil prepare oil-in-water (O/W) carvacrol nanoemulsions (CA-NEs) with a spontaneous emulsification method since carvacrol itself is exactly the oil. We speculate that the discarding of carrier oil can not only simplify production technique and reduce manufacturing cost of nanoemulsions, but also avoid the influence of introducing carrier oil on the application of nanoemulsions. Carboxymethyl chitosan (CMC) is a water-soluble, non-toxic, biodegradable and biocompatible chitosan derivative, which has good abilities to form hydrogels and films and has better biological activity compared with chitosan [13,14]. Polymer-based materials, such as micro- and nanoparticles, hydrogels, and films, are capable of encapsulating bioactive molecules for various applications [15,16]. Among them, polymer films (or coatings) are particularly suitable as bioactive molecules-loaded carriers deposited on solid substrates [17–20]. Electrospray deposition is an efficient and greatly materialsaving technique for the fabrication of functional films (or coatings) [21–23]. The electrospray technique employs an electric field between a film-forming solution contained within a syringe and a conductive substrate to direct produce nearly dry films on the substrates. Compared with other film-preparation methods, the electrospray technique
Corresponding author. E-mail address: [email protected] (X. Li).
https://doi.org/10.1016/j.colsurfb.2018.12.054 Received 10 October 2018; Received in revised form 17 December 2018; Accepted 18 December 2018 Available online 19 December 2018 0927-7765/ © 2018 Elsevier B.V. All rights reserved.
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works quickly and can fabricate large area films on non-flat surfaces [24–26]. In addition, our previous studies demonstrated that using polymeric microgels as film-forming raw materials could more rapidly produce continuous and dense polymeric films [25]. Microbial contamination is a quite frequent phenomenon for food products, which seriously threatens the health of humans. In the past, adding chemical preservatives and using plastic packaging materials were common methods to avoid food contaminate and prolong shelf life of food. However, consumers worry increasingly about negative consequences of preservatives nowadays, in addition, poor biodegradation of plastic materials has generated a massive accumulation of plastic waste in the environment [27]. The development of antimicrobial and biodegradable packaging has been the focus of recent research [28–30]. Therefore, the aims of this work were as follows: oil-in-water (O/W) CA-NEs are prepared using spontaneous emulsification method without introducing carrier oil and the preparation parameters of the nanoemulsions are optimized; CMC microgels are fabricated using a solvent exchange method and are employed as film-forming raw materials to produce the CMC films on the surface of tin foil via an electrospray technique; CA-NEs are introduced into the CMC films for the preparation of CA-NEs@CMC composite films and the performances of the composite films as active packaging are studied in detail.
0.1 mL·min-1and 1 h, respectively, for the fabrication of the CMC films. The films obtained were immersed in CaCl2 solution (pH 7.1, 1.0 mol L1 ) for 3 h to crosslink CMC in films. 2.4. Film’s stability in water The stability of the CMC films in water was analyzed via film’s solubility in water (SW). For SW measurements, the cross-linked CMC films deposited on tin foil (10 × 10 cm2) were dried at 105 °C for 24 h and then determined the initial weight using an analytical balance. Dried films were immersed in 50 mL of deionized water for 24 h at 25 °C. Film samples were then removed from water, and dried in an oven at 105 °C for 24 h to determine their final dry matter. Film solubility was calculated based on the difference between the initial and final film weight with respect to the initial film weight. 2.5. Fabrication of CA-NEs@CMC films The cross-linked CMC films fabricated according to method in Section 2.3 were dried to a constant weight and accurately weighed 5.0 mg of the dried CMC films. Immersed the CMC films into carvacrol nanoemulsions for 10 min, then dried naturally, and the CA-NEs@CMC films were obtained. In subsequent experiments and discussion, the investigated CA-NEs@CMC film samples are labeled as CA-NEs-x@CMC which is obtained by immersing CMC films into nanoemulsion containing x % of carvacrol for 10 min.
2. Materials and methods 2.1. Materials Carboxymethyl chitosan (CMC, Mw ca. 20,000–30,000, degree of substitution over 80%) was purchased from Zhejiang Aoxing Biotechnology Co., Ltd. 1,1-diphenyl-2-picrylhydrazide (DPPH) was purchased from Solarbio. Absolute ethanol (99.7%) was purchased from Shanghai Titan Scientific Co., Ltd. Carvacrol (99%) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Surfactants including fatty alcohol polyoxyethylene ether carboxylic acid, primary alcobol ethoxylate, cocoamidopropyl betaine-35, polyquaternium-39, polyquaternium-7, Tween 80 and dodecyl dimethyl betaine were purchased from Shandong Usolf Chemical Technology Co., Ltd. Escherichia coli (E. coli, Gram-negative) ATCC25922 and Staphylococcus aureus (S. aureus, Gram-positive) ATCC25923 were used as model bacteria in antibacterial tests. The other chemicals and solvents were of analytical reagent grade and used as received. Deionized water was used for all the experiments. A high voltage supply source (DW-P403-2 ACDF) was procured from Tianjin Dongwen High Coltage Power Supply Co., Ltd.
2.6. Drug loading amount determination To determine the drug loading amount of CMC films, CA release experiments were carried out. Absolute ethanol was employed as the release medium. The cumulative amount of CA released from CA-NEsx@CMC films into absolute ethanol were monitored by a Shimadzu UV1800 spectrophotometer until CA were completely released from the films. More specifically, first, the calibration curve was acquired for CA using eight concentrations of standard CA ethanol solution. This step was repeated over 3 times in order to ensure the repeatability of the calibration curve (Fig. S1). Then the CA-NEs-x@CMC films fabricated according to method in Section 2.5 were immersed into a beaker containing 10.0 mL of absolute ethanol, which was replaced by a fresh one after a fixed time (10 min) of immersion to ensure constant release conditions until no CA could be detected in absolute ethanol. The concentration of CA released at each time point was determined by measuring the absorbance of CA ethanol solution at 276 nm based on the Beer-Lambert law which is linearity between the concentration and absorbance of the light at a specified wavelength. The actual weight of the released CA was calculated based on the measured concentration and the absolute ethanol volume. Drug loading amount of CMC films was calculated according to the following equation:
2.2. Preparation of CA-NEs CA-NEs were prepared using a method based on a spontaneous emulsification procedure. First, carvacrol and surfactant were mixed together under magnetic stirring to form a homogeneous organic phase. The organic phase was then added dropwise into deionized water while magnetically stirring the system at ambient temperature for 5 min.
Drug loading amount = (mc /mf) × 100% where mc (mg) and mf (mg) are the weight of the completely released CA from CA-NEs-x@CMC films and weight of dry CMC film, respectively.
2.3. Fabrication of CMC films CMC microgels were firstly prepared by mixing 5.0 mL of aqueous CMC solution (pH 8.9, 40 mg mL−1) and 5.0 mL of absolute ethanol with continuous stirring for 10 min at ambient temperature. The CMC microgels solution obtained was used for the preparation of CMC films. The CMC films were fabricated using an electrospray deposition setup with horizontal configuration. The setup was equipped with a high voltage supply source, a syringe (10.0 mL) and a stainless steel needle (φ 0.30 mm) on the syringe pump to control the feed rate. The tin foil (10 × 10 cm2) was fixed on the collector surface 10 cm away from the needle. The CMC microgels solution was drew into the syringe and the electrospray process was carried out in air at about 25 °C. The positive voltage, the flow rate and electrospray time were set to 25 kV,
2.7. Antioxidant activity The antioxidant activity of CA-NEs-x@CMC films was evaluated using 1,1-diphenyl-2-picrylhydrazide (DPPH) radical. Concretely, pure CMC film (5.0 mg) and CA-NEs-1.5@CMC, CA-NEs-2.5@CMC, CA-NEs4.0@CMC and CA-NEs-5.0@CMC films (weight of dry CMC film is 5.0 mg) were immersed in 3.0 mL of DPPH solution (10 μg mL−1), the system was then kept aside in the dark for 0.5 h at room temperature. At the end of the 0.5 h, the films were removed from DPPH solution and the absorbance of DPPH solution was measured at 517 nm. The 689
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antioxidant activity (AOA) of film samples was calculated using the following equation:
2.11. Characterization The weight of samples was weighed using a Shimadzu AUY220D hundred thousandth analytical balance. Digital camera images of were captured using a Nikon D3100 camera. Viscosities were measured with a Shanghai Bangxi NDJ-8S digital rotary viscometer. Surface tension was measured by a Shanghai Huake DMP-2C Micro differential pressure measuring instrument using maximum bubble pressure method. Measurement of particle size and morphology of samples was described as below.
AOA = [(A0–As)/A0] × 100% where A0 and As are the absorbance values of the DPPH solution without and with the presence of the film samples. 2.8. Antibacterial activity of films using colony counting S. aureus and E.coli were selected as model gram-positive and gramnegative bacteria. The antibacterial activities of CA-NEs-x@CMC films and pure CMC film (as control group) were assayed using colony counting. The film samples (1 × 1 cm2), 2 mL of PBS buffer solution and 10 μL of the standardized bacterial suspension (5 × 107 cfu·mL−1) were successively added into a centrifugal tube and then incubated for 1 h at 37 °C in the incubation chamber. After incubation, the film samples were removed from solution and the mixture solution was diluted to one-tenth of original concentration with PBS buffer solution. 20 μL of the diluted bacterial suspension was spread on nutrient agar plates, and the plates were incubated at 37 °C for 12 h. The numbers of viable colonies were manually counted and expressed as the mean cfu·mL−1. The bactericidal efficiencies of films could be calculated according to the following equation:
2.11.1. Particle size measurements The particle size distributions, mean particle diameters and zeta potential of nanoemulsions and microgels were measured at room temperature using a dynamic light scattering instrument (Malvern ZS90, Worcester-Shire, UK). This instrument determines the particle size from intensity − time fluctuations of a laser beam (633 nm) scattered from a sample at an angle of 173°. Each individual measurement for particle diameter was an average of 10 runs, while for zeta potential was an average of 50 runs. To avoid multiple scattering effects, samples were diluted appropriately before the particle size measurements with deionized water for CA-NEs and aqueous ethanol solution (50% v/v) for CMC microgels.
Sterilizing ratio = [(number of original colony‒number of survival colony)/umber of original colony] × 100% [31].
2.11.2. Morphology of samples The morphologies of CA-NEs and film samples were examined by a field emission scanning electron microscope (SEM; s-4800, Hitachi, Japan) at an operating voltage of 10.0 kV. CA-NEs coated on silicon wafer and film samples deposited on tin foil were dried at 4 °C and 25 °C, respectively for 24; the silicon wafers and tin foil were then fixed on the support using a double-sided adhesive tape and coated with platinum prior to examination.
2.9. Study of effectiveness of the films to preserve bread Wheat breads without adding preservative were employed to study effectiveness of the CA-NEs-x@CMC films to preserve food. Bread slices (about 5.0 g) were placed on the base of petri dishes (inside dimensions: 85 mm diameter, 15 mm height). Film samples (5 × 5 cm2) were cut and adhered to the top of the lid of petri dishes to ensure no direct contact between bread and film. Petri dishes were sealed with parafilm and then incubated in an incubation chamber at 25 °C for 7 days. Experiments were carried out in duplicate. After 7 days, microbiological quality of bread samples was evaluated by determining total aerobic and yeast and mold counts. Bread samples were homogenized with 45 mL of 0.1% peptone water for 30 min on a magnetic stirrer. Serial dilutions (10−1–10-6) were performed using peptone water. 30 μL of dilution was spread onto plate count agar for a total aerobic count and potato dextrose agar for yeast and mold count. The plates were incubated at 37 °C for 12 h and at 25 °C for 48 h for aerobic bacteria count and yeast and mold count, respectively [32]. The results are expressed in colony forming unit (cfu g−1). The growth inhibition rate (%) was calculated according to the following equation: Growth inhibition rate = [(Ncontrol－N
sample)/
3. Results and discussion 3.1. Preparation and characterization of CA-NEs Oil-in-water CA-NEs were prepared using a method based on spontaneous emulsification procedure. In order to simplify production process and reduce cost, here we designed using carvacrol as the oil phase without other carrier oil, because carvacrol itself is the hydrophobic oil. To produce a stable nanoemulsion, the effects of surfactant type, surfactant to oil ratio (SOR), carvacrol and surfactant contents in the system on the formation and stability of CA-NEs were investigated in detail. Initially, we examined the influence of surfactant type on the dispersity of CA-NEs produced using spontaneous emulsification. The surfactants including fatty alcohol polyoxyethylene ether carboxylic acid (AEC), primary alcobol ethoxylate, cocoamidopropyl betaine-35, polyquaternium-39, polyquaternium-7, Tween 80 and dodecyl dimethyl betaine were researched. A series of carvacrol nanoemulsions with fixed carvacrol (2.5%) and surfactant (8.8%) contents were prepared using different surfactants. Among them, AEC could be used for the preparation of homogeneous and transparent CA-NEs (Fig. 1A). In contrast, when other surfactants were used, the droplet diameter was relatively large, and phase separation rapidly occurred. Thus, in the subsequent experiments, AEC was selected as an emulsifier to produce carvacrol nanoemulsions. The influence of SOR on the stability of CA-NEs was then studied. As clearly showed in Fig. 1B, AEC content was 8.8% in the system, the formation of stable nanoemulsions could sustain the maximal content of 2.5% for carvacrol. However, when the carvacrol content exceeded 2.5% obvious phase separation occurred in the system. Namely, when the stable nanoemulsions with maximal carvacrol content were formed, the SOR was 3.5:1. Further, the effects of AEC and carvacrol contents in the system on transparency and particle size of CA-NEs were
Ncontrol] × 100%
where Ncontrol and Nsample are the numbers of colonies (cfu g −1) of the bread sample without films and with films stored at 25 °C for 7 days. In addition, to investigate the effects of carvacrol released from the CA-NEs-x@CMC films into headspace on sensory properties of bread, the smell and color of bread samples with CA-NEs-5.0@CMC film protection were monitored everyday for 7 days. Experiments were carried out in triplicate. 2.10. Statistical analysis All experiments were carried out three times using freshly prepared samples, and the results were reported as the mean and standard deviation of these measurements. Statistical analysis were performed using a one-way analysis of variance (ANOVA) using the SPSS 19.0. The mean values were compared using a LSD-Duncan’s multiple range test and a p value of < 0.05 was considered to be statistically significant. 690
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Fig. 1. (A) Effects of surfactant type on the formation and physical stability of carvacrol emulsions. From left to right, surfactants used in the photograph are AEC, primary alcobol ethoxylate, cocoamidopropyl betaine-35, polyquaternium-39, polyquaternium-7, Tween 80 and dodecyl dimethyl betaine, respectively. (B) Effects of surfactant to oil ratio (SOR) on the physical stability of carvacrol emulsions. AEC was 8.8 wt % in the system. From left to right, the carvacrol contents in the system are 1.5%, 2.0%, 2.5%, 3.0% and 3.5%, respectively.
investigated by preparing a series of CA-NEs with a fixed SOR (3.5:1). With increasing surfactant and carvacrol contents in the system, the transparency of CA-NEs enhanced (Fig. 2A) but mean droplet diameter of CA-NEs increased slightly in both the aqueous phase and dry state (Fig. 2B) and the nanoemulsions in four case were all spherical (Fig. 2D). For example, the nanoemulsions containing 5.0% of carvacrol also had relatively small initial droplet diameters (about 180 nm in dry state) and the nanoemulsions solution was highly transparent and stable to no phase separation over a few months. However, we also observed that the nanoemulsions prepared using exceed 5.0% carvacrol, were unstable to droplet growth, the mean droplet diameter increased rapidly over a few days, and even phase separation occurred (data not shown). The physicochemical origin of droplet growth at high surfactant content is currently unknown and may have been owing to droplet coalescence and/or Ostwald ripening at elevated surfactant levels. In addition, Fig. 2C showed that CA-NEs were negatively charged in aqueous solution and the zeta potential values of CA-NEs enhanced slowly with the increase of surfactant and carvacrol contents in the system, indicating that the charge amount on the surface of CANEs decreased. This is because that AEC is a weak acid whose degree of dissociation decrease with the increase of the concentration. In a word, the above experimental results demonstrated that the
present method using carvacrol as the oil phase without introducing other carrier oil could be used for preparation of stable CA-NEs and the size of CA-NEs (dry state), which was almost constant as long as the SOR keep 3.5:1 unchanged and the content of carvacrol does not exceed 5.0% in the system, although the possible reasons for some above experimental phenomenon is currently unknown. 3.2. Fabrication and characterization of CMC microgels and films Our previous studies have demonstrated that the polymeric microgels could be rapidly deposited on the surface of solid substrates to fabricate film (or coating) materials using an electrospray technique [25]. Here, CMC microgels were first synthesized as briefly explained in the Method section. We found that mixing aqueous CMC solution with absolute ethanol could form CMC hydrosol. The CMC hydrosols obtained were homogeneous and transparent and had conspicuous Tyndall effect, while aqueous CMC solution had no Tyndall effect (Fig. 3A). In addition, the viscosity number of CMC hydrosols (80.9 ± 0.8 mPa·s) significantly increased when compared to the viscosity number of aqueous CMC solution (31.4 ± 0.9 mPa·s) indicating that the entanglement of CMC chains strengthened due to intermolecular crosslinking. The formation of CMC hydrosols can be explained as follow: 691
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Fig. 2. Effect of surfactant and CA contents on (A) transparency, (B) mean particle diameter, (C) zeta potential (D) top-view SEM images of CA-NEs produced by spontaneous emulsification. The surfactant used is AEC. SOR in the system was fixed at 3.5:1. From left to right in (A) and (D), the CA content in system are 1.5%, 2.5%, 4.0% and 5.0%, respectively.
migration of the encapsulated active compounds in its environment [33]. Therefore, it is important to further enhance stability of CMC films in water for practical application due to high solubility of CMC in water. It was reported that CMC could be crosslinked with Ca2+ to form hydrogel beads [34]. Thus, the CMC films were further crosslinked via Ca2+ by immersing the CMC films into Ca2+ solution for 3 h. The results showed that the weight of the crosslinked CMC films increased by 2.3 ± 0.4% compared to pure CMC films (Table S1), indicating that Ca2+ was introduced in the films and the CMC films have not been solubilized in Ca2+ solution in the process of crosslinking. Further, the stability of the CMC films in water was analyzed via film’s solubility in water. The solubility value for the CMC films before crosslinked by Ca2+ was (82.6 ± 1.0) % (p > 0.05), in contrast, the solubility value for the crosslinked CMC films was (3.8 ± 0.4) % (p > 0.05). Thus, the crosslinked CMC films showed highly stability in water compared to non-crosslinked CMC films. In addition, as shown in Fig. 4A, the crosslinked CMC film was visually continuous and dense with no bubbles, cracks and its thickness was 9.28 ± 0.10 μm, which was marked by a yellow arrow in the inset of Fig. 4B, indicated that the crosslinked CMC films should be a good candidate for practical applications, such as the packaging materials.
water and ethanol which are mutually soluble, but CMC is hardly soluble in ethanol, thus when aqueous CMC solution was mixed with a mountain of absolute ethanol, the CMC molecules aggregated to form the particles in aqueous ethanol solution based on intermolecular hydrogen bonds. If assumed that particles are spherical in shape, DLS measurements indicated that the average diameter of CMC microgels was around 825 nm, while the average diameter of CMC molecules in aqueous solution was only 58.8 nm (Fig. 3B), these results verified the above explaining for the formation of CMC microgels. In addition, surface tension of CMC microgels solution (29.36 ± 0.12 × 10−3 N·m1 ) was lower than that of aqueous CMC solution (72.31 ± 0.05 × 10−3 N·m-1) due to that ethanol weakened the intermolecular interaction between CMC microgel and H2O as well as H2O and H2O. The low surface tension of CMC microgels which is beneficial to prepare the CMC films using the CMC microgels as the film-forming raw material via the electrospray technique. The preparation experiments of films showed that CMC microgels could be rapidly deposited on the surface of tin foil to fabricate the CMC films by the electrospray technique. However using aqueous CMC solution as the film-forming solution to fabricate CMC films, which was infeasible due to CMC’s high surface tension in aqueous solution. Stability of the films in water was an important performance for practical application, which could impact film integrity as well as the
Fig. 3. (A) Optical properties and (B) particle size of CMC molecules and CMC microgels in aqueous solution. 692
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Fig. 4. Top-view and cross-sectional SEM images of film samples: (A, B), (C, D), (E, F), (G, H), and (I, J) are CMC, CA-NEs-1.5@CMC, CA-NEs-2.5@CMC, CA-NEs4.0@CMC and CA-NEs-5.0@CMC films, respectively. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)
3.3. Fabrication and performances of CA-NEs-x@CMC films 3.3.1. Fabrication and characterization of CA-NEs-x@CMC films The crosslinked CMC films were immersed in CA-NEs solution with different carvacrol level for 10 min and then dried naturally to fabricate CA-NEs-x@CMC films. Fig. 4 showed that four films of CA-NEs1.5@CMC, CA-NEs-2.5@CMC, CA-NEs-4.0@CMC and CA-NEs5.0@CMC were all continuous and dense. However, there were some wrinkles on the surface of these films, and thickness of four films all increased when they compared to the pure CMC films since CMC films became swelling after immersing them in CA-NEs solution and CA-NEs were absorbed into the CMC films. More specifically, after immersing the CMC films in CA-NEs solution, the surface morphology and thickness of the CA-NEs-x@CMC films showed the change rules as following: with the increasing of carvacrol level in nanoemulsions solution, wrinkles on the surface of CA-NEs-x@CMC films became less and flatter, and the increment of the thickness of CA-NEs-x@CMC films also decreased. This is due to the fact that the osmotic pressure of CA-NEs solution increased with the increase of carvacrol level in nanoemulsions solution, resulting in the decrease of swelling level of CMC films in nanoemulsions solution. The reduced swelling level of CMC films necessarily made the decreasing of film thickness increment.
4.0@CMC and CA-NEs-5.0@CMC were the same and could be well fitted by Korsmeyer–Peppas model. This indicated that the CA release from the CA-NEs-x@CMC films was a diffusion controlled process. At the initial 10 min, over 95% of CA were rapidly diffused into absolute ethanol, indicating a burst release in the release process, and thereafter slowed down until the remaining CA were completely released in about 60 min. The absorbance of the CA released in absolute ethanol at 276 nm obeyed Beer’s law. The concentration of the CA released from the film was determined using a calibration curve for CA in absolute ethanol (Fig. S1). The mass of the released CA was calculated based on the measured concentration and the absolute ethanol volume. It could be obtained that after immersed 5 mg of dry CMC films into 1.5%, 2.5%, 4.0% and 5.0% of CA-NEs solution for 10 min, the CA-loading amounts of CMC films are 4.74 ± 0.15, 6.98 ± 0.57, 19.19 ± 1.60, 26.96 ± 1.33 mg/g, respectively; in other words, for above four films, weight ratio of CA to CMC film were about 0.47%, 0.70%, 1.92%, 2.70%, respectively. Obviously, CA loading amount within CMC films increased with increasing the concentration CA-NEs in aqueous solution in the same loading time. This is because that CA-NEs mainly depends on its diffusion to go into the CMC films, while the high concentration difference provided greater driving force for CA-NEs to diffuse into inside of swollen CMC films.
3.3.2. Drug-loading amounts In order to determine the drug loading amount of CA-NEs-x@CMC films, carvacrol’s good solvent, absolute ethanol was employed as medium to study release kinetics of CA from the CA-NEs-x@CMC films and the results were summarized in Fig. 5A. The release patterns of CA from four films of CA-NEs-1.5@CMC, CA-NEs-2.5@CMC, CA-NEs-
3.3.3. Antioxidant activity of CA-NE-x@CMC films DPPH radical scavenging assay is widely employed to evaluate the antioxidant activity (AOA) of phenolic compounds. In the presence of an H-donating antioxidant, the dark purple DPPH radical can be reduced to yellow DPPH-H in an ethanol solution [35]. Thus, the AOA of CA-NEs-x@CMC films was tested by DPPH assay. Results in Fig. 5B
Fig. 5. (A) Time-dependent release profiles of carvacrol from CA-NEs-x@CMC films. (B) Antioxidant activity of film samples. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.) 693
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Fig. 6. Photographs for the bacterial culture plates of (A) S. aureus and (B) E. coli upon a 60 min contact of film samples.
antibacterial activity, and the antimicrobial activity of films enhanced with increasing the carvacrol amount within the films (Table 1). For example, nearly no survival colonies were detected on the culture plate when two bacteria come into contact with CA-NEs-5.0@CMC films for 1 h. The experimental results above demonstrated that the CA-NEs5.0@CMC films can almost completely inactivate E. coli and S. aureus with bacterial concentrations of 5 × 107 cfu·mL−1 within 1 h. Moreover, the results also indicated that the antibacterial activity of the CANEs-x@CMC films is mainly benefited from carvacrol within the films. Indeed, previous reports expounded that the mechanism of antibacterial activity of carvacrol consisted in a directly bind of the peptidoglycan layer in the membrane of Gram-positive bacteria [36]; for E. coli, carvacrol exerts its effect via inducing changes in cell membrane permeability, releasing of proteins and nucleic acid and reduction in ATP levels [37].
showed that pure CMC films almost have no radical scavenging activity on DPPH since CMC has no the performance of H-donating, in contrast to CMC films, CA-NEs-x@CMC films possessed good antioxidant activity. Furthermore, the AOA of CA-NEs-x@CMC films significantly increased with increasing the carvacrol levels within CMC films and this increase in radical scavenging activity was significant (p < 0.05). In other words, the AOA of CA-NEs-x@CMC films are strongly dependent on the carvacrol levels within CMC films, for example, AOA of CA-NEs1.5@CMC film was 21.7%, while AOA of CA-NEs-5.0@CMC film was 53.7%. In addition to this, the results also indicated that the antioxidant activity of the CA-NEs-x@CMC films is completely attributed to carvacrol, which is capable of acting as strong donor of hydrogen atoms. Therefore, the CA-NEs-x@CMC films have potential applications as an antioxidant food packaging. 3.3.4. Antibacterial activity of CA-NEs-x@CMC films Antibacterial activities of CA-NEs-x@CMC films against S. aureus and E. coli with a bacterial concentration of 5 × 107 cfu·mL−1 were evaluated via plate counting measurements. These bacteria are the common cause of various humans and animals infections thus they were selected as the study subjects. As seen in Fig. 6A and B, the photographs of the culture plates visualize the survival case of bacteria in which survival colonies are small white dots. Bacteria both S. aureus and E. coli present dense colonies on the control plate, indicating that their robust growth in the absence of the CA-NEs-x@CMC films. Similarly, slightly decrease yet still dense colonies are detected on the culture plate of two bacteria being treated with pure CMC films, suggesting that the pure CMC films only possess low antibacterial activities against S. aureus and E. coli. In contrast, when carvacrol nanoemulsions were introduced into the films, the CA-NEs-x@CMC films showed excellent
3.3.5. Study of effectiveness of the films to preserve bread Currently, adding preservatives are common method to prolong shelf life of bread. However, now, there are increasing consumer concerns about negative consequence of preservatives to human health. Therefore, there is a demand to develop a new strategy to prolong shelf life of bread without preservatives. Here, ability of CA-NEs-x@CMC films to preserve bread samples was evaluated. Fig. 7 shows the photographs of bread samples without or with CA-NEs-x@CMC film protection after storage at 25 °C for 7 days. The growth of microorganism was distinctly seen in bread samples without CA-NEs-x@CMC film protection (Fig. 7A). In contrast, the decrease of microbial growth was visually observed in bread samples with CMC and CA-NEs-x@CMC films protection. Especially, using CA-NEs-4.0@CMC and CA-NEs5.0@CMC films to protect bread sample, no visual evidence of
Table 1 Sterilizing ratio of film samples against S. aureus and E. coli and growth inhibition rates (%) of film samples against aerobic bacteria, mold and yeast in wheat bread samples. Film samples
Sterilizing ratio S. aureus
CMC CA-NEs-1.5@CMC CA-NEs-2.5@CMC CA-NEs-4.0@CMC CA-NEs-5.0@CMC
22.2 80.3 92.3 99.7 99.9
± ± ± ± ±
2.0%a 1.1%b 1.4%c 0.3%d 0.1%d
Growth inhibition rate E.coli 21.4 55.4 67.7 98.2 99.0
Aerobic bacteria ± ± ± ± ±
2.8%a 0.6%b 8.5%c 0.5%d 0.2%d
13.1 43.5 74.3 98.9 99.8
± ± ± ± ±
2.1a 3.2b 3.3c 0.3d 0.4d
Mold and yeast 22.9 55.5 81.5 95.7 99.4
Mean values ± standard deviation (n = 3). Different letters in the same column indicate significant differences (p < 0.05). 694
± ± ± ± ±
1.9a 3.4b 1.7c 0.6d 1.0e
11.2 27.0 69.1 90.0 98.8
± ± ± ± ±
1.6a 2.8b 3.8c 2.5d 1.0e
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Fig. 7. Images of wheat bread samples (A) without and with (B) CMC, (C) CA-NEs-1.5@CMC, (D) CA-NEs-2.5@CMC, (E) CA-NEs-4.0@CMC and (F) CA-NEs5.0@CMC films protection after storage at 25 °C for 7 days.
ability to extend the shelf life of wheat bread. In addition, we anticipate that the distinct advantages of the present method for the preparation of CA-NEs and CMC microgels, and loading of CA-NEs into CMC microgel films will include the following: (i) The low-energy spontaneous emulsification method with essential oils itself as the oil phase without introducing carrier oil for the preparation of physically stable O/W nanoemulsions, which possesses the advantages of simple production procedure and low cost. (ii) A solvent exchange method using ethanol as the poor solvent to fabricate polymeric microgels, which not only is facile and effective, but also can adjustment physical properties of the microgels system, such as surface tension. (iii) Different from the solution blend, immersing dry films in nanoemulsions solution for loading of nanoemulsions into polymeric microgel films, which does not alter initial physical properties and performance of nanoemulsions, such as size, biological activity, etc. More importantly, we believe that the present methods to prepare essential oil nanoemulsions, polymeric microgels and films, as well as polymeric films loaded with nanoemulsions for active packaging can be extended to other types of polymers and antimicrobial essential oils. The results reported in this work have important implications for the preparation and utilization of essential oil nanoemulsions and polymeric microgels in the food and other industries.
microbial growth was observed in bread samples. The growth inhibition rate of CA-NEs-x@CMC films on aerobic mesophilic bacteria and mold and yeast was also researched. As shown in Table 1, the growth inhibition of both aerobic mesophilic bacteria and mold and yeast in bread samples increased with the increasing of carvacrol content in the CA-NEs-x@CMC films. The results confirmed what have been shown in the photographs of bread samples in Fig. 7. Especially, CA-NEs-5.0@CMC films showed the excellent inhibitory effects on aerobic mesophilic bacteria, mold and yeast, and exhibited 99.8%, 99.4% and 98.8% growth inhibition rate against aerobic bacteria, mold and yeast, respectively, whereas pure CMC films showed very low inhibitory effects on the above bacteria. It has been reported that the inhibitory effect of carvacrol on bacteria is attributed to the alteration of cell membrane permeability by the action of phenols [38]. On the other hand, the effects of released carvacrol into headspace on organoleptic properties of bread were also investigated. The smell and color of bread samples with CA-NEs-5.0@CMC film protection were monitored everyday. The results showed that in 3 days before the storage, there was no carvacrol’s smell in bread samples, while from the fourth day to the seventh day, only slight carvacrol’s scent exhaled from the bread samples, indicating that a small number of carvacrol could migrate into bread; in addition, color of the bread samples had hardly any change within 7 days (image data not shown). The results above demonstrated the CA-NEs-x@CMC films are promising alternatives as food packaging to prolong shelf life of bread.
Acknowledgment This work was supported by the National Natural Science Foundations of China (Grant No. 21808188) and the Natural Science Foundation of Shaanxi Province (Grant No. 2017JQ5043).
4. Conclusion In summary, the facile and innovative strategies established in this work to fabricate CA-NEs, CMC microgels followed by subsequent loading of CA-NEs into the CMC films were all feasible. The CANEs@CMC films obtained exhibited good antioxidant activity, desirable antibacterial activity against S. aureus and E. coli, as well as superior
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2018.12.054. 695
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
The innovative fabrication and applications of carvacrol nanoemulsions, carboxymethyl chitosan microgels and their composite films
T
⁎
Kai Lei, Xinran Wang, Xiaozhou Li , Lin Wang College of Chemistry & Pharmacy, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Nanoemulsions Carvacrol Polymeric Microgels Films Active packaging
Carvacrol (CA) was firstly made into physically stable nanoemulsions using an innovative spontaneous emulsification method with carvacrol itself as oil phase without introducing other carrier oils. Self-crosslinking carboxymethyl chitosan (CMC) microgels via intermolecular hydrogen bonds were then fabricated using a simple solvent exchange method with ethanol as poor solvent. The CMC microgels obtained were rapidly deposited on the surface of tin foil using an electrospray technique to produce the CMC films. The stability of the films in water was further intensified by using Ca2+ as cross-linking agent. Also, the carvacrol nanoemulsions (CA-NEs) were loaded into the CMC films by utilizing swelling property of CMC microgels in aqueous solution. The results showed that the CA-NEs@CMC composite films possess satisfactory antioxidant activity, good antibacterial activity against S. aureus and E. coli, and excellent ability to extend the shelf life of wheat bread. We believe that the present strategies to prepare CA-NEs, CMC microgels and their composite films also apply to other types of essential oils and polymers, and the CA-NEs@CMC composite film is an excellent candidate for the active packaging.
1. Introduction Natural antimicrobial agents, essential oils have low extract cost, wide-spectrum antimicrobial activity and outstanding antioxidant performance [1,2] and therefore have attracted considerable scientific interests as functional ingredients in pharmaceutical, cosmetic, and food applications [3]. Carvacrol (CA) is a major component of oregano and thyme oil, which has been proven to be a very active antimicrobial agent against fungi, food-borne pathogens, yeast and molds, etc., besides, it is also an excellent antioxidant [4–6]. More importantly, carvacrol is classified as Generally Recognized as Safe (GRAS) substance by the Food and Drug Administration [7] and is registered by the European Commission as healthy with no risk to the consumers [8]. However, carvacrol is highly lipophilic with poor solubility and stability in aqueous media that limits its application in many industries [9,10]. Nanoemulsion can be generally classified into oil-in-water and water-in-oil types, is a colloidal nanodispersion of oil and water being stabilized by interfacial layer of surfactant. The size of the droplets in nanoemulsions is often much smaller, which means that they have much better stability to gravitational separation, flocculation, and coalescence than macroemulsions [11,12]. The major components of nanoemulsions include oil phase, emulsifying agent and aqueous phase. Among them, carrier oil within the oil phase is employed to dissolve active component, ⁎
however, the inappropriate select of carrier oil would seriously impact the formation, stability and performance of a nanoemulsion [12]. For that reason, we design that using carvacrol itself as the oil phase without introducing other carrier oil prepare oil-in-water (O/W) carvacrol nanoemulsions (CA-NEs) with a spontaneous emulsification method since carvacrol itself is exactly the oil. We speculate that the discarding of carrier oil can not only simplify production technique and reduce manufacturing cost of nanoemulsions, but also avoid the influence of introducing carrier oil on the application of nanoemulsions. Carboxymethyl chitosan (CMC) is a water-soluble, non-toxic, biodegradable and biocompatible chitosan derivative, which has good abilities to form hydrogels and films and has better biological activity compared with chitosan [13,14]. Polymer-based materials, such as micro- and nanoparticles, hydrogels, and films, are capable of encapsulating bioactive molecules for various applications [15,16]. Among them, polymer films (or coatings) are particularly suitable as bioactive molecules-loaded carriers deposited on solid substrates [17–20]. Electrospray deposition is an efficient and greatly materialsaving technique for the fabrication of functional films (or coatings) [21–23]. The electrospray technique employs an electric field between a film-forming solution contained within a syringe and a conductive substrate to direct produce nearly dry films on the substrates. Compared with other film-preparation methods, the electrospray technique
Corresponding author. E-mail address: [email protected] (X. Li).
https://doi.org/10.1016/j.colsurfb.2018.12.054 Received 10 October 2018; Received in revised form 17 December 2018; Accepted 18 December 2018 Available online 19 December 2018 0927-7765/ © 2018 Elsevier B.V. All rights reserved.
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works quickly and can fabricate large area films on non-flat surfaces [24–26]. In addition, our previous studies demonstrated that using polymeric microgels as film-forming raw materials could more rapidly produce continuous and dense polymeric films [25]. Microbial contamination is a quite frequent phenomenon for food products, which seriously threatens the health of humans. In the past, adding chemical preservatives and using plastic packaging materials were common methods to avoid food contaminate and prolong shelf life of food. However, consumers worry increasingly about negative consequences of preservatives nowadays, in addition, poor biodegradation of plastic materials has generated a massive accumulation of plastic waste in the environment [27]. The development of antimicrobial and biodegradable packaging has been the focus of recent research [28–30]. Therefore, the aims of this work were as follows: oil-in-water (O/W) CA-NEs are prepared using spontaneous emulsification method without introducing carrier oil and the preparation parameters of the nanoemulsions are optimized; CMC microgels are fabricated using a solvent exchange method and are employed as film-forming raw materials to produce the CMC films on the surface of tin foil via an electrospray technique; CA-NEs are introduced into the CMC films for the preparation of CA-NEs@CMC composite films and the performances of the composite films as active packaging are studied in detail.
0.1 mL·min-1and 1 h, respectively, for the fabrication of the CMC films. The films obtained were immersed in CaCl2 solution (pH 7.1, 1.0 mol L1 ) for 3 h to crosslink CMC in films. 2.4. Film’s stability in water The stability of the CMC films in water was analyzed via film’s solubility in water (SW). For SW measurements, the cross-linked CMC films deposited on tin foil (10 × 10 cm2) were dried at 105 °C for 24 h and then determined the initial weight using an analytical balance. Dried films were immersed in 50 mL of deionized water for 24 h at 25 °C. Film samples were then removed from water, and dried in an oven at 105 °C for 24 h to determine their final dry matter. Film solubility was calculated based on the difference between the initial and final film weight with respect to the initial film weight. 2.5. Fabrication of CA-NEs@CMC films The cross-linked CMC films fabricated according to method in Section 2.3 were dried to a constant weight and accurately weighed 5.0 mg of the dried CMC films. Immersed the CMC films into carvacrol nanoemulsions for 10 min, then dried naturally, and the CA-NEs@CMC films were obtained. In subsequent experiments and discussion, the investigated CA-NEs@CMC film samples are labeled as CA-NEs-x@CMC which is obtained by immersing CMC films into nanoemulsion containing x % of carvacrol for 10 min.
2. Materials and methods 2.1. Materials Carboxymethyl chitosan (CMC, Mw ca. 20,000–30,000, degree of substitution over 80%) was purchased from Zhejiang Aoxing Biotechnology Co., Ltd. 1,1-diphenyl-2-picrylhydrazide (DPPH) was purchased from Solarbio. Absolute ethanol (99.7%) was purchased from Shanghai Titan Scientific Co., Ltd. Carvacrol (99%) was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. Surfactants including fatty alcohol polyoxyethylene ether carboxylic acid, primary alcobol ethoxylate, cocoamidopropyl betaine-35, polyquaternium-39, polyquaternium-7, Tween 80 and dodecyl dimethyl betaine were purchased from Shandong Usolf Chemical Technology Co., Ltd. Escherichia coli (E. coli, Gram-negative) ATCC25922 and Staphylococcus aureus (S. aureus, Gram-positive) ATCC25923 were used as model bacteria in antibacterial tests. The other chemicals and solvents were of analytical reagent grade and used as received. Deionized water was used for all the experiments. A high voltage supply source (DW-P403-2 ACDF) was procured from Tianjin Dongwen High Coltage Power Supply Co., Ltd.
2.6. Drug loading amount determination To determine the drug loading amount of CMC films, CA release experiments were carried out. Absolute ethanol was employed as the release medium. The cumulative amount of CA released from CA-NEsx@CMC films into absolute ethanol were monitored by a Shimadzu UV1800 spectrophotometer until CA were completely released from the films. More specifically, first, the calibration curve was acquired for CA using eight concentrations of standard CA ethanol solution. This step was repeated over 3 times in order to ensure the repeatability of the calibration curve (Fig. S1). Then the CA-NEs-x@CMC films fabricated according to method in Section 2.5 were immersed into a beaker containing 10.0 mL of absolute ethanol, which was replaced by a fresh one after a fixed time (10 min) of immersion to ensure constant release conditions until no CA could be detected in absolute ethanol. The concentration of CA released at each time point was determined by measuring the absorbance of CA ethanol solution at 276 nm based on the Beer-Lambert law which is linearity between the concentration and absorbance of the light at a specified wavelength. The actual weight of the released CA was calculated based on the measured concentration and the absolute ethanol volume. Drug loading amount of CMC films was calculated according to the following equation:
2.2. Preparation of CA-NEs CA-NEs were prepared using a method based on a spontaneous emulsification procedure. First, carvacrol and surfactant were mixed together under magnetic stirring to form a homogeneous organic phase. The organic phase was then added dropwise into deionized water while magnetically stirring the system at ambient temperature for 5 min.
Drug loading amount = (mc /mf) × 100% where mc (mg) and mf (mg) are the weight of the completely released CA from CA-NEs-x@CMC films and weight of dry CMC film, respectively.
2.3. Fabrication of CMC films CMC microgels were firstly prepared by mixing 5.0 mL of aqueous CMC solution (pH 8.9, 40 mg mL−1) and 5.0 mL of absolute ethanol with continuous stirring for 10 min at ambient temperature. The CMC microgels solution obtained was used for the preparation of CMC films. The CMC films were fabricated using an electrospray deposition setup with horizontal configuration. The setup was equipped with a high voltage supply source, a syringe (10.0 mL) and a stainless steel needle (φ 0.30 mm) on the syringe pump to control the feed rate. The tin foil (10 × 10 cm2) was fixed on the collector surface 10 cm away from the needle. The CMC microgels solution was drew into the syringe and the electrospray process was carried out in air at about 25 °C. The positive voltage, the flow rate and electrospray time were set to 25 kV,
2.7. Antioxidant activity The antioxidant activity of CA-NEs-x@CMC films was evaluated using 1,1-diphenyl-2-picrylhydrazide (DPPH) radical. Concretely, pure CMC film (5.0 mg) and CA-NEs-1.5@CMC, CA-NEs-2.5@CMC, CA-NEs4.0@CMC and CA-NEs-5.0@CMC films (weight of dry CMC film is 5.0 mg) were immersed in 3.0 mL of DPPH solution (10 μg mL−1), the system was then kept aside in the dark for 0.5 h at room temperature. At the end of the 0.5 h, the films were removed from DPPH solution and the absorbance of DPPH solution was measured at 517 nm. The 689
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antioxidant activity (AOA) of film samples was calculated using the following equation:
2.11. Characterization The weight of samples was weighed using a Shimadzu AUY220D hundred thousandth analytical balance. Digital camera images of were captured using a Nikon D3100 camera. Viscosities were measured with a Shanghai Bangxi NDJ-8S digital rotary viscometer. Surface tension was measured by a Shanghai Huake DMP-2C Micro differential pressure measuring instrument using maximum bubble pressure method. Measurement of particle size and morphology of samples was described as below.
AOA = [(A0–As)/A0] × 100% where A0 and As are the absorbance values of the DPPH solution without and with the presence of the film samples. 2.8. Antibacterial activity of films using colony counting S. aureus and E.coli were selected as model gram-positive and gramnegative bacteria. The antibacterial activities of CA-NEs-x@CMC films and pure CMC film (as control group) were assayed using colony counting. The film samples (1 × 1 cm2), 2 mL of PBS buffer solution and 10 μL of the standardized bacterial suspension (5 × 107 cfu·mL−1) were successively added into a centrifugal tube and then incubated for 1 h at 37 °C in the incubation chamber. After incubation, the film samples were removed from solution and the mixture solution was diluted to one-tenth of original concentration with PBS buffer solution. 20 μL of the diluted bacterial suspension was spread on nutrient agar plates, and the plates were incubated at 37 °C for 12 h. The numbers of viable colonies were manually counted and expressed as the mean cfu·mL−1. The bactericidal efficiencies of films could be calculated according to the following equation:
2.11.1. Particle size measurements The particle size distributions, mean particle diameters and zeta potential of nanoemulsions and microgels were measured at room temperature using a dynamic light scattering instrument (Malvern ZS90, Worcester-Shire, UK). This instrument determines the particle size from intensity − time fluctuations of a laser beam (633 nm) scattered from a sample at an angle of 173°. Each individual measurement for particle diameter was an average of 10 runs, while for zeta potential was an average of 50 runs. To avoid multiple scattering effects, samples were diluted appropriately before the particle size measurements with deionized water for CA-NEs and aqueous ethanol solution (50% v/v) for CMC microgels.
Sterilizing ratio = [(number of original colony‒number of survival colony)/umber of original colony] × 100% [31].
2.11.2. Morphology of samples The morphologies of CA-NEs and film samples were examined by a field emission scanning electron microscope (SEM; s-4800, Hitachi, Japan) at an operating voltage of 10.0 kV. CA-NEs coated on silicon wafer and film samples deposited on tin foil were dried at 4 °C and 25 °C, respectively for 24; the silicon wafers and tin foil were then fixed on the support using a double-sided adhesive tape and coated with platinum prior to examination.
2.9. Study of effectiveness of the films to preserve bread Wheat breads without adding preservative were employed to study effectiveness of the CA-NEs-x@CMC films to preserve food. Bread slices (about 5.0 g) were placed on the base of petri dishes (inside dimensions: 85 mm diameter, 15 mm height). Film samples (5 × 5 cm2) were cut and adhered to the top of the lid of petri dishes to ensure no direct contact between bread and film. Petri dishes were sealed with parafilm and then incubated in an incubation chamber at 25 °C for 7 days. Experiments were carried out in duplicate. After 7 days, microbiological quality of bread samples was evaluated by determining total aerobic and yeast and mold counts. Bread samples were homogenized with 45 mL of 0.1% peptone water for 30 min on a magnetic stirrer. Serial dilutions (10−1–10-6) were performed using peptone water. 30 μL of dilution was spread onto plate count agar for a total aerobic count and potato dextrose agar for yeast and mold count. The plates were incubated at 37 °C for 12 h and at 25 °C for 48 h for aerobic bacteria count and yeast and mold count, respectively [32]. The results are expressed in colony forming unit (cfu g−1). The growth inhibition rate (%) was calculated according to the following equation: Growth inhibition rate = [(Ncontrol－N
sample)/
3. Results and discussion 3.1. Preparation and characterization of CA-NEs Oil-in-water CA-NEs were prepared using a method based on spontaneous emulsification procedure. In order to simplify production process and reduce cost, here we designed using carvacrol as the oil phase without other carrier oil, because carvacrol itself is the hydrophobic oil. To produce a stable nanoemulsion, the effects of surfactant type, surfactant to oil ratio (SOR), carvacrol and surfactant contents in the system on the formation and stability of CA-NEs were investigated in detail. Initially, we examined the influence of surfactant type on the dispersity of CA-NEs produced using spontaneous emulsification. The surfactants including fatty alcohol polyoxyethylene ether carboxylic acid (AEC), primary alcobol ethoxylate, cocoamidopropyl betaine-35, polyquaternium-39, polyquaternium-7, Tween 80 and dodecyl dimethyl betaine were researched. A series of carvacrol nanoemulsions with fixed carvacrol (2.5%) and surfactant (8.8%) contents were prepared using different surfactants. Among them, AEC could be used for the preparation of homogeneous and transparent CA-NEs (Fig. 1A). In contrast, when other surfactants were used, the droplet diameter was relatively large, and phase separation rapidly occurred. Thus, in the subsequent experiments, AEC was selected as an emulsifier to produce carvacrol nanoemulsions. The influence of SOR on the stability of CA-NEs was then studied. As clearly showed in Fig. 1B, AEC content was 8.8% in the system, the formation of stable nanoemulsions could sustain the maximal content of 2.5% for carvacrol. However, when the carvacrol content exceeded 2.5% obvious phase separation occurred in the system. Namely, when the stable nanoemulsions with maximal carvacrol content were formed, the SOR was 3.5:1. Further, the effects of AEC and carvacrol contents in the system on transparency and particle size of CA-NEs were
Ncontrol] × 100%
where Ncontrol and Nsample are the numbers of colonies (cfu g −1) of the bread sample without films and with films stored at 25 °C for 7 days. In addition, to investigate the effects of carvacrol released from the CA-NEs-x@CMC films into headspace on sensory properties of bread, the smell and color of bread samples with CA-NEs-5.0@CMC film protection were monitored everyday for 7 days. Experiments were carried out in triplicate. 2.10. Statistical analysis All experiments were carried out three times using freshly prepared samples, and the results were reported as the mean and standard deviation of these measurements. Statistical analysis were performed using a one-way analysis of variance (ANOVA) using the SPSS 19.0. The mean values were compared using a LSD-Duncan’s multiple range test and a p value of < 0.05 was considered to be statistically significant. 690
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Fig. 1. (A) Effects of surfactant type on the formation and physical stability of carvacrol emulsions. From left to right, surfactants used in the photograph are AEC, primary alcobol ethoxylate, cocoamidopropyl betaine-35, polyquaternium-39, polyquaternium-7, Tween 80 and dodecyl dimethyl betaine, respectively. (B) Effects of surfactant to oil ratio (SOR) on the physical stability of carvacrol emulsions. AEC was 8.8 wt % in the system. From left to right, the carvacrol contents in the system are 1.5%, 2.0%, 2.5%, 3.0% and 3.5%, respectively.
investigated by preparing a series of CA-NEs with a fixed SOR (3.5:1). With increasing surfactant and carvacrol contents in the system, the transparency of CA-NEs enhanced (Fig. 2A) but mean droplet diameter of CA-NEs increased slightly in both the aqueous phase and dry state (Fig. 2B) and the nanoemulsions in four case were all spherical (Fig. 2D). For example, the nanoemulsions containing 5.0% of carvacrol also had relatively small initial droplet diameters (about 180 nm in dry state) and the nanoemulsions solution was highly transparent and stable to no phase separation over a few months. However, we also observed that the nanoemulsions prepared using exceed 5.0% carvacrol, were unstable to droplet growth, the mean droplet diameter increased rapidly over a few days, and even phase separation occurred (data not shown). The physicochemical origin of droplet growth at high surfactant content is currently unknown and may have been owing to droplet coalescence and/or Ostwald ripening at elevated surfactant levels. In addition, Fig. 2C showed that CA-NEs were negatively charged in aqueous solution and the zeta potential values of CA-NEs enhanced slowly with the increase of surfactant and carvacrol contents in the system, indicating that the charge amount on the surface of CANEs decreased. This is because that AEC is a weak acid whose degree of dissociation decrease with the increase of the concentration. In a word, the above experimental results demonstrated that the
present method using carvacrol as the oil phase without introducing other carrier oil could be used for preparation of stable CA-NEs and the size of CA-NEs (dry state), which was almost constant as long as the SOR keep 3.5:1 unchanged and the content of carvacrol does not exceed 5.0% in the system, although the possible reasons for some above experimental phenomenon is currently unknown. 3.2. Fabrication and characterization of CMC microgels and films Our previous studies have demonstrated that the polymeric microgels could be rapidly deposited on the surface of solid substrates to fabricate film (or coating) materials using an electrospray technique [25]. Here, CMC microgels were first synthesized as briefly explained in the Method section. We found that mixing aqueous CMC solution with absolute ethanol could form CMC hydrosol. The CMC hydrosols obtained were homogeneous and transparent and had conspicuous Tyndall effect, while aqueous CMC solution had no Tyndall effect (Fig. 3A). In addition, the viscosity number of CMC hydrosols (80.9 ± 0.8 mPa·s) significantly increased when compared to the viscosity number of aqueous CMC solution (31.4 ± 0.9 mPa·s) indicating that the entanglement of CMC chains strengthened due to intermolecular crosslinking. The formation of CMC hydrosols can be explained as follow: 691
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Fig. 2. Effect of surfactant and CA contents on (A) transparency, (B) mean particle diameter, (C) zeta potential (D) top-view SEM images of CA-NEs produced by spontaneous emulsification. The surfactant used is AEC. SOR in the system was fixed at 3.5:1. From left to right in (A) and (D), the CA content in system are 1.5%, 2.5%, 4.0% and 5.0%, respectively.
migration of the encapsulated active compounds in its environment [33]. Therefore, it is important to further enhance stability of CMC films in water for practical application due to high solubility of CMC in water. It was reported that CMC could be crosslinked with Ca2+ to form hydrogel beads [34]. Thus, the CMC films were further crosslinked via Ca2+ by immersing the CMC films into Ca2+ solution for 3 h. The results showed that the weight of the crosslinked CMC films increased by 2.3 ± 0.4% compared to pure CMC films (Table S1), indicating that Ca2+ was introduced in the films and the CMC films have not been solubilized in Ca2+ solution in the process of crosslinking. Further, the stability of the CMC films in water was analyzed via film’s solubility in water. The solubility value for the CMC films before crosslinked by Ca2+ was (82.6 ± 1.0) % (p > 0.05), in contrast, the solubility value for the crosslinked CMC films was (3.8 ± 0.4) % (p > 0.05). Thus, the crosslinked CMC films showed highly stability in water compared to non-crosslinked CMC films. In addition, as shown in Fig. 4A, the crosslinked CMC film was visually continuous and dense with no bubbles, cracks and its thickness was 9.28 ± 0.10 μm, which was marked by a yellow arrow in the inset of Fig. 4B, indicated that the crosslinked CMC films should be a good candidate for practical applications, such as the packaging materials.
water and ethanol which are mutually soluble, but CMC is hardly soluble in ethanol, thus when aqueous CMC solution was mixed with a mountain of absolute ethanol, the CMC molecules aggregated to form the particles in aqueous ethanol solution based on intermolecular hydrogen bonds. If assumed that particles are spherical in shape, DLS measurements indicated that the average diameter of CMC microgels was around 825 nm, while the average diameter of CMC molecules in aqueous solution was only 58.8 nm (Fig. 3B), these results verified the above explaining for the formation of CMC microgels. In addition, surface tension of CMC microgels solution (29.36 ± 0.12 × 10−3 N·m1 ) was lower than that of aqueous CMC solution (72.31 ± 0.05 × 10−3 N·m-1) due to that ethanol weakened the intermolecular interaction between CMC microgel and H2O as well as H2O and H2O. The low surface tension of CMC microgels which is beneficial to prepare the CMC films using the CMC microgels as the film-forming raw material via the electrospray technique. The preparation experiments of films showed that CMC microgels could be rapidly deposited on the surface of tin foil to fabricate the CMC films by the electrospray technique. However using aqueous CMC solution as the film-forming solution to fabricate CMC films, which was infeasible due to CMC’s high surface tension in aqueous solution. Stability of the films in water was an important performance for practical application, which could impact film integrity as well as the
Fig. 3. (A) Optical properties and (B) particle size of CMC molecules and CMC microgels in aqueous solution. 692
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Fig. 4. Top-view and cross-sectional SEM images of film samples: (A, B), (C, D), (E, F), (G, H), and (I, J) are CMC, CA-NEs-1.5@CMC, CA-NEs-2.5@CMC, CA-NEs4.0@CMC and CA-NEs-5.0@CMC films, respectively. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.)
3.3. Fabrication and performances of CA-NEs-x@CMC films 3.3.1. Fabrication and characterization of CA-NEs-x@CMC films The crosslinked CMC films were immersed in CA-NEs solution with different carvacrol level for 10 min and then dried naturally to fabricate CA-NEs-x@CMC films. Fig. 4 showed that four films of CA-NEs1.5@CMC, CA-NEs-2.5@CMC, CA-NEs-4.0@CMC and CA-NEs5.0@CMC were all continuous and dense. However, there were some wrinkles on the surface of these films, and thickness of four films all increased when they compared to the pure CMC films since CMC films became swelling after immersing them in CA-NEs solution and CA-NEs were absorbed into the CMC films. More specifically, after immersing the CMC films in CA-NEs solution, the surface morphology and thickness of the CA-NEs-x@CMC films showed the change rules as following: with the increasing of carvacrol level in nanoemulsions solution, wrinkles on the surface of CA-NEs-x@CMC films became less and flatter, and the increment of the thickness of CA-NEs-x@CMC films also decreased. This is due to the fact that the osmotic pressure of CA-NEs solution increased with the increase of carvacrol level in nanoemulsions solution, resulting in the decrease of swelling level of CMC films in nanoemulsions solution. The reduced swelling level of CMC films necessarily made the decreasing of film thickness increment.
4.0@CMC and CA-NEs-5.0@CMC were the same and could be well fitted by Korsmeyer–Peppas model. This indicated that the CA release from the CA-NEs-x@CMC films was a diffusion controlled process. At the initial 10 min, over 95% of CA were rapidly diffused into absolute ethanol, indicating a burst release in the release process, and thereafter slowed down until the remaining CA were completely released in about 60 min. The absorbance of the CA released in absolute ethanol at 276 nm obeyed Beer’s law. The concentration of the CA released from the film was determined using a calibration curve for CA in absolute ethanol (Fig. S1). The mass of the released CA was calculated based on the measured concentration and the absolute ethanol volume. It could be obtained that after immersed 5 mg of dry CMC films into 1.5%, 2.5%, 4.0% and 5.0% of CA-NEs solution for 10 min, the CA-loading amounts of CMC films are 4.74 ± 0.15, 6.98 ± 0.57, 19.19 ± 1.60, 26.96 ± 1.33 mg/g, respectively; in other words, for above four films, weight ratio of CA to CMC film were about 0.47%, 0.70%, 1.92%, 2.70%, respectively. Obviously, CA loading amount within CMC films increased with increasing the concentration CA-NEs in aqueous solution in the same loading time. This is because that CA-NEs mainly depends on its diffusion to go into the CMC films, while the high concentration difference provided greater driving force for CA-NEs to diffuse into inside of swollen CMC films.
3.3.2. Drug-loading amounts In order to determine the drug loading amount of CA-NEs-x@CMC films, carvacrol’s good solvent, absolute ethanol was employed as medium to study release kinetics of CA from the CA-NEs-x@CMC films and the results were summarized in Fig. 5A. The release patterns of CA from four films of CA-NEs-1.5@CMC, CA-NEs-2.5@CMC, CA-NEs-
3.3.3. Antioxidant activity of CA-NE-x@CMC films DPPH radical scavenging assay is widely employed to evaluate the antioxidant activity (AOA) of phenolic compounds. In the presence of an H-donating antioxidant, the dark purple DPPH radical can be reduced to yellow DPPH-H in an ethanol solution [35]. Thus, the AOA of CA-NEs-x@CMC films was tested by DPPH assay. Results in Fig. 5B
Fig. 5. (A) Time-dependent release profiles of carvacrol from CA-NEs-x@CMC films. (B) Antioxidant activity of film samples. (For interpretation of the references to colour in the text, the reader is referred to the web version of this article.) 693
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Fig. 6. Photographs for the bacterial culture plates of (A) S. aureus and (B) E. coli upon a 60 min contact of film samples.
antibacterial activity, and the antimicrobial activity of films enhanced with increasing the carvacrol amount within the films (Table 1). For example, nearly no survival colonies were detected on the culture plate when two bacteria come into contact with CA-NEs-5.0@CMC films for 1 h. The experimental results above demonstrated that the CA-NEs5.0@CMC films can almost completely inactivate E. coli and S. aureus with bacterial concentrations of 5 × 107 cfu·mL−1 within 1 h. Moreover, the results also indicated that the antibacterial activity of the CANEs-x@CMC films is mainly benefited from carvacrol within the films. Indeed, previous reports expounded that the mechanism of antibacterial activity of carvacrol consisted in a directly bind of the peptidoglycan layer in the membrane of Gram-positive bacteria [36]; for E. coli, carvacrol exerts its effect via inducing changes in cell membrane permeability, releasing of proteins and nucleic acid and reduction in ATP levels [37].
showed that pure CMC films almost have no radical scavenging activity on DPPH since CMC has no the performance of H-donating, in contrast to CMC films, CA-NEs-x@CMC films possessed good antioxidant activity. Furthermore, the AOA of CA-NEs-x@CMC films significantly increased with increasing the carvacrol levels within CMC films and this increase in radical scavenging activity was significant (p < 0.05). In other words, the AOA of CA-NEs-x@CMC films are strongly dependent on the carvacrol levels within CMC films, for example, AOA of CA-NEs1.5@CMC film was 21.7%, while AOA of CA-NEs-5.0@CMC film was 53.7%. In addition to this, the results also indicated that the antioxidant activity of the CA-NEs-x@CMC films is completely attributed to carvacrol, which is capable of acting as strong donor of hydrogen atoms. Therefore, the CA-NEs-x@CMC films have potential applications as an antioxidant food packaging. 3.3.4. Antibacterial activity of CA-NEs-x@CMC films Antibacterial activities of CA-NEs-x@CMC films against S. aureus and E. coli with a bacterial concentration of 5 × 107 cfu·mL−1 were evaluated via plate counting measurements. These bacteria are the common cause of various humans and animals infections thus they were selected as the study subjects. As seen in Fig. 6A and B, the photographs of the culture plates visualize the survival case of bacteria in which survival colonies are small white dots. Bacteria both S. aureus and E. coli present dense colonies on the control plate, indicating that their robust growth in the absence of the CA-NEs-x@CMC films. Similarly, slightly decrease yet still dense colonies are detected on the culture plate of two bacteria being treated with pure CMC films, suggesting that the pure CMC films only possess low antibacterial activities against S. aureus and E. coli. In contrast, when carvacrol nanoemulsions were introduced into the films, the CA-NEs-x@CMC films showed excellent
3.3.5. Study of effectiveness of the films to preserve bread Currently, adding preservatives are common method to prolong shelf life of bread. However, now, there are increasing consumer concerns about negative consequence of preservatives to human health. Therefore, there is a demand to develop a new strategy to prolong shelf life of bread without preservatives. Here, ability of CA-NEs-x@CMC films to preserve bread samples was evaluated. Fig. 7 shows the photographs of bread samples without or with CA-NEs-x@CMC film protection after storage at 25 °C for 7 days. The growth of microorganism was distinctly seen in bread samples without CA-NEs-x@CMC film protection (Fig. 7A). In contrast, the decrease of microbial growth was visually observed in bread samples with CMC and CA-NEs-x@CMC films protection. Especially, using CA-NEs-4.0@CMC and CA-NEs5.0@CMC films to protect bread sample, no visual evidence of
Table 1 Sterilizing ratio of film samples against S. aureus and E. coli and growth inhibition rates (%) of film samples against aerobic bacteria, mold and yeast in wheat bread samples. Film samples
Sterilizing ratio S. aureus
CMC CA-NEs-1.5@CMC CA-NEs-2.5@CMC CA-NEs-4.0@CMC CA-NEs-5.0@CMC
22.2 80.3 92.3 99.7 99.9
± ± ± ± ±
2.0%a 1.1%b 1.4%c 0.3%d 0.1%d
Growth inhibition rate E.coli 21.4 55.4 67.7 98.2 99.0
Aerobic bacteria ± ± ± ± ±
2.8%a 0.6%b 8.5%c 0.5%d 0.2%d
13.1 43.5 74.3 98.9 99.8
± ± ± ± ±
2.1a 3.2b 3.3c 0.3d 0.4d
Mold and yeast 22.9 55.5 81.5 95.7 99.4
Mean values ± standard deviation (n = 3). Different letters in the same column indicate significant differences (p < 0.05). 694
± ± ± ± ±
1.9a 3.4b 1.7c 0.6d 1.0e
11.2 27.0 69.1 90.0 98.8
± ± ± ± ±
1.6a 2.8b 3.8c 2.5d 1.0e
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Fig. 7. Images of wheat bread samples (A) without and with (B) CMC, (C) CA-NEs-1.5@CMC, (D) CA-NEs-2.5@CMC, (E) CA-NEs-4.0@CMC and (F) CA-NEs5.0@CMC films protection after storage at 25 °C for 7 days.
ability to extend the shelf life of wheat bread. In addition, we anticipate that the distinct advantages of the present method for the preparation of CA-NEs and CMC microgels, and loading of CA-NEs into CMC microgel films will include the following: (i) The low-energy spontaneous emulsification method with essential oils itself as the oil phase without introducing carrier oil for the preparation of physically stable O/W nanoemulsions, which possesses the advantages of simple production procedure and low cost. (ii) A solvent exchange method using ethanol as the poor solvent to fabricate polymeric microgels, which not only is facile and effective, but also can adjustment physical properties of the microgels system, such as surface tension. (iii) Different from the solution blend, immersing dry films in nanoemulsions solution for loading of nanoemulsions into polymeric microgel films, which does not alter initial physical properties and performance of nanoemulsions, such as size, biological activity, etc. More importantly, we believe that the present methods to prepare essential oil nanoemulsions, polymeric microgels and films, as well as polymeric films loaded with nanoemulsions for active packaging can be extended to other types of polymers and antimicrobial essential oils. The results reported in this work have important implications for the preparation and utilization of essential oil nanoemulsions and polymeric microgels in the food and other industries.
microbial growth was observed in bread samples. The growth inhibition rate of CA-NEs-x@CMC films on aerobic mesophilic bacteria and mold and yeast was also researched. As shown in Table 1, the growth inhibition of both aerobic mesophilic bacteria and mold and yeast in bread samples increased with the increasing of carvacrol content in the CA-NEs-x@CMC films. The results confirmed what have been shown in the photographs of bread samples in Fig. 7. Especially, CA-NEs-5.0@CMC films showed the excellent inhibitory effects on aerobic mesophilic bacteria, mold and yeast, and exhibited 99.8%, 99.4% and 98.8% growth inhibition rate against aerobic bacteria, mold and yeast, respectively, whereas pure CMC films showed very low inhibitory effects on the above bacteria. It has been reported that the inhibitory effect of carvacrol on bacteria is attributed to the alteration of cell membrane permeability by the action of phenols [38]. On the other hand, the effects of released carvacrol into headspace on organoleptic properties of bread were also investigated. The smell and color of bread samples with CA-NEs-5.0@CMC film protection were monitored everyday. The results showed that in 3 days before the storage, there was no carvacrol’s smell in bread samples, while from the fourth day to the seventh day, only slight carvacrol’s scent exhaled from the bread samples, indicating that a small number of carvacrol could migrate into bread; in addition, color of the bread samples had hardly any change within 7 days (image data not shown). The results above demonstrated the CA-NEs-x@CMC films are promising alternatives as food packaging to prolong shelf life of bread.
Acknowledgment This work was supported by the National Natural Science Foundations of China (Grant No. 21808188) and the Natural Science Foundation of Shaanxi Province (Grant No. 2017JQ5043).
4. Conclusion In summary, the facile and innovative strategies established in this work to fabricate CA-NEs, CMC microgels followed by subsequent loading of CA-NEs into the CMC films were all feasible. The CANEs@CMC films obtained exhibited good antioxidant activity, desirable antibacterial activity against S. aureus and E. coli, as well as superior
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2018.12.054. 695
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