Complexation of methyl salicylate with β-cyclodextrin and its release characteristics for active food packaging

Abstract

A series of methyl salicylate (MeSA)/β-cyclodextrin (β-CD) inclusion complexes (ICs) were prepared at different MeSA concentrations by the co-precipitation method using methyl salicylate for maintaining the quality of fresh produce. The formation of IC was confirmed through FTIR, ¹H NMR, TGA, and SEM measurements. Among the grades applied, IC with 1:1 grade showed the highest MeSA entrapment efficiency (59%). The release rate of MeSA from an IC was greater at higher temperature and higher relative humidity. In addition, the MeSA release from ICs of all grades followed a diffusive nature and first-order kinetics at 25 °C under all RH conditions, except at 7 °C. These results indicate that the use of a MeSA/β-CD IC in active packaging applications can effective maintain the quality of fresh produce.

Introduction

Cyclodextrin (CD) belongs to the family of cyclic oligosaccharides, which comprise non-reducing d-glucopyranose units linked by α-d-(1 → 4) bonds in a cyclic manner, forming a cage-type molecule. In general, three types of CDs with different numbers of constitute units are reported: α-CD (six units), β-CD (seven units), and δ-CD (eight units), with the corresponding inner cavity diameters being 4.7–5.2, 6.0–6.4, and 7.5–8.3 Ǻ (Crini 2014; Szejtli 1998).

CD, which can be obtained from the enzymatic degradation of starch, is the most essential polysaccharide and is nontoxic in nature. Therefore, it is used in several sectors including the pharmaceutical, food, agriculture, environmental, and chemical industries (Cheng et al. 2019; Devi et al. 2010; Jeon et al. 2012; Li and Purdy 1992; Szente et al. 1999; Szente and Szemán 2013). The inner cavity of CD is dimensionally stable and hydrophobic, and is hydrophilic on the outside (Tanwar et al. 2019). Thus, CD is used for the formation of a “host–guest” interaction with various types of small molecules (Aytac et al. 2016; Bouchemela et al. 2019; Hu et al. 2014; Li et al. 2019). Enhancing the controlled release properties of a hydrophobic molecule in a solid–liquid or solid–gas system is significant, and thus, its application can be considerably expanded (Jin et al. 2018).

Consumers have shown high demand for fresh fruits and vegetables as part of a healthy diet. However, most fresh produce has short shelf life after harvesting, owing to fungal infection and physiological deterioration during distribution (Almenar et al. 2007; Duran et al. 2016), which can be overcome by using an effective processing technology. Active packaging technology is currently being developed to maintain the quality of fresh produce and meet customer demand.

Methyl salicylate (MeSA) is a volatile hydrophobic organic compound produced by various plants (Shulaev et al. 1997). It is used as a fragrant ingredient in the cosmetics industry and can be found in various types of cleaning products. MeSA is also nontoxic and has thus been used as a food additive (Min et al. 2018). Since MeSA has a strong ability to fight against pathogens (Ryals et al. 1996), it can be extremely useful in maintaining the quality of fresh fruit products (Habibi et al. 2019). The treatment process for fresh fruits when applying MeSA is extremely complicated owing to the volatile and hydrophobic nature of this compound; this problems can be effective overcome by using inclusion complexes (ICs).

In this study, we aimed to prepare and characterize a MeSA/β-CD IC. To prepare the MeSA/β-CD IC, we followed a co-precipitation method, and evaluated the entrapment efficiency of MeSA in a β-CD cavity. The formation of MeSA/β-CD ICs was characterized by using Fourier-transform IR (FTIR) spectroscopy, ¹NMR spectroscopy, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM). In addition, we have focused on evaluating the controlled release of MeSA from the MeSA/β-CD IC at different temperatures and relative humidity levels. Based on the experimental results, the MeSA/β-CD IC can be safely applied to an active packaging system containing fresh produce.

Materials and methods

Chemicals

The β-CD (98%) used and ethanol (99.5%) were purchased from Daejung Co., (Gyeonggi, Korea). MeSA (98%) was purchased from Junsei Co., Ltd. (Tokyo, Japan). All the reagents were used as received without any further purification.

Preparation of MeSA/β-CD IC

The MeSA/β-CD IC was prepared according to the following reported co-precipitation method (Abarca et al. 2016; Wang et al. 2011). In this procedure, 10 g of β-CD was added to a 250-mL beaker and mixed with 100 mL of ethanol and distilled water (1:2, v/v) at 60 °C under 1 h of stirring to create a homogeneous mixture. The mixture was then cooled to 40 °C under stirring. Different amounts of MeSA were slowly added to the mixture to obtain β-CD: MeSA molar ratios of 1:0.5; 1:1; 1:2, and 1:5 for the preparation of ICs of different grades, viz. 1:0.5; IC 1:1; IC 1:2, and IC 1:5, respectively, where IC indicates MeSA/β-CD IC. Next, the mixture was stirred for 4 h at 40 °C. The reaction mixture was then left undisturbed overnight at 4 °C. The cold precipitates of MeSA/β-CD ICs were collected via vacuum-filtration and washed with ethanol. The collected precipitates were kept in a hot air oven at 50 °C until a constant weight was obtained. Finally, the product was stored in a desiccator at room temperature for further use. The details of all the parameters used in the ICs applied are listed in Table 1.

Table 1.

Synthesis of MeSA/β-CD ICs and MeSA entrapment in β-CD

Sample name	Compositions		Total MeSA (μL/g dry matter)	Entrapment efficiency (%)
	β-CD (g)	MeSA (mL)
IC 1:0.5	10	0.57	6.29	52.5
IC 1:1	10	1.15	14.24	59.0
IC 1:2	10	2.30	13.31	27.6
IC 1:5	10	5.75	14.89	11.3

Determination of MeSA concentration in MeSA/β-CD ICs

The entrapment efficiency (EE) of MeSA in an IC was determined spectrophotometrically at 305 nm using a UV–Vis spectrometer (V-650, Jasco, Tokyo, Japan) following the standard protocol (Abarca et al. 2016; Hill et al. 2013). During this procedure, 20 mg of the MeSA/β-CD ICs of different grades was placed in a 250-mL glass beaker and mixed with 100 mL of ethanol and distilled water (1:2 v/v) under stirring for 24 h to allow all the entrapped active compounds to be included in the solution. The solution was then centrifuged at 5000 rpm for 15 min to separate β-CD from the MeSA/β-CD IC in the solution and the absorbance of MeSA was monitored at 305 nm. The EE was calculated using the following formula (Abarca et al. 2016):

$$\text{Entrapment}\text{efficiency}(\text{EE})=\frac{\text{Amount}\text{of}\text{entrapped}\text{MeSA}}{\text{Initial}\text{MeSA}\text{amount}}\times 100$$ 1

where “Amount of entrapped MeSA” is the amount of compound present in the IC particles, and “Initial MeSA amount” is the amount of compound initially used to produce such particles.

Controlled release of MeSA from MeSA/β-CD ICs

The controlled release of MeSA from a MeSA/β-CD IC was investigated by following the standard protocol (Li et al. 2007; Nguyen and Yoshii 2018; Zhang et al. 2015) at temperatures of 7 °C and 25 °C, at relative humidities of 27%, 57%, and 95%. The release rate of MeSA was evaluated using a UV–Vis spectrophotometer (V-650, Jasco) at 305 nm. A definite amount (1 mg) of MeSA/β-CD complex powder was taken in multiple watch glasses and spread flatly. Each watch glass was then placed in two different sets of three desiccators each that maintain different relative humidity conditions of 27%, 57%, and 95%, controlled using standard salt solutions of magnesium nitrate, sodium chloride, and potassium sulfate. The two sets of desiccators were then stored at 7 °C and 25 °C for 720 min. After a 180-min interval, 20 mg of the MeSA/β-CD ICs of different grades was collected from each watch glass. Each powder sample was mixed with 10 mL of a water–ethanol mixture (2:1 v/v) solvent with intermittent shaking for 30 min. The upper portion of the solvent was separated by a pipette and collected. The residual amount of MeSA was measured on a UV–Vis spectrophotometer using the separated solvent. The percentage of MeSA release was calculated using the following equation (Nguyen and Yoshii 2018). The experiment was carried out twice for each of the samples.

$$\text{MeSA}\text{Release}\text{Rate}(\text{\%) =}\left(1-\frac{\text{Residual}\text{MeSA}\text{amount}\text{of}\text{ICs}}{\text{Initial}\text{MeSA}\text{amount}\text{of}\text{ICs}}\right)\times 100$$ 2

Characterization of ICs

Fourier-transform infrared spectroscopy (FT-IR)

FT-IR spectra of the MeSA, β-CD, and MeSA/β-CD IC were recorded on a Spectrum 65 FT-IR spectrometer (PerkinElmer Co., Waltham, USA) operated in the attenuated total reflection (ATR) mode, within a wavenumber range from 4000 to 400 cm⁻¹ range using 16 scans at a resolution of 1 cm⁻¹.

¹H Nuclear magnetic resonance (¹H NMR) spectroscopy

The ¹H NMR spectra of MeSA, β-CD, and MeSA/β-CD ICs were recorded on an AVANCE III HD 400 FT-NMR spectrometer (Bruker Biospin, MA, USA) at 400 MHz and 295 K using dimethyl sulfoxide (DMSO). The results were reported as chemical shifts (δ) of the β-CD protons before and after encapsulation.

Thermal analysis of IC

The thermal stabilities of MeSA, β-CD, and MeSA/β-CD IC were investigated using a TGA 4000 thermogravimetric analyzer (Perkin Elmer Co. Ltd., MA, USA). The experiment was conducted in a nitrogen environment by heating 10-mg samples from 30 to 700 °C at a heating rate of 10 °C/min.

Scanning electron microscopy (SEM)

The surface morphology and dimensions of the ICs were examined using a Quanta FEG250 scanning electron microscope (FEI Co. Ltd., Oregon, USA). The samples were prepared by mounting approximately 1 mg of powder onto an aluminum stub. The prepared samples were then coated with a thin layer of Pt/Pd and examined at an accelerating voltage of 30 kV.

Results and discussion

Entrapment efficiency (EE)

The entrapment efficiency was calculated based on the experimental results, which are listed in Table 1. The entrapment efficiency for ICs of different grades ranged between 11.3% (obtained for an IC grade of 1:1) and 59.0% (obtained for an IC grade of 1:5). Another research group reported an EE of 90% for a eugenol/β-CD IC with a ratio of 1:1 prepared by using a freeze drying method at low temperatures (Hill et al. 2013). Our results suggest that the entrapment efficiency of the ICs was increased with increasing MeSA concentration up to a maximum value; any further increase in MeSA concentration reduced the entrapment efficiency. These results are dependent on the chemical structure and physical properties of the guest molecule (Assaf et al. 2016). The molecular volume of a MeSA molecule is 0.209 nm³, and the cavity volume of β-CD is 0.262 nm³ (Crini 2014; Szejtli 1998). Thus, the cavity size of β-CD is sufficient for the entrapment of a MeSA molecule. MeSA is an aromatic molecule bearing polar hydroxyl and ester groups, and a nonpolar methyl group. The entrapment phenomenon is dependent on the dipole–dipole and van der Waals interactions that can develop inside the β-CD cavity due to the substitutes on a MeSA molecule. The vapor pressure of a guest molecule is an important parameter for the preparation of ICs because the entrapment efficiency is dependent upon the vapor pressure. The vapor pressure of MeSA was 1 mmHg at 54 °C, which is favorable for evaporation during the synthesis period and the drying steps (Abarca et al. 2016). This difference in vapor pressure can be associated with the loss of a MeSA molecule owing to volatilization during the complexation process and the drying step, which were carried out in an open container at room temperature.

FT-IR analysis

FI-IR analysis is a popular technique for the confirming the formation of an IC. The FT-IR spectra of β-CD, MeSA, and the MeSA/β-CD ICs of different grades are shown in Fig. 1(A). The spectrum of β-CD showed absorption bands at 3320 cm⁻¹ for O–H stretching, 2918 cm⁻¹ for C–H stretching, 1648 cm⁻¹ for H–O–H bending, 1151 cm⁻¹ for C–O stretching, and 1013 cm⁻¹ for C–O–C stretching vibrations. The MeSA spectrum shows prominent absorption bands at 3186 cm⁻¹ for hydroxyl group stretching vibration, 2958 cm⁻¹ for C–H stretching, 1668 cm⁻¹ for the carbonyl group, and 1616–1440 cm⁻¹ for the C=C stretching vibrations. However, all the spectra of the MeSA/β-CD ICs of different grades were dissimilar to the MeSA spectrum. The absorption bands at 3320 and 2916 cm⁻¹ were shifted to slightly lower frequencies for ICs of the all the grades due to the formation of intra-molecular hydrogen bonds between the functional groups of a MeSA molecule with the functional groups of β-CD (Wang et al. 2011). Therefore, the MeSA molecule was entrapped inside the β-CD cavity via intra-molecular interactions with different functional groups.

Fig. 1.

(A) FTIR spectra and (B) ¹NMR spectra of β-CD, MeSA, and MeSA/β-CD ICs of different grades

¹H NMR analysis

The ¹H NMR spectra provide direct evidence for the formation of the IC. If a guest molecule of MeSA is entrapped inside the β-CD cavity, environment inside the β-CD cavity should be changed for the protons of H-3 and H-5. The environment outside the β-CD molecule should remain the same for protons H-1, H-2, and H-4 (Polyakov et al. 2004). This leads to changes in the chemical shift for the inside protons owing to the entrapment of a MeSA molecule inside the β-CD cavity. The ¹H-NMR spectra of the MeSA, β-CD, and MeSA/β-CD (IC 1:5) complexes are shown in Fig. 1(B). The ¹H-NMR spectrum of MeSA showed a chemical shift of the protons. After complexation, the chemical environmental of H_a, H_b, H_d, H_e, and H_f were changed, while H_c remained in the same environment. The H_a, H_d, and H_f protons were shifted to lower ppm, i.e., an upfield zone, after complexation owing to the increase in electron density around the protons inside the β-CD cavity. Furthermore, the H_b and H_e protons were shifted to higher ppm, i.e., a downfield zone, after complexation because of the decrease in electron density around these protons inside the β-CD cavity owing to a hexatomic-ring generated van der Waals force (Yuan et al. 2012). As shown in Fig. 1(B), the chemical environments of the H-3 and H-5 protons of β-CD are changed with Δδ of -0.02 ppm, indicating that the chemical shift changed to a lower ppm, i.e., an upfield zone, after complexation because the electron density around these two protons was increased owing to the entrapment of the MeSA molecule inside the β-CD cavity. After complexation, the outside protons (H-2 and H-4) remained in the same chemical environment. Thus, formation of β-CD/MeSA IC is confirmed by analysis of the ¹H-NMR spectra.

Thermogravimetric analysis

The thermal degradation behaviors of MeSA, β-CD, and the MeSA/β-CD ICs of different grades were determined through TGA. The results are shown in Fig. 2. The pyrolysis patterns of the MeSA/β-CD ICs of different grades differ from that of β-CD. It was confirmed that 90% degradation occurred at 241 °C. A 10% weight loss was observed at 317, 357, 355, 322, and 355 °C, a 20% weight loss was observed at 374, 365, 366, 361, and 366 °C, a 50% weight loss was observed at 383, 376, 376, 373, and 376 °C, and a 90% weight loss was observed at 526, 556, 517, 663, and 517 °C for β-CD, and the IC 1:0.5, IC 1:1, IC 1:2, and IC 1:5 grade complexes, respectively. For each IC complex, the degradation temperatures for 10%, 20%, and 50% weight loss were lower than the corresponding values for β-CD owing to the highly volatile nature of the MeSA molecule. However, the opposite results were obtained for the IC 1:0.5 and IC 1:2 grade complexes, whereas similar trends were observed for the IC 1:1 and IC 1:5 grade complexes. This phenomenon was due to the van der Waals, hydrophobic and dipole–dipole interactions of the functional groups of the MeSA molecule with those inside the β-CD molecule (Liu et al. 2001). Thus, the stability of the IC may increase due to presence of MeSA molecule. The derivative thermogravimetry (DTG) was determined based on the TGA data and the results are shown in Fig. 2(B), which indicates that the degradation of MeSA began at 211.2 °C. The degradation of the inclusion complex started at 309.5 °C, which is lower than the degradation temperature of β-CD (316.8 °C). This phenomenon was due to the volatile nature of the MeSA molecule.

Fig. 2.

Results of (A) thermogravimetric analysis and (B) derivative thermogravimetry of β-CD, MeSA, and MeSA/β-CD ICs of different grades

Scanning electron microscopy

SEM is a qualitative method for determining the surface morphology when studying the structural features of β-CD and a guest molecule. The surface morphology of β-CD and MeSA/β-CD ICs of the different grades are shown in Fig. 3. The SEM morphology of β-CD showed a square structure with a size ranging from 20 to 110 µm. This indicates that the size of the β-CD was not uniform. All the inclusion complexes demonstrated rougher spherical morphologies, indicating that the crystalline nature of the β-CD molecule was upon the formation of IC. The size of each inclusion complex was 30–35 µm, smaller than that of β-CD.

Fig. 3.

SEM images of (A) β-CD, (B) IC 1:0.5, (C) IC 1:1, (D) IC 1:2, and (E) IC 1:5

Controlled release of MeSA from MeSA/β-CD inclusion complexes

The release behavior of MeSA from ICs of different grades was determined at 25 °C and 7 °C, and at different humidity levels of 27%, 57%, and 95%. As shown in Fig. 4, the MeSA release rate gradually increased with increasing relative humidity and temperature. It has been observed (Kant et al. 2004; Yoshii et al. 1994) that water is an essential component for the formation of the ICs between β-CD and the hydrophobic guest molecule MeSA.

Fig. 4.

Controlled release of MeSA for ICs of different grades: (a) 27% RH, (b) 57% RH, and (c) 95% RH at (A) 25 °C and (B) 7 °C

The reaction following the substitution pathway and the reversible behavior of a guest molecule of water in the cavity of a β-CD molecule and vice versa are shown in Eq. (3).

The release of a MeSA molecule from the β-CD cavity was clearly affected by environmental humidity.

$$\mathrm{\beta }{\text{-CD-MeSA + xH}}_2\text{O}\underset{{\text{k}}_2}{\stackrel{{\text{k}}_1}{⟷}}\mathrm{\beta }{\text{-CD-xH}}_{2}O+MeSA$$ 3

Avrami’s equation (Li et al. 2007; Rehmann et al. 2003), i.e., Equation (4), was used for an estimation of the release rate constant.

$$\text{R}\text{or}\frac{{\text{C}}_{\text{t}}}{{\text{C}}_{\text{o}}}=exp[-{(\text{kt})}^{\text{n}}]$$ 4

where R is the percentage retention of MeSA, given by the ratio of C_t to C_o, where C_t is the residual MeSA concentration in the IC at time t and C_o is the initial MeSA concentration in the IC. In addition, k is the rate constant and n is the parameter determining the release mechanism; n = 1, corresponds to first-order release kinetics, whereas n = 0.54 indicates that the diffusion-limited system is followed.

Equation (4) is simplified by taking logarithm on both sides twice, which yields Eq. (5):

$$ln(-ln\text{R})=\text{n}ln(\text{k})+\text{n}ln(\text{t})$$ 5

From Eq. (5), n can be estimated as the slope of the plot of ln(-lnR) along the Y axis and ln(t) along the X axis; k can also be determined from the plot (Shiga et al. 2001).

The data on n and k are summarized in Table 2 for all the β-CD/MeSA ICs of different grades.

Table 2.

n and k values for the release of MeSA from MeSA/β-CD ICs

Sample name	Relative humidity [27%]			Relative humidity [57%]			Relative humidity [95%]
	n	K [S−1] × 10−4	r2	n	K [S−1] × 10−4	r2	n	K [S−1] × 10−4	r2
25 °C
IC 1:0.5	0.582	2.46	0.899	0.806	5.304	0.980	0.805	7.958	0.939
IC 1:1	0.656	3.84	0.920	0.653	9.166	0.955	0.580	9.120	0.944
IC 1:2	0.634	3.88	0.924	0.614	8.705	0.923	0.595	10.37	0.926
IC 1:5	0.542	3.58	0.716	0.520	9.095	0.918	0.648	11.25	0.959
7 °C
IC 1:0.5	0.721	0.622	0.993	0.620	1.13	0.766	1.308	4.17	0.972
IC 1:1	0.924	1.78	0.728	0.455	1.14	0.645	0.756	3.44	0.639
IC 1:2	1.317	4.02	0.973	0.468	0.679	0.944	0.569	1.57	0.750
IC 1:5	0.862	1.83	0.958	0.443	0.736	0.971	0.490	1.31	0.679

The results indicate that the k values of all the grades increased with increasing relative humidity at 25 °C, but the same trends were not obtained for IC 1:1 at 57% and 95% RH. Similar trends were not observed at a lower temperature of 7 °C. This indicates that the MeSA release rate increased with increasing relative humidity for the IC 1:0.5 grade. The k values at 25 °C for all the grades were higher than the corresponding k values at 7 °C, but similar trends were not observed for IC 1:2 at 27% RH. The MeSA release rate was also higher at 25 °C than at 7 °C. This confirmed that the MeSA release rate from the β-CD/MeSA IC increased at 25 °C owing to the volatile nature of MeSA. The n value lies between 1 and 0.54 at 25 °C for all the grades of IC, but IC 1:5 at 57% RH showed n < 0.54. This suggests that MeSA release from the β-CD/MeSA ICs of different grades at 25 °C under all the relative humidity conditions followed diffusion-limited system. At 7 °C, the n value was n > 1 for IC 1:0.5 at 27% and 57% RH, IC 1:1 at 27% and 95% RH, IC 1:2 at 95% RH, and IC 1:5 at 27% RH followed first-order release kinetics. It also showed n < 0.54 for IC 1:0.5 at 27% and 57% RH, IC 1:1 at 27% and 95% RH, IC 1:2 at 95% RH, and IC 1:5 at 27% RH followed diffusion-limited system. These results indicated that MeSA release was diffusion-limited when n < 0.54 and followed first-order release kinetics when n > 1.