Antimicrobial and Mechanical Properties of β-Cyclodextrin Inclusion

Aug 14, 2014 - Agriculture (USDA), 2001 South Rock Road, Fort Pierce, Florida 34945, United States. ABSTRACT: Chitosan films incorporated with various...
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Antimicrobial and Mechanical Properties of β‑Cyclodextrin Inclusion with Essential Oils in Chitosan Films Xiuxiu Sun,† Siyao Sui,‡ Christopher Ference,§ Yifan Zhang,† Shi Sun,† Ninghui Zhou,† Wenjun Zhu,† and Kequan Zhou*,† †

Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan 48202, United States School of Biological and Agricultural Engineering, Jilin University, Number 5988 Renmin Street, Changchun, Jilin 130025, People’s Republic of China § United States Horticultural Research Laboratory (USHRL), Agricultural Research Service (ARS), United States Department of Agriculture (USDA), 2001 South Rock Road, Fort Pierce, Florida 34945, United States ‡

ABSTRACT: Chitosan films incorporated with various concentrations of the complex of β-cyclodextrin and essential oils (βCD/EO) were prepared and investigated for antimicrobial, mechanical, and physical properties. Four bacterial strains that commonly contaminate food products were chosen as target bacteria to evaluate the antimicrobial activity of the prepared films. The incorporation of β-CD/EO significantly increased the antimicrobial activities of the chitosan films against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes. It was also found that tensile strength (TS) of chitosan film was significantly increased with the incorporation of the β-cyclodextrin and 0.75% essential oils complex. The elongation at break (EB) decreased with the increasing concentrations of essential oils. Inclusion of the complex of β-cyclodextrin and 0.25% essential oils also significantly decreased water vapor permeability (WVP) of chitosan films. Our results suggest that chitosan films containing β-CD/EO could be used as active food-packaging material. KEYWORDS: chitosan, β-cyclodextrin, essential oils, antimicrobial activity, mechanical properties, physical properties



INTRODUCTION Foodborne illness, because of its potential to be lifethreatening, has been given ever increasing attention. It is a major problem with enormous associated costs.1 Some important pathogens, such as Escherichia coli and Salmonella typhimutium, are main causes of foodborne illnesses. The use of antimicrobial agents in food packaging can play an important role in the controlling of foodborne illnesses.2 Nowadays, chemicals are commonly used as commercial preservatives in the food industry.3 Even though chemical preservatives have become widely accepted, the undesirable side effects cannot be ignored.4 Concerns regarding food safety and health standards have led to increased consumer awareness of the presence of chemical residues in the food products.5 Attention has shifted to naturally occurring substances, such as plant extracts, because of negative consumer perception of chemical preservatives.6 Plant essential oils, such as carvacrol (CAR), transcinnamaldehyde (CIN), and eugenol (EUG), are good antimicrobial compounds.7 Chitosan film incorporated with 1.5% CAR showed antibacterial activity against S. typhimurium and E. coli O157:H7.8 The incorporation of 0.4% cinnamon essential oil improved the antibacterial properties of chitosan films against Listeria monocytogenes, E. coli, Lactobacillus plantarum, Lactobacillus sakei, and Pseudomonas fluorescens.9 However, the volatile property of essential oils limits their application in the food industry. Cyclodextrins are a family of compounds made up of α-Dglucopyranoside units bound together in a ring.10 One of the important characteristics of cyclodextrins is the formation of © XXXX American Chemical Society

inclusion compounds in both the solution and solid states, in which each guest molecule is surrounded by the hydrophobic environment of the cyclodextrin cavity.11 This can lead to changes of physical, chemical, and biological properties of guest molecules and can eventually have considerable pharmaceutical potential.12 The α-, β-, and γ-cyclodextrins are widely used natural cyclodextrins, consisting of six, seven, and eight Dglucopyranose residues, respectively, linked by R-1,4 glycosidic bonds into a macrocycle.12 Cyclodextrins can mask the flavor of essential oils to be used as antimicrobials and can also protect them against oxidation or heat damage, allowing for the essential oils to remain effective as antimicrobial agents under a wide variety of environmental conditions and for longer time periods.13 The inclusion of essential oils into cyclodextrins with a very high incorporation rate has been investigated.11 However, to our knowledge, the inclusion of essential oils with cyclodextrins into chitosan film has not been studied thus far. The objective of this work was to develop β-cyclodextrin inclusion with essential oils in chitosan edible films and study their antimicrobial, mechanical, and physical properties.



MATERIALS AND METHODS

Materials. Chitosan (95−98% deacetylated, CS), β-cyclodextrin (β-CD), and glacial acetic acid (99%, analytical reagent grade) were obtained from Sigma-Aldrich Co. (St. Louis, MO). Glycerol, as a Received: June 11, 2014 Revised: August 14, 2014 Accepted: August 14, 2014

A

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Table 1. Antimicrobial Activity of β-CD/EO in CS Filmsa EO

concentration (%)

CIN

0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1 0 0.25 0.5 0.75 1

CAR

EUG

a

E. coli 0.51 1.28 1.59 1.79 2.11 0.51 1.38 1.99 2.08 2.24 0.51 1.07 1.35 1.51 1.69

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.12 0.03 0.21 0.19 0.06 0.13 0.54 0.52 0.13 0.06 0.03 0.01 0.03 0.04

S. typhimurium d c b,c a,b a b a a a a e d c b a

0.69 0.94 1.19 1.42 1.88 0.69 0.87 1.08 1.31 1.82 0.69 0.43 0.61 0.75 1.13

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.12 0.01 0.02 0.27 0.03 0.12 0.06 0.35 0.11 0.56 0.12 0.12 0.05 0.14 0.04

S. aureus

e d c b a c b,c b,c a,b a c b b b a

0.82 1.54 1.78 1.83 2.52 0.82 1.58 2.01 2.24 2.78 0.82 1.48 1.65 1.75 2.04

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.12 0.25 0.04 0.14 0.07 0.12 0.15 0.24 0.29 0.11 0.12 0.12 0.03 0.11 0.03

L. monocytogenes d c b b a d c b b a e d c b a

0.71 0.87 0.97 1.13 1.31 0.71 0.88 0.97 1.18 1.30 0.71 0.70 0.74 0.76 0.81

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.02 0.06 0.04 0.14 0.04 0.02 0.08 0.04 0.14 0.04 0.04 0.07 0.07 0.01

e d c b a e d c b a d d c b a

Within each column and for each essential oil, means having the same letters are not significantly different (p > 0.05) from each other.

plasticizing agent, and CAR, CIN, and EUG, which are antimicrobial agents, were purchased from Fisher Scientific, Inc. (Pittsburgh, PA). All other chemicals and solvents were of analytical grade. Preparation of Essential Oils and β-CD Complex. The complex of β-CD and essential oils (β-CD/EO) was prepared using a coprecipitation method described by Ayala-Zavala et al.,14 with minor modification. Briefly, 5 g (± 0.01) of β-CD was dissolved in 100 mL of an ethanol/distilled water (1:2, v/v) mixture and maintained at 60 ± 2 °C on a hot plate. After cooling the β-CD solution to 40 ± 2 °C, a portion of 0.5 g of EO dissolved in ethanol (1:1, v/v) was then slowly added to the solution with continuous agitation. The resultant mixture was treated by ultrasonic cleaners at 90 W for 4 h. The final solution was maintained overnight at 4 °C. The cold precipitated β-CD/EO complex was recovered by vacuum filtration. The precipitate was washed twice with 30% ethanol solution to clear EO, which was absorbed on the surface of β-CD and dried in a vacuum oven at 40 °C for 4 h until the weight was kept constant. The final dry complex powders were stored in an airtight glass desiccator at room temperature for further analysis. Film Preparation. The edible films were prepared by dissolving 1% (w/v) of CS in a 1% acetic acid solution and stirred, at room temperature, until CS was completely dissolved. Glycerol at 30% (w/w of CS) was added as a plasticizer. Films without β-CD/EO were designated as the control sample. To make the antimicrobial films, a constant amount (150 mL) of the film solutions with varying concentrations of β-CD/EO (0.25, 0.5, 0.75, and 1%) were spread in a glass plate (200 × 200 mm2) and dried at 35 ± 2 °C in an incubator (New Brunswick Scientific Excella E24, Fisher Scientific, Inc., Pittsburgh, PA). The films were removed from the glass plate with a thin spatula and conditioned at 23 ± 2 °C and 50 ± 2% relative humidity (RH) before running further tests.15 Bacterial Strains and Cultures. Four types of bacterial strains, E. coli ATCC 25922 and S. typhimurium ATCC 19585 were used representing Gram-negative bacteria. Staphylococcus aureus NCTC 8325 and L. monocytogenes ATCC 23074 were used representing Gram-positive bacteria. They were incubated in nutrient broth media at 37 °C for 24 h. Antimicrobial Activity. Antimicrobial properties of the crafted films were determined by the log reduction method, with a slight modification.16 Briefly, culture medium broth was inoculated with 1% suspension of bacteria. The bacterial concentration in the seeding culture was approximately 6 × 108 colony forming units (CFU)/mL. Serial dilutions of the suspension were performed, and the optical density values were tested to achieve a standard curve. Square film pieces (20 × 20 mm2) were sterilized and introduced into a test tube containing 5 mL of fresh suspension of bacteria and incubated at 37 °C for 24 h. The optical density of culture media was measured at 620 nm using a PerkinElmer HTS 7000 Bio Assay reader, and cell

concentrations were determined. All samples/standards were run in triplicate. Film Thickness (FT).17 FT was measured with a 0−25 mm dial thickness gauge with an accuracy of ± 0.01 mm in five random locations for each film. The average was calculated for mechanical properties, water vapor permeability (WVP), and oxygen permeability. Mechanical Properties. Tensile strength (TS) and elongation at break (EB) tests were performed at room temperature (23 ± 2 °C) using a universal testing machine [PARAM XLW (B) Auto Tensile Tester, Jinan, China] according to the standard testing method ASTM D882-01. Sample films, previously equilibrated at 23 ± 2 °C and 50 ± 2% RH, were cut into strips 15 mm wide and 130 mm long. Five specimens from each film were tested. The initial grip separation and mechanical crosshead speed were set at 80 mm and 50 mm/min, respectively.18 TS19 was calculated using the following equation: TS = Fmax /A where Fmax is the maximum load (N) needed to pull the sample apart and A is the cross-sectional area (m2) of the samples. EB (%) was calculated using the following equation:

EB = (L /80) × 100 where L is the film elongation (mm) at the moment of rupture and 80 is the initial grip length (mm) of samples. WVP. The WVP of the films was determined by a WVP tester (PERME TSY-TIL, Labthink Instruments Co., Ltd., Jinan, China) according to the standard testing method ASTM E-96-95. Test cups were 2/3 filled with distilled water. The circular sample was tightly covering the cup. The different water vapor pressures between the inside and outside of the cup causes the water vapor diffusion through the sample. Water vapor transmission rates were determined at 23 ± 2 °C and 50 ± 2% RH using saturated salt solutions of MgCl2. The weight of the cups was measured at 1 h intervals for 24 h to determine the water vapor transmission rate and transmission coefficient. For each sample, at least five replicates were tested. Statistical Analysis. Analysis of variance (ANOVA) was carried out by SPSS software (version 17). A significance level was set at 0.05.



RESULTS AND DISCUSSION Antimicrobial Activity. The antimicrobial activity of βCD/EO in CS films against E. coli, S. typhimurium, S. aureus, and L. monocytogenes was tested, and the results are shown in Table 1. The CS films incorporated with different concentrations of β-CD/EO significantly improved the antimicrobial activities of the CS film against all of the tested bacteria (p < B

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0.05). The log reduction increases with the increase of the βCD/EO concentration, which illustrates the antimicrobial activity of β-CD/EO. The results show that the log reductions for β-CD/CIN CS and β-CD/CAR CS films were higher than β-CD/EUG CS films. The β-CD/CIN CS films at 1% CIN could reduce the number of E. coli and S. aureus up to 2.11 and 2.52 logs, respectively. The β-CD/CAR CS films showed the best inhibition to S. aureus, and the log reduction of it was about 3. β-CD/CAR CS films were able to inhibit E. coli and S. aureus growth significantly. Essential oils capable of preventing the growth of foodborne bacteria have been well-documented.7 Studies have shown that essential oils are able to disrupt and penetrate the lipid structure of the cell membrane and the mitochondrial membrane, leading to the disruption of the cell membrane, cytoplasmatic leakage, cell lysis, and eventually, cell death.20 It has been suggested that the antimicrobial activity of essential oils may be related to their involvement with enzymatic reactions, which regulate wall synthesis.21 The lipophilic properties of essential oil components might have also aided its ability to penetrate the plasma membrane. 22 The observations made with light microscopy are in accordance with previous studies, in which essential oils of aromatic plants caused morphological alterations on the fungal hyphae.21 The antibacterial activities of CIN, EUG, and CAR against E. coli were compared in a previous study, and the minimum inhibitory concentrations (MIC) of CIN and CAR were 400 mg/L; however, MIC of EUG was 1600 mg/L, indicating that EUG has weaker antimicrobial activity against E. coli.23 It is possible that this resulted from the lower aqueous solubility of EUG that would prevent maximum contact with the pathogens in solution.24 CIN may act upon the pathogens without being fully released from the inclusion complex.25 The inhibition zones of CAR and EUG against S. typhimurium were larger than L. monocytogenes indicating that L. monocytogenes was more resistant to CAR and EUG than S. typhimurium.24 E. coli, L. monocytogenes, and S. typhymurium were destroyed completely by four essential oils at concentrations of ≤ 0.8% (v/v), whereas S. aureus was destroyed completely by 26 essential oils at concentrations of ≤ 0.4% (v/v), demonstrating that S. aureus was the most sensitive bacteria to essential oils.26 Our results shown in Table 1 are in agreement with those results. The β-CD/EO complexes were shown to be able to inhibit Salmonella enterica and Listeria innocua at low active compound concentrations, and the MIC values for free essential oils, such as EUG and CIN, were significantly higher than β-CD/EO complexes (p < 0.05), which is probably due to the increased water solubility of β-CD/EO complexes that led to increased contact between pathogens and essential oils, thus improving the antimicrobial activity of EO at lower concentrations of active compound.11 The cinnamon bark and clove bud extract β-CD complexes were the most powerful antimicrobials, despite showing the lowest entrapment efficiencies among the oils.11 Results suggest that the application of these antimicrobial complexes in food systems may be effective at inhibiting pathogens. Furthermore, the films showed a higher effectiveness against S. aureus compared to E. coli and S. typhimurium, which may be due to the characteristic difference of the outer membrane between Gram-positive bacteria and Gram-negative bacteria.27 It was reported that Gram-negative bacteria were more resistant to the essential oils.6 However, L. monocytogenes (Gram-positive

bacteria) was more resistant than other bacteria tested, which is in agreement with the previous work.24 Mechanical Properties. The results of TS and EB are shown in Figures 1 and 2. The effect on these properties of the type and concentration of β-CD/EO in CS films were evaluated.

Figure 1. TS of β-CD/EO in CS films at four different concentrations (0.25, 0.5, 0.75, or 1%) of β-CD/EO (CIN, CAR, or EUG) were incorporated into 1% chitosan solution, and the one without β-CD/ EO was taken as the control.

Figure 2. EB of β-CD/EO in CS films at four different concentrations (0.25, 0.5, 0.75, or 1%) of β-CD/EO (CIN, CAR, or EUG) were incorporated into 1% chitosan solution, and the one without β-CD/ EO was taken as the control.

The CS control film had TS and EB values of 17.82 ± 3.75 MPa and 31.46 ± 5.31%, respectively (Figures 1 and 2). These values are comparable to the previous reports with TS and EB in the range of 12−20 MPa and 17−42%, respectively.28 The addition of β-CD/EO to the CS films produced changes in their mechanical properties. The films exhibited the highest TS when the concentration of β-CD/EO was 0.75% (Figure 1). However, the EB was significantly (p < 0.05) reduced by the incorporation of β-CD/EO at all four concentrations. The EB values of the films decreased significantly (p < 0.05) with increasing β-CD/EO concentrations (Figure 2). The TS and EB of CS films are affected by the type of CS used, the presence of glycerol, and the temperature during film drying.29 Interestingly, the incorporation of 0.75% β-CD/EO C

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into CS films significantly increased the TS of the films with 1% β-CD/EO (p < 0.05). The addition of a relatively lower dose of β-CD/EO (0.75%) exhibited the highest TS among the films, which could be attributed to the formation of intermolecular hydrogen bonding between NH3+ of the CS backbone and OH− of β-CD/EO.30 The intermolecular hydrogen bonding between CS and β-CD/EO could enhance the cross-linkage, which decreases the molecular mobility and the free volume of CS.31 This phenomenon was reported by other researchers in similar systems. For example, the cross-linking of microcrystalline wheat-bran cellulose and soy protein isolate films resulted in an increased TS because of the enhancement of the structural bonds in the polymer network.32 However, when the added concentration of β-CD/EO is higher than 0.75%, the TS of the resulting films decreased with increasing the β-CD/EO concentration. It is possible that the excessive β-CD/EO could aggregate and destroy the inner structure of β-CD/EO and the CS matrix. The decrease of EB values in β-CD/EO in CS films indicated that β-CD/EO acted as fillers for the CS matrix, decreasing the strain of the films. The incorporation of β-CD/EO into the CS film resulted in a strong reaction between fillers and matrix, which decreased EB by the motion restriction of the matrix.10 It has been reported that the filler cellulose whiskers produce a significant decrease in the EB of CS films, indicating that the strong interactions between matrix and filler restricted the motion of the matrix.33 WVP. The effect of different concentrations of EO on the resistance to permeability of water vapor was evaluated for the films, and the results are presented in Figure 3.

create steric hindrance and decrease segmental mobility, restricting water vapor diffusion between the CS matrix and β-CD/EO. The fillers provide an excellent water vapor barrier property because they contain a large number of hydrogen bonds between the matrix, which has a strong holding property to keep water trapped and prevent water diffusion through the films,32 thus reducing WVP values. However, when the concentration of β-CD/EO was higher than 0.5%, the WVP of the film increased (p < 0.05), which may be related to the excessive β-CD/EO scattered in the film, which subsequently decreased the intermolecular forces between polymer chains and increased the free volume and segmental motions.34 In conclusion, it was shown that incorporation of low amounts of β-CD/EO in the CS film significantly improves its antimicrobial properties. The mechanical properties of films incorporated with β-CD/EO in CS were confirmed to be at least as effective for active food packaging as films without the additional incorporation of β-CD/EO in CS. The interaction between CS and β-CD/EO could improve tensile strength of the CS films. The CS films with a lower concentration of βCD/EO improved the barrier properties by reducing WVP. Because of the antimicrobial, mechanical, and physical properties, the films developed could be used as functional food packaging.



AUTHOR INFORMATION

Corresponding Author

*Telephone: +1-313-577-3444. Fax: +1-313-577-8616. E-mail: [email protected]. Funding

The authors recognize and appreciate the financial support from Wayne State University Rumble Fellowship. Notes

The authors declare no competing financial interest.



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Figure 3. WVP of β-CD/EO in CS films at four different concentrations (0.25, 0.5, 0.75, or 1%) of β-CD/EO (CIN, CAR, or EUG) were incorporated into 1% chitosan solution, and the one without β-CD/EO was taken as the control.

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