Antimicrobial films prepared with e

Antimicrobial films prepared with essential oil emulsions often have a high degree of opacity. This may be prevented using transparent microemulsions. The objective of this study was to characterize physical, mechanical, and antimicrobial properties of films prepared from mixtures with 1% w/w chitosan solution and microemulsions containing 1:0, 2:1, and 4:1 mass ratios of cinnamon bark oil (CBO) (1, 2 and 3% w/ w) and soybean oil. Changes in solvent polarity after mixing chitosan solution and microemulsions increased droplet dimension from 88 nm of the filmeforming mixtures and induced different extents of coalescence after film formation. Despite these physical changes, films prepared from microemulsions were transparent and had low opacity. The incorporation of micro- emulsions increased the thickness and water vapor permeability of films and significantly reduced the moisture content and swelling ratio. The retention of CBO was improved for films prepared from microemulsions with 2 and 3% CBO immediately following film formation and after ambient storage, when compared to control films prepared with emulsions with less Tween 80. Large zones of inhibition against foodborne pathogens were observed for film discs prepared with 2 and 3% CBO. These charac- teristics show the potential of using microemulsions as an easy approach to incorporate EOs in biopolymer antimicrobial films to improve microbiological safety and film transparency.

Introduction

Fresh produce is perishable and is sometimes associated with outbreaks of foodborne illnesses due to contamination by microbial pathogens (CDC, 2012a, 2012b). Many strategies to improve the microbiological safety and quality of fresh produce have been studied, including films or coatings with and without antimicro- bials. Polysaccharides (Perdones, Vargas, Atare s, & Chiralt, 2014; Zivanovic, Chi, & Draughon, 2005), proteins (Go mez-Estaca, Montero, & Go mez-Guille n, 2014; Moditsi, Lazaridou, Moschakis, & Biliaderis, 2014) and lipids (Arcan & Yemeniciog!lu, 2013) have been utilized as edible filmeforming or coating materials. Chitosan, a copolymer consisting of b-(1e4)-2-acetamido-D-glucose and b- (1e4)-2-amino-D-glucose units, is an excellent filmeforming ma- terial derived from chitin by N-deacetylation (Domard & Domard, 2001; Elsabee & Abdou, 2013). Chitosan films have good mechanical properties and a selective permeability (higher permeability to CO2 and lower to O2) (Despond, Espuche, & Domard, 2001; Elsabee et al., 2013). In addition, chitosan itself has antibacterial and antifungal activity (Kim, Thomas, Lee, & Park, 2003; Tsai, Su, Chen, & Pan, 2002). Thus, coating with chitosan may be a good strategy to improve the microbiological safety and quality of fresh produce (Sangsuwan, Rattanapanone, & Rachtanapun, 2008). Various antimicrobials have been incorporated into films and plant essential oils (EOs) are frequently studied due to their broad antimicrobial activity (Chen, Zhang, & Zhong, 2014; Ma, Davidson, & Zhong, 2013). Incorporating EOs into chitosan films has been shown to enhance antimicrobial activity and lower water vapor permeability (WVP) (Ojagh, Rezaei, Razavi, & Hosseini, 2010; Pereda, Amica, & Marcovich, 2012; Zivanovic et al., 2005). How- ever, adding EOs into chitosan films can also increase the opacity thus affecting the appearance of products (Hosseini, Razavi, & Mousavi, 2009; Pereda et al., 2012). This largely results from the low-water solubility of EOs that form particulate structures in the film to scatter visible light. In addition, the volatile nature of EOs causes their significant loss during film formation and storage (Chi, Zivanovic, & Penfield, 2006). Much work is still needed to improve the properties of antimicrobial films/coatings with EOs. Colloidal systems are used to improve various functional prop- erties of oil and water mixtures. Microemulsions are thermody- namically stable isotropic mixtures of water, oil, surfactants, and co-surfactants (Danielsson & Lindman, 1981). Microemulsions are transparent because their droplets are from 1 to 100 nm, typically 10e50 nm (Moulik & Paul, 1998; Slomkowski et al., 2011). The interfacial tension in microemulsions is very low, which enables their easy preparation without using high mechanical energy as in conventional emulsification (Klossek, Marcus, Touraud, & Kunz, 2014). Therefore, transparent films may be obtained by incorpo- rating EO microemulsions in a biopolymer matrix. Furthermore, many fresh produce products, such as cantaloupes, have irregular and rough surfaces and entrapment of bacteria in the cavities on the produce surface can reduce or eliminate the effectiveness of antimicrobial films and coatings. This was demonstrated in a study on the influence of surface roughness of fresh produce on the adhesion rate of Escherichia coli O157:H7 (Wang, Feng, Liang, Luo, & Malyarchuk, 2009). A positive linear correlation was found be- tween adhesion rate of the bacterium and surface roughness but a negative correlation existed between the surface roughness and inactivation efficacy by acidified electrolyzed water and peroxy- acetic acid. In another study, the improved inactivation of E. coli O157:H7 on spinach leaves was reported after adding the surfac- tant, sucrose monolaurate, to a sodium hypochlorite wash solution (Xiao et al., 2011). This study illustrated that surfactants can lower the solid/liquid interfacial tension to facilitate access of antimi- crobials to bacteria which are protected by the heterogeneous structures of fresh produce. Because surfactants are a part of microemulsions, coatings prepared from EO microemulsions may have the potential to enhance antimicrobial activity. Additionally, microemulsions can be formulated to dissolve long-chain tri- acylglycerols such as soybean oil (SBO) that may change evapora- tion properties of volatile compounds (Kim, Wu, Kubota, & Kobayashi, 1995) and film properties. In a previous study (Ma & Zhong, 2015), we formulated micro- emulsions with an oil phase consisting of various mass ratios of cinnamon bark oil (CBO) and SBO using polysorbate 80 (TweenTM 80) as the surfactant and an equal mass of water and propylene glycol (PG) as the polar phase. The objective of the present work was to characterize physical, mechanical, and antimicrobial prop- erties of films cast from mixtures of chitosan solution and the microemulsions formulated with various mass ratios of CBO and SBO.


Materials and methods

2.1. Materials Low molecular weight chitosan (75e85% deacetylated) and CBO were purchased from SigmaeAldrich Corp. (St. Louis, MO). PG, SBO, glycerol, acetic acid, tryptic soy broth (TSB) and TweenTM 80 were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA).

2.2. Preparation of microemulsions Microemulsions were prepared by simple mixing as previously described (Ma & Zhong, 2015). Microemulsions contained TweenTM 80, a polar phase with equal mass of PG and water, and an oil phase comprised of CBO and SBO at mass ratios of 1:0, 2:1, or 4:1 (abbreviated as microemulsion 1:0, 2:1, and 4:1, hereafter). Specific compositions of microemulsions are listed in Table 1. TweenTM 80, the oil phase and the polar phase were added together and mixed by hand shaking until a transparent appearance with no further visible changes.

2.3. Film preparation A chitosan stock solution was prepared at 2% w/w in 1% w/w acetic acid solution. The impurities were removed by filtering the solution through a microcloth (CalbiochemeNovabiochem Corp., San Diego, CA). Microemulsions were mixed with the 2% w/w chitosan stock solution, glycerol (at 20% mass of chitosan), and deionized water to final CBO concentrations of 1, 2, and 3% w/w. The final concentrations of chitosan and acetic acid were 1% and 0.5% w/ w, respectively. Films were prepared by casting 30 g of the mixtures on 17.8 cm 17.8 cm glass plates and drying at ambient conditions (21 C) for 24 h. Unless stated otherwise, the dried films were peeled and conditioned for about 48 h at room temperature in a desiccator with 57% relative humidity (RH) controlled by a satu- rated sodium bromide solution prior to physical characterizations. Films without microemulsions were prepared at the same chitosan concentration as a control.

2.4. Hydrodynamic diameters (Dh) of filmeforming mixtures The Dh of fresh filmeforming mixtures was measured using dynamic light scattering. A Delsa Nano analyzer (Beckman Coulter, Atlanta, GA) with a scattering angle of 165 was used. Measure- ments were done in triplicate for each sample.

2.5. Viscosity of filmeforming mixtures The viscosities of filmeforming mixtures were measured using an AR2000 rheometer (TA Instruments, Inc., New Castle, DE). About 20 mL of each sample was loaded into a bobecup geometry with the outer diameter of bob and the inner diameter of cup being 28 and 30 mm, respectively. Shear rate ramps were conducted at a shear rate range of 0.1e100 s 1 at 25 C, and each sample was sheared for 1 min. Each test was repeated at least once.

2.6. Physical and mechanical properties of films
2.6.1. Thickness The thickness of films was measured using a digital microcaliper (Mitutoyo Corp., Kawasaki, Japan) with 0.001 mm precision. A total of 12 points were measured for each film. Means from two film replicates were reported.

2.6.2. Color and opacity The color and opacity of films were measured using a MiniScan XE Plus Hunter colorimeter (Hunter Associates Laboratory, Inc., Reston, VA). The color of films was measured for lightness (L) and chromaticity parameters a (redegreen) and b (yelloweblue) in the Hunter Lab scale. Color measurements were performed over the standard white tile. Opacity was measured over the standard white tile and black glass. For each treatment, two film replicates were measured, each tested in triplicate.
2.6.3. Moisture content and swelling ratio To determine the moisture content (Eq. (1)) and sw

ผลลัพธ์ (ไทย) 1:

ฟิล์มจุลินทรีย์พร้อม emulsions น้ำมันมักจะมีความทึบแสงในระดับสูง นี้อาจจะไม่สามารถใช้ microemulsions โปร่งใส วัตถุประสงค์ของการศึกษานี้คือการ กำหนดลักษณะทางกายภาพ เครื่องจักรกล และคุณสมบัติการต้านจุลชีพของฟิล์มเตรียมจากส่วนผสมกับ 1% w/w ไคโตซานโซลูชัน microemulsions ประกอบด้วย 1:0, 2:1 และ 4:1 อัตราส่วนมวลของเปลือกอบเชยน้ำมัน (CBO) (1, 2 และ 3% พร้อม w) และน้ำมันถั่วเหลือง เปลี่ยนแปลงในตัวทำละลายขั้วหลังผสมไคโตซานโซลูชั่นและ microemulsions หยดเพิ่มขนาดจาก < 30 nm microemulsions ไป > 88 nm ส่วนผสม filmeforming และเหนี่ยวนำให้ขอบเขตโดยรวมแตกต่างกันของ coalescence หลังจากการก่อตัวของฟิล์ม แม้ มีการเปลี่ยนแปลงเหล่านี้มีอยู่จริง ฟิล์มที่เตรียมจาก microemulsions ได้โปร่งใส และมีความทึบแสงน้อย จดทะเบียนของ micro emulsions เพิ่ม permeability ไอน้ำและความหนาของฟิล์ม และลดเนื้อหาความชื้นและอัตราการบวม เก็บรักษาของ CBO ถูกปรับปรุงสำหรับฟิล์มที่เตรียมจาก microemulsions 2 และ 3% CBO ทันทีต่อฟิล์มก่อตัว และหลัง จาก เก็บสภาวะ เมื่อเทียบกับฟิล์มควบคุมพร้อม emulsions กับ Tween 80 น้อย โซนใหญ่ของยับยั้งกับโรค foodborne สุภัคสำหรับดิสก์ฟิล์มพร้อม 2 และ 3% CBO Charac-teristics เหล่านี้แสดงศักยภาพของการใช้ microemulsions เป็นวิธีง่ายเพื่อรวม EOs ในฟิล์มจุลินทรีย์ biopolymer เพื่อปรับปรุงความปลอดภัยทางจุลชีววิทยาและความโปร่งใสฟิล์มแนะนำ Fresh produce is perishable and is sometimes associated with outbreaks of foodborne illnesses due to contamination by microbial pathogens (CDC, 2012a, 2012b). Many strategies to improve the microbiological safety and quality of fresh produce have been studied, including films or coatings with and without antimicro- bials. Polysaccharides (Perdones, Vargas, Atare s, & Chiralt, 2014; Zivanovic, Chi, & Draughon, 2005), proteins (Go mez-Estaca, Montero, & Go mez-Guille n, 2014; Moditsi, Lazaridou, Moschakis, & Biliaderis, 2014) and lipids (Arcan & Yemeniciog!lu, 2013) have been utilized as edible filmeforming or coating materials. Chitosan, a copolymer consisting of b-(1e4)-2-acetamido-D-glucose and b- (1e4)-2-amino-D-glucose units, is an excellent filmeforming ma- terial derived from chitin by N-deacetylation (Domard & Domard, 2001; Elsabee & Abdou, 2013). Chitosan films have good mechanical properties and a selective permeability (higher permeability to CO2 and lower to O2) (Despond, Espuche, & Domard, 2001; Elsabee et al., 2013). In addition, chitosan itself has antibacterial and antifungal activity (Kim, Thomas, Lee, & Park, 2003; Tsai, Su, Chen, & Pan, 2002). Thus, coating with chitosan may be a good strategy to improve the microbiological safety and quality of fresh produce (Sangsuwan, Rattanapanone, & Rachtanapun, 2008). Various antimicrobials have been incorporated into films and plant essential oils (EOs) are frequently studied due to their broad antimicrobial activity (Chen, Zhang, & Zhong, 2014; Ma, Davidson, & Zhong, 2013). Incorporating EOs into chitosan films has been shown to enhance antimicrobial activity and lower water vapor permeability (WVP) (Ojagh, Rezaei, Razavi, & Hosseini, 2010; Pereda, Amica, & Marcovich, 2012; Zivanovic et al., 2005). How- ever, adding EOs into chitosan films can also increase the opacity thus affecting the appearance of products (Hosseini, Razavi, & Mousavi, 2009; Pereda et al., 2012). This largely results from the low-water solubility of EOs that form particulate structures in the film to scatter visible light. In addition, the volatile nature of EOs causes their significant loss during film formation and storage (Chi, Zivanovic, & Penfield, 2006). Much work is still needed to improve the properties of antimicrobial films/coatings with EOs. Colloidal systems are used to improve various functional prop- erties of oil and water mixtures. Microemulsions are thermody- namically stable isotropic mixtures of water, oil, surfactants, and co-surfactants (Danielsson & Lindman, 1981). Microemulsions are transparent because their droplets are from 1 to 100 nm, typically 10e50 nm (Moulik & Paul, 1998; Slomkowski et al., 2011). The interfacial tension in microemulsions is very low, which enables their easy preparation without using high mechanical energy as in conventional emulsification (Klossek, Marcus, Touraud, & Kunz, 2014). Therefore, transparent films may be obtained by incorpo- rating EO microemulsions in a biopolymer matrix. Furthermore, many fresh produce products, such as cantaloupes, have irregular and rough surfaces and entrapment of bacteria in the cavities on the produce surface can reduce or eliminate the effectiveness of antimicrobial films and coatings. This was demonstrated in a study on the influence of surface roughness of fresh produce on the adhesion rate of Escherichia coli O157:H7 (Wang, Feng, Liang, Luo, & Malyarchuk, 2009). A positive linear correlation was found be- tween adhesion rate of the bacterium and surface roughness but a negative correlation existed between the surface roughness and inactivation efficacy by acidified electrolyzed water and peroxy- acetic acid. In another study, the improved inactivation of E. coli O157:H7 on spinach leaves was reported after adding the surfac- tant, sucrose monolaurate, to a sodium hypochlorite wash solution (Xiao et al., 2011). This study illustrated that surfactants can lower the solid/liquid interfacial tension to facilitate access of antimi- crobials to bacteria which are protected by the heterogeneous structures of fresh produce. Because surfactants are a part of microemulsions, coatings prepared from EO microemulsions may have the potential to enhance antimicrobial activity. Additionally, microemulsions can be formulated to dissolve long-chain tri- acylglycerols such as soybean oil (SBO) that may change evapora- tion properties of volatile compounds (Kim, Wu, Kubota, & Kobayashi, 1995) and film properties. In a previous study (Ma & Zhong, 2015), we formulated micro- emulsions with an oil phase consisting of various mass ratios of cinnamon bark oil (CBO) and SBO using polysorbate 80 (TweenTM 80) as the surfactant and an equal mass of water and propylene glycol (PG) as the polar phase. The objective of the present work was to characterize physical, mechanical, and antimicrobial prop- erties of films cast from mixtures of chitosan solution and the microemulsions formulated with various mass ratios of CBO and SBO. Materials and methods 2.1. Materials Low molecular weight chitosan (75e85% deacetylated) and CBO were purchased from SigmaeAldrich Corp. (St. Louis, MO). PG, SBO, glycerol, acetic acid, tryptic soy broth (TSB) and TweenTM 80 were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA). 2.2. Preparation of microemulsions Microemulsions were prepared by simple mixing as previously described (Ma & Zhong, 2015). Microemulsions contained TweenTM 80, a polar phase with equal mass of PG and water, and an oil phase comprised of CBO and SBO at mass ratios of 1:0, 2:1, or 4:1 (abbreviated as microemulsion 1:0, 2:1, and 4:1, hereafter). Specific compositions of microemulsions are listed in Table 1. TweenTM 80, the oil phase and the polar phase were added together and mixed by hand shaking until a transparent appearance with no further visible changes. 2.3. Film preparation A chitosan stock solution was prepared at 2% w/w in 1% w/w acetic acid solution. The impurities were removed by filtering the solution through a microcloth (CalbiochemeNovabiochem Corp., San Diego, CA). Microemulsions were mixed with the 2% w/w chitosan stock solution, glycerol (at 20% mass of chitosan), and deionized water to final CBO concentrations of 1, 2, and 3% w/w. The final concentrations of chitosan and acetic acid were 1% and 0.5% w/ w, respectively. Films were prepared by casting 30 g of the mixtures on 17.8 cm 17.8 cm glass plates and drying at ambient conditions (21 C) for 24 h. Unless stated otherwise, the dried films were peeled and conditioned for about 48 h at room temperature in a desiccator with 57% relative humidity (RH) controlled by a satu- rated sodium bromide solution prior to physical characterizations. Films without microemulsions were prepared at the same chitosan concentration as a control. 2.4. Hydrodynamic diameters (Dh) of filmeforming mixtures The Dh of fresh filmeforming mixtures was measured using dynamic light scattering. A Delsa Nano analyzer (Beckman Coulter, Atlanta, GA) with a scattering angle of 165 was used. Measure- ments were done in triplicate for each sample. 2.5. Viscosity of filmeforming mixtures The viscosities of filmeforming mixtures were measured using an AR2000 rheometer (TA Instruments, Inc., New Castle, DE). About 20 mL of each sample was loaded into a bobecup geometry with the outer diameter of bob and the inner diameter of cup being 28 and 30 mm, respectively. Shear rate ramps were conducted at a shear rate range of 0.1e100 s 1 at 25 C, and each sample was sheared for 1 min. Each test was repeated at least once.
2.6. Physical and mechanical properties of films
2.6.1. Thickness The thickness of films was measured using a digital microcaliper (Mitutoyo Corp., Kawasaki, Japan) with 0.001 mm precision. A total of 12 points were measured for each film. Means from two film replicates were reported.

2.6.2. Color and opacity The color and opacity of films were measured using a MiniScan XE Plus Hunter colorimeter (Hunter Associates Laboratory, Inc., Reston, VA). The color of films was measured for lightness (L) and chromaticity parameters a (redegreen) and b (yelloweblue) in the Hunter Lab scale. Color measurements were performed over the standard white tile. Opacity was measured over the standard white tile and black glass. For each treatment, two film replicates were measured, each tested in triplicate.
2.6.3. Moisture content and swelling ratio To determine the moisture content (Eq. (1)) and sw

ผลลัพธ์ (ไทย) 3:

ฟิล์มต่อต้านจุลินทรีย์เตรียมอิมัลชันน้ำมันมักจะมีระดับสูงของความทึบ นี้อาจป้องกันโดยใช้ไมโครอิมัลชันที่โปร่งใส การวิจัยครั้งนี้มีวัตถุประสงค์เพื่อศึกษาสมบัติทางกายภาพทางกลและการดื้อยาต้านจุลชีพของเชื้อผสมกับฟิล์มที่เตรียมจากสารละลายไคโตซาน 1 % w / w และ ไมโครอิมัลชันที่ประกอบด้วย 1 : 0 , 2 : 1 และ 2 :1 มวลอัตราส่วนของน้ำมันเปลือกอบเชย ( CBO ) ( 1 , 2 และ 3 % w / w ) และน้ำมันถั่วเหลือง การเปลี่ยนแปลงในตัวทำละลายขั้วหลังจากผสมสารละลายไคโตแซนไมโครอิมัลชันเพิ่มขึ้นมิติและหยดจาก < 30 nm ของไมโครอิมัลชันเพื่อ > 88 nm ของ filmeforming แตกต่างกัน extents ผสมและการรวมตัวหลังจากการก่อตัวของฟิล์ม แม้จะมีการเปลี่ยนแปลงทางกายภาพเหล่านี้ฟิล์มที่เตรียมจากไมโครอิมัลชันมีความโปร่งใสและมีตัวเลือกน้อย การรวมตัวของไมโครอิมัลชันจะทำให้ความหนาของฟิล์มและลดการซึมผ่านของไอน้ำและความชื้นและมีอัตราส่วนการบวมน้ำ การคงอยู่ของการปรับปรุงสำหรับฟิล์มที่เตรียมจาก CBO CBO ปี 2 และ 3% ทันทีต่อไปนี้ภาพยนตร์รูปแบบและหลังจากรอบกระเป๋า ,เมื่อเทียบกับการควบคุมภาพยนตร์เตรียมอิมัลชันที่มีน้อยกว่า Tween 80 โซนขนาดใหญ่ของการต่อต้านเชื้อโรคอาหารเป็นพิษที่พบในแผ่นภาพยนตร์เตรียม 2 และ 3% CBO . เหล่านี้ charac - teristics โชว์ศักยภาพของการใช้อนุภาคเป็นวิธีการที่ง่ายในการรวมฟังก์ชั่นในฟิล์มต่อต้านจุลินทรีย์ ไบโอโพลีเมอร์เพื่อปรับปรุงคุณภาพและความปลอดภัยเบื้องต้น


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